# Patent

## Title
Micrococcineae serine protease polypeptides and compositions thereof

## Abstract

The present invention provides novel serine proteases, novel genetic material encoding these enzymes, and proteolytic proteins obtained from Micrococcineae spp., including but not limited to Cellulomonas spp. and variant proteins developed therefrom. In particular, the present invention provides protease compositions obtained from a Cellulomonas spp, DNA encoding the protease, vectors comprising the DNA encoding the protease, host cells transformed with the vector DNA, and an enzyme produced by the host cells. The present invention also provides cleaning compositions (e.g., detergent compositions), animal feed compositions, and textile and leather processing compositions comprising protease(s) obtained from a Micrococcineae spp., including but not limited to Cellulomonas spp. In alternative embodiments, the present invention provides mutant (i.e., variant) proteases derived from the wild-type proteases described herein. These mutant proteases also find use in numerous applications.

## Background
The present application claims priority under 35 U.S.C. §119, to co-pending U.S. Provisional Patent Application Ser. No. 60/523,609, filed Nov. 19, 2003.


SEQUENCE LISTING
The sequence listing submitted via EFS, in compliance with 37 C.F.R. §1.52(e), is incorporated herein by reference. The sequence listing text file submitted via EFS contains the file “SequenceListing_amd” created on May 7, 2012, which is 315,161 bytes in size.
FIELD OF THE INVENTION
The present invention provides novel serine proteases, novel genetic material encoding these enzymes, and proteolytic proteins obtained from Micrococcineae spp., including but not limited to Cellulomonas spp. and variant proteins developed therefrom. In particular, the present invention provides protease compositions obtained from a Cellulomonas spp, DNA encoding the protease, vectors comprising the DNA encoding the protease, host cells transformed with the vector DNA, and an enzyme produced by the host cells. The present invention also provides cleaning compositions (e.g., detergent compositions), animal feed compositions, and textile and leather processing compositions comprising protease(s) obtained from a Micrococcineae spp., including but not limited to Cellulomonas spp. In alternative embodiments, the present invention provides mutant (i.e., variant) proteases derived from the wild-type proteases described herein. These mutant proteases also find use in numerous applications.
BACKGROUND OF THE INVENTION
Serine proteases are a subgroup of carbonyl hydrolases comprising a diverse class of enzymes having a wide range of specificities and biological functions (See e.g., Stroud, Sci. Amer., 131:74-88). Despite their functional diversity, the catalytic machinery of serine proteases has been approached by at least two genetically distinct families of enzymes: 1) the subtilisins; and 2) the mammalian chymotrypsin-related and homologous bacterial serine proteases (e.g., trypsin and S. griseus trypsin). These two families of serine proteases show remarkably similar mechanisms of catalysis (See e.g., Kraut, Ann. Rev. Biochem., 46:331-358 [1977]). Furthermore, although the primary structure is unrelated, the tertiary structure of these two enzyme families brings together a conserved catalytic triad of amino acids consisting of serine, histidine and aspartate. The subtilisins and chymotrypsin-related serine proteases both have a catalytic triad comprising aspartate, histidine and serine. In the subtilisin-related proteases the relative order of these amino acids, reading from the amino to carboxy terminus, is aspartate-histidine-serine. However, in the chymotrypsin-related proteases, the relative order is histidine-aspartate-serine. Much research has been conducted on the subtilisins, due largely to their usefulness in cleaning and feed applications. Additional work has been focused on the adverse environmental conditions (e.g., exposure to oxidative agents, chelating agents, extremes of temperature and/or pH) which can adversely impact the functionality of these enzymes in various applications. Nonetheless, there remains a need in the art for enzyme systems that are able to resist these adverse conditions and retain or have improved activity over those currently known in the art.
SUMMARY OF THE INVENTION
The present invention provides novel serine proteases, novel genetic material encoding these enzymes, and proteolytic proteins obtained from Micrococcineae spp., including but not limited to Cellulomonas spp. and variant proteins developed therefrom. In particular, the present invention provides protease compositions obtained from a Cellulomonas spp, DNA encoding the protease, vectors comprising the DNA encoding the protease, host cells transformed with the vector DNA, and an enzyme produced by the host cells. The present invention also provides cleaning compositions (e.g., detergent compositions), animal feed compositions, and textile and leather processing compositions comprising protease(s) obtained from a Micrococcineae spp., including but not limited to Cellulomonas spp. In alternative embodiments, the present invention provides mutant (i.e., variant) proteases derived from the wild-type proteases described herein. These mutant proteases also find use in numerous applications.
The present invention provides isolated serine proteases obtained from a member of the Micrococcineae. In some embodiments, the proteases are cellulomonadins. In some preferred embodiments, the protease is obtained from an organism selected from the group consisting of Cellulomonas, Oerskovia, Cellulosimicrobium, Xylanibacterium, and Promicromonospora. In some particularly preferred embodiments, the protease is obtained so from Cellulomonas 69B4. In further embodiments, the protease comprises the amino acid sequence set forth in SEQ ID NO:8. In additional embodiments, the present invention provides isolated serine proteases comprising at least 45% amino acid identity with serine protease comprising SEQ ID NO:8. In some embodiments, the isolated serine proteases comprise at least 50% identity, preferably at least 55%, more preferably at least 60%, yet more preferably at least 65%, even more preferably at least 70%, more preferably at least 75%, still more preferably at least 80%, more preferably 85%, yet more preferably 90%, even more preferably at least 95%, and most preferably 99% identity.
The present invention also provides compositions comprising isolated serine proteases having immunological cross-reactivity with the serine proteases obtained from the Micrococcineae. In some preferred embodiments, the serine proteases have immunological cross-reactivity with serine protease obtained from Cellulomonas 69B4. In alternative embodiments, the serine proteases have immunological cross-reactivity with serine protease comprising the amino acid sequence set forth in SEQ ID NO:8. In still further embodiments, the serine proteases have cross-reactivity with fragments (i.e., portions) of any of the serine proteases obtained from the Micrococcineae, the Cellulomonas 69B4 protease, and/or serine protease comprising the amino acid sequence set forth in SEQ ID NO:8.
In some embodiments, the present invention provides the amino acid sequence set forth in SEQ ID NO:8, wherein the sequence comprises substitutions at least one amino acid position selected from the group comprising positions 2, 8, 10, 11, 12, 13, 14, 15, 16, 24, 26, 31, 33, 35, 36, 38, 39, 40, 43, 46, 49, 51, 54, 61, 64, 65, 67, 70, 71, 76, 78, 79, 81, 83, 85, 86, 90, 93, 99, 100, 105, 107, 109, 112, 113, 116, 118, 119, 121, 123, 127, 145, 155, 159, 160, 163, 165, 170, 174, 179, 183, 184, 185, 186, 187, and 188. In alternative embodiments, the sequence comprises substitutions at least one amino acid position selected from the group comprising positions 1, 4, 22, 27, 28, 30, 32, 41, 47, 48, 55, 59, 63, 66, 69, 75, 77, 80, 84, 87, 88, 89, 92, 96, 110, 111, 114, 115, 117, 128, 134, 144, 143, 146, 151, 154, 156, 158, 161, 166, 176, 177, 181, 182, 187, and 189.
In some preferred embodiments, the present invention provides protease variants having an amino acid sequence comprising at least one substitution of an amino acid made at a position equivalent to a position in a Cellulomonas 69B4 protease comprising the amino acid sequence set forth in SEQ ID NO:8. In alternative embodiments, the present invention provides protease variants having an amino acid sequence comprising at least one substitution of an amino acid made at a position equivalent to a position in a Cellulomonas 69B4 protease comprising at least a portion of SEQ ID NO:8. In some embodiments, the substitutions are made at positions equivalent to positions 2, 8, 10, 11, 12, 13, 14, 15, 16, 24, 26, 31, 33, 35, 36, 38, 39, 40, 43, 46, 49, 51, 54, 61, 64, 65, 67, 70, 71, 76, 78, 79, 81, 83, 85, 86, 90, 93, 99, 100, 105, 107, 109, 112, 113, 116, 118, 119, 121, 123, 127, 145, 155, 159, 160, 163, 165, 170, 174, 179, 183, 184, 185, 186, 187, and 188 in a Cellulomonas 69B4 protease having an amino acid sequence set forth in SEQ ID NO:8. In alternative embodiments, the substitutions are made at positions equivalent to positions 1, 4, 22, 27, 28, 30, 32, 41, 47, 48, 55, 59, 63, 66, 69, 75, 77, 80, 84, 87, 88, 89, 92, 96, 110, 111, 114, 115, 117, 128, 134, 144, 143, 146, 151, 154, 156, 158, 161, 166, 176, 177, 181, 182, 187, and 189, in a Cellulomonas 69B4 protease having an amino acid sequence set forth in SEQ ID NO:8. In some preferred embodiments, the protease variants comprise the amino acid sequence comprising SEQ ID NO:8, wherein at least one amino acid position at positions selected from the group consisting of 14, 16, 35, 36, 65, 75, 76, 79, 123, 127, 159, and 179, are substituted with another amino acid. In some particularly preferred embodiments, the proteases comprise at least one mutation selected from the group consisting of R14L, R161, R16L, R16Q, R35F, T36S, G65Q, Y75G, N76L, N76V, R79T, R123L, R123Q, R127A, R127K, R127Q, R159K, R159Q, and R179Q. In some alternative preferred embodiments, the proteases comprise multiple mutations selected from the group consisting of R16Q/R35F/R159Q, R16Q/R123L, R14LJR127Q/R159Q, R14L/R179Q, R123L/R127Q/R179Q, R16Q/R79T/R127Q, and R16Q/R79T. In some particularly preferred embodiments, the proteases comprise the following mutations R123L, R127Q, and R179Q.
The present invention also provides protease variants having amino acid sequences comprising at least one substitution selected from the group consisting of T36I, A38R, N170Y, N73T, G77T, N24A, T36G, N24E, L69S, T36N, T36S, E119R, N74G, T36W, S76W, N24T, N24Q, T36P, S76Y, T36H, G54D, G78A, S187P, R179V, N24V, V90P, T36D, L69H, G65P, G65R, N7L, W103M, N55F G186E, A70H, S76V, G186V, R159F, T36Y, T36V, G65V, N24M, S51A, G65Y, Q71I, V66H, P118A, T116F, A38F, N24H, V66D, S76L, G177M, G186I, H85Q, Q71K, Q71G, G65S, A38D, P118F, A38S, G65T, N67G, T36R, P118R, S114G, Y75I, I181H, G65Q, Y75G, T36F, A38H, R179M, T183I, G78S, A64W, Y75F, G77S, N24L, W103I, V3L, Q81V, R179D, G54R, T36L, Q71M, A70S, G49F, G54L, G54H, G78H, R179I, Q81K, V90I, A38L, N67L, T109I, R179N, V66I, G78T, R179Y, S187T, N67K, N73S, E119K, V3I, Q71H, I1Q, A64H, R14E, R179T, L69V, V150L, Q71A, G65L, Q71N, V905, A64N, I11A, N145I, H85T, A64Y, N145Q, V66L, S92G, S188M, G78D, N67A, N7S, V80H, G54K, A70D, P118H, D2G, G54M, Q81H, D2Q, V66E, I479P, A38N, N145E, R179L, T109H, R179K, V66A, G54A, G78N, T109A, R179A, N7A, R179E, H104K, A64R, and V80L. In further embodiments, wherein the amino acid sequence of the protease variants so comprise at least one substitution selected from the group consisting of H85R, H85L, T62I, N67H, G54I, N24F, T40V, T86A, G63V, G54Q, A64F, G77Y, R35F, T129S, R61M, I126L, S76N, T182V, R79G, T109P, R127F, R123E, P118I, T109R, I71S, T183K, N67T, P89N, F1T, A64K, G78I, T109L, G78V, A64M, A64S, T10G, G77N, A64L, N67D, S76T, N42H, D184F, D184R, S76I, S78R, A38K, W2I, V3T, T107S, A38V, F47I, N55Q, S76E, P118Q, ss T109G, Q71D, P118K, N67S, Q167N, N145G, I28L, I11T, A64I, G49K, G49A, G65A, N170D, H85K, S185I, I181N, V80F, L69W, S76R, D184H, V150M, T183M, N67Q, S51Q, A38Y, T107V, N145T, Q71F, A83N, S76A, N67R, T151L, T163L, S51F, Q81I, F47M, A41N, P118E, N67Y, T107M, N73H, 67V, G63W, T10K, I181G, S187E, T107H, D2A, L142V, A143N, A8G, S187L, V90A, G49L, N170L, G65H, T36C, Q12W, S76Q, A143S, F1A, N7H, S185V, A110T, N55K, N67F, N7I, A110S, N170A, Q81D, A64Q, Q71L, A38I, N112I, V90T, N145L, A64T, I11S, A30S, R123I, D2H, V66M, Q71R, V90L, L68W, N24S, R159E, V66N, D184Q, E133Q, A64V, D2N, G13M, T40S, S76K, G177S, G63Q, S15F, A8K, A70G, and A38G. In some preferred embodiments, these variants have improved casein hydrolysis performance as compared to wild-type Cellulomonas 69B4 protease.
The present invention also provides protease variants having amino acid sequences comprising at least one substitution selected from the group consisting of R35E, R35D, R14E, R14D, Q167E, G49C, S15R, S15H, I11W, S15C, G49Q, R35Q, R35V, G49E, R123D, R123Y, G49H, A38D, R35S, F47R, R123c, T151L, R14T, R35T, R123E, G49A, G49V, D56L, R35N, R35A, G12D, R35c, R123N, T46V, R123H, S155C, T121E, R127E, S113C, R123T, R16E, T46F, T121L, A38C, T46E, R123W, T44E, N55G, A8G, E119G, R35P, R14G, F59W, R127S, R61E, R14S, S155W, R123F, R123S, G49N, R127D, E119Y, A48E, N170D, R159T, S99A, G120, P118R, F165W, R127G, R35H, G12N, A22C, G12V, R16T, Y57G, T100A, T46Y, R159E, E119R, T107R, T151C, G54C, E119T, R61V, I11E, R14I, R61M, S15E, A22S, R16C, T36C, R16V, L125Q, M180L, R123Q, R14A, R14Q, R35M, R127K, R159Q, N112P, G124D, R179E, G49L, A41D, G177D, R123V, E119V, T10L, T109E, R179D, G12S, T10C, G91Q, S15Y, S155Y, R14C, T163D, T121F, R14N, F165E, N24E, A41C, R61T, G12I, P118K, T46C, I11T, R159D, N170C, R159V, S155I, I11Q, D2P, T100R, R159S, S114C, R16D, and P134R. In alternative embodiments, the protease variants have amino acid sequences comprising at least one substitution selected from the group consisting of S99G, T100K, R127A, F1P, S155V, T128A, F165H, G177E, A70M, S140P, A87E, D2I, R159K, T36V, R179c, E119N, T10Y, I172A, ABT, F47V, W103L, R61K, D2V, R179V, D2T, R159N, E119A, G54E, R16Q, G49S, R16I, S51L, S155E, S15M, R179I, T10Q, G12H, R159c, R179T, T163C, R159A, A132S, N157D, G13E, L141M, A41T, R123M, R14M, ABR, Q81P, N24T, T10D, A88F, R61Q, S99K, R179Y, T121A, N112E, S155T, T151V, S99Q, T10E, S92T, T109K, T44C, R123A, A87C, S15F, S155F, D56F, T10F, A83H, R179M, T121D, G13D, P118C, G49F, Q174C, S114E, T86E, F1N, T115C, R127c, R123K, V66N, G12Y, S113A, S15N, A175T, R79T, R123G, R179S, R179N, R123I, P118A, S187E, N112D, A70G, E119L, E119S, R159M, R14H, R179F, A64C, A41S, R179W, N24G, T100Q, P118W, Q81G, G49K, R14L, N55A, R35K, R79V, D2M, T160D, A83D, R179L, S51A, G12P, S99H, N42D, S188E, T10M, L125M, T116N, A70P, Q174S, G65D, S113D, E119Q, A83E, N170L, Q81A, S51C, P118G, Q174T, I28V, S15G, and T116G. In some preferred embodiments, these variants have improved LAS stability as compared to wild-type Cellulomonas 69B4 protease.
The present invention also provides protease variants having amino acid sequences comprising at least one substitution selected from the group consisting of G26I, G26K, G26Q, G26V, G26W, F27V, F27W, I28P, T29E, T129W, T40D, T40Q, R43D, P43H, P43K, P43L, A22C, T40H, P89W, G91L, S18E, F59K, A30M, A30N, G31M, C33M, G161L, G161V, P43N, G26E, N73P, G84C, G84P, G45V, C33L, Y9E, Y9P, A147E, C158H, I28W, A48P, A22S, T62R, S137R, S155P, S155R, G156I, G156L, Q81A, R96c, I4D, I4P, A70P, C105E, C105G, C105K, C105M, C105N, C105S, T128A, T128V, T128G, S140P, G12D, C33N, C33E, T164G, G45A, G156P, S99A, Q167L, S155W, I28T, R96F, A30P, R123W, T40P, T39R, C105P, T100A, C105W, S155K, T46Y, R123F, I4G, S155Y, T46V, A93S, Y57N, Q81S, G186S, G31H, T10Y, G31V, A83H, A38D, R123Y, R79T, C158G, G31Y, Q81P, R96E, A30Y, R159K, A22T, T40N, Y57M, G31N, Q81G, T164L, T121E, T10F, Q146P, R123N, V3R, P43G, Q81H, Q81D, G161I, C158M; N24T, T10W, T128S, T160I, Y176P, S155F, T128C, L125A, P168Y, T62G, F166S, S188A, Q81F, T46W, A70G, and A38G. In alternative embodiments, the protease variants have amino acid sequences comprising at least one substitution selected from the group consisting of S188E, S188V, Y117K, Y117Q, Y117R, Y117V, R127K, R127Q, R123L, T86S, R123I, Q81E, L125M, H32A, S188T, N74F, C33D, F27I, A83M, Q71Y, R123T, V90A, F59W, L141C, N170E, T46F, S51V, G162P, S185R, A41S, R79V, T151C, T107S, T129Y, M180L, F166C, C105T, T160E, P89A, R159T, T183P, S188M, T10L, G25S, N24S, E119L, T107L, T107Q, G161K, G15Q, S15R, G153K, G153V, S188G, A83E, G186P, T121D, G49A, S15C, C105Y, C105A, R127F, Q71A, T10C, R179K, T86I, W103N, A87S, F166A, A83F, R123Q, A132C, A143H, T163I, T39V, A93D, V90M, R123K, P134W, G177N, V115I, S155T, T110D, G105L, N170D, T107A, G84V, G84M, L111K, P168I, G154L, T183I, S99G, S15T, A8G, S15N, P189S, S188C, T100Q, A110G, A121A, G12A, R159V, G31A, G154R, T182L, V15L, T160Q, T107F, R159Q, G144A, S92T, T101S, A83R, G12HM S15H, T116Q, T36V, G154, Q81C, V130T, T183A, P118T, A87E, T86M, V150N, and N24E. In some preferred embodiments, these variants have improved thermostability as compared to wild-type Cellulomonas 69B4 protease.
The present invention also provides protease variants having amino acid sequences comprising at least one substitution selected from the group consisting of T36I, I172T, N24E, N170Y, G77T, G186N, I181L, N73T, A38R, N74G, N24A, G54D, S76D, R123E, I59E, N112E, R35E, R179V, R123D, N24T, R179T, R14L, A38D, V90P, R14Q, R123I, R179D, S76V, R79G, R35L, S76E, S76Y, R79D, R79P, R35Q, R179N, N112D, R179E, G65P, Y75G, V90S, R179M, R35F, R123F, A64I, N24Q, R14I, R179A, R127A, R179I, N170D, R35A, R159F, T109E, R14D, N67D, G49A, N112Q, G78D, T121E, L69S, T116E, V90I, T36S, T36G, N145E, T86D, S51D, R179K, T107E, T129S, L142V, R79A, R79E, A38H, T107S, R123A, N55E, R123L, R159N, G65D, R14N, G65Q, R123Q, N24V, R14G, T116Q, A38N, R159Q, R179Y, A83E, N112L, S99N, G78A, T10N, H85Q, R35Q, N24L, N24H, G49S, R79L, S76T, S76L, G65S, N55F, R79V, G65T, R123N, T86E, Y75F, F1T, S76N, S99V, R79T, N112V, R79M, T107V, R79S, G54E, G65V, R127Q, R159D, T107H, H85T, R35T, T36N, Q81E, R123H, S76I, A38F, V90T, and R14T. In alternative embodiments, the protease variants have amino acid sequences comprising at least one substitution selected from the group consisting of G65L, S99D, T107M, S113T, S99T, G77S, R14M, A64N, R61M, A70D, Q71G, A93D, S92G, N112Y, S15W, R159K, N67G, T10E, R127H, A64Y, R159c, A38L, T160E, T183E, R127S, A8E, S51Q, N7L, G63D, A38S, R35H, R14K, T107I, G12D, A64L, S76W, A41N, R35M, A64V, A38Y, T183I, W103M, A41D, R127K, T36D, R61T, G65Y, G13S, R35Y, R123T, A64H, G49H, A70H, A64F, R127Y, R61E, A64P, T121D, V115A, R123Y, T101S, T182V, H85L, N24M, R127E, N145D, Q71H, S76Q, A64T, G49F, A64Q, T10D, F1D, A70G, R35W, Q71D, N121I, A64M, T36H, A8G, T107N, R35S, N67T, S92A, N170L, N67E, S114A, R14A, R14S, Q81D, S51H, R123S, A93S, R127F, I19V, T40V, S185N, R123G, R179L, S51V, T163D, T109I, A64S, V721, N67S, R159S, H85M, T109G, Q71S, R61H, T107A, Q81V, V90N, T109A, A38T, N145T, R159A, A110S, Q81H, A48E, S51T, A64W, R159L, N67H, A93E, T116F, R61S, R123V, V3L, and R159Y. In some preferred embodiments, these variants have improved keratin hydrolysis activity as compared to wild-type Cellulomonas 69B4 protease.
The present invention also provides protease variants having amino acid sequences comprising at least one substitution selected from the group consisting of T36I, P89D, A93T, A93S, T36N, N73T, T36G, R159F, T36S, A38R, S99W, S76W, T36P, G77T, G54D, R127A, R159E, H85Q, T36D, S76L, S99N, Y75G, S76Y, R127S, N24E, R127Q, D184F, N170Y, N24A, S76T, H85L, Y75F, S76V, L69S, R159K, R127K, G65P, N74G, R159H, G65Q, G186V, A48Q, T36H, N67L, F114I, R127L, T36Y, S76I, S114G, R127H, S187P, V3L, G78D, R123I, I181Q, R35F, H85R, R127Y, N67S, Q81P, R123F, R159N, S99A, S76D, A132V, R127F, A143N, S92A, N24T, R79P, S76N, R14M, G186E, N24Q, N67A, R127T, H85K, G65T, G65Y, R179V, Y75, I11Q, A38L, T36L, R159Y, R159D, N24V, G65S, N157D, G186I, G54Q, N67Y, R127G, S76A, A38S, T109E, V66H, T116F, R123L, G49A, A64H, T36W, D184H, S99D, G161K, P134E, A64F, N67G, S99T, D2Q, S76E, R16Q, G54N, N67V, R35L, Q71I, N7L, N112E, L69H, N24H, G54I, R16L, N24M, A64Y, S113A, H85F, R79G, I11A, T121D, R61V, and G65L. In alternative embodiments, the protease variants have amino acid sequences comprising at least one substitution selected from the group consisting of N67Q, S187Q, Q71H, T163D, R61K, R159V, Q71F, V31F, V90I, R79D, T160E, R123Q, A38Y, S113G, A88F, A70G, I11T, G78A, N24L, S92G, R14L, D184R, G54L, N112L, H85Y, R16N, G77S, R179T, V80L, G65V, T121E, Q71D, R16G, P89N, N42H, G49F, I11S, R61M, R159c, G65R, T183I, A93D, L111E, S51Q, G78N, N67T, A38N, T40V, A64W, R159L, T10E, R179K, R123E, V90P, A64N, G161E, H85T, ABG, L142V, A41N, S185I, Q71L, A64T, R16I, A38D, G54M, N112Q, R16A, R14E, V80H, N170D, S99G, R179N, S15E, G49H, A70P, A64S, G54A, S185W, R61H, T10Q, A38F, N170L, T10L, N67F, G12D, D184T, R14N, S187E, R14P, N112D, S140A, N112G G49S, L111D, N67M, V150L, G12Y, R123K, P89V, V66D, G77N, S51T, A8D, I181H, T86N, R179D, N55F, N24S, D184L, R61S, N67K, G186L, F1T, R159A, I11L, R61T, D184Q, A93E, Q71T, R179E, L69W, T163I, S188Q, L125V, A38V, R35A, P134G, A64V, N145D, V90T, and A143S. In some preferred embodiments, these variants have improved BMI performance as compared to wild-type Cellulomonas 69B4 protease.
The present invention also provides protease variants having amino acid sequences comprising at least one substitution selected from the group consisting of T36I, N170Y, A38R, R79P, G77T, L69S, N73T, S76V, S76Y, R179V, T36N, N55F, R159F, G54D, G65P, L69H, T36G, G177M, N24E, N74G, R159E, T36S, Y75G, S76I, S76D, ABR, A24A, V90P, R159c, G65Q, T121E, A8V, S76L, T109E, R179M, ABT, T107N, G186E, S76W, R123E, A38F, T36P, N67G, Y75F, S76N, R179I, S187P, N67V, V90S, R127A, R179Y, R35F, N145S, G65S, R61M, S51A, R179N, R123D, N24T, N55E, R79c, G186V, R123I, G161E, G65Y, A38S, R14L, V90I, R79G, N145E, N67L, R127S, R150Y, M180D, N67T, A93D, T121D, Q81V, I109I, A93E; T107S, R179T, R179L, R179K, R159D; R179A, R79E, R123F, R79D, T36D, A64N, L142V, T109A, I172V, A83N, T85A, R179D, A38L, I126L, R127Q, R127L, L69W, R127K, G65T, R127H, P134A, N67D, R14M, N24Q, A143N, N55S, N67M, S51D, S76E, T163D, A38D, R159K, T183I, G63V, ABS, T107M, H85Q, N112E, N67F, N67S, A64H, T86I, P134E, T182V, N67Y, A64S, G78D, V90T, R61T, R16Q, G65R, T86L, V90N, R159Q, G54I, S76C, R179E, V66D, L69V, R127Y, R35L, R14E, and T86F. In alternative embodiments, the protease variants have amino acid sequences comprising at least one substitution selected from the group consisting of G186I, A64Q, T109G, G64L, N24L, A8E, N112D, A38H, R179W, S114G, R123L, ABL, T129S, N170D, R159N, N67C, S92C, T107A, G54E, T107E, T36V, R127T, ABN, H85L, A110S, N170C, A64R, A132V, T36Y, G63D, W103M, T151V, R123P, W103Y, S76T, S1871′′, R127F, N67A, P171M, A70S, R159H, S76Q, L125V, G54Q, G49L, R14I, R14Q, A83I, V90L, T183E, R159A, T101S, G65D, G54A, T107Q, Q71M, T86E, N24M, N55Q, R61V, P134D, R96K, A88F, N145Q, A64M, A64T, N24V, S140A, A8H, A64I, R123Q, T183Q, N24H, A64W, T62I, T129G, R35A, T40V, I11T, A38N, N145G, A175T, G77Q, T109H, A8P, R35E, T109N, A110T, N67Q, G63P, H859, S92G, A175V, S51Q, G63Q, T116F, G65A, R79L, N145P, L69Q, Q146D, A83D, F166Y, R123A, T121L, R123H, A70P, T182W, S76A, A64F, T107H, G186L, Q81I, R123K, A64L, N67R, V3L, S187E, S161K, T86M, I4M, G77N, G49A, A41N, G54M, T107V, Q81E, A38I, T109L, T183K, A70G, Q71D, T183L, Q81H, A64V, A93Q, S188E, S51F, G186P, G186T, R159L, P134G, N145T, N55V, V66E, R159V, Y176L, and R16L. In some preferred embodiments, these variants have improved BMI performance under low pH conditions, as compared to wild-type Cellulomonas 69B4 protease.
The present invention also provides serine proteases comprising at least a portion of an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:9. In some embodiments, the nucleotide sequences encoding these serine proteases comprise a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. In some embodiments, the serine proteases are variants having amino acid sequences that are similar to that set forth in SEQ ID NO:8. In some preferred embodiments, the proteases are obtained from a member of the Micrococcineae. In some particularly preferred embodiments, the proteases are obtained from an organism selected from the group consisting of Cellulomonas, Oerskovia, Cellulosimicrobium, Xylanibacterium, and Promicromonospora. In some particularly preferred embodiments, the protease is obtained from variants of Cellulomonas 69B4.
The present invention also provides isolated protease variants having amino acid sequences comprising at least one substitution of an amino acid made at a position equivalent to a position in a Cellulomonas 69B4 protease comprising the amino acid sequence set forth in SEQ ID NO:8, wherein the amino acid of the protease comprises Arg14, Ser15, Arg16, Cys17, His32, Cys33, Phe52, Asp56, Thr100, Val115, Thr116, Tyr117, Pro118, Glu119, Ala132, Glu133, Pro134, Gly135, Asp136, Ser137, Thr151, Ser152, Gly153, Gly154, Ser155, Gly156, Asn157, Thr164, and Phe165. In some embodiments, the catalytic triad of the proteases comprises His 32, Asp56, and Ser137. In alternative embodiments, the proteases comprise Cys13I, Ala132, Glu133, Pro134, Gly135, Thr15I, Ser152, Gly153, Gly154, Ser155, Gly156, Asn157 and Gly 162, Thr 163, and Thr164. In some preferred embodiments, the amino acid sequence of the proteases comprise Phe52, Tyr117, Pro118 and Glu119. In some particularly preferred embodiments, the amino acids sequences of the proteases have main-chain to main-chain hydrogen bonding from Gly 154 to the substrate main-chain.
In embodiments, the proteases of the present invention comprise three disulfide bonds. In some preferred embodiments, the disulfide bonds are located between C17 and C38, C95 and C105, and C131 and C158. In some particularly preferred embodiments, the disulfide bonds are located between C17 and C38, C95 and C105, and C131 and C158 of SEQ ID NO:8. In alternative protease variant embodiments, the disulfide bonds are located at positions equivalent to the disulfide bonds in SEQ ID NO:8.
The present invention also provides isolated protease variants having amino acid sequences comprising at least one substitution of an amino acid made at a position equivalent to a position in a Cellulomonas 69B4 protease comprising the amino acid sequence set forth in SEQ ID NO:8, wherein the variants have altered substrate specificities as compared to wild-type Cellulomonas 69B4 protease. In some further preferred embodiments, the variants have altered pls as compared to wild-type Cellulomonas 69B4 protease. In additional preferred embodiments, the variants have improved stability as compared to wild-type Cellulomonas 69B4 protease. In still further preferred embodiments, the variants exhibit altered surface properties. In some particularly preferred embodiments, the variants exhibit altered surface properties as compared to wild-type Cellulomonas 69B4 protease. In additional particularly preferred embodiments, the variants comprise mutations at least one substitution at sites selected from the group consisting of 1, 2, 4, 7, 8, 10, 11, 12, 13, 14, 15, 16, 22, 24, 25, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 57, 59, 61, 62, 63, 64, 65, 66, 67, 68, 69, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 95, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 123, 124, 126, 127, 128, 130, 131, 132, 133, 134, 135, 137, 143, 144, 145, 146, 147, 148, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, and 184.
The present invention also provides protease variants having at least one improved property as compared to the wild-type protease. In some particularly preferred embodiments, the variants are variants of a serine protease obtained from a member of the Micrococcineae. In some particularly preferred embodiments, the proteases are obtained from an organism selected from the group consisting of Cellulomonas, Oerskovia, Cellulosimicrobium, Xylanibacterium, and Promicromonospora. In some particularly preferred embodiments, the protease is obtained from variants of Cellulomonas 69B4. In some preferred embodiments, at least one improved property is selected from the group consisting of acid stability, thermostability, casein hydrolysis, keratin hydrolysis, cleaning performance, and LAS stability.
The present invention also provides expression vectors comprising a polynucleotide sequence encoding protease variants having amino acid sequences comprising at least one substitution of an amino acid made at a position equivalent to a position in a Cellulomonas 69B4 protease comprising the amino acid sequence set forth in SEQ ID NO:8. In further embodiments, the present invention provides host cells comprising these expression vectors. In some particularly preferred embodiments, the host cells are selected from the group consisting of Bacillus sp., Streptomyces sp., Aspergillus sp., and Trichoderma sp. The present invention also provides the serine proteases produced by the host cells.
The present invention also provides variant proteases comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78, In some preferred embodiments, the amino acid sequence is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NOS:53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, and 77. In further embodiments, the present invention provides expression vectors comprising a polynucleotide sequence encoding at least one protease variant. In additional embodiments, the present invention provides host cells comprising these expression vectors. In some particularly preferred embodiments, the host cells are selected from the group consisting of Bacillus. sp., Streptomyces sp., Aspergillus sp., and Trichoderma sp. The present invention also provides the serine proteases produced by the host cells.
The present invention also provides compositions comprising at least a portion of an isolated serine protease of obtained from a member of the Micrococcineae, wherein the protease is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In some preferred embodiments, the sequence comprises at least a portion of SEQ ID NO:1. In further embodiments, the present invention provides host cells comprising these expression vectors. In some particularly preferred embodiments, the host cells are selected from the group consisting of Bacillus sp., Streptomyces sp., Aspergillus sp., and Trichoderma sp. The present invention also provides the serine proteases produced by the host cells.
The present invention also provides variant serine proteases, wherein the proteases comprise at least one substitution corresponding to the amino acid positions in SEQ ID NO:8, and wherein variant proteases have better performance in at least one property selected from the group consisting of keratin hydrolysis, thermostability, casein activity, LAS stability, and cleaning, as compared to wild-type Cellulomonas 69B4 protease.
The present invention also provides isolated polynucleotides comprising a nucleotide sequence (i) having at least 70% identity to SEQ ID NO:4, or (ii) being capable of hybridizing to a probe derived from the nucleotide sequence set forth in SEQ ID NO:4, under conditions of intermediate to high stringency, or (iii) being complementary to the nucleotide sequence set forth in SEQ ID NO:4. In embodiments, the present invention provides expression vectors encoding at least one such polynucleotide. In further embodiments, the present invention provides host cells comprising these expression vectors. In some particularly preferred embodiments, the host cells are selected from the group consisting of Bacillus sp., Streptomyces sp., Aspergillus sp., and Trichoderma sp. The present invention also provides the serine proteases produced by the host cells. In further embodiments, the present invention provides polynucleotides that are complementary to at least a portion of the sequence set forth in SEQ ID NO:4.
The present invention also provides methods of producing an enzyme having protease activity, comprising: transforming a host cell with an expression vector comprising a polynucleotide having at least 70% sequence identity to SEQ ID NO:4; cultivating the transformed host cell under conditions suitable for host cell. In some embodiments, the host cell is selected from the group consisting of Streptomyces, Aspergillus, Trichoderma and Bacillus species.
The present invention also provides probes comprising 4 to 150 nucleotide sequence substantially identical to a corresponding fragment of SEQ ID NO:4, wherein the probe is used to detect a nucleic acid sequence coding for an enzyme having proteolytic activity, and wherein the nucleic acid sequence is obtained from a member of the Micrococcineae. In some embodiments, the Micrococcineae is a Cellulomonas spp. In some preferred embodiments, the Cellulomonas is Cellulomonas strain 69B4.
The present invention also provides cleaning compositions comprising at least one serine protease obtained from a member of the Micrococcineae. In some embodiments, ate least one protease is obtained from an organism selected from the group consisting of Cellulomonas, Oerskovia, Cellulosimicrobium, Xylanibacterium, and Promicromonospora. In some preferred embodiments, the protease is obtained from Cellulomonas 69B4. In some particularly preferred embodiments, at least one protease comprises the amino acid sequence set forth in SEQ ID NO:8. In some further embodiments, the present invention provides isolated serine proteases comprising at least 45% amino acid identity with serine protease comprising SEQ ID NO:8. In some embodiments, the isolated serine proteases comprise at least 50% identity, preferably at least 55%, more preferably at least 60%, yet more preferably at least 65%, even more preferably at least 70%, more preferably at least 75%, still more preferably at least 80%, more preferably 85%, yet more preferably 90%, even more preferably at least 95%, and most preferably 99% identity. 75.
The present invention further provides cleaning compositions comprising at least one serine protease, wherein at least one of the serine proteases has immunological cross-reactivity with the serine protease obtained from a member of the Micrococcineae. In some preferred embodiments, the serine proteases have immunological cross-reactivity with serine protease obtained from Cellulomonas 69B4. In alternative embodiments, the serine proteases have immunological cross-reactivity with serine protease comprising the amino acid sequence set forth in SEQ ID NO:8. In still further embodiments, the serine proteases have cross-reactivity with fragments (i.e., portions) of any of the serine proteases obtained from the Micrococcineae, the Cellulomonas 69B4 protease, and/or serine protease comprising the amino acid sequence set forth in SEQ ID NO:8.
The present invention further provides cleaning compositions comprising at least one serine protease, wherein the protease is a variant protease having an amino acid sequence comprising at least one substitution of an amino acid made at a position equivalent to a position in a Cellulomonas 69B4 protease having an amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the substitutions are made at positions equivalent to positions 2, 8, 10, 11, 12, 13, 14, 15, 16, 24, 26, 31, 33, 35, 36, 38, 39, 40, 43, 46, 49, 51, 54, 61, 64, 65, 67, 70, 71, 76, 78, 79, 81, 83, 85, 86, 90, 93, 99, 100, 105, 101, 109, 112, 113, 116, 118, 119, 121, 123, 127, 145, 155, 159, 160, 163, 165, 170, 174, 179, 183, 184, 185, 186, 187, and 188 in a Cellulomonas 69B4 protease comprising an amino acid sequence set forth in SEQ ID NO:8. In alternative embodiments, the substitutions are made at positions equivalent to positions 1, 4, 22, 27, 28, 30, 32, 41, 47, 48, 55, 59, 63, 66, 69, 75, 77, 80, 84, 87, 88, 89, 92, 96, 110, 111, 114, 115, 117, 128, 134, 144, 143, 146, 151, 154, 156, 158, 161, 166, 176, 177, 181, 182, 187, and 189, in a Cellulomonas 69B4 protease comprising an amino acid sequence set forth in SEQ ID NO:8. In further embodiments, the protease comprises at least one amino acid substitutions at positions 14, 16, 35, 36, 65, 75, 76, 79, 123, 127, 159, and 179, in an equivalent amino acid sequence to that set forth in SEQ ID NO:8. In still further embodiments, the protease comprises at least one mutation selected from the group consisting of R14L, R16I, R16L, R16Q, R35F, T36S, G65Q, Y75G, N76L, N76V, R79T, R123L, R123Q, R127A, R127K, R127Q, R159K, R159Q, and R179Q. In yet additional embodiments, the protease comprises a set of mutations selected from the group consisting of the sets R16Q/R35F/R159Q, R16Q/R123L, R14L/R127Q/R159Q, R14L/R179Q, R123L/R9127Q/R179Q, R16Q/R79T/R127Q, and R16Q/R79T. In some particularly preferred embodiments, the protease comprises the following mutations R123L, R127Q, and R179Q. In some particularly preferred embodiments, the variant serine as proteases comprise at least one substitution corresponding to the amino acid positions in SEQ ID NO:8, and wherein the variant proteases have better performance in at least one property selected from the group consisting of keratin hydrolysis, thermostability, casein activity, LAS stability, and cleaning, as compared to wild-type Cellulomonas 69B4 protease. In some embodiments, the variant protease comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78. In alternative embodiments, the variant protease amino acid sequence is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NOS:53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, and 77.
The present invention also provides cleaning compositions comprising a cleaning effective amount of a proteolytic enzyme, the enzyme comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:4, and a suitable cleaning formulation. In some preferred embodiments, the cleaning compositions further comprise one or more additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases.
The present invention also provides compositions comprising at least one serine protease obtained from a member of the Micrococcineae, wherein the compositions further comprise at least one stabilizer. In some embodiments, the stabilizer is selected from the group consisting of borax and glycerol. In some embodiments, the present invention provides competitive inhibitors suitable to stabilize the enzyme of the present invention to anionic surfactants. In some embodiments, at least one protease is obtained from an organism selected from the group consisting of Cellulomonas, Oerskovia, Cellulosimicrobium, Xylanibacterium, and Promicromonospora. In some preferred embodiments, the protease is obtained from Cellulomonas 69B4. In some particularly preferred embodiments, at least one protease comprises the amino acid sequence set forth in SEQ ID NO:8.
The present invention further provides compositions comprising at least one serine protease obtained obtained from a member of the Micrococcineae, wherein the serine protease is an autolytically stable variant. In some embodiments, at least one variant protease is obtained from an organism selected from the group consisting of Cellulomonas, Oerskovia, Cellulosimicrobium, Xylanibacterium, and Promicromonospora. In some preferred embodiments, the variant protease is obtained from Cellulomonas 69B4. In some particularly preferred embodiments, at least one variant protease comprises the amino acid sequence set forth in SEQ ID NO:8.
The present invention also provides cleaning compositions comprising at least 0.0001 weight percent of the serine protease of the present invention, and optionally, an adjunct ingredient. In some embodiments, the composition comprises an adjunct ingredient. In some preferred embodiments, the composition comprises a sufficient amount of a pH modifier to provide the composition with a neat pH of from about 3 to about 5, the composition being essentially free of materials that hydrolyze at a pH of from about 3 to about 5. In some particularly preferred embodiments, the materials that hydrolyze comprise a surfactant material. In additional embodiments, the cleaning composition is a liquid composition. In further embodiments, the surfactant material comprises a sodium alkyl sulfate surfactant that comprises an ethylene oxide moiety.
The present invention additionally provides cleaning compositions that comprise at least one acid stable enzyme, the cleaning composition comprising a sufficient amount of a pH modifier to provide the composition with a neat pH of from about 3 to about 5, the composition being essentially free of materials that hydrolyze at a pH of from about 3 to about 5. In further embodiments, the materials that hydrolyze comprise a surfactant material. In some preferred embodiments, the cleaning composition being a liquid composition. In yet additional embodiments, the surfactant material comprises a sodium alkyl sulfate surfactant that comprises an ethylene oxide moiety. In some embodiments, the cleaning composition comprises a suitable adjunct ingredient. In some additional embodiments, the composition comprises a suitable adjunct ingredient. In some preferred embodiments, the composition comprises from about 0.001 to about 0.5 weight % of ASP.
In some alternatively preferred embodiments, the composition comprises from about 0.01 to about 0.1 weight percent of ASP.
The present invention also provides methods of cleaning, the comprising the steps of: a) contacting a surface and/or an article comprising a fabric with the cleaning composition comprising the serine protease of the present invention at an appropriate concentration; and b) optionally washing and/or rinsing the surface or material. In alternative embodiments, any suitable composition provided herein finds use in these methods.
The present invention also provides animal feed comprising at least one serine protease obtained from a member of the Micrococcineae. In some embodiments, at least one protease is obtained from an organism selected from the group consisting of Cellulomonas, Oerskovia, Cellulosimicrobium, Xylanibacterium, and Promicromonospora. In some preferred embodiments, the protease is obtained from Cellulomonas 69B4. In some particularly preferred embodiments, at least one protease comprises the amino acid sequence set forth in SEQ ID NO:8.
The present invention provides an isolated polypeptide having proteolytic activity, (e.g., a protease) having the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the present invention provides isolated polypeptides having approximately 40% to 98% identity with the sequence set forth in SEQ ID NO:8. In some preferred embodiments, the polypeptides have approximately 50% to 95% identity with the sequence set forth in SEQ ID NO:8. In some additional preferred embodiments, the polypeptides have approximately 60% to 90% identity with the sequence set forth in SEQ ID NO:8. In yet additional embodiments, the polypeptides have approximately 65% to 85% identity with the sequence set forth in SEQ ID NO:8. In some particularly preferred embodiments, the polypeptides have approximately 90% to 95% identity with the sequence set forth in SEQ ID NO:8.
The present invention further provides proteases obtained from bacteria of the suborder Micrococcineae. In some preferred embodiments, the proteases are obtained from members of the family Promicromonosporaceae. In yet further embodiments; the proteases are obtained from any member of the genera Xylanimicrobium, Xylanibacterium, Xylanimonas, Myceligenerans, and Promicromonospora. In some preferred embodiments, the proteases are obtained from members of the family Cellulomonadaceae. In some particularly preferred embodiments, the proteases are obtained from members of the genera Cellulomonas and Oerskovia. In some further preferred embodiments, the proteases are derived from Cellulomonas spp. In some embodiments, the Cellulomonas spp. is selected from Cellulomonas fimi, Cellulomonas biazotea, Cellulomonas cellasea, Cellulomonas hominis, Cellulomonas flavigena, Cellulomonas persica, Cellulomonas iranensis, Cellulomonas gelida, Cellulomonas humilata, Cellulomonas turbata, Cellulomonas uda, Cellulomonas fermentans, Cellulomonas xylanilytica, Cellulomonas humilata and Cellulomonas strain 69B4 (DSM 16035).
In alternative embodiments, the proteases are derived from Oerskovia spp. In some preferred embodiments, the Oerskovia spp. is selected from Oerskovia jenensis, Oerskovia paurometabola, Oerskovia enterophila, Oerskovia turbata and Oerskovia turbata strain DSM so 20577.
In some embodiments, the proteases have apparent molecular weights of about 17 kD to 21 kD as determined by a matrix assisted laser desorption/ionizaton—time of flight (“MALDI-TOF”) spectrophotometer.
The present invention further provides isolated polynucleotides that encode proteases comprise an amino acid sequence comprising at least 40% amino acid sequence identity to SEQ ID NO:8. In some embodiments, the proteases have at least 50% amino acid sequence identity to SEQ ID NO:8. In some embodiments, the proteases have at least 60% amino acid sequence identity to SEQ ID NO:8. In some embodiments, the proteases have at least 70% amino acid sequence identity to SEQ ID NO:8. In some embodiments, the proteases have at least 80% amino acid sequence identity to SEQ ID NO:8. In some embodiments, the proteases have at least 90% amino acid sequence identity to SEQ ID NO:8. In some embodiments, the proteases have at least 95% amino acid sequence identity to SEQ ID NO:8. The present invention also provides expression vectors comprising any of the polynucleotides provided above.
The present invention further provides host cells transformed with the expression vectors of the present invention, such that at least one protease is expressed by the host cells. In some embodiments, the host cells are bacteria, while in other embodiments, the host cells are fungi. In some preferred embodiments, the bacterial host cells are selected from the group consisting of the genera Bacillus and Streptomyces. In some alternative preferred embodiments, the fungal host cells are members of the genus Trichoderma, while in other alternative preferred embodiments, the fungal host cells are members of the genus Aspergillus. 
The present invention also provides isolated polynucleotides comprising a nucleotide sequence (i) having at least 70% identity to SEQ ID NOS:3 or 4, or (ii) being capable of hybridizing to a probe derived from the nucleotide sequence disclosed in SEQ ID NOS: 3 or 4, under conditions of medium to high stringency, or (iii) being complementary to the nucleotide sequence disclosed in SEQ ID NOS:3 or 4. In some embodiments, the present invention provides vectors comprising such polynucleotide. In further embodiments, the present invention provides host cells transformed with such vector.
The present invention further provides methods for producing at least one enzyme having protease activity, comprising: the steps of transforming a host cell with an expression vector comprising a polynucleotide comprising at least 70% sequence identity to SEQ ID NO:4, cultivating the transformed host cell under conditions suitable for the host cell to produce the protease; and recovering the protease. In some preferred embodiments, the host cell is a Streptomyces spp, while in other embodiments, the host cell is a Bacillus spp, a Trichoderma spp., and/or a Aspergillus spp. In some embodiments, the Streptomyces spp. is Streptomyces lividans. In alternative embodiments, the host cell is T. reesei. In further embodiments, the Aspergillus spp. is A. niger. 
The present invention also provides fragments (i.e., portions) of the DNA encoding the proteases provided herein. These fragments find use in obtaining partial length DNA fragments capable of being used to isolate or identify polynucleotides encoding mature protease enzyme described herein from Cellulomonas 69B4, or a segment thereof having proteolytic activity. In some embodiments, portions of the DNA provided in SEQ ID NO:1 find use in obtaining homologous fragments of DNA from other species, and particularly from Micrococcineae spp. which encode a protease or portion thereof having proteolytic activity.
The present invention further provides at least one probe comprising a polynucleotide substantially identical to a fragment of SEQ ID NOS:1, 2, 3 or 4, wherein the probe is used to detect a nucleic acid sequence coding for an enzyme having proteolytic activity, and wherein the nucleic acid sequence is obtained from a bacterial source. In some embodiments, the bacterial source is a Cellulomonas spp. In some preferred embodiments, the bacterial source is Cellulomonas strain 69B4.
The present invention further provides compositions comprising at least one of the proteases provided herein. In some preferred embodiments, the compositions are cleaning compositions. In some embodiments, the present invention provides cleaning compositions comprising a cleaning effective amount of at least one protease comprising an amino acid sequence having at least 40% sequence identity to SEQ ID NO:8, at least 90% sequence identity to SEQ ID NO:8, and/or having an amino acid sequence of SEQ ID NO:8. In some embodiments, the cleaning compositions further comprise at least one suitable cleaning adjunct. In some embodiments, the protease is derived from a Cellulomonas sp. In some preferred embodiments, the Cellulomonas spp. is selected from Cellulomonas fimi, Cellulomonas biazotea, Cellulomonas cellasea, Cellulomonas hominis, Cellulomonas flavigena, Cellulomonas persica, Cellulomonas iranensis, Cellulomonas gelida, Cellulomonas humilata, Cellulomonas turbata, Cellulomonas uda, and Cellulomonas strain 69B4 (DSM 16035). In some particularly preferred embodiments, the Cellulomonas spp is Cellulomonas strain 69B4. In still further embodiments, the cleaning composition further comprises at least one additional enzymes or enzyme derivatives selected from the group consisting of protease, amylase, lipase, mannanase and cellulase.
The present invention also provides isolated naturally occurring proteases comprising an amino acid sequence having at least 45% sequence identity to SEQ ID NO:8, at least 60% sequence identity to SEQ ID NO:8, at least 75% sequence identity to SEQ ID NO:8, at least 90% sequence identity to SEQ ID NO:8, at least 95% sequence identity to SEQ ID NO:8, and/or having the sequence identity of SEQ ID NO:8, the protease being isolated from a Cellulomonas spp. In some embodiments, the protease is isolated from Cellulomonas strain 69B4 (DSM 16035).
In additional embodiments, the present invention provides engineered variants of the serine proteases of the present invention. In some embodiments, the engineered variants are genetically modified using recombinant DNA technologies, while in other embodiments, the variants are naturally occurring. The present invention further encompasses engineered variants of homologous enzymes. In some embodiments, the engineered variant homologous proteases are genetically modified using recombinant DNA technologies, while in other embodiments, the variant homologous proteases are naturally occurring.
The present invention also provides serine proteases that immunologically cross-react with the Cellulomonas 69B4 protease (i.e., ASP) of the present invention. Indeed, it is intended that the present invention encompass fragments (e.g., epitopes) of the ASP protease that stimulate an immune response in animals (including, but not limited to humans) and/or are recognized by antibodies of any class. The present invention further encompasses epitopes on proteases that are cross-reactive with ASP epitopes. In some embodiments, the ASP epitopes are recognized by antibodies, but do not stimulate an immune response in animals (including, but not limited to humans), while in other embodiments, the ASP epitopes stimulate an immune response in at least one animal species (including, but not limited to humans) and are recognized by antibodies' of any class. The present invention also provides means and compositions for identifying and assessing cross-reactive epitopes.
The present invention further provides at least one polynucleotide encoding a signal peptide (i) having at least 70% sequence identity to SEQ ID NO:9, or (ii) being capable of hybridizing to a probe derived from the polypeptide sequence encoding SEQ ID NO:9, under conditions of medium to high stringency, or (iii) being complementary to the polypeptide sequence provided in SEQ ID NO:9. In further embodiments, the present invention provides at vectors comprising the polynucleotide described above. In yet additional embodiments, a host cell is provided that is transformed with the vector.
The present invention also provides methods for producing proteases, comprising: (a) transforming a host cell with an expression vector comprising a polynucleotide having at least 70% sequence identity to SEQ ID NO:4, at least 95% sequence identity to SEQ ID NO:4, and/or having a polynucleotide sequence of SEQ ID NO:4; (b) cultivating the transformed host cell under conditions suitable for the host cell to produce the protease; and
(c) recovering the protease. In some embodiments, the host cell is a Bacillus species (e.g., B. subtilis, B. clausii, or B. licheniformis). In alternative embodiments, the host cell is a Streptomyces spp., (e.g., Streptomyces lividans). In additional embodiments, the host cell is a Trichoderma spp., (e.g., Trichoderma reesei). In yet further embodiments, the host cell is a Aspergillus spp. (e.g., Aspergillus niger).
As will be appreciated, an advantage of the present invention is that a polynucleotide has been isolated which provides the capability of isolating further polynucleotides which encode proteins having serine protease activity, wherein the backbone is substantially identical to that of the Cellulomonas protease of the present invention.
In further embodiments, the present invention provides means to produce host cells that are capable of producing the serine proteases of the present invention in relatively large quantities. In particularly preferred embodiments, the present invention provides means to produce protease with various commercial applications where degradation or synthesis of polypeptides are desired, including cleaning compositions, as well as feed components, textile processing, leather finishing, grain processing, meat processing, cleaning, preparation of protein hydrolysates, digestive aids, microbicidal compositions, bacteriostatic composition, fungistatic compositions, personal care products, including oral care, hair care, and/or skin care.
The present invention further provides enzyme compositions have comparable or improved wash performance, as compared to presently used subtilisin proteases. Other objects and advantages of the present invention are apparent from the present Specification.
The present invention provides an isolated polypeptide having proteolytic activity, (e.g., a protease) having the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the present invention provides isolated polypeptides having approximately 40% to 98% identity with the sequence set forth in SEQ ID NO:8. In some preferred embodiments, the polypeptides have approximately 50% to 95% identity with the sequence set forth in SEQ ID NO:8. In some additional preferred embodiments, the polypeptides have approximately 60% to 90% identity with the sequence set forth in SEQ ID NO:8. In yet additional embodiments, the polypeptides have approximately 65% to 85% identity with the sequence set forth in SEQ ID NO:8. In some particularly preferred embodiments, the polypeptides have approximately 90% to 95% identity with the sequence set forth in SEQ ID NO:8.
The present invention further provides proteases obtained from bacteria of the suborder Micrococcineae. In some preferred embodiments, the proteases are obtained from members of the family Promicromonosporaceae. In yet further embodiments, the proteases are obtained from any member of the genera Xylanimicrobium, Xylanibacterium, Xylanimonas, Myceligenerans, and Promicromonospora. In some preferred embodiments, the proteases are obtained from members of the family Cellulomonadaceae. In some particularly preferred embodiments, the proteases are obtained from members of the genera Cellulomonas and Oerskovia. In some further preferred embodiments, the proteases are derived from Cellulomonas spp. In some embodiments, the Cellulomonas spp. is selected from Cellulomonas fimi, Cellulomonas biazotea, Cellulomonas cellasea, Cellulomonas hominis, Cellulomonas flavigena, Cellulomonas persica, Cellulomonas iranensis, Cellulomonas gelida, Cellulomonas humilata, Cellulomonas turbata, Cellulomonas uda, Cellulomonas fermentans, Cellulomonas xylanilytica, Cellulomonas humilata and Cellulomonas strain 69B4 (DSM 16035).
In alternative embodiments, the proteases are derived from Oerskovia spp. In some preferred embodiments, the Oerskovia spp. is selected from Oerskovia jenensis, Oerskovia paurometabola, Oerskovia enterophila, Oerskovia turbata and Oerskovia turbata strain DSM 20577.
In some embodiments, the proteases have apparent molecular weights of about 17 kD to 21 kD as determined by a matrix assisted laser desorption/ionizaton—time of flight (“MALDI-TOF”) spectrophotometer.
The present invention further provides isolated polynucleotides that encode proteases comprise an amino acid sequence comprising at least 40% amino acid sequence identity to SEQ ID NO:8. In some embodiments, the proteases have at least 50% amino acid sequence identity to SEQ ID NO:8. In some embodiments, the proteases have at least 60% amino acid sequence identity to SEQ ID NO:8. In some embodiments, the proteases have at least 70% amino acid sequence identity to SEQ ID NO:8. In some embodiments, the proteases have at least 80% amino acid sequence identity to SEQ ID NO:8. In some embodiments, the proteases have at least 90% amino acid sequence identity to SEQ ID NO:8. In some embodiments, the proteases have at least 95% amino acid sequence identity to SEQ ID NO:8. The present invention also provides expression vectors comprising any of the polynucleotides provided above.
The present invention further provides host cells transformed with the expression vectors of the present invention, such that at least one protease is expressed by the host cells. In some embodiments, the host cells are bacteria, while in other embodiments, the host cells are fungi. In some preferred embodiments, the bacterial host cells are selected from the group consisting of the genera Bacillus and Streptomyces. In some alternative preferred embodiments, the fungal host cells are members of the genus Trichoderma, while in other alternative preferred embodiments, the fungal host cells are members of the genus Aspergillus. 
The present invention also provides isolated polynucleotides comprising a nucleotide sequence (i) having at least 70% identity to SEQ ID NOS:3 or 4, or (ii) being capable of hybridizing to a probe derived from the nucleotide sequence disclosed in SEQ ID NOS: 3 or 4, under conditions of medium to high stringency, or (iii) being complementary to the nucleotide sequence disclosed in SEQ ID NOS:3 or 4. In some embodiments, the present invention provides vectors comprising such polynucleotide. In further embodiments, the present invention provides host cells transformed with such vector.
The present invention further provides methods for producing at least one enzyme having protease activity, comprising: the steps of transforming a host cell with an expression vector comprising a polynucleotide comprising at least 70% sequence identity to SEQ ID NO:4, cultivating the transformed host cell under conditions suitable for the host cell to produce the protease; and recovering the protease. In some preferred embodiments, the host cell is a Streptomyces spp, while in other embodiments, the host cell is a Bacillus spp, a Trichoderma spp., and/or a Aspergillus spp. In some embodiments, the Streptomyces spp. is Streptomyces lividans. In alternative embodiments, the host cell is T. reesei. In further embodiments, the Aspergillus spp. is A. niger. 
The present invention also provides fragments (i.e., portions) of the DNA encoding the proteases provided herein: These fragments find use in obtaining partial length DNA fragments capable of being used to isolate or identify polynucleotides encoding mature protease enzyme described herein from Cellulomonas 69B4, or a segment thereof having proteolytic activity. In some embodiments, portions of the DNA provided in SEQ ID NO:1 find use in obtaining homologous fragments of DNA from other species, and particularly from Micrococcineae spp. which encode a protease or portion thereof having proteolytic activity.
The present invention further provides at least one probe comprising a polynucleotide substantially identical to a fragment of SEQ ID NOS:1, 2, 3 or 4, wherein the probe is used to detect a nucleic acid sequence coding for an enzyme having proteolytic activity, and wherein the nucleic acid sequence is obtained from a bacterial source. In some embodiments, the bacterial source is a Cellulomonas spp. In some preferred embodiments, the bacterial source is Cellulomonas strain 69B4.
The present invention further provides compositions comprising at least one of the proteases provided herein. In some preferred embodiments, the compositions are cleaning compositions. In some embodiments, the present invention provides cleaning compositions comprising a cleaning effective amount of at least one protease comprising an amino acid sequence having at least 40% sequence identity to SEQ ID NO:8, at least 90% sequence identity to SEQ ID NO:8, and/or having an amino acid sequence of SEQ ID NO:8. In some embodiments, the cleaning compositions further comprise at least one suitable cleaning adjunct. In some embodiments, the protease is derived from a Cellulomonas sp. In some preferred embodiments, the Cellulomonas spp. is selected from Cellulomonas fimi, Cellulomonas biazotea, Cellulomonas cellasea, Cellulomonas hominis, Cellulomonas flavigena, Cellulomonas persica, Cellulomonas iranensis, Cellulomonas gelida, Cellulomonas humilata, Cellulomonas turbata, Cellulomonas uda, and Cellulomonas strain 69B4 (DSM 16035). In some particularly preferred embodiments, the Cellulomonas spp is Cellulomonas strain 69B4. In still further embodiments, the cleaning composition further comprises at least one additional enzymes or enzyme derivatives selected from the group consisting of protease, amylase, lipase, mannanase and cellulase.
The present invention also provides isolated naturally occurring proteases comprising an amino acid sequence having at least 45% sequence identity to SEQ ID NO:8, at least 60% sequence identity to SEQ ID NO:8, at least 75% sequence identity to SEQ ID NO:8, at least 90% sequence identity to SEQ ID NO:8, at least 95% sequence identity to SEQ ID NO:8, and/or having the sequence identity of SEQ ID NO:8, the protease being isolated from a Cellulomonas spp. In some embodiments, the protease is isolated from Cellulomonas strain 69B4 (DSM 16035).
In additional embodiments, the present invention provides engineered variants of the serine proteases of the present invention. In some embodiments, the engineered variants are genetically modified using recombinant DNA technologies, while in other embodiments, the variants are naturally occurring. The present invention further encompasses engineered variants of homologous enzymes. In some embodiments, the engineered variant homologous proteases are genetically modified using recombinant DNA technologies, while in other embodiments, the variant homologous proteases are naturally occurring.
The present invention also provides serine proteases that immunologically cross-react with the ASP protease of the present invention. Indeed, it is intended that the present invention encompass fragments (e.g., epitopes) of the ASP protease that stimulate an immune response in animals (including, but not limited to humans) and/or are recognized by antibodies of any class. The present invention further encompasses epitopes on proteases that are cross-reactive with ASP epitopes. In some embodiments, the ASP epitopes are recognized by antibodies, but do not stimulate an immune response in animals (including, but not limited to humans), while in other embodiments, the ASP epitopes stimulate an immune response in at least one animal species (including, but not limited to humans) and are recognized by antibodies of any class. The present invention also provides means and compositions for identifying and assessing cross-reactive epitopes.
The present invention further provides at least one polynucleotide encoding a signal peptide (i) having at least 70% sequence identity to SEQ ID NO:9, or (ii) being capable of hybridizing to a probe derived from the polypeptide sequence encoding SEQ ID NO:9, under conditions of medium to high stringency, or (iii) being complementary to the polypeptide sequence provided in SEQ ID NO:9. In further embodiments, the present invention provides at vectors comprising the polynucleotide described above. In yet additional embodiments, a host cell is provided that is transformed with the vector.
The present invention also provides methods for producing proteases, comprising: (a) transforming a host cell with an expression vectorcomprising a polynucleotide having at least 70% sequence identity to SEQ ID NO:4, at least 95% sequence identity to SEQ ID NO:4, and/or having a polynucleotide sequence of SEQ ID NO:4; (b) cultivating the transformed host cell under conditions suitable for the host cell to produce the protease; and
(c) recovering the protease. In some embodiments, the host cell is a Bacillus species (e.g., B. subtilis, B. clausii, or B. licheniformis). In alternative embodiments, the host cell is a Streptomyces spp., (e.g., Streptomyces lividans). In additional embodiments, the host cell is a Trichoderma spp., (e.g., Trichoderma reesei). In yet further embodiments, the host cell is a Aspergillus spp., (e.g., Aspergillus niger).
As will be appreciated, an advantage of the present invention is that a polynucleotide has been isolated which provides the capability of isolating further polynucleotides which encode proteins having serine protease activity, wherein the backbone is substantially identical to that of the Cellulomonas protease of the invention.
In further embodiments, the present invention provides means to produce host cells that are capable of producing the serine proteases of the present invention in relatively large quantities. In particularly preferred embodiments, the present invention provides means to produce protease with various commercial applications where degradation or synthesis of polypeptides are desired, including cleaning compositions, as well as feed components, textile processing, leather finishing, grain processing, meat processing, cleaning, preparation of protein hydrolysates, digestive aids, microbicidal compositions, bacteriostatic composition, fungistatic compositions, personal care products, including oral care, hair care, and/or skin care.
The present invention further provides enzyme compositions have comparable or improved wash performance, as compared to presently used subtilisin proteases. Other objects and advantages of the present invention are apparent from the present Specification.



DESCRIPTION OF THE FIGURES
FIG. 1 provides an unrooted phylogenetic tree illustrating the relationship of novel strain 69B4 to members of the family Cellulomonadaceae and other related genera of the suborder Micrococcineae. 
FIG. 2 provides a phylogenetic tree for ASP protease.
FIG. 3 provides a MALDI TOF spectrum of a protease derived from Cellulomonas strain 69B4
FIG. 4 shows the sequence of N-terminal most tryptic peptide from C. flavigena 
FIG. 5 provides the plasmid map of the pSEGCT vector.
FIG. 6 provides the plasmid map of the pSEGCT69B4 vector.
FIG. 7 provides the plasmid map of the pSEA469BCT vector.
FIG. 8 provides the plasmid map of the pHPLT-Asp-C1-1 vector.
FIG. 9 provides the plasmid map of the pHPLT-Asp-C1-2 vector.
FIG. 10 provides the plasmid map of the pHPLT-Asp-C2-1 vector.
FIG. 11 provides the plasmid map of the pHPLT-Asp-C2-2 vector.
FIG. 12 provides the plasmid map of the pHPLT-ASP-III vector.
FIG. 13 provides the plasmid map of the pHPLT-ASP-IV vector.
FIG. 14 provides the plasmid map of the pHPLT-ASP-VII vector.
FIG. 15 provides the plasmid map of the pXX-KpnI vector.
FIG. 16 provides the plasmid map of the p2JM103-DNNP1 vector.
FIG. 17 provides the plasmid map of the pHPLT vector.
FIG. 18 provides the map and MXL-prom sequences for the opened pHPLT-ASP-C1-2.
FIG. 19 provides the plasmid map of the pENMx3 vector.
FIG. 20 provides the plasmid map of the pICatH vector.
FIG. 21 provides the plasmid map of the pTREX4 vector.
FIG. 22 provides the plasmid map of the pSLGAMpR2 vector.
FIG. 23 provides the plasmid map of the pRAXdes2-ASP vector.
FIG. 24 provides the plasmid map of the pAPDI vector.
FIG. 25 provides graphs showing ASP autolysis. Panel A provides a graph showing the ASP autolysis peptides observed in a buffer without LAS. Panel B provides a graph showing the ASP autolysis peptides observed in a buffer with 0.1% LAS.
FIG. 26 compares the cleaning activity (absorbance at 405 nm) dose (ppm) response curves of certain serine proteases (69B4 [-x-]; PURAFECT® [-♦-]; RELASETM [-▴]; and OPTIMASETM [-▪-] in liquid TIDE® detergent under North American wash conditions.
FIG. 27 provides a graph that compares the cleaning activity (absorbance at 405 nm) dose (ppm) response curves of certain serine proteases (69B4 [-x-]; PURAFECT® [-♦-]; RELASETM [-▴-]; and OPTIMASETM [-▪-] in Detergent Composition III powder detergent (0.66 g/l) North American concentration/detergent formulation under Japanese wash conditions.
FIG. 28 provides a graph that compares the cleaning activity (absorbance at 405 nm) dose (ppm) response curves of certain serine proteases (69B4 [-x-]; PURAFECT® [-♦-]; RELASETM [-▴]; and OPTIMASETM [-▪-] in ARIEL® REGULAR detergent powder under European wash conditions.
FIG. 29 provides a graph that compares the cleaning activity (absorbance at 405 nm) dose (ppm) response curves of certain serine protease (69B4 [-x-]; PURAFECT® [-♦-]; RELASETM [-▴-]; and OPTIMASETM [-▪-] in PURE CLEAN detergent powder under Japanese conditions.
FIG. 30 provides a graph that compares the cleaning activity (absorbance at 405 nm) dose (ppm) response curves of certain serine proteases (69B4 [-x-]; PURAFECT® [-♦-]; RELASETM [-▴-]; and OPTIMASETM [-▪] in Detergent Composition III powder (1.00 g/l) under North American conditions.
FIG. 31 provides a graph that shows comparative oxidative inactivation of various serine proteases (100 ppm) as a measure of percent enzyme activity over time (minutes) (69B4 [-x-]; BPN′ variant 1 [-♦-]; PURAFECT® [-▴-]; and GG36-variant 1 [-▪-]) with 0.1 M H2O2 at pH 9.45, 25° C.
FIG. 32 provides a graph that shows comparative chelator inactivation of various serine proteases (100 ppm) as a measure of percent enzyme activity over time (minutes) (69B4 [-x-]; BPN′-variant 1 [-♦-]; PURAFECT® [-▴-]; and GG36-variant 1 [-▪-] with 10 mM EDTA at pH 8.20, 45° C.
FIG. 33 provides a graph that shows comparative thermal inactivation of various serine proteases (100 ppm) as a measure of percent enzyme activity over time (minutes) (69B4 [-x-]; BPN′-variant [-♦-]; PURAFECT® [-▴-]; and GG36-variant 1 [-▪-] with 50 mM Tris at pH 8.0, 45° C.
FIG. 34 provides a graph that shows comparative thermal inactivation of certain serine proteases (69B4 [-x-]; BPN′-variant [-♦]; PURAFECT® [-▴-]; and GG36-variant-1 [▪-] at pH 8.60, over a temperature gradient of 57° C. to 62° C.
FIG. 35 provides a graph that shows enzyme activity (hydrolysis of di-methyl casein measured by absorbance at 405 nm) of certain serine proteases (2.5 ppm) (69B4 [▪-]; BPN′-variant [-♦-PURAFECT® [-▴-]; and GG36-variant 1[-●-] at pH's ranging from 5 to 12 at 37° C.
FIG. 36 provides a bar graph that shows enzyme stability as indicated by % remaining activity (hydrolysis of di-methyl casein measured by absorbance at 405 nm) of certain serine proteases (2.5 ppm) (69B4, BPN′-variant; PURAFECT® and GG36-variant 1 at pHs ranging from 3 (), 4 (), 5 () to 6 () at 25°, 35°, and 45° C., respectively.
FIG. 37 provides a graph that shows enzyme stability as indicated by % remaining activity of a BPN′-variant at pH ranges from 3 (-▾-), 4 (--▪--), 5 (--▴--) to 6 (--X--) at 25°, 35°, and 45° C., respectively
FIG. 38 provides a graph that shows enzyme stability as indicated by % remaining activity of PURAFECT® TM protease at pH ranges from 3 (-♦-), 4 (--▪--), 5 (--▴--) to 6 (--X--) at 25°, 35°, and 45° C., respectively
FIG. 39 provides a graph that shows enzyme stability as indicated by % remaining activity of 69B4 protease at pH ranges from 3 (-♦-), 4 (--▪--), 5 (--▴--) to 6 (--X--) at 25°, 35° and 45° C., respectively



DESCRIPTION OF THE INVENTION
The present invention provides novel serine proteases, novel genetic material encoding these enzymes, and proteolytic proteins obtained from Micrococcineae spp., including but not limited to Cellulomonas spp. and variant proteins developed therefrom. In particular, the present invention provides protease compositions obtained from a Cellulomonas spp, DNA encoding the protease, vectors comprising the DNA encoding the protease, host cells transformed with the vector DNA, and an enzyme produced by the host cells. The present invention also provides cleaning compositions (e.g., detergent compositions), animal feed compositions, and textile and leather processing compositions comprising protease(s) obtained from a Micrococcineae spp., including but not limited to Cellulomonas spp. In alternative embodiments, the present invention provides mutant (i.e., variant) proteases derived from the wild-type proteases described herein. These mutant proteases also find use in numerous applications:
Gram-positive alkalophilic bacteria have been isolated from in and around alkaline soda lakes (See e.g., U.S. Pat. No. 5,401,657, herein incorporated by reference). These alkalophilic were analyzed according to the principles of numerical taxonomy with respect to each other and also a collection of known bacteria, and taxonomically characterized. Six natural clusters or phenons of alkalophilic bacteria were generated. Amongst the strains isolated was a strain identified as 69B4.
Cellulomonas spp. are Gram-positive bacteria classified as members of the family Cellulomonadaceae, Suborder Micrococcineae, Order Actinomycetales, Class Actinobacteria. Cellulomonas grows as slender, often irregular rods that may occasionally show branching, but no mycelium is formed. In addition, there is no aerial growth and no spores are formed. Cellulomonas and Streptomyces are only distantly related at a genetic level. The large genetic (genomic) distinction between Cellulomonas and Streptomyces is reflected in a great difference in phenotypic properties. While serine proteses in Streptomyces have been previously examined, there apparently have been no reports of any serine proteases (approx. MW 18,000 to 20,000) secreted by Cellulomonas spp. In addition, there apparently have been no previous reports of Cellulomonas proteases being used in the cleaning and/or feed industry.
Streptomyces are Gram-positive bacteria classified as members of the Family Streptomycetaceae, Suborder Streptomycineae, Order Actinomycetales, class Actinobacteria. Streptomyces grows as an extensively branching primary or substrate mycelium and an abundant aerial mycelium that at maturity bear characteristic spores. Streptogrisins are serine proteases secreted in large amounts from a wide variety of Streptomyces species. The amino acid sequences of Streptomyces proteases have been determined from at least 9 different species of Streptomyces including Streptomyces griseus Streptogrisin C (accession no. P52320); alkaline proteinase (EC 3.4.21.-) from Streptomyces sp. (accession no. PC2053); alkaline serine proteinase I from Streptomyces sp. (accession no. S34672), serine protease from Streptomyces lividans (accession no. CAD4208); putative serine protease from Streptomyces coelicolor A3(2) (accession no. NP—625129); putative serine protease from Streptomyces avermitilis MA-4680 (accession no. NP—822175); serine protease from Streptomyces lividans (accession no. CAD42809); putative serine protease precursor from Streptomyces coelicolor A3(2) (accession no. NP—628830)). A purified native alkaline protease having an apparent molecular weight of 19,000 daltons and isolated from Streptomyces griseus var. alcalophilus protease and cleaning compositions comprised thereof have been described (See e.g., U.S. Pat. No. 5,646,028, incorporated herein by reference).
The present invention provides protease enzymes produced by these organisms. Importantly, these enzymes have good stability and proteolytic activity. These enzymes find use in various applications, including but not limited to cleaning compositions, animal feed, textile processing and etc. The present invention also provides means to produce these enzymes. In some preferred embodiments, the proteases of the present invention are in pure or relatively pure form.
The present invention also provides nucleotide sequences which are suitable to produce the proteases of the present invention in recombinant organisms. In some embodiments, recombinant production provides means to produce the proteases in quantities that are commercially viable.
Unless otherwise indicated, the practice of the present invention involves conventional techniques commonly used in molecular biology, microbiology, and recombinant DNA, which are within the skill of the art. Such techniques are known to those of skill in the art and are described in numerous texts and reference works (See e.g., Sambrook et al., “Molecular Cloning: A Laboratory Manual”, Second Edition (Cold Spring Harbor), [1989]); and Ausubel et al., “Current Protocols in Molecular Biology” [1987]). All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby expressly incorporated herein by reference.
Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, N.Y. (1991) provide those of skill in the art with a general dictionaries of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular “a”, “an” and “the” includes the plural reference unless the context clearly indicates otherwise. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.
The practice of the present invention employs, unless otherwise indicated, conventional techniques of protein purification, molecular biology, microbiology, recombinant DNA techniques and protein sequencing, all of which are within the skill of those in the art.
Furthermore, the headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole. Nonetheless, in order to facilitate understanding of the invention, a number of terms are defined below.
I. Definitions
As used herein, the terms “protease,” and “proteolytic activity” refer to a protein or peptide exhibiting the ability to hydrolyze peptides or substrates having peptide linkages. Many well known procedures exist for measuring proteolytic activity (Kalisz, “Microbial Proteinases,” In: Fiechter (ed.), Advances in Biochemical Engineering/Biotechnology, [1988]). For example, proteolytic activity may be ascertained by comparative assays which analyze the respective protease's ability to hydrolyze a commercial substrate. Exemplary substrates useful in the such analysis of protease or protelytic activity, include, but are not limited to di-methyl casein (Sigma C-9801), bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625), and bovine keratin (ICN Biomedical 902111). Colorimetric assays utilizing these substrates are well known in the art (See e.g., WO 99/34011; and U.S. Pat. No. 6,376,450, both of which are incorporated herein by reference. The pNA assay (See e.g., Del Mar et al., Anal. Biochem., 99:316-320 [1979]) also finds use in determining the active enzyme concentration for fractions collected during gradient elution. This assay measures the rate at which p-nitroaniline is released as the enzyme hydrolyzes the soluble synthetic substrate, succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (sAAPF-pNA). The rate of production of yellow color from the hydrolysis reaction is measured at 410 nm on a spectrophotometer and is proportional to the active enzyme concentration. In addition, absorbance measurements at 280 nm can be used to determine the total protein concentration. The active enzyme/total-protein ratio gives the enzyme purity.
As used herein, the terms “ASP protease,” “Asp protease,” and “Asp,” refer to the serine proteases described herein. In some preferred embodiments, the Asp protease is the protease designed herein as 69B4 protease obtained from Cellulomonas strain 69B4. Thus, in preferred embodiments, the term “69B4 protease” refers to a naturally occurring mature protease derived from Cellulomonas strain 69B4 (DSM 16035) having substantially identical amino acid sequences as provided in SEQ ID NO:8. In alternative embodiments, the present invention provides portions of the ASP protease.
The term “Cellulomonas protease homologues” refers to naturally occurring proteases having substantially identical amino acid sequences to the mature protease derived from Cellulomonas strain 69B4 or polynucleotide sequences which encode for such naturally occurring proteases, and which proteases retain the functional characteristics of a serine protease encoded by such nucleic acids. In some embodiments, these protease homologues are referred to as “cellulomonadins.”
As used herein, the terms “protease variant,” “ASP variant,” “ASP protease variant,” and “69B protease variant” are used in reference to proteases that are similar to the wild-type ASP, particularly in their function, but have mutations in their amino acid sequence that make them different in sequence from the wild-type protease.
As used herein, “Cellulomonas ssp.” refers to all of the species within the genus “Cellulomonas,” which are Gram-positive bacteria classified as members of the Family Cellulomonadaceae, Suborder Micrococcineae, Order Actinomycetales, Class Actinobacteria. It is recognized that the genus Cellulomonas continues to undergo taxonomical reorganization. Thus, it is intended that the genus include species that have been reclassified
As used herein, “Streptomyces ssp.” refers to all of the species within the genus “Streptomyces,” which are Gram-positive bacteria classified as members of the Family Streptomycetaceae, Suborder Streptomycineae, Order Actinomycetales, class Actinobacteria. It is recognized that the genus Streptomyces continues to undergo taxonomical reorganization. Thus, it is intended that the genus include species that have been reclassified
As used herein, “the genus Bacillus” includes all species within the genus “Bacillus,” as known to those of skill in the art, including but not limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis. It is recognized that the genus Bacillus continues to undergo taxonomical reorganization. Thus, it is intended that the genus include species that have been reclassified, including but not limited to such organisms as B. stearothermophilus, which is now named “Geobacillus stearothermophilus.” The production of resistant endospores in the presence of oxygen is considered the defining feature of the genus Bacillus, although this characteristic also applies to the recently named Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus, and Virgibacillus. 
The terms “polynucleotide” and “nucleic acid”, used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. These terms include, but are not limited to, a single-, double- or triple-stranded DNA genomic DNA, cDNA, RNA, DNA-RNA hybrid, or a polymer comprising purine and pyrimidine bases, or other natural, chemically, biochemically modified, non-natural or derivatized nucleotide bases. The following are non-limiting examples of polynucleotides: genes, gene fragments, chromosomal fragments, ESTs, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. In some embodiments, polynucleotides comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracil, other sugars and linking groups such as fluororibose and thioate, and nucleotide branches. In alternative embodiments, the sequence of nucleotides is interrupted by non-nucleotide components.
As used herein, the terms “DNA construct” and “transforming DNA” are used interchangeably to refer to DNA used to introduce sequences into a host cell or organism. The DNA may be generated in vitro by PCR or any other suitable technique(s) known to those in the art. In particularly preferred embodiments, the DNA construct comprises a sequence of interest (e.g., as an incoming sequence). In some embodiments, the sequence is operably linked to additional elements such as control elements (e.g., promoters, etc.). The DNA construct may further comprise a selectable marker. It may further comprise an incoming sequence flanked by homology boxes. In a further embodiment, the transforming DNA comprises other non-homologous sequences, added to the ends (e.g., stuffer sequences or flanks). In some embodiments, the ends of the incoming sequence are closed such that the transforming DNA forms a closed circle. The transforming sequences may be wild-type, mutant or modified. In some embodiments, the DNA construct comprises sequences homologous to the host cell chromosome. In other embodiments, the DNA construct comprises non-homologous sequences. Once the DNA construct is assembled in vitro it may be used to: 1) insert heterologous sequences into a desired target sequence of a host cell, and/or 2) mutagenize a region of the host cell chromosome (i.e., replace an endogenous sequence with a heterologous sequence), 3) delete target genes; and/or introduce a replicating plasmid into the host.
As used herein, the terms “expression cassette” and “expression vector” refer to nucleic acid constructs generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In preferred embodiments, expression vectors have the ability to incorporate and express heterologous DNA fragments in a host cell. Many prokaryotic and eukaryotic expression vectors are commercially available. Selection of appropriate expression vectors is within the knowledge of those of skill in the art. The term “expression cassette” is used interchangeably herein with “DNA construct,” and their grammatical equivalents. Selection of appropriate expression vectors is within the knowledge of those of skill in the art.
As used herein, the term “vector” refers to a polynucleotide construct designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, cassettes and the like. In some embodiments, the polynucleotide construct comprises a DNA sequence encoding the protease (e.g., precursor or mature protease) that is operably linked to a suitable prosequence (e.g., secretory, etc.) capable of effecting the expression of the DNA in a suitable host.
As used herein, the term “plasmid” refers to a circular double-stranded (ds) DNA construct used as a cloning vector, and which forms an extrachromosomal self-replicating genetic element in some eukaryotes or prokaryotes, or integrates into the host chromosome.
As used herein in the context of introducing a nucleic acid sequence into a cell, the term “introduced” refers to any method suitable for transferring the nucleic acid sequence into the cell. Such methods for introduction include but are not limited to protoplast fusion, transfection, transformation, conjugation, and transduction (See e.g., Ferrari et al., “Genetics,” in Hardwood et al, (eds.), Bacillus, Plenum Publishing Corp., pages 57-72, [1989]).
As used herein, the terms “transformed” and “stably transformed” refers to a cell that has a non-native (heterologous) polynucleotide sequence integrated into its genome or as an episomal plasmid that is maintained for at least two generations.
As used herein, the term “selectable marker-encoding nucleotide sequence” refers to a nucleotide sequence which is capable of expression in the host cells and where expression of the selectable marker confers to cells containing the expressed gene the ability to grow in the presence of a corresponding selective agent or lack of an essential nutrient.
As used herein, the terms “selectable marker” and “selective marker” refer to a nucleic acid (e.g., a gene) capable of expression in host cell which allows for ease of selection of those hosts containing the vector. Examples of such selectable markers include but are not limited to antimicrobials. Thus, the term “selectable marker” refers to genes that provide an indication that a host cell has taken up an incoming DNA of interest or some other reaction has occurred. Typically, selectable markers are genes that confer antimicrobial resistance or a metabolic advantage on the host cell to allow cells containing the exogenous DNA to be distinguished from cells that have not received any exogenous sequence during the transformation. A “residing selectable marker” is one that is located on the chromosome of the microorganism to be transformed. A residing selectable marker encodes a gene that is different from the selectable marker on the transforming DNA construct. Selective markers are well known to those of skill in the art. As indicated above, preferably the marker is an antimicrobial resistant marker (e.g., ampR; phleoR; specR; kanR; eryR; tetR; cmpR; and neoR; See e.g., Guerot-Fleury, Gene, 167:335-337 [1995]; Palmeros et al., Gene 247:255-264 [2000]; and Trieu-Cuot et al., Gene, 23:331-341 [1983]). Other markers useful in accordance with the invention include, but are not limited to auxotrophic markers, such as tryptophan; and detection markers, such asp-galactosidase.
As used herein, the term “promoter” refers to a nucleic acid sequence that functions to direct transcription of a downstream gene. In preferred embodiments, the promoter is appropriate to the host cell in which the target gene is being expressed. The promoter, together with other transcriptional and translational regulatory nucleic acid sequences (also termed “control sequences”) is necessary to express a given gene. In general, the transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA encoding a secretory leader a signal peptide), is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
As used herein the term “gene” refers to a polynucleotide (e.g., a DNA segment), that encodes a polypeptide and includes regions preceding and following the coding regions as well as intervening sequences (introns) between individual coding segments (exons).
As used herein, “homologous genes” refers to a pair of genes from different, but usually related species, which correspond to each other and which are identical or very similar to each other. The term encompasses genes that are separated by speciation (i.e., the development of new species) (e.g., orthologous genes), as well as genes that have been separated by genetic duplication (e.g., paralogous genes).
As used herein, “ortholog” and “orthologous genes” refer to genes in different species that have evolved from a common ancestral gene (i.e., a homologous gene) by speciation. Typically, orthologs retain the same function during the course of evolution. Identification of orthologs finds use in the reliable prediction of gene function in newly sequenced genomes.
As used herein, “paralog” and “paralogous genes” refer to genes that are related by duplication within a genome. While orthologs retain the same function through the course of evolution, paralogs evolve new functions, even though some functions are often related to the original one. Examples of paralogous genes include, but are not limited to genes encoding trypsin, chymotrypsin, elastase, and thrombin, which are all serine proteinases and occur together within the same species.
As used herein, “homology” refers to sequence similarity or identity, with identity being preferred. This homology is determined using standard techniques known in the art (See e.g., Smith and Waterman, Adv. Appl. Math., 2:482 [1981]; Needleman and Wunsch, J. Mol. Biol., 48:443 [1970]; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988]; programs such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, Wis.); and Devereux et al., Nucl. Acid Res., 12:387-395 [1984]).
As used herein, an “analogous sequence” is one wherein the function of the gene is essentially the same as the gene based on the Cellulomonas strain 69B4 protease. Additionally, analogous genes include at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity with the sequence of the Cellulomonas strain 69B4 protease. Alternately, analogous sequences have an alignment of between 70 to 100% of the genes found in the Cellulomonas strain 69B4 protease region and/or have at least between 5-10 genes found in the region aligned with the genes in the Cellulomonas strain 69B4 chromosome. In additional embodiments more than one of the above properties applies to the sequence. Analogous sequences are determined by known methods of sequence alignment. A commonly used alignment method is BLAST, although as indicated above and below, there are other methods that also find use in aligning sequences.
One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pair-wise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (Feng and Doolittle, J. Mol. Evol., 35:351-360 [1987]). The method is similar to that described by Higgins and Sharp (Higgins and Sharp, CABIOS 5:151-153 [1989]). Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.
Another example of a useful algorithm is the BLAST algorithm, described by Altschul et al., (Altschul et al., J. Mol. Biol., 215:403-410, [1990]; and Karlin et al., Proc. Natl. Acad. Sci. USA 90:5873-5787 [1993]). A particularly useful BLAST program is the WU-BLAST-2 program (See, Altschul et al., Meth. Enzymol., 266:460-480 [1996]). WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span=1, overlap fraction=0.125, word threshold (T)=11. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched. However, the values may be adjusted to increase sensitivity. A % amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the “longer” sequence in the aligned region. The “longer” sequence is the one having the most actual residues in the aligned region (gape introduced by WU-Blast-2 to maximize the alignment score are ignored).
Thus, “percent (%) nucleic acid sequence identity” is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues of the starting sequence (i.e., the sequence of interest). A preferred method utilizes the so BLASTN module of WU-BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.
As used herein, the term “hybridization” refers to the process by which a strand of nucleic acid joins with a complementary strand through base pairing, as known in the art.
A nucleic acid sequence is considered to be “selectively hybridizable” to a reference nucleic acid sequence if the two sequences specifically hybridize to one another under moderate to high stringency hybridization and wash conditions. Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex or probe. For example, “maximum stringency” typically occurs at about Tm-5° C. (5° below the Tm of the probe); “high stringency” at about 5-10° C. below the Tm; “intermediate stringency” at about 10-20° C. below the Tm of the probe; and “low stringency” at about 20-25° C. below the Tm. Functionally, maximum stringency conditions may be used to identify sequences having strict identity or near-strict identity with the hybridization probe; while an intermediate or low stringency hybridization can be used to identify or detect polynucleotide sequence homologs.
Moderate and high stringency hybridization conditions are well known in the art. An example of high stringency conditions includes hybridization at about 42° C. in 50% formamide, 5×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured carrier DNA followed by washing two times in 2×SSC and 0.5% SDS at room temperature and two additional times in 0.1×SSC and 0.5% SDS at 42° C. An example of moderate stringent conditions include an overnight incubation at 37° C. in a solution comprising 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1×SSC at about 37-50° C. Those of skill in the art know how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
As used herein, “recombinant” includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid sequence or that the dell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all as a result of deliberate human intervention. “Recombination,” “recombining,” and generating a “recombined” nucleic acid are generally the assembly of two or more nucleic acid fragments wherein the assembly gives rise to a chimeric gene.
In a preferred embodiment, mutant DNA sequences are generated with site saturation mutagenesis in at least one codon. In another preferred embodiment, site saturation mutagenesis is performed for two or more codons. In a further embodiment, mutant DNA sequences have more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% homology with the wild-type sequence. In alternative embodiments, mutant DNA is generated in vivo using any known mutagenic procedure such as, for example, radiation, nitrosoguanidine and the like. The desired DNA sequence is then isolated and used in the methods provided herein.
As used herein, the term “target sequence” refers to a DNA sequence in the host cell that encodes the sequence where it is desired for the incoming sequence to be inserted into the host cell genome. In some embodiments, the target sequence encodes a functional wild-type gene or operon, while in other embodiments the target sequence encodes a functional mutant gene or operon, or a non-functional gene or operon.
As used herein, a “flanking sequence” refers to any sequence that is either upstream or downstream of the sequence being discussed (e.g., for genes A-B-C, gene B is flanked by the A and C gene sequences). In a preferred embodiment, the incoming sequence is flanked by a homology box on each side. In another embodiment, the incoming sequence and the homology boxes comprise a unit that is flanked by stuffer sequence on each side. In some embodiments, a flanking sequence is present on only a single side (either 3′ or 5′), but in preferred embodiments, it is on each side of the sequence being flanked. In some embodiments, a flanking sequence is present on only a single side (either 3′ or 5′), while in preferred embodiments, it is present on each side of the sequence being flanked.
As used herein, the term “stuffer sequence” refers to any extra DNA that flanks homology boxes (typically vector sequences). However, the term encompasses any non-homologous DNA sequence. Not to be limited by any theory, a stuffer sequence provides a noncritical target for a cell to initiate DNA uptake.
As used herein, the terms “amplification” and “gene amplification” refer to a process by which specific DNA sequences are disproportionately replicated such that the amplified gene becomes present in a higher copy number than was initially present in the genome. In some embodiments, selection of cells by growth in the presence of a drug (e.g., an inhibitor of an inhibitable enzyme) results in the amplification of either the endogenous gene encoding the gene product required for growth in the presence of the drug or by amplification of exogenous (i.e., input) sequences encoding this gene product, or both.
“Amplification” is a special case of nucleic acid replication involving template specificity. It is to be contrasted with non-specific template replication (i.e., replication that is template-dependent but not dependent on a specific template). Template specificity is here distinguished from fidelity of replication (i.e., synthesis of the proper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-) specificity. Template specificity is frequently described in terms of “target” specificity. Target sequences are “targets” in the sense that they are sought to be sorted out from other nucleic acid. Amplification techniques have been designed primarily for this sorting out.
As used herein, the term “co-amplification” refers to the introduction into a single cell of an amplifiable marker in conjunction with other gene sequences (i.e., comprising one or more non-selectable genes such as those contained within an expression vector) and the application of appropriate selective pressure such that the cell amplifies both the amplifiable marker and the other, non-selectable gene sequences. The amplifiable marker may be physically linked to the other gene sequences or alternatively two separate pieces of DNA, one containing the amplifiable marker and the other containing the non-selectable marker, may be introduced into the same cell.
As used herein, the terms “amplifiable marker,” “amplifiable gene,” and “amplification vector” refer to a gene or a vector encoding a gene which permits the amplification of that gene under appropriate, growth conditions.
“Template specificity” is achieved in most amplification techniques by the choice of enzyme. Amplification enzymes are enzymes that, under conditions they are used, will process only specific sequences of nucleic acid in a heterogeneous mixture of nucleic acid. For example, in the case of Qβ replicase, MDV-1 RNA is the specific template for the replicase (See e.g., Kacian et al., Proc. Natl. Acad. Sci. USA 69:3038 [1972]). Other nucleic acids are not replicated by this amplification enzyme. Similarly, in the case of T7 RNA polymerase, this amplification enzyme has a stringent specificity for its own promoters (See, Chamberlin et al., Nature 228:227 [1970]). In the case of T4 DNA ligase; the enzyme will not ligate the two oligonucleotides or polynucleotides, where there is a mismatch between the oligonucleotide or polynucleotide substrate and the template at the ligation junction (See, Wu and Wallace, Genomics 4:560 [1989]). Finally, Taq and Pfu polymerases, by virtue of their ability to function at high temperature, are found to display high specificity for the sequences bounded and thus defined by the primers; the high temperature results in thermodynamic conditions that favor primer hybridization with the target sequences and not hybridization with non-target sequences.
As used herein, the term “amplifiable nucleic acid” refers to nucleic acids which may be amplified by any amplification method. It is contemplated that “amplifiable nucleic acid” will usually comprise “sample template.”
As used herein, the term “sample template” refers to nucleic acid originating from a sample which is analyzed for the presence of “target” (defined below). In contrast, “background template” is used in reference to nucleic acid other than sample template which may or may not be present in a sample. Background template is most often inadvertent. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample.
As used herein, the term “primer” refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.
As used herein, the term “probe” refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, which is capable of hybridizing to another oligonucleotide of interest. A probe may be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular gene sequences. It is contemplated that any probe used in the present invention will be labeled with any “reporter molecule,” so that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.
As used herein, the term “target,” when used in reference to the polymerase chain reaction, refers to the region of nucleic acid bounded by the primers used for polymerase chain reaction. Thus, the “target” is sought to be sorted out from other nucleic acid sequences. A “segment” is defined as a region of nucleic acid within the target sequence.
As used herein, the term “polymerase chain reaction” (“PCR”) refers to the methods of U.S. Pat. Nos. 4,683,195 4,683,202, and 4,965,188, hereby incorporated by reference, which include methods for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded target sequence. To effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one “cycle”; there can be numerous “cycles”) to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative position's of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the “polymerase chain reaction” (hereinafter “PCR”). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be “PCR amplified”.
As used herein, the term “amplification reagents” refers to those reagents (deoxyribonucleotide triphosphates, buffer, etc.), needed for amplification except for primers, nucleic acid template and the amplification enzyme. Typically, amplification reagents along with other reaction components are placed and contained in a reaction vessel (test tube, microwell, etc.).
With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of 32P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide or polynucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.
As used herein, the terms “PCR product,” “PCR fragment,” and “amplification product” refer to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing and extension are complete. These terms encompass the case where there has been amplification of one or more segments of one or more target sequences.
As used herein, the term “RT-PCR” refers to the replication and amplification of RNA sequences. In this method, reverse transcription is coupled to PCR, most often using a one enzyme procedure in which a thermostable polymerase is employed, as described in U.S. Pat. No. 5,322,770, herein incorporated by reference. In RT-PCR, the RNA template is converted to cDNA due to the reverse transcriptase activity of the polymerase, and then amplified using the polymerizing activity of the polymerase (i.e., as in other PCR methods).
As used herein, the terms “restriction endonucleases” and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
A “restriction site” refers to a nucleotide sequence recognized and cleaved by a given restriction endonuclease and is frequently the site for insertion of DNA fragments. In certain embodiments of the invention restriction sites are engineered into the selective marker and into 5′ and 3′ ends of the DNA construct.
As used herein, the term “chromosomal integration” refers to the process whereby an incoming sequence is introduced into the chromosome of a host cell. The homologous regions of the transforming DNA align with homologous regions of the chromosome. Subsequently, the sequence between the homology boxes is replaced by the incoming sequence in a double crossover (i.e., homologous recombination). In some embodiments of the present invention, homologous sections of an inactivating chromosomal segment of a DNA construct align with the flanking homologous regions of the indigenous chromosomal region of the Bacillus chromosome. Subsequently, the indigenous chromosomal region is deleted by the DNA construct in a double crossover (i.e., homologous recombination).
“Homologous recombination” means the exchange of DNA fragments between two DNA molecules or paired chromosomes at the site of identical or nearly identical nucleotide sequences. In a preferred embodiment, chromosomal integration is homologous recombination.
“Homologous sequences” as used herein means a nucleic acid or polypeptide sequence having 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 88%, 85%, 80%, 75%, or 70% sequence identity to another nucleic acid or polypeptide sequence when optimally aligned for comparison. In some embodiments, homologous sequences have between 85% and 100% sequence identity, while in other embodiments there is between 90% and 100% sequence identity, and in more preferred embodiments, there is 95% and 100% sequence identity.
As used herein “amino acid” refers to peptide or protein sequences or portions thereof. The terms “protein,” “peptide,” and “polypeptide” are used interchangeably.
As used herein, “protein of interest” and “polypeptide of interest” refer to a protein/polypeptide that is desired and/or being assessed. In some embodiments, the protein of interest is expressed intracellularly, while in other embodiments, it is a secreted polypeptide. In particularly preferred embodiments, these enzyme include the serine proteases of the present invention. In some embodiments, the protein of interest is a secreted polypeptide which is fused to a signal peptide (i.e., an amino-terminal extension on a protein to be secreted). Nearly all secreted proteins use an amino-terminal protein extension which plays a crucial role in the targeting to and translocation of precursor proteins across the membrane. This extension is proteolytically removed by a signal peptidase during or immediately following membrane transfer.
As used herein, the term “heterologous protein” refers to a protein or polypeptide that does not naturally occur in the host cell. Examples of heterologous proteins include enzymes such as hydrolases including proteases. In some embodiments, the gene encoding the proteins are naturally occurring genes, while in other embodiments, mutated and/or synthetic genes are used.
As used herein, “homologous protein” refers to a protein or polypeptide native or naturally occurring in a cell. In preferred embodiments, the cell is a Gram-positive cell, while in particularly preferred embodiments, the cell is a Bacillus host cell. In alternative embodiments, the homologous protein is a native protein produced by other organisms, including but not limited to E. coli, Streptomyces, Trichoderma, and Aspergillus. The invention encompasses host cells producing the homologous protein via recombinant DNA technology.
As used herein, an “operon region” comprises a group of contiguous genes that are transcribed as a single transcription unit from a common promoter, and are thereby subject to co-regulation. In some embodiments, the operon includes a regulator gene. In most preferred embodiments, operons that are highly expressed as measured by RNA levels, but have an unknown or unnecessary function are used.
As used herein, an “antimicrobial region” is a region containing at least one gene that encodes an antimicrobial protein.
A polynucleotide is said to “encode” an RNA or a polypeptide if, in its native state or when manipulated by methods known to those of skill in the art, it can be transcribed and/or translated to produce the RNA, the polypeptide or a fragment thereof. The anti-sense strand of such a nucleic acid is also said to encode the sequences.
As is known in the art, a DNA can be transcribed by an RNA polymerase to produce RNA, but an RNA can be reverse transcribed by reverse transcriptase to produce a DNA. Thus a DNA can encode a RNA and vice versa.
The term “regulatory segment” or “regulatory sequence” or “expression control sequence” refers to a polynucleotide sequence of DNA that is operatively linked with a polynucleotide sequence of DNA that encodes the amino acid sequence of a polypeptide chain to effect the expression of the encoded amino acid sequence. The regulatory sequence can inhibit, repress, or promote the expression of the operably linked polynucleotide sequence encoding the amino acid.
“Host strain” or “host cell” refers to a suitable host for an expression vector comprising DNA according to the present invention.
An enzyme is “overexpressed” in a host cell if the enzyme is expressed in the cell at a higher level that the level at which it is expressed in a corresponding wild-type cell.
The terms “protein” and “polypeptide” are used interchangeability herein. The 3-letter code for amino acids as defined in conformity with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) is used through out this disclosure. It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.
A “prosequence” is an amino acid sequence between the signal sequence and mature protease that is necessary for the secretion of the protease. Cleavage of the pro sequence will result in a mature active protease.
The term “signal sequence” or “signal peptide” refers to any sequence of nucleotides and/or amino acids which may participate in the secretion of the mature or precursor forms of the protein. This definition of signal sequence is a functional one, meant to include all those amino acid sequences encoded by the N-terminal portion of the protein gene, which participate in the effectuation of the secretion of protein. They are often, but not universally, bound to the N-terminal portion of a protein or to the N-terminal portion of a precursor protein. The signal sequence may be endogenous or exogenous. The signal sequence may be that normally associated with the protein (e.g., protease), or may be from a gene encoding another secreted protein. One exemplary exogenous signal sequence comprises the first seven amino acid residues of the signal sequence from Bacillus subtilis subtilisin fused to the remainder of the signal sequence of the subtilisin from Bacillus lentus (ATCC 21536).
The term “hybrid signal sequence” refers to signal sequences in which part of sequence is obtained from the expression host fused to the signal sequence of the gene to be expressed. In some embodiments, synthetic sequences are utilized.
The term “substantially the same signal activity” refers to the signal activity, as indicated by substantially the same secretion of the protease into the fermentation medium, for example a fermentation medium protease level being at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% of the secreted protease levels in the fermentation medium as provided by the signal sequence of SEQ ID NOS:5 and/or 9.
The term “mature” form of a protein or peptide refers to the final functional form of the protein or peptide. To exemply, a mature form of the protease of the present invention at least includes the amino acid sequence identical to residue positions 1-189 of SEQ ID NO:8.
The term “precursor” form of a protein or peptide refers to a mature form of the protein having a prosequence operably linked to the amino or carbonyl terminus of the protein. The precursor may also have a “signal” sequence operably linked, to the amino terminus of the prosequence. The precursor may also have additional polynucleotides that are involved in post-translational activity (e.g., polynucleotides cleaved therefrom to leave the mature form of a protein or peptide).
“Naturally occurring enzyme” refers to an enzyme having the unmodified amino acid sequence identical to that found in nature. Naturally occurring enzymes include native enzymes, those enzymes naturally expressed or found in the particular microorganism.
The terms “derived from” and “obtained from” refer to not only a protease produced or producible by a strain of the organism in question, but also a protease encoded by a DNA sequence isolated from such strain and produced in a host organism containing such DNA sequence. Additionally, the term refers to a protease which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the protease in question. To exemplify, “proteases derived from Cellulomonas” refers to those enzymes having proteolytic activity which are naturally-produced by Cellulomonas, as well as to serine proteases like those produced by Cellulomonas sources but which through the use of genetic engineering techniques are produced by non-Cellulomonas organisms transformed with a nucleic acid encoding said serine proteases.
A “derivative” within the scope of this definition generally retains the characteristic proteolytic activity observed in the wild-type, native or parent form to the extent that the derivative is useful for similar purposes as the wild-type, native or parent form. Functional derivatives of serine protease encompass naturally occurring, synthetically or recombinantly produced peptides or peptide fragments which have the general characteristics of the serine protease of the present invention.
The term “functional derivative” refers to a derivative of a nucleic acid which has the functional characteristics of a nucleic acid which encodes serine protease. Functional derivatives of a nucleic acid which encode serine protease of the present invention encompass naturally occurring, synthetically or recombinantly produced nucleic acids or fragments and encode serine protease characteristic of the present invention. Wild type nucleic acid encoding serine proteases according to the invention include naturally occurring alleles and homologues based on the degeneracy of the genetic code known in the art.
The term “identical” in the context of two nucleic acids or polypeptide sequences refers to the residues in the two sequences that are the same when aligned for maximum correspondence, as measured using one of the following sequence comparison or analysis algorithms.
The term “optimal alignment” refers to the alignment giving the highest percent identity score.
“Percent sequence identity,” “percent amino acid sequence identity,” “percent gene sequence identity,” and/or “percent nucleic acid/polynucloetide sequence identity,” with respect to two amino acid, polynucleotide and/or gene sequences (as appropriate), refer to the percentage of residues that are identical in the two sequences when the sequences are optimally aligned. Thus, 80% amino acid sequence identity means that 80% of the amino acids in two optimally aligned polypeptide sequences are identical.
The phrase “substantially identical” in the context of two nucleic acids or polypeptides thus refers to a polynucleotide or polypeptide that comprising at least 70% sequence identity, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97%, preferably at least 98% and preferably at least 99% sequence identity as compared to a reference sequence using the programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters. One indication that two polypeptides are substantially identical is that the first polypeptide is immunologically cross-reactive with the second polypeptide. Typically, polypeptides that differ by conservative amino acid substitutions are immunologically cross-reactive. Thus, a polypeptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions (e.g., within a range of medium to high stringency).
The phrase “equivalent,” in this context, refers to serine proteases enzymes that are encoded by a polynucleotide capable of hybridizing to the polynucleotide having the sequence as shown in SEQ ID NO:1, under conditions of medium to maximal stringency. For example, being equivalent means that an equivalent mature serine protease comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and/or at least 99% sequence identity to the mature Cellulomonas serine protease having as the amino acid sequence of SEQ ID NO:8.
The term “isolated” or “purified” refers to a material that is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, the material is said to be “purified” when it is present in a particular composition in a higher or lower concentration than exists in a naturally occurring or wild type organism or in combination with components not normally present upon expression from a naturally occurring or wild type organism. For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector, and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment. In preferred embodiments, a nucleic acid or protein is said to be purified, for example, if it gives rise to essentially one band in an electrophoretic gel or blot.
The term “isolated”, when used in reference to a DNA sequence, refers to a DNA sequence that has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5′ and 3′ untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (See e.g., Dynan and Tijan, Nature 316:774-78 [1985]). The term “an isolated DNA sequence” is alternatively referred to as “a cloned DNA sequence”.
The term “isolated,” when used in reference to a protein, refers to a protein that is found in a condition other than its native environment. In a preferred form, the isolated protein is substantially free of other proteins, particularly other homologous proteins. An isolated protein is more than 10% pure, preferably more than 20% pure, and even more preferably more than 30% pure, as determined by SDS-PAGE. Further aspects of the invention encompass the protein in a highly purified form (i.e., more than 40% pure, more than 60% pure, more than 80% pure, more than 90% pure, more than 95% pure, more than 97% pure, and even more than 99% pure), as determined by SDS-PAGE.
As used herein, the term, “combinatorial mutagenesis” refers to methods in which libraries of variants of a starting sequence are generated. In these libraries, the variants contain one or several mutations chosen from a predefined set of mutations. In addition, the methods provide means to introduce random mutations which were not members of the predefined set of mutations. In some embodiments, the methods include those set forth in U.S. patent application Ser. No. 09/699,250, filed Oct. 26, 2000, hereby incorporated by reference. In alternative embodiments, combinatorial mutagenesis methods encompass commercially available kits (e.g., QuikChange® Multisite, Stratagene, San Diego, Calif.).
As used herein, the term “library of mutants” refers to a population of cells which are identical in most of their genome but include different homologues of one or more genes. Such libraries can be used, for example, to identify genes or operons with improved traits.
As used herein, the term “starting gene” refers to a gene of interest that encodes a protein of interest that is to be improved and/or changed using the present invention.
As used herein, the term “multiple sequence alignment” (“MSA”) refers to the sequences of multiple homologs of a starting gene that are aligned using an algorithm (e.g., Clustal W).
As used herein, the terms “consensus sequence” and “canonical sequence” refer to an archetypical amino acid sequence against which all variants of a particular protein or sequence of interest are compared. The terms also refer to a sequence that sets forth the nucleotides that are most often present in a DNA sequence of interest. For each position of a gene, the consensus sequence gives the amino acid that is most abundant in that position in the MSA.
As used herein, the term “consensus mutation” refers to a difference in the sequence of a starting gene and a consensus sequence. Consensus mutations are identified by comparing the sequences of the starting gene and the consensus sequence resulting from an MSA. In some embodiments, consensus mutations are introduced into the starting gene such that it becomes more similar to the consensus sequence. Consensus mutations also include amino acid changes that change an amino acid in a starting gene to an amino acid that is more frequently found in an MSA at that position relative to the frequency of that amino acid in the starting gene. Thus, the term consensus mutation comprises all single amino acid changes that replace an amino acid of the starting gene with an amino acid that is more abundant than the amino acid in the MSA.
As used herein, the term “initial hit” refers to a variant that was identified by screening a combinatorial consensus mutagenesis library. In preferred embodiments, initial hits have improved performance characteristics, as compared to the starting gene.
As used herein, the term “improved hit” refers to a variant that was identified by screening an enhanced combinatorial consensus mutagenesis library.
As used herein, the terms “improving mutation” and “performance-enhancing mutation” refer to a mutation that leads to improved performance when it is introduced into the starting gene. In some preferred embodiments, these mutations are identified by sequencing hits that were identified during the screening step of the method. In most embodiments, mutations that are more frequently found in hits are likely to be improving mutations, as compared to an unscreened combinatorial consensus mutagenesis library.
As used herein, the term “enhanced combinatorial consensus mutagenesis library” refers to a CCM library that is designed and constructed based on screening and/or sequencing results from an earlier round of CCM mutagenesis and screening. In some embodiments, the enhanced CCM library is based on the sequence of an initial hit resulting from an earlier round of CCM. In additional embodiments, the enhanced CCM is designed such that mutations that were frequently observed in initial hits from earlier rounds of mutagenesis and screening are favored. In some preferred embodiments, this is accomplished by omitting primers that encode performance-reducing mutations or by increasing the concentration of primers that encode performance-enhancing mutations relative to other primers, that were used in earlier CCM libraries.
As used herein, the term “performance-reducing mutations” refer to mutations in the combinatorial consensus mutagenesis library that are less frequently found in hits resulting from screening as compared to an unscreened combinatorial consensus mutagenesis library. In preferred embodiments, the screening process removes and/or reduces the abundance of variants that contain “performance-reducing mutations.”
As used herein, the term “functional assay” refers to an assay that provides an indication of a protein's activity. In particularly preferred embodiments, the term refers to assay systems in which a protein is analyzed for its ability to function in its usual capacity. For example, in the case of enzymes, a functional assay involves determining the effectiveness of the enzyme in catalyzing a reaction.
As used herein, the term “target property” refers to the property of the starting gene that is to be altered. It is not intended that the present invention be limited to any particular target property. However, in some preferred embodiments, the target property is the stability of a gene product (e.g., resistance to denaturation, proteolysis or other degradative factors), while in other embodiments, the level of production in a production host is altered. Indeed, it is contemplated that any property of a starting gene will find use in the present invention.
The term “property” or grammatical equivalents thereof in the context of a nucleic acid, as used herein, refer to any characteristic or attribute of a nucleic acid that can be selected or detected. These properties include, but are not limited to, a property affecting binding to a polypeptide, a property conferred on a cell comprising a particular nucleic acid, a property affecting gene transcription (e.g., promoter strength, promoter recognition, promoter regulation, enhancer function), a property affecting RNA processing (e.g., RNA splicing, RNA stability, RNA conformation, and post-transcriptional modification), a property affecting translation (e.g., level, regulation, binding of mRNA to ribosomal proteins, post-translational modification). For example, a binding site for a transcription factor, polymerase, regulatory factor, etc., of a nucleic acid may be altered to produce desired characteristics or to identify undesirable characteristics.
The term “property” or grammatical equivalents thereof in the context of a polypeptide, as used herein, refer to any characteristic or attribute of a polypeptide that can be selected or detected. These properties include, but are not limited to oxidative stability, substrate specificity, catalytic activity, thermal stability, alkaline stability, pH activity profile, resistance to proteolytic degradation, KM, Kcat, Kcat/kM ratio, protein folding, inducing an immune response, ability to bind to a ligand, ability to bind to a receptor, ability to be secreted, ability to be displayed on the surface of a cell, ability to oligomerize, ability to signal, ability to stimulate cell proliferation, ability to inhibit cell proliferation, ability to induce apoptosis, ability to be modified by phosphorylation or glycosylation, ability to treat disease.
As used herein, the term “screening” has its usual meaning in the art and is, in general a multi-step process. In the first step, a mutant nucleic acid or variant polypeptide therefrom is provided. In the second step, a property of the mutant nucleic acid or variant polypeptide is determined. In the third step, the determined property is compared to a property of the corresponding precursor nucleic acid, to the property of the corresponding naturally occurring polypeptide or to the property of the starting material (e.g., the initial sequence) for the generation of the mutant nucleic acid.
It will be apparent to the skilled artisan that the screening procedure for obtaining a nucleic acid or protein with an altered property depends upon the property of the starting material the modification of which the generation of the mutant nucleic acid is intended to facilitate. The skilled artisan will therefore appreciate that the invention is not limited to any specific property to be screened for and that the following description of properties lists illustrative examples only. Methods for screening for any particular property are generally described in the art. For example, one can measure binding, pH, specificity, etc., before and after mutation, wherein a change indicates an alteration. Preferably, the screens are performed in a high-throughput manner, including multiple samples being screened simultaneously, including, but not limited to assays utilizing chips, phage display, and multiple substrates and/or indicators.
As used herein, in some embodiments, screens encompass selection steps in which variants of interest are enriched from a population of variants. Examples of these embodiments include the selection of variants that confer a growth advantage to the host organism, as well as phage display or any other method of display, where variants can be captured from a population of variants based on their binding or catalytic properties. In a preferred embodiment, a library of variants is exposed to stress (heat, protease, denaturation) and subsequently variants that are still intact are identified in a screen or enriched by selection. It is intended that the term encompass any suitable means for selection. Indeed, it is not intended that the present invention be limited to any particular method of screening.
As used herein, the term “targeted randomization” refers to a process that produces a plurality of sequences where one or several positions have been randomized. In some embodiments, randomization is complete (i.e., all four nucleotides, A, T, G, and C can occur at a randomized position. In alternative embodiments, randomization of a nucleotide is limited to a subset of the four nucleotides. Targeted randomization can be applied to one or several codons of a sequence, coding for one or several proteins of interest. When expressed, the resulting libraries produce protein populations in which one or more amino acid positions can contain a mixture of all 20 amino acids or a subset of amino acids, as determined by the randomization scheme of the randomized codon. In some embodiments, the individual members of a population resulting from targeted randomization differ in the number of amino acids, due to targeted or random insertion or deletion of codons. In further embodiments, synthetic amino acids are included in the protein populations produced. In some preferred embodiments, the majority of members of a population resulting from targeted randomization show greater sequence homology to the consensus sequence than the starting gene. In some embodiments, the sequence encodes one or more proteins fo interest. In alternative embodiments, the proteins have differing biological functions. In some preferred embodiments, the incoming sequence comprises at least one selectable marker.
The terms “modified sequence” and “modified genes” are used interchangeably herein to refer to a sequence that includes a deletion, insertion or interruption of naturally occurring nucleic acid sequence. In some preferred embodiments, the expression product of the modified sequence is a truncated protein (e.g., if the modification is a deletion or interruption of the sequence). In some particularly preferred embodiments, the truncated protein retains biological activity. In alternative embodiments, the expression product of the modified sequence is an elongated protein (e.g., modifications comprising an insertion into the nucleic acid sequence). In some embodiments, an insertion leads to a truncated protein (e.g., when the insertion results in the formation of a stop codon). Thus, an insertion may result in either a truncated protein or an elongated protein as an expression product.
As used herein, the terms “mutant sequence” and “mutant gene” are used interchangeably and refer to a sequence that has an alteration in at least one codon occurring in a host cell's wild-type sequence. The expression product of the mutant sequence is a protein with an altered amino acid sequence relative to the wild-type. The expression product may have an altered functional capacity (e.g., enhanced enzymatic activity).
The terms “mutagenic primer” or “mutagenic oligonucleotide” (used interchangeably herein) are intended to refer to oligonucleotide compositions which correspond to a portion of the template sequence and which are capable of hybridizing thereto. With respect to mutagenic primers, the primer will not precisely match the template nucleic acid, the mismatch or mismatches in the primer being used to introduce the desired mutation into the nucleic acid library. As used herein, “non-mutagenic primer” or “non-mutagenic oligonucleotide” refers to oligonucleotide compositions which will match precisely to the template nucleic acid. In one embodiment of the invention, only mutagenic primers are used. In another preferred embodiment of the invention, the primers are designed so that for at least one region at which a mutagenic primer has been included, there is also non-mutagenic primer included in the oligonucleotide mixture. By adding a mixture of mutagenic primers and non-mutagenic primers corresponding to at least one of the mutagenic primers, it is possible to produce a resulting nucleic acid library in which a variety of combinatorial mutational patterns are presented. For example, if it is desired that some of the members of the mutant nucleic acid library retain their precursor sequence at certain positions while other members are mutant at such sites, the non-mutagenic primers provide the ability to obtain a specific level of non-mutant members within the nucleic acid library for a given residue. The methods of the invention employ mutagenic and non-mutagenic oligonucleotides which are generally between 10-50 bases in length, more preferably about 15-45 bases in length. However, it may be necessary to use primers that are either shorter than 10 bases or longer than 50 bases to obtain the mutagenesis result desired. With respect to corresponding mutagenic and non-mutagenic primers, it is not necessary that the corresponding oligonucleotides be of identical length, but only that there is overlap in the region corresponding to the mutation to be added.
Primers may be added in a pre-defined ratio according to the present invention. For example, if it is desired that the resulting library have a significant level of a certain specific mutation and a lesser amount of a different mutation at the same or different site, by adjusting the amount of primer added, it is possible to produce the desired biased library. Alternatively, by adding lesser or greater amounts of non-mutagenic primers, it is possible to adjust the frequency with which the corresponding mutation(s) are produced in the mutant nucleic acid library.
As used herein, the phrase “contiguous mutations” refers to mutations which are presented within the same oligonucleotide primer. For example, contiguous mutations may be adjacent or nearby each other, however, they will be introduced into the resulting mutant template nucleic acids by the same primer.
As used herein, the phrase “discontiguous mutations” refers to mutations which are presented in separate oligonucleotide primers. For example, discontiguous mutations will be introduced into the resulting mutant template nucleic acids by separately prepared oligonucleotide primers.
The terms “wild-type sequence,” or “wild-type gene” are used interchangeably herein, to refer to a sequence that is native or naturally occurring in a host cell. In some embodiments, the wild-type sequence refers to a sequence of interest that is the starting point of a protein engineering project. The wild-type sequence may encode either a homologous or heterologous protein. A homologous protein is one the host cell would produce without intervention. A heterologous protein is one that the host cell would not produce but for the intervention.
As used herein, the term “antibodies” refers to immunoglobulins. Antibodies include but are not limited to immunoglobulins obtained directly from any species from which it is desirable to produce antibodies. In addition, the present invention encompasses modified antibodies. The term also refers to antibody fragments that retain the ability to bind to the epitope that the intact antibody binds and include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, anti-idiotype (anti-ID) antibodies. Antibody fragments include, but are not limited to the complementarity-determining regions (CDRs), single-chain fragment variable regions (scFv), heavy chain variable region (VH), light chain variable region (VL). Polyclonal and monoclonal antibodies are also encompassed by the present invention. Preferably, the antibodies are monoclonal antibodies.
The term “oxidation stable” refers to proteases of the present invention that retain a specified amount of enzymatic activity over a given period of time under conditions prevailing during the proteolytic, hydrolyzing, cleaning or other process of the invention, for example while exposed to or contacted with bleaching agents or oxidizing agents. In some embodiments, the proteases retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% proteolytic activity after contact with a bleaching or oxidizing agent over a given time period, for example, at least 1 minute, 3 minutes, 5 minutes, 8 minutes, 12 minutes, 16 minutes, 20 minutes, etc. In some embodiments, the stability is measured as described in the Examples.
The term “chelator stable” refers to proteases of the present invention that retain a specified amount of enzymatic activity over a given period of time under conditions prevailing during the proteolytic, hydrolyzing, cleaning or other process of the invention, for example while exposed to or contacted with chelating agents. In some embodiments, the proteases retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% proteolytic activity after contact with a chelating agent over a given time period, for example, at least 10 minutes, 20 minutes, 40 minutes, 60 minutes, 100 minutes, etc. In some embodiments, the chelator stability is measured as described in the Examples.
The terms “thermally stable” and “thermostable” refer to proteases of the present invention that retain a specified amount of enzymatic activity after exposure to identified temperatures over a given period of time under conditions prevailing during the proteolytic, hydrolyzing, cleaning or other process of the invention, for example while exposed altered temperatures. Altered temperatures includes increased or decreased temperatures. In some embodiments, the proteases retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% proteolytic activity after exposure to altered temperatures over a given time period, for example, at least 60 minutes, 120 minutes, 180 minutes, 240 minutes, 300 minutes, etc. In some embodiments, the thermostability is determined as described in the Examples.
The term “enhanced stability” in the context of an oxidation, chelator, thermal and/or pH stable protease refers to a higher retained proteolytic activity over time as compared to other serine proteases (e.g., subtilisin proteases) and/or wild-type enzymes.
The term “diminished stability” in the context of an oxidation, chelator, thermal and/or pH stable protease refers to a lower retained proteolytic activity over time as compared to other serine proteases (e.g., subtilisin proteases) and/or wild-type enzymes.
As used herein, the term “cleaning composition” includes, unless otherwise indicated, granular or powder-form all-purpose or “heavy-duty” washing agents, especially cleaning detergents; liquid, gel or paste-form all-purpose washing agents, especially the so-called heavy-duty liquid types; liquid fine-fabric detergents; hand dishwashing agents or light duty dishwashing agents, especially those of the high-foaming type; machine dishwashing agents, including the various tablet, granular, liquid and rinse-aid types for household and institutional use; liquid cleaning and disinfecting agents, including antibacterial hand-wash types, cleaning bars, mouthwashes, denture cleaners, car or carpet shampoos, bathroom cleaners; hair shampoos and hair-rinses; shower gels and foam baths and metal cleaners; as well as cleaning auxiliaries such as bleach additives and “stain-stick” or pre-treat types.
It is to be understood that the test methods described in the Examples herein are used to determine the respective values of the parameters of the present invention, as such invention is described and claimed herein.
Unless otherwise noted, all component or composition levels are in reference to the active level of that component or composition, and are exclusive of impurities, for example, residual solvents or by-products, which may be present in commercially available sources.
Enzyme components weights are based on total active protein. All percentages and ratios are calculated by weight unless otherwise indicated. All percentages and ratios are calculated based on the total composition unless otherwise indicated.
It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
The term “cleaning activity” refers to the cleaning performance achieved by the protease under conditions prevailing during the proteolytic, hydrolyzing, cleaning or other process of the invention. In some embodiments, cleaning performance is determined by the application of various cleaning assays concerning enzyme sensitive stains, for example grass, blood, milk, or egg protein as determined by various chromatographic, spectrophotometric or other quantitative methodologies after subjection of the stains to standard wash conditions. Exemplary assays include, but are not limited to those described in WO 99/34011, and U.S. Pat. No. 6,605,458 (both of which are herein incorporated by reference), as well as those methods included in the Examples.
The term “cleaning effective amount” of a protease refers to the quantity of protease described hereinbefore that achieves a desired level of enzymatic activity in a specific cleaning composition. Such effective amounts are readily ascertained by one of ordinary skill in the art and are based on many factors, such as the particular protease used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g., granular, bar) composition is required, etc.
The term “cleaning adjunct materials,” as used herein, means any liquid, solid or gaseous material selected for the particular type of cleaning composition desired and the form of the product (e.g., liquid, granule, powder, bar, paste, spray, tablet, gel; or foam composition), which materials are also preferably compatible with the protease enzyme used in the composition. In some embodiments, granular compositions are in “compact” form, while in other embodiments, the liquid compositions are in a “concentrated” form.
The term “enhanced performance” in the context of cleaning activity refers to an increased or greater cleaning activity of certain enzyme sensitive stains such as egg, milk, grass or blood, as determined by usual evaluation after a standard wash cycle and/or multiple wash cycles.
The term “diminished performance” in the context of cleaning activity refers to an decreased or lesser cleaning activity of certain enzyme sensitive stains such as egg, milk, grass or blood, as determined by usual evaluation after a standard wash cycle.
The term “comparative performance” in the context of cleaning activity refers to at least 60%, at least 70%, at least 80% at least 90% at least 95% of the cleaning activity of a comparative subtilisin protease (e.g., commercially available proteases), including but not limited to OPTIMASETM protease (Genencor), PURAFECTTM protease products (Genencor), SAVINASETM protease (Novozymes), BPN′-variants (See e.g., U.S. Pat. No. Re 34,606), RELASETM, DURAZYMTM, EVERLASETM, KANNASETM protease (Novozymes), MAXACALTM; MAXAPEMTM, PROPERASETM proteases (Genencor; See also, U.S. Pat. No. Re 34,606, U.S. Pat. Nos. 5,700,676; 5,955,340; 6,312,936; 6,482,628), and B. lentus variant protease products [for example those described in WO 92/21760, WO 95/23221 and/or WO 97/07770 (Henkel). Exemplary subtilisin protease variants include, but are not limited to those having substitutions or deletions at residue positions equivalent to positions 76, 101, 103, 104, 120, 159, 167, 170, 194, 195, 217, 232, 235, 236, 245, 248, and/or 252 of BPN′. Cleaning performance can be determined by comparing the proteases of the present invention with those subtilisin proteases in various cleaning assays concerning enzyme sensitive stains such as grass, blood or milk as determined by usual spectrophotometric or analytical methodologies after standard wash cycle conditions.
As used herein, a “low detergent concentration” system includes detergents where less than about 800 ppm of detergent components are present in the wash water. Japanese detergents are typically considered low detergent concentration systems, as they have usually have approximately 667 ppm of detergent components present in the wash water.
As used herein, a “medium detergent concentration” systems includes detergents wherein between about 800 ppm and about 2000 ppm of detergent components are present in the wash water. North American detergents are generally considered to be medium detergent concentration systems as they have usually approximately 975 ppm of detergent components present in the wash water. Brazilian detergents typically have approximately 1500 ppm of detergent components present in the wash water.
As used herein, “high detergent concentration” systems includes detergents wherein greater than about 2000 ppm of detergent components are present in the wash water. European detergents are generally considered to be high detergent concentration systems as they have approximately 3000-8000 ppm of detergent components in the wash water.
As used herein, “fabric cleaning compositions” include hand and machine laundry detergent compositions including laundry additive compositions and compositions suitable for use in the soaking and/or pretreatment of stained fabrics (e.g., clothes, linens, and other textile materials).
As used herein, “non-fabric cleaning compositions” include non-textile (i.e., fabric) surface cleaning compositions, including but not limited to dishwashing detergent compositions, oral cleaning compositions, denture cleaning compositions, and personal cleansing compositions.
The “compact” form of the cleaning compositions herein is best reflected by density and, in terms of composition, by the amount of inorganic filler salt. Inorganic filler salts are conventional ingredients of detergent compositions in powder form. In conventional detergent compositions, the filler salts are present in substantial amounts, typically 17-35% by weight of the total composition. In contrast, in compact compositions, the filler salt is present in amounts not exceeding 15% of the total composition. In some embodiments, the filler salt is present in amounts that do not exceed 10%, or more preferably, 5%, by weight of the composition. In some embodiments, the inorganic filler salts are selected from the alkali and alkaline-earth-metal salts of sulfates and chlorides. A preferred filler salt is sodium sulfate.
II. Serine Protease Enzymes and Nucleic Acid Encoding Serine Protease Enzymes
The present invention provides isolated polynucleotides encoding amino acid sequences, encoding proteases. In some embodiments, these polynucleotides comprise at least 65% amino acid sequence identity, preferably at least 70% amino acid sequence identity, more preferably at least 75% amino acid sequence identity, still more preferably at least 80% amino acid sequence identity, more preferably at least 85% amino acid sequence identity, even more preferably at least 90% amino acid sequence identity, more preferably at least 92% amino acid sequence identity, yet more preferably at least 95% amino acid sequence identity, more preferably at least 97% amino acid sequence identity, still more preferably at least 98% amino acid sequence identity, and most preferably at least 99% amino acid sequence identity to an amino acid sequence as shown in SEQ ID NOS:6-8, (e.g., at least a portion of the amino acid sequence encoded by the polynucleotide having proteolytic activity, including the mature protease catalyzing the hydrolysis of peptide linkages of substrates), and/or demonstrating comparable or enhanced washing performance under identified wash conditions.
In some embodiments, the percent identity (amino acid sequence, nucleic acid sequence, gene sequence) is determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs find use in these analysis, such as those described above. Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (Genetics Computer Group, Madison, Wis.) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above.
An example of an algorithm that is suitable for determining sequence similarity is the BLAST algorithm, which is described in Altschul, et al., J. Mol. Biol., 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. These initial neighborhood word hits act as starting points to find longer HSPs containing them. The word hits are expanded in both directions along each of the two sequences being compared for as far as the cumulative alignment score can be increased. Extension of the word hits is stopped when: the cumulative alignment score falls off by the quantity X from a maximum achieved value; the cumulative score goes to zero or below; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (See, Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M′S, N′-4, and a comparison of both strands.
The BLAST algorithm then performs a statistical analysis of the similarity between two sequences (See e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 [1993]). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a serine protease nucleic acid of this invention if the smallest sum probability in a comparison of the test nucleic acid to a serine protease nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. Where the test nucleic acid encodes a serine protease polypeptide, it is considered similar to a specified serine protease nucleic acid if the comparison results in a smallest sum probability of less than about 0.5, and more preferably less than about 0.2.
In some embodiments of the present invention, sequences were analyzed by BLAST and protein translation sequence tools. In some experiments, the preferred version was BLAST (Basic BLAST version 2.0). The program'chosen was “BlastX”, and the database chosen was “nr”. Standard/default parameter values were employed.
In some preferred embodiments, the present invention encompasses the approximately 1621 base pairs in length polynucleotide set forth in SEQ. ID NO:1. A start codon is shown in bold in SEQ ID NO:1. In another embodiment of the present invention, the polynucleotides encoding these amino acid sequences comprise a 1485 base pair portion (residues 1-1485 of SEQ ID NO:2) that, if expressed, is believed to encode a signal sequence (nucleotides 1-84 of SEQ ID NO:5) encoding amino acids 1-28 of SEQ ID NO:9; an N-terminal prosequence (nucleotides 84-594 encoding amino acid residues 29-198 of SEQ ID NO:6); a mature protease sequence (nucleotides 595-1161 of SEQ ID NO:2 encoding amino acid residues 1-189 of SEQ ID NO:8); and a C-terminal pro-sequence (nucleotides 1162-1486 encoding amino acid residues 388-495 of SEQ ID NO:6). Alternatively, the signal peptide, the N-terminal pro-sequence, mature serine protease sequence and C-terminal pro-sequence is numbered in relation to the amino acid residues of the mature protease of SEQ ID NO:6 being numbered 1-189, i.e., signal peptide (residues −198 to −171), an N-terminal pro sequence (residues −171 to −1), the mature serine protease sequence (residues 1-189) and a C-terminal pro-sequence (residues 190-298). In another embodiment of the present invention, the polynucleotide encoding an amino acid sequence having proteolytic activity comprises a nucleotide sequence of nucleotides 1 to 1485 of the portion of SEQ ID NO:2 encoding the signal peptide and precursor protease. In another embodiment of the present invention, the polynucleotide encoding an amino acid sequence comprises the sequence of nucleotides 1 to 1412 of the polynucleotide encoding the precursor Cellulomonas protease (SEQ ID NO:3). In yet another embodiment, the polynucleotide encoding an amino acid sequence comprises the sequence of nucleotides 1 to 587 of the portion of the polynucleotide encoding the mature Cellulomonas protease (SEQ ID NO:4).
As will be understood by the skilled artisan, due to the degeneracy of the genetic code, a variety of polynucleotides can encode the signal peptide, precursor protease and/or mature protease provided in SEQ ID NOS:6, 7, and/or 8, respectively, or a protease having the % sequence identity described above. Another embodiment of the present invention encompasses a polynucleotide comprising a nucleotide sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 92% sequence identity, at least 95% sequence identity, at least 97% sequence identity, at least 98% sequence identity and at least 99% sequence identity to the polynucleotide sequence of SEQ ID NOS:2, 3, and/or 4, respectively, encoding the signal peptide and precursor protease, the precursor protease and/or the mature protease, respectively.
In additional embodiments, the present invention provides fragments or portions of DNA that encodes proteases, so long as the encoded fragment retains proteolytic activity. Another embodiment of the present invention encompasses polynucleotides having at least 20% of the sequence length, at least 30% of the sequence length, at least 40% of the sequence length, at least 50% of the sequence length, at least 60% of the sequence length, 70% of the sequence length, at least 75% of the sequence length, at least 80% of the sequence length, at least 85% of the sequence length, at least 90% of the sequence length, at least 92% of the sequence length, at least 95% of the sequence length, at least 97% of the sequence length, at least 98% of the sequence length and at least 99% of the sequence of the polynucleotide sequence of SEQ ID NO:2, or residues 185-1672 of SEQ ID NO:1, encoding the precursor protease. In alternative embodiments, these fragments or portions of the sequence length are contiguous portions of the sequence length, useful for shuffling of the DNA sequence in recombinant DNA sequences (See e.g., U.S. Pat. No. 6,132,970)
Another embodiment of the invention includes fragments of the DNA described herein that find use according to art recognized techniques in obtaining partial length DNA fragments capable of being used to isolate or identify polynucleotides encoding mature protease enzyme described herein from Cellulomonas 6964, or a segment thereof having proteolytic activity. Moreover, the DNA provided in SEQ ID NO:1 finds use in identifying homologous fragments of DNA from other species, and particularly from Cellulomonas spp. which encode a protease or portion thereof having proteolytic activity.
In addition, the present invention encompasses using primer or probe sequences constructed from SEQ ID NO:1, or a suitable portion or fragment thereof (e.g., at least about 5-20 or 10-15 contiguous nucleotides), as a probe or primer for screening nucleic acid of either genomic or cDNA origin. In some embodiments, the present invention provides DNA probes of the desired length (i.e., generally between 100 and 1000 bases in length), based on the sequences in SEQ ID NOS1, 2, 3, and/or 4.
In some embodiments, the DNA fragments are electrophoretically isolated, cut from the gel, and recovered from the agar matrix of the gel. In preferred embodiments, this purified fragment of DNA is then labeled (using, for example, the Megaprime labeling system according to the instructions of the manufacturer) to incorporate P32 in the DNA. The labeled probe is denatured by heating to 95° C. for a given period of time (e.g., 5 minutes), and immediately added to the membrane and prehybridization solution. The hybridization reaction proceeds for an appropriate time and under appropriate conditions (e.g., 18 hours at 37° C.), with gentle shaking or rotation. The membrane is rinsed (e.g., twice in SSC/0.3% SDS) and then washed in an appropriate wash solution with gentle agitation. The stringency desired is a reflection of the conditions under which the membrane (filter) is washed. In some embodiments herein, “low-stringency” conditions involve washing with a solution of 0.2×SSC/0.1% SDS at 20° C. for 15 minutes, while in other embodiments, “medium-stringency” conditions, involve a further washing step comprising washing with a solution of 0.2×SSC/0.1% SDS at 37° C. for 30 minutes, while in other embodiments, “high-stringency” conditions involve a further washing step comprising washing with a solution of 0.2×SSC/0.1% SDS at 37° C. for 45 minutes, and in further embodiments, “maximum-stringency” conditions involve a further washing step comprising washing with a solution of 0.2×SSC/0.1% SDS at 37° C. for 60 minutes. Thus, various embodiments of the present invention provide polynucleotides capable of hybridizing to a probed derived from the nucleotide sequence provided in SEQ ID NOS:1, 2, 3, 4, and/or 5, under conditions of medium, high and/or maximum stringency.
After washing, the membrane is dried and the bound probe detected. If P32 or another radioisotope is used as the labeling agent, the bound probe is detected by autoradiography. Other techniques for the visualization of other probes are well-known to those of skill in the art. The detection of a bound probe indicates a nucleic acid sequence has the desired homology, and therefore identity to SEQ ID NOS:1, 2, 3, 4, and/or 5, and is encompassed by the present invention. Accordingly, the present invention provides methods for the detection of nucleic acid encoding a protease encompassed by the present invention which comprises hybridizing part or all of a nucleic acid sequence of SEQ ID NOS:1, 2, 3, 4, and/or 5 with other nucleic acid of either genomic or cDNA origin.
As indicated above, in other embodiments, hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, to confer a defined “stringency” as explained below. “Maximum stringency” typically occurs at about Tm-5° C. (5° C. below the Tm of the probe); “high stringency” at about 5° C. to 10° C. below Tm; “intermediate stringency” at about 10° C. to 20° C. below Tm; and “low stringency” at about 20° C. to 25° C. below Tm. As known to those of skill in the art, medium, high and/or maximum stringency hybridization are chosen such that conditions are optimized to identify or detect polynucleotide sequence homologues or equivalent polynucleotide sequences.
In yet additional embodiments, the present invention provides nucleic acid constructs (i.e., expression vectors) comprising the polynucleotides encoding the proteases of the present invention. In further embodiments, the present invention provides host cells transformed with at least one of these vectors.
In further embodiments, the present invention provides polynucleotide sequences further encoding a signal sequence. In some embodiments, invention encompasses polynucleotides having signal activity comprising a nucleotide sequence having at least 65% sequence identity, at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, still further preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, more preferably at least 95% sequence identity, more preferably at least 97% sequence identity, at least 98% sequence identity, and most preferably at least 99% sequence identity to SEQ ID NO:5. Thus, in these embodiments, the present invention provides a sequence with a putative signal sequence, and polynucleotides being capable of hybridizing to a probe derived from the nucleotide sequence disclosed in SEQ ID NO:5 under conditions of medium, high and/or maximal stringency, wherein the signal sequences have substantially the same signal activity as the signal sequence encoded by the polynucleotide of the present invention.
In some embodiments, the signal activity is indicated by substantially the same level of secretion of the protease into the fermentation medium, as the starting material. For example, in some embodiments, the present invention provides fermentation medium protease levels at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% of the secreted protease levels in the fermentation medium as provided by the signal sequence of SEQ ID NO:3. In some embodiments, the secreted protease levels are ascertained by protease activity analyses such as the pNA assay (See e.g., Del Mar, [1979], infra). Additional means for determining the levels of secretion of a heterologous or homologous protein in a Gram-positive host cell and detecting secreted proteins include using either polyclonal or monoclonal antibodies specific for the protein. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS), as well-known those in the art.
In further embodiments, the present invention provides polynucleotides, encoding an amino acid sequence of a signal peptide (nucleotides 1-84 of SEQ ID NO:5), as shown in SEQ ID NO:9, nucleotide residue positions 1 to 85 of SEQ ID NO:2, and for SEQ ID NO:5. The invention further encompasses nucleic acid sequences which hybridize to the nucleic acid sequence shown in SEQ ID NO:5 under low, medium, high stringency and/or maximum stringency conditions, but which have substantially the same signal activity as the sequence. The present invention encompasses all such polynucleotides.
In further embodiments, the present invention provides polynucleotides that are complementary to the nucleotide sequences described herein. Exemplary complementary nucleotide sequences include those that are provided in SEQ ID NOS:1-5.
Further aspects of the present invention encompass polypeptides having proteolytic activity comprising 65% amino acid sequence identity, at least 70% sequence identity, at least 75% amino acid sequence identity, at least 80% amino acid sequence identity, at least 85% amino acid sequence identity, at least 90% amino acid sequence identity, at least 92% amino acid sequence identity, at least 95% amino acid sequence identity, at least 97% amino acid sequence identity, at least 98% amino acid sequence identity and at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 6 (i.e., the signal and precursor protease), SEQ ID NO:7 (i.e., the precursor protease), and/or of SEQ ID NO:8 (i.e., the mature protease). The proteolytic activity of these polypeptides is determined using methods known in the art and include such methods as those used to assess detergent function. In further embodiments, the polypeptides are isolated. In additional embodiments of the present invention, the polypeptides comprise amino acid sequences that identical to amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOS:6, 7, or 8. In some further embodiments, the polypeptides are identical to portions of SEQ ID NOS:6, 7 or 8.
In some embodiments, the present invention provides isolated polypeptides having proteolytic activity, comprising the amino acid sequence approximately 495 amino acids in length, as provided in SEQ ID NO:6. In further embodiments, the present invention encompasses polypeptides having proteolytic activity comprising the amino acid sequence approximately 467 amino acids in length provided in SEQ ID NO:7. In some embodiments, these amino acid sequences comprise a signal sequence (amino acids 1-28 of SEQ ID NO:9); and a precursor protease (amino acids 1-467 of SEQ ID NO:7). In additional embodiments, the present invention encompasses polypeptides comprising an N-terminal prosequence (amino acids 1-170 of SEQ ID NO:7), a mature protease sequence (amino acids 1-189 of SEQ ID NO:8), and a C-terminal prosequence (amino acids 360-467 of SEQ ID NO:7). In still further embodiments, the present invention encompasses polypeptides comprising a precursor protease sequence (e.g., amino acids 1-467 of SEQ ID NO:7). In yet another embodiment, the present invention encompasses polypeptides comprising a mature protease sequence comprising amino acids (e.g., 1-189 of SEQ ID NO:8).
In further embodiments, the present invention provides polypeptides and/or proteases comprising amino acid sequences of the above described sequence derived from bacterial species including, but not limited to Micrococcineae which are identified through amino acid sequence homology studies. In some embodiments, an amino acid residue of a precursor Micrococcineae protease is equivalent to a residue of Cellulomonas strain 69B4, if it is either homologous (i.e., corresponding in position in either primary or tertiary structure) or analogous to a specific residue or portion of that residue in Cellulomonas strain 69B4 protease (i.e., having the same or similar functional capacity to combine, react, or interact chemically).
In some preferred embodiments, in order to establish homology to primary structure, the amino acid sequence of a precursor protease is directly compared to the Cellulomonas strain 69B4 mature protease amino acid sequence and particularly to a set of conserved residues which are discerned to be invariant in all or a large majority of Cellulomonas like proteases for which sequence is known. After aligning the conserved residues, allowing for necessary insertions and deletions in order to maintain alignment (i.e., avoiding the elimination of conserved residues through arbitrary deletion and insertion), the residues corresponding to particular amino acids in the mature protease (SEQ ID NO:8) and Cellulomonas 69B4 protease are determined. Alignment of conserved residues preferably should conserve 100% of such residues. However, alignment of greater than 75% or as little as 45% of conserved residues is also adequate to define equivalent residues. However, conservation of the catalytic triad, His32/Asp56/Ser137 of SEQ ID NO:8 should be maintained.
For example, in some embodiments, the amino acid sequence of proteases from Cellulomonas strain 69B4, and other Micrococcineae spp. described above are aligned to provide the maximum amount of homology between amino acid sequences. A comparison of these sequences indicates that there are a number of conserved residues contained in each sequence. These are the residues that are identified and utilized to establish the equivalent residue positions of amino acids identified in the precursor or mature Micrococcineae protease in question.
These conserved residues are used to ascertain the corresponding amino acid residues of Cellulomonas strain 69B4 protease in one or more in Micrococcineae homologues (e.g., Cellulomonas cellasea (DSM 20118) and/or a Cellulomonas homologue herein). These particular amino acid sequences are aligned with the sequence of Cellulomonas 69B4 protease to produce the maximum homology of conserved residues. By this alignment, the sequences and particular residue positions of Cellulomonas 69B4 are observed in comparison with other Cellulomonas spp. Thus, the equivalent amino acid for the catalytic triad (e.g., in Cellulomonas 69B4 protease) is identifiable in the other Micrococcineae spp. In some embodiments of the present invention, the protease homologs comprise the equivalent of His32/Asp56/Ser137 of SEQ ID NO:8.
Another indication that two polypeptides are substantially identical is that the first polypeptide is immunologically cross-reactive with the second polypeptide. Methodologies for determining immunological cross-reactivity are described in the art and are described in the Examples herein. Typically, polypeptides that differ by conservative amino acid substitutions are immunologically cross-reactive. Thus, a polypeptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution.
The present invention encompasses proteases obtained from various sources. In some preferred embodiments, the proteases are obtained from bacteria, while in other embodiments, the proteases are obtained from fungi.
In some particularly preferred embodiments, the bacterial source is selected from the members of the suborder Micrococcineae. In some embodiments, the bacterial source is the family Promicromonosporaceae. In some preferred embodiments, the Promicromonosporaceae spp. includes and/or is selected from the group consisting of Promicromonospora citrea (DSM 431.10), Promicromonospora sukumoe (DSM 44121), Promicromonospora aerolata (CCM 7043), Promicromonospora vindobonensis (CCM 7044), Myceligenerans xiligouense (DSM 15700), Isoptericola variabilis (DSM 10177, basonym Cellulosimicrobium variabile), Cellulosimicrobium cellulans (DSM 20424, basonym Nocardia cellulans, Cellulomonas cellulans), Cellulosimicrobium funkei, Xylanimonas cellulosilytica (LMG 20990), Xylanibacterium ulmi (LMG 21721), and Xylanimicrobium pachnodae (DSM 12657, basonym Promicromonospora pachnodae).
In other particularly preferred embodiments, the bacterial source is the family Cellulomonadaceae. In some preferred embodiments, the Cellulomonadaceae spp. includes and/or is selected from the group of Cellulomonas fimi (ATCC 484, DSM 20113), Cellulomonas biazotea (ATCC 486, DSM 20112), Cellulomonas cellasea (ATCC 487, 21681, DSM 20118), Cellulomonas denverensis, Cellulomonas hominis (DSM 9581), Cellulomonas flavigena (ATCC 482, DSM 20109), Cellulomonas persica (ATCC 700642, DSM 14784), Cellulomonas iranensis (ATCC 700643, DSM 14785); Cellulomonas fermentans (ATCC 43279, DSM 3133), Cellulomonas gelida (ATCC 488, DSM 20111, DSM 20110), Cellulomonas humilata (ATCC 25174, basonym Actinomyces humiferus), Cellulomonas uda (ATCC 491, DSM 20107), Cellulomonas xylanilytica (LMG 21723), Cellulomonas septica, Cellulomonas parahominis, Oerskovia turbata (ATCC 25835, DSM 20577, synonym Cellulomonas turbata), Oerskovia jenensis (DSM 46000), Oerskovia enterophila (ATCC 35307, DSM 43852, basonym Promicromonospora enterophila), Oerskovia paurometabola (DSM 14281), and Cellulomonas strain 69B4 (DSM 16035). In further embodiments, the bacterial source also includes and/or is selected from the group of Thermobifida spp., Rarobacter spp., and/or Lysobacter spp. In yet additional embodiments, the Thermobifida spp. is Thermobifida fusca (basonym Thermomonospora fusca) (tfpA, AAC23545; See, Lao et. al, Appl. Environ. Microbiol., 62: 4256-4259 [1996]). In an alternative embodiment, the Rarobacter spp. is Rarobacter faecitabidus (RP1, A45053; See e.g., Shimoi et al., J. Biol. Chem., 267:25189-25195 [1992]). In yet another embodiment, the Lysobacter spp. is Lysobacter enzymogenes. 
In further embodiments, the present invention provides polypeptides and/or polynucleotides obtained and/or isolated from fungal sources. In some embodiments, the fungal source includes a Metarhizium spp. In some preferred embodiments, the fungal source is a Metarhizium anisopliae (CHY1 (CAB60729).
In another embodiment, the present invention provides polypeptides and/or polynucleotides derived from a Cellulomonas strain selected from cluster 2 of the taxonomic classification described in U.S. Pat. No. 5,401,657, herein incorporated by reference. In U.S. Pat. No. 5,401,657, twenty strains of bacteria isolated from in and around alkaline lakes were assigned to the type of bacteria known as Gram-positive bacteria on the basis of: (1) the Dussault modification of the Gram's staining reaction (Dussault, J. Bacteriol., 70:484-485 [1955]); (2) the KOH sensitivity test (Gregersen, Eur. J. Appl. Microbiol. Biotechnol., 5:123-127 [1978]; Halebian et al., J. Clin. Microbiol., 13:444-448 [1981]; and (3) the aminopeptidase reaction (Cerny, Eur. J. Appl. Microbiol., 3:223-225 [1976]; Cerny, Eur. J. Appl. Microbiol., 5:113-122 [1978]). In addition, in most cases, confirmation was also made on the basis of quinone analysis (Collins and Jones, Microbiol. Rev., 45:316-354 [1981]) using the method described by Collins (See, Collins, In Goodfellow and Minnikin (eds), Chemical Methods in Bacterial Systematics, Academic Press, London [1985], pp. 267-288). In addition, strains can be tested for 200 characters and the results analyzed using the principles of numerical taxonomy (See e.g., Sneath and Sokal, Numerical Taxonomy, W.H. Freeman & Co., San Francisco, Calif. [1973]). Exemplary characters tested, testing methods, and codification methods are also described in U.S. Pat. No. 5,401,657.
As described in U.S. Pat. No. 5,401,657, the phenetic data, consisting of 200 unit characters was scored and set out in the form of an “n.times.t” matrix, whose t columns represent the “t” bacterial strains to be grouped on the basis of resemblances, and whose “n” rows are the unit characters. Taxonomic resemblance of the bacterial strains was estimated by means of a similarity coefficient (Sneath and Sokal, supra, pp. 114-187). Although many different coefficients have been used for biological classification, only a few have found regular use in bacteriology. Three association Coefficients (See e.g., Sneath and Sokal, supra, at p. 129), namely, the Gower, Jaccard and Simple Matching coefficients were applied. These have been frequently applied to the analysis of bacteriological data and are widely accepted by those skilled in the art, as they have been shown to result in robust classifications.
The coded data were analyzed using the TAXPAK program package (Sackin; Meth. Microbiol., 19:459-494 [1987]), run on a DEC VAX computer at the University of Leicester, U.K.
A similarity matrix was constructed for all pairs of strains using the Gower Coefficient (SG) with the option of permitting negative matches (See, Sneath and Sokal, supra, at pp. 135-136), using the RTBNSIM program in TAXPAK. As the primary instrument of analysis and the one upon which most of the taxonomic data presented herein are based, the Gower Coefficient was chosen over other coefficients for generating similarity matrices because it is applicable to all types of characters or data, namely, two-state, multistate (ordered and qualitative), and quantitative.
Cluster analysis of the similarity matrix was accomplished using the Unweighted Pair Group Method with Arithmetic Averages (UPGMA) algorithm, also known as the Unweighted Average Linkage procedure, by running the SMATCLST sub-routine in TAXPAK.
Dendrograms illustrate the levels of similarity between bacterial strains In some embodiments, dendrograms are obtained by using the DENDGR program in TAXPAK. The phenetic data were re-analyzed using the Jaccard Coefficient (SJ) (Sneath and Sokal, supra, at p. 131) and Simple Matching Coefficient (SSM) (Sneath, P. H. A. and Sokal, R. R., ibid, P. 132) by running the RTBNSIM program in TAXPAK. An additional two dendrograms were obtained by using the SMATCLST with UPGMA option and DENDGR sub-routines in TAXPAK.
Using the SG/UPGMA method, six natural clusters or phenons of alkalophilic bacteria were generated at the 79% similarity level. These six clusters included 15 of the 20 alkalophilic bacteria isolated from alkaline lakes. Although the choice of 79% for the level of delineation was arbitrary, it was in keeping with current practices in numerical taxonomy (See e.g., Austin Priest, Modern Bacterial Taxonomy, Van Nostrand Reinhold, Wokingham, U.K., [1986], p. 37). Placing the delineation at a lower percentage would combine groups of clearly unrelated organisms whose definition is not supported by the data. At the 79% level, 3 of the clusters exclusively contain novel alkalophilic bacteria representing 13 of the newly isolated strains (potentially representing new taxa). Protease 69B4 was classified as in cluster 2 by this method.
The significance of the clustering at this level was supported by the results of the TESTDEN program. This program tests the significance of all dichotomous pairs of clusters (comprising 4 or more strains) in a UPGMA generated dendrogram with Squared Euclidean distances, or their complement as a measurement and assuming that the clusters are hyperspherical. The critical overlap was set at 0.25%. The separation of the clusters is highly significant.
The SJ coefficient is a useful adjunct to the SG coefficient, as it can be used to detect phenons in the latter that are based on negative matches or distortions owing to undue weight being put on potentially subjective qualitative data. Consequently, the SJ coefficient is useful for confirming the validity of clusters defined initially by the use of the SG coefficient. The Jaccard Coefficient is particularly useful in comparing biochemically unreactive organisms (Austin and Priest, supra, at p. 37). In addition, there may be some question about the admissibility of matching negative character states (See, Sneath and Sokal, supra, at p. 131), in which case the Simple Matching Coefficient is a widely applied alternative. Strain 69B4 was classified as in cluster 2 by this method.
In the main, all of the clusters (especially the clusters of the new bacteria) generated by the SG/UPGMA method were recovered in the dendrograms produced by the SJ/UPGMA method (cophenetic correlation, 0.795), and the SSM/UPGMA method (cophenetic correlation, 0.814). The main effect of these transformations was to gather all the Bacillus strains in a single large cluster which further serves to emphasize the separation between the alkalophilic Bacillus species and the new alkalophilic bacteria, and the uniqueness of the latter. Based on these methodologies, 6984 is considered to be a cluster 2 bacterium.
In other aspects of the present invention, the polynucleotide is derived from a bacteria having a 16S rRNA gene nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 98% sequence identity with the 16S rRNA gene nucleotide sequence of Cellulomonas strain 69B4. The sequence of the 16S rRNA gene is deposited at GenBank under Accession Number X92152.
FIG. 1 provides an unrooted phylogenetic tree illustrating the relationship of novel strain 69B4 to members of the family Cellulomonadaceae (including Cellulomonas strain 69B4) and other related genera of the suborder Micrococcineae. The dendrogram was constructed from aligned 16S rDNA sequences (1374 nt) using TREECONW v.1.3b (Van de Peer and De Wachter, Comput. Appl. Biosci., 10: 569-570 [1994]). Distance estimations were calculated using the substitution rate calibration of Jukes and Cantor (Jukes and Cantor, “Evolution of protein molecules,” In, Munro (ed.), Mammalian Protein Metabolism, Academic Press, NY, at pp. 21-132, [1969]) and tree topology inferred by the Neighbor-Joining algorithm (Saitou and Nei, Mol. Biol. Evol., 4:406-425 [1987]). The numbers at the nodes refer to bootstrap values from 100 resampled data sets (Felsenstein, Evol., 39:783-789 [1985]) and the scale bar indicates 2 nucleotide substitutions in 100 nt.
The strain 69B4 exhibits the closest 16S rDNA relationship to members of Cellulomonas and Oerskovia of the family Cellulomonadaceae. The closest relatives are believed to be C. cellasea (DSM 20118) and C. fimi (DSM 20113), with at least 95% sequence identity with the 16S rRNA gene nucleotide sequence of Cellulomonas strain 69B4 (e.g., 96% and 95% identity respectively) to strain 69B4 16S rRNA gene sequence.
In some preferred embodiments of the present invention, the Cellulomonas spp. is Cellulomonas strain 69B4 (DSM16035). This strain was originally isolated from a sample of sediment and water from the littoral zone of Lake Bogoria, Kenya at Acacia Camp (Lat. 0° 12′N, Long. 36° 07′E) collected on 10 Oct. 1988. The water temperature was 33° C., pH 10.5 with a conductivity of 44 mS/cm. Cellulomonas strain 69B4 was determined to have the phenotypic characteristics described below. Fresh cultures were Gram-positive, slender, generally straight, rod-shaped bacteria, approximately 0.5-0.7 μm×1.8-4 μm. Older cultures contained mainly short rods and coccoid cells. Cells occasionally occurred in pairs or as V-forms, but primary branching was not observed. Endospores were not detected. On alkaline GAM agar the strain forms opaque, glistening, pale-yellow coloured, circular and convex or domed colonies, with entire margins, about 2 mm in diameter after 2-3 days incubation at 37° C. The colonies were viscous or slimy with a tendency to dump when scraped with a loop. On neutral Tryptone Soya Agar, strain growth was less vigorous, giving translucent yellow colonies, generally <1 mm in diameter. The cultures were facultatively anaerobic, as they were capable of growth under strictly anaerobic conditions. However, growth under anaerobic conditions was markedly reduced compared to aerobic growth. The strain also appeared to be negative in standard oxidase, urease, aminopeptidase, and KOH tests. In addition, nitrate was not reduced, although the organisms were catalase positive and DNase was produced under alkaline conditions. The preferred temperature range for growth was 20-37° C., with an optimum temperature at around 30-37° C. No growth was observed at 15° C. or 45° C.
The strain is alkalophilic and slightly halophilic. The strain may also be characterized as having growth occurring at pH values between 6.0 and 10.5 with an optimum around pH 9-10. No growth was observed at pH 11 or pH 5.5. Growth below pH 7 was less vigorous and abundant than that of cultures grown at the optimal temperature. The strain was observed to grow in medium containing 0-8% (w/v) NaCl. Furthermore, the strain may also be characterized as a chemo-organotroph, since it grew on complex substrates such as yeast extract and peptone; and hydrolyzed starch, gelatin, casein, carboxymethylcellulose and amorphous cellulose.
The strain was observed to have metabolism that was respiratory and also fermentative. Acid was produced both aerobically and anaerobically from (API 50CH): L-arabinose, D-xylose, D-glucose, D-fructose, D-mannose, rhamnose'(weak), cellobiose, maltose, sucrose, trehalose, gentiobiose, D-turanose, D-lyxose and 5-keto-gluconate (weak). Amygdalin, arbutin, salicin and esculin are also utilized. The strain was unable to utilize: ribose, lactose, galactose, melibiose, D-raffinose, glycogen, glycerol, erythritol, inositol, mannitol, sorbitol, xylitol, arabitol, gluconate and lactate.
The strain was determined to be susceptible to ampicillin, chloramphenicol, erythromycin, fusidic acid, methicillin, novobiocin, streptomycin, tetracycline, sulphafurazole, oleandomycin, polymixin, rifampicin, vancomycin and bacitracin; but resistant to gentamicin, nitrofurantoin, nalidixic acid, sulphmethoxazole, trimethoprim, penicillin G, neomycin and kanamycin.
The following enzymes, aside from the protease of the present invention, were observed to be produced (ApiZym, API Coryne); C4-esterase, C8-esterase/lipase, leucine arylamidase, alpha-chymotrypsin, alpha-glucosidase, beta-glucosidase and pyrazinamidase.
The strain was observed to exhibit the following chemotaxonomic characteristics. Major fatty acids (>10% of total) were C16:1 (28.1%), C18:0 (31.1%), C18:1 (13.9%). N-saturated (79.1%), n-unsaturated (19.9%). Fatty acids with even numbers of carbons accounted for 98%. Main polar lipid components: phosphatidylglycerol (PG) and 3 unidentified glycolipids (alpha-napthol positive) were present; DPG, PGP, PI and PE were not detected. Menaquinones MK-4, MK-6, MK-7 and MK-9 were the main isoprenoids present. The cell wall peptidoglycan type was A4β with L-ornithine as diamino acid and D-aspartic acid in the interpeptide bridge. With regard to toxicity evaluation, there are no known toxicity or pathogenicity issues associated with bacteria of the genus Cellulomonas. 
Although there may be variations in the sequence of a naturally occurring enzyme within a given species of organism, enzymes of a specific type produced by organisms of the same species generally are substantially identical with respect to substrate specificity and/or proteolytic activity levels under given conditions (e.g., temperature, pH, water hardness, oxidative conditions, chelating conditions, and concentration), etc. Thus, for the purposes of the present invention, it is contemplated that other strains and species of Cellulomonas also produce the Cellulomonas protease of the present invention and thus provide useful sources for the proteases of the present invention. Indeed, as presented herein, it is contemplated that other members of the Micrococcineae will find use in the present invention.
In some embodiments, the proteolytic polypeptides of this invention are characterized physicochemically, while in other embodiments, they are characterized based on their functionally, while in further embodiments, they are characterized using both sets of properties. Physicochemical characterization takes advantages of well known techniques such as SDS electrophoresis, gel filtration, amino acid composition, mass spectrometry (e.g., MALDI-TOF-MS, LC-ES-MS/MS, etc.), and sedimentation to determine the molecular weight of proteins, isoelectric focusing to determine the pl of proteins, amino acid sequencing to determine the amino acid sequences of protein, crystallography studies to determine the tertiary structures of proteins, and antibody binding to determine antigenic epitopes present in proteins.
In some embodiments, functional characteristics are determined by techniques well known to the practitioner in the protease field and include, but are not limited to, hydrolysis of various commercial substrates, such as di-methyl casein (“DMC”) and/or AAPF-pNA. This preferred technique for functional characterization is described in greater detail in the Examples provided herein.
In some embodiments of the present invention, the protease has a molecular weight of about 17 kD to about 21 kD, for example about 18 kD to 19 kD, for example 18700 daltons to 18800 daltons, for example about 18764 daltons, as determined by MALDI-TOF-MS). In another aspect of the present invention, the protease measured MALDI-TOF-MS spectrum as set forth in FIG. 3.
The mature protease also displays proteolytic activity (e.g., hydrolytic activity on a substrate having peptide linkages) such as DMC. In further embodiments, proteases of the present invention provide enhanced wash performance under identified conditions. Although the present invention encompasses the protease 69B as described herein, in some embodiments, the proteases of the present invention exhibit at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% proteolytic activity as compared to the proteolytic activity of 69B4. In some embodiments, the proteases display at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% proteolytic activity as compared to the proteolytic activity of proteases sold under the tradenames SAVINASE® (Novzymes) or PURAFECT® (Genencor) under the same conditions. In some embodiments, the proteases of the present invention display comparative or enhanced wash performance under identified conditions as compared to 69B4 under the same conditions. In some preferred embodiments, the proteases of the present invention display comparative or enhanced wash performance under identified conditions, as compared to proteases sold under the tradenames SAVINASE® (Novozymes) or PURAFECT® (Genencor) under the same conditions.
In yet further embodiments, the proteases and/or polynucleotides encoding the proteases of the present invention are provided purified form (i.e., present in a particular composition in a higher or lower concentration than exists in a naturally occurring or wild type organism), or in combination with components not normally present upon expression from a naturally occurring or wild-type organism. However, it is not intended that the present invention be limited to proteases of any specific purity level, as ranges of protease purity find use in various applications in which the proteases of the present inventing are suitable.
III. Obtaining Polynucleotides Encoding Micrococcineae (e.g., Cellulomonas) Proteases of the Present Invention
In some embodiments, nucleic acid encoding a protease of the present invention is obtained by standard procedures known in the art from, for example, cloned DNA (e.g., a DNA ulibraryn), chemical synthesis, cDNA cloning, PCR, cloning of genomic DNA or fragments thereof, or purified from a desired cell, such as a bacterial or fungal species (See, for example, Sambrook et al., supra [1989]; and Glover and Hames (eds.), DNA Cloning: A Practical Approach, Vols 1 and 2, Second Edition). Synthesis of polynucleotide sequences is well known in the art (See e.g., Beaucage and Caruthers, Tetrahedron Lett., 22:1859-1862 [1981]), including the use of automated synthesizers (See e.g., Needham-VanDevanter et al., Nucl. Acids Res., 12:6159-6168 [1984]). DNA sequences can also be custom made and ordered from a variety of commercial sources. As described in greater detail herein, in some embodiments, nucleic acid sequences derived from genomic DNA contain regulatory regions in addition to coding regions.
In some embodiments involving the molecular cloning of the gene from genomic DNA, DNA fragments are generated, some of which comprise at least a portion of the desired gene. In some embodiments, the DNA is cleaved at specific sites using various restriction enzymes. In some alternative embodiments, DNAse is used in the presence of manganese to fragment the DNA, or the DNA is physically sheared (e.g., by sonication). The linear DNA fragments created are then be separated according to size and amplified by standard techniques, including but not limited to, agarose and polyacrylamide gel electrophoresis, PCR and column chromatography.
Once nucleic acid fragments are generated, identification of the specific DNA fragment encoding a protease may be accomplished in a number of ways. For example, in some embodiments, a proteolytic hydrolyzing enzyme encoding the asp gene or its specific RNA, or a fragment thereof, such as a probe or primer, is isolated, labeled, and then used in hybridization assays well known to those in the art, to detect a generated gene (See e.g., Benton and Davis, Science 196:180 [1977]; and Grunstein and Hogness, Proc. Natl. Acad. Sci. USA 723961 [1975]). In preferred embodiments, DNA fragments sharing substantial sequence similarity to the probe hybridize under medium to high stringency.
In some preferred embodiments, amplification is accomplished using PCR, as known in the art. In some preferred embodiments, a nucleic acid sequence of at least about 4 nucleotides and as many as about 60 nucleotides from SEQ ID NOS:1, 2, 3 and/or 4 (i.e., fragments), preferably about 12 to 30 nucleotides, and more preferably about 25 nucleotides are used in any suitable combinations as PCR primer. These same fragments also find use as probes in hybridization and product detection methods.
In some embodiments, isolation of nucleic acid constructs of the invention from a cDNA or genomic library utilizes PCR with using degenerate oligonucleotide primers prepared on the basis of the amino acid sequence of the protein having the amino acid sequence as shown in SEQ ID NOS:1-5. The primers can be of any segment length, for example at least 4, at least 5, at least 8, at least 15, at least 20, nucleotides in length. Exemplary probes in the present application utilized a primer comprising a TTGWHCGT and a GDSGG polynucleotide sequence as more fully described in Examples.
In view of the above, it will be appreciated that the polynucleotide sequences provided herein and based on the polynucleotide sequences provided in SEQ ID NOS:1-5 are useful for obtaining identical or homologous fragments of polynucleotides from other species, and particularly from bacteria that encode enzymes having the serine protease activity expressed by protease 69B4.
IV. Expression and Recovery of Serine Proteases of the Present Invention
Any suitable means for expression and recovery of the serine proteases of the present invention find use herein. Indeed, those of skill in the art know many methods suitable for cloning a Cellulomonas-derived polypeptide having proteolytic activity, as well as an additional enzyme (e.g., a second peptide having proteolytic activity, such as a protease, cellulase, mannanase, or amylase, etc.). Numerous methods are also known in the art for introducing at least one (e.g., multiple) copies of the polynucleotide(s) encoding the enzyme(s) of the present invention in conjunction with any additional sequences desired, into the genes or genome of host cells.
In general, standard procedures for cloning of genes and introducing exogenous proteases encoding, regions (including multiple copies of the exogenous encoding regions) into said genes find use in obtaining a Cellulomonas 69B4 protease derivative or homologue thereof. Indeed, the present Specification, including the Examples provides such teaching: However, additional methods known in the art are also suitable (See e.g., Sambrook et al. supra (1989); Ausubel et al., supra [1995]; and Harwood and Cutting, (eds.) Molecular Biological Methods for Bacillus,” John Wiley and Sons, [1990]; and WO 96/34946).
In some preferred embodiments, the polynucleotide sequences of the present invention are expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employed by that expression vector to transform an appropriate host according to techniques well established in the art. In some embodiments, the polypeptides produced on expression of the DNA sequences of this invention are isolated from the fermentation of cell cultures and purified in a variety of ways according to well established techniques in the art. Those of skill in the art are capable of selecting the most appropriate isolation and purification techniques.
More particularly, the present invention provides constructs, vectors comprising polynucleotides described herein, host cells transformed with such vectors, proteases expressed by such host cells, expression methods and systems for the production of serine protease enzymes derived from microorganisms, in particular, members of the Micrococcineae, including but not limited to Cellulomonas species. In some embodiments, the polynucleotide(s) encoding serine protease(s) are used to produce recombinant host cells suitable for the expression of the serine protease(s). In some preferred embodiments, the expression hosts are capable of producing the protease(s) in commercially viable quantities.
IV. Recombinant Vectors
As indicated above, in some embodiments, the present invention provides vectors comprising the aforementioned polynucleotides. In some embodiments, the vectors (i.e., constructs) of the invention encoding the protease are of genomic origin (e.g., prepared though use of a genomic library and screening for DNA sequences coding for all or part of the protease by hybridization using synthetic oligonucleotide probes in accordance with standard techniques). In some preferred embodiments, the DNA sequence encoding the protease is obtained by isolating chromosomal DNA from the Cellulomonas strain 69B4 and amplifying the sequence by PCR methodology (See, the Examples).
In alternative embodiments, the nucleic acid construct of the invention encoding the protease is prepared synthetically by established standard methods (See e.g., Beaucage and Caruthers, Tetra. Lett. 22:1859-1869 [1981]; and Matthes et al., EMBO J., 3:801-805 [1984]). According to the phosphoramidite method, oligonucleotides are synthesized (e.g., in an automatic DNA synthesizer), purified, annealed, ligated and cloned in suitable vectors.
In additional embodiments, the nucleic acid construct is of mixed synthetic and genomic origin. In some embodiments, the construct is prepared by ligating fragments of synthetic or genomic DNA (as appropriate), wherein the fragments correspond to various parts of the entire nucleic acid construct, in accordance with standard techniques.
In further embodiments, the present invention provides vectors comprising at least one DNA construct of the present invention. In some embodiments, the present invention encompasses recombinant vectors. It is contemplated that any suitable vector will find use in the present invention, including autonomously replicating vector a well as vectors that integrate (either transiently or stably) within the host cell genome). Indeed, a wide variety of vectors, and expression cassettes suitable for the cloning, transformation and expression in fungal (mold and yeast), bacterial, insect and plant cells are known to those of skill in the art. Typically, the vector or cassette contains sequences directing transcription and translation of the nucleic acid, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. In some embodiments, suitable vectors comprise a region 5′ of the gene which harbors transcriptional initiation controls and a region 3′ of the DNA fragment which controls transcriptional termination. These control regions may be derived from genes homologous or heterologous to the host as long as the control region selected is able to function in the host cell.
The vector is preferably an expression vector in which the DNA sequence encoding the protease of the invention is operably linked to additional segments required for transcription of the DNA. In some preferred embodiments, the expression vector is derived from plasmid or viral DNA, or in alternative embodiments, contains elements of both. Exemplary vectors include, but are not limited to pSEGCT, pSEACT, and/or pSEA4CT, as well as all of the vectors described in the Examples herein. Construction of such vectors is described herein, and methods are well known in the art (See e.g., U.S. Pat. No. 6,287,839; and WO 02/50245). In some preferred embodiments, the vector pSEGCT (about 8302 bp; See, FIG. 5) finds use in the construction of a vector comprising the polynucleotides described herein (e.g., pSEG69B4T; See, FIG. 6). In alternative preferred embodiments, the vector pSEA469B4CT (See, FIG. 7) finds use in the construction of a vector comprising the polynucleotides described herein. Indeed, it is intended that all of the vectors described herein will find use in the present invention.
In some embodiments, the additional segments required for transcription include regulatory segments (e.g., promoters, secretory segments, inhibitors, global regulators, etc.), as known in the art. One example includes any DNA sequence that shows transcriptional activity in the host cell of choice and is derived from genes encoding proteins either homologous or heterologous to the host cell. Specifically, examples of suitable promoters for use in bacterial host cells include but are not limited to the promoter of the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus amyloliquefaciens (BAN) amylase gene, the Bacillus subtilis alkaline protease gene, the Bacillus clausii alkaline protease gene the Bacillus pumilus xylosidase gene, the Bacillus thuringiensis cryIIIA, and the Bacillus licheniformis alpha-amylase gene. Additional promoters include the A4 promoter, as described herein. Other promoters that find use in the present invention include, but are not limited to phage Lambda PR or PL promoters, as well as the E. coli lac, trp or tac promoters.
In some embodiments, the promoter is derived from a gene encoding said protease or a fragment thereof having substantially the same promoter activity as said sequence. The invention further encompasses nucleic acid sequences which hybridize to the promoter sequences under intermediate, high, and/or maximum stringency conditions, or which have at least about 90% homology and preferably about 95% homology to such promoter, but which have substantially the same promoter activity. In some embodiments, this promoter is used to promote the expression of either the protease and/or a heterologous DNA sequence (e.g., another enzyme in addition to the protease of the present invention). In additional embodiments, the vector also comprises at least one selectable marker.
In some embodiments, the recombinant vectors of the invention further comprise a DNA sequence enabling the vector to replicate in the host cell. In some preferred embodiments involving bacterial host cells, these sequences comprise all the sequences needed to allow plasmid replication (e.g., ori and/or rep sequences).
In some particularly preferred embodiments, signal sequences (e.g., leader sequence or pre sequence) are also included in the vector, in order to direct a polypeptide of the present invention into the secretory pathway of the host cells. In some more preferred embodiments, a secretory signal sequence is joined to the-DNA sequence encoding the precursor protease in the correct reading frame (See e.g., SEQ ID NOS:1 and 2). Depending on whether the protease is to be expressed intracellularly or is secreted, a polynucleotide sequence or expression vector of the invention is engineered with or without a natural polypeptide signal sequence or a signal sequence which functions in bacteria (e.g., Bacillus sp.), fungi (e.g., Trichoderma), other prokaryoktes or eukaryotes. In some embodiments, expression is achieved by either removing or partially removing the signal sequence
In some embodiments involving secretion from bacterial cells, the signal peptide is a naturally occurring signal peptide, or a functional part thereof, while in other embodiments, it is a synthetic peptide. Suitable signal peptides include but are not limited to sequences derived from Bacillus licheniformis alpha-amylase, Bacillus clausii alkaline protease, and Bacillus amyloliquefaciens amylase. One preferred signal sequence is the signal peptide derived from Cellulomonas strain 69B4, as described herein. Thus, in some particularly preferred embodiments, the signal peptide comprises the signal peptide from the protease described herein. This signal finds use in facilitating the secretion of the 69B4 protease and/or a heterologous DNA sequence (e.g. a second protease, such as another wild-type protease, a BPN′ variant protease, a GG36 variant protease, a lipase, a cellulase, a mannanase, etc.). In some embodiments, these second enzymes are encoded by the DNA sequence and/or the amino acid sequences known in the art (See e.g., U.S. Pat. Nos. 6,465,235, 6,287,839, 5,965,384, and 5,795,764; as well as WO 98/22500, WO 92/05249, EP 0305216B1, and WO 94/25576). Furthermore, it is contemplated that in some embodiments, the signal sequence peptide is also be operatively linked to an endogenous sequence to activate and secrete such endogenous encoded protease.
The procedures used to ligate the DNA sequences coding for the present protease, the promoter and/or secretory signal sequence, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to those skilled in the art. As indicated above, in some embodiments, the nucleic acid construct is prepared using PCR with specific primers.
V. Host Cells
As indicated above, in some embodiments, the present invention also provides host cells transformed with the vectors described above. In some embodiments, the polynucleotide encoding the protease(s) of the present invention that is introduced into the host cell is homologous, while in other embodiments, the polynucleotide is heterologous to the host. In some embodiments in which the polynucleotide is homologous to the host cell (e.g., additional copies of the native protease produced by the host cell are introduced), it is operably connected to another homologous or heterologous promoter sequence. In alternative embodiments, another secretory signal sequence, and/or terminator sequence find use in the present invention. Thus, in some embodiments, the polypeptide DNA sequence comprises multiple copies of a homologous polypeptide sequence, a heterologous polypeptide sequence from another organism, or synthetic polypeptide sequence(s). Indeed, it is not intended that the present invention be limited to any particular host cells and/or vectors.
Indeed, the host cell into which the DNA construct of the present invention is introduced may be any cell which is capable of producing the present alkaline protease, including, but not limited to bacteria, fungi, and higher eukaryotic cells.
Examples of bacterial host cells which find use in the present invention include, but are not limited to Gram-positive bacteria such as Bacillus, Streptomyces, and Thermobifida, for example strains of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. clausii, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megaterium, B. thuringiensis, S. griseus, S. lividans, S. coelicolor, S. avermitilis and T. fusca; as well as Gram-negative bacteria such as members of the Enterobacteriaceae (e.g., Escherichia coli). In some particularly preferred embodiments, the host cells are B. subtilis, B. clausii, and/or B. licheniformis. In additional preferred embodiments, the host cells are strains of S. lividans (e.g., TK23 and/or TK21). Any suitable method for transformation of the bacteria find use in the present invention, including but not limited to protoplast transformation, use of competent cells, etc., as known in the art. In some preferred embodiments, the method provided in U.S. Pat. No. 5,264,366 (incorporated by reference herein), finds used in the present invention. For S. lividans, one preferred means for transformation and protein expression is that described by Fernandez-Abalos et al. (See, Fernandez-Abalos et al., Microbiol., 149:1623-1632 [2003]; See also, Hopwood, et al., Genetic Manipulation of Streptomyces: Laboratory Manual, Innis [1985], both of which are incorporated by reference herein). Of course, the methods described in the Example herein find use in the present invention.
Examples of fungal host cells which find use in the present invention include, but are not limited to Trichoderma spp. and Aspergillus spp. In some particularly preferred embodiments, the host cells are Trichoderma reesei and/or Aspergillus niger. In some embodiments, transformation and expression in Aspergillus is performed as described in U.S. Pat. No. 5,364,770, herein incorporated by reference. Of course, the methods described in the Example herein find use in the present invention.
In some embodiments, particular promoter and signal sequences are needed to provide effective transformation and expression of the protease(s) of the present invention. Thus, in some preferred embodiments involving the use of Bacillus host cells, the aprE promoter is used in combination with known Bacillus-derived signal and other regulatory sequences. In some preferred embodiments involving expression in Aspergillus, the glaA promoter is used. In some embodiments involving Streptomyces host cells, the glucose isomerase (GI) promoter of Actinoplanes missouriensis is used, while in other embodiments, the A4 promoter is used.
In some embodiments involving expression in bacteria such as E. coli, the protease is retained in the cytoplasm, typically as insoluble granules (i.e., inclusion bodies). However, in other embodiments, the protease is directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured after which the protease is refolded by diluting the denaturing agent. In the latter case, the protease is recovered from the periplasmic space by disrupting the cells (e.g., by sonication or osmotic shock), to release the contents of the periplasmic space and recovering the protease.
In preferred embodiments, the transformed host cells of the present invention are cultured in a suitable nutrient medium under conditions permitting the expression of the present protease, after which the resulting protease is recovered from the culture. The medium used to culture the cells comprises any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g., in catalogues of the American Type Culture Collection). In some embodiments, the protease produced by the cells is recovered from the culture medium by conventional procedures, including, but not limited to separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt (e.g., ammonium sulfate), chromatographic purification (e.g., ion exchange, gel filtration, affinity, etc.). Thus, any method suitable for recovering the protease(s) of the present invention will find use. Indeed, it is not intended that the present invention be limited to any particular purification method.
VI. Applications for Serine Protease Enzymes
As described in greater detail herein, the proteases of the present invention have important characteristics that make them very suitable for certain applications. For example, the proteases of the present invention have enhanced thermal stability, enhanced oxidative stability, and enhanced chelator stability, as compared to some currently used proteases.
Thus, these proteases find use in cleaning compositions. Indeed, under certain wash conditions, the present proteases exhibit comparative or enhanced wash performance as compared with currently used subtilisin proteases. Thus, it is contemplated that the cleaning and/or enzyme compositions of the present invention will be provided in a variety of cleaning compositions. In some embodiments, the proteases of the present invention are utilized in the same manner as subtilisin proteases (i.e., proteases currently in use). Thus, the present proteases find use in various cleaning compositions, as well as animal feed applications, leather processing (e.g., bating), protein hydrolysis, and in textile uses. The identified proteases also find use in personal care applications.
Thus, the proteases of the present invention find use in a number of industrial applications, in particular within the cleaning, disinfecting, animal feed, and textile/leather industries. In some embodiments, the protease(s) of the present invention are combined with detergents, builders, bleaching agents and other conventional ingredients to produce a variety of novel cleaning compositions useful in the laundry and other cleaning arts such as, for example, laundry detergents (both powdered and liquid), laundry pre-soaks, all fabric bleaches, automatic dishwashing detergents (both liquid and powdered), household cleaners, particularly bar and liquid soap applications, and drain openers. In addition, the protease find use in the cleaning of contact lenses, as well as other items, by contacting such materials with an aqueous solution of the cleaning composition. In addition these naturally occurring proteases can be used, for example in peptide hydrolysis, waste treatment, textile applications, medical device cleaning, biofilm removal and as fusion-cleavage enzymes in protein production, etc. The composition of these products is not critical to the present invention, as long as the protease(s) maintain their function in the setting used. In some embodiments, the compositions are readily prepared by combining a cleaning effective amount of the protease or an enzyme composition comprising the protease enzyme preparation with the conventional components of such compositions in their art recognized amounts.
A. Cleaning Compositions
The cleaning composition of the present invention may be advantageously employed for example, in laundry applications, hard surface cleaning, automatic dishwashing applications, as well as cosmetic applications such as dentures, teeth, hair and skin. However, due to the unique advantages of increased effectiveness in lower temperature solutions and the superior color-safety profile, the enzymes of the present invention are ideally suited for laundry applications such as the bleaching of fabrics. Furthermore, the enzymes of the present invention may be employed in both granular and liquid compositions.
The enzymes of the present invention may also be employed in a cleaning additive product. A cleaning additive product including the enzymes of the present invention is ideally suited for inclusion in a wash process when additional bleaching effectiveness is desired. Such instances may include, but are not limited to low temperature solution cleaning application. The additive product may be, in its simplest form, one or more proteases, including ASP. Such additive may be packaged in dosage form for addition to a cleaning process where a source of peroxygen is employed and increased bleaching effectiveness is desired. Such single dosage form may comprise a pill, tablet, gelcap or other single dosage unit such as pre-measured powders or liquids. A filler or carrier material may be included to increase the volume of such composition. Suitable filler or carrier materials include, but are not limited to, various salts of sulfate, carbonate and silicate as well as talc, clay and the like. Filler or carrier materials for liquid compositions may be water or low molecular weight primary and secondary alcohols including polyols and diols. Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol and isopropanol. The compositions may contain from about 5% to about 90% of such materials. Acidic fillers can be used to reduce pH. Alternatively, the cleaning additive may include activated peroxygen source defined below or the adjunct ingredients as fully defined below.
The present cleaning compositions and cleaning additives require an effective amount of the ASP enzyme and/or variants provided herein. The required level of enzyme may be achieved by the addition of one or more species of the enzymes of the present invention. Typically the present cleaning compositions will comprise at least 0.0001 weight percent, from about 0.0001 to about 1, from about 0.001 to about 0.5, or even from about 0.01 to about 0.1 weight percent of at least one of the enzymes of the present invention.
The cleaning compositions herein will typically be formulated such that, during use in aqueous cleaning operations, the wash water will have a pH of from about 5.0 to about 11.5 or even from about 7.5 to about 10.5. Liquid product formulations are typically formulated to have a neat pH from about 3.0 to about 9.0 or even from about 3 to about 5. Granular laundry products are typically formulated to have a pH from about 9 to about 11. Techniques for controlling pH at recommended usage levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art.
Suitable low pH cleaning compositions typically have a neat pH of from about 3 to about 5, and are typically free of surfactants that hydrolyze in such a pH environment. Such surfactants include sodium alkyl sulfate surfactants that comprise at least one ethylene oxide moiety or even from about 1 to 16 moles of ethylene oxide. Such cleaning compositions typically comprise a sufficient amount of a pH modifier, such as sodium hydroxide, monoethanolamine or hydrochloric acid, to provide such cleaning composition with a neat pH, of from about 3 to about 5. Such compositions typically comprise at least one acid stable enzyme. Said compositions may be liquids or solids. The pH of such liquid compositions is measured as a neat pH. The pH of such solid compositions is measured as a 10% solids solution of said composition wherein the solvent is distilled water. In these embodiments, all pH measurements are taken at 20° C.
When the serine protease(s) is/are employed in a granular composition or liquid, it may be desirable for the enzyme to be in the form of an encapsulated particle to protect such enzyme from other components of the granular composition during storage. In addition, encapsulation is also a means of controlling the availability of the enzyme during the cleaning process and may enhance performance of the enzymes provided herein. In this regard, the serine proteases of the present invention may be encapsulated with any encapsulating material known in the art.
The encapsulating material typically encapsulates at least part of the catalyst for the enzymes of the present invention. Typically, the encapsulating material is water-soluble and/or water-dispersible. The encapsulating material may have a glass transition temperature (Tg) of 0° C. or higher. Glass transition temperature is described in more detail in WO 97/11151, especially from page 6, line 25 to page 7, line 2.
The encapsulating material is may be selected from the group consisting of carbohydrates, natural or synthetic gums, chitin and chitosan, cellulose and cellulose derivatives, silicates, phosphates, borates, polyvinyl alcohol, polyethylene glycol, paraffin waxes and combinations thereof. When the encapsulating material is a carbohydrate, it may be typically selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and combinations thereof. Typically, the encapsulating material is a starch. Suitable starches are described in EP 0 922 499; U.S. Pat. No. 4,977,252; U.S. Pat. No. 5,354,559 and U.S. Pat. No. 5,935,826.
The encapsulating material may be a microsphere made from plastic such as thermoplastics, acrylonitrile, methacrylonitrile, polyacrylonitrile, polymethacrylonitrile and mixtures thereof; commercially available microspheres that can be used are those supplied by Expancel of Stockviksverken, Sweden under the trademark Expancel®, and those supplied by PQ Corp. of Valley Forge, Pa. U.S.A. under the tradename PM 6545, PM 6550, PM 7220, PM 7228, Extendospheres®, Luxsil®, Q-cel® and Sphericel®.
As described herein, the proteases of the present invention find particular use in the cleaning industry, including, but not limited to laundry and dish detergents. These applications place enzymes under various environmental stresses. The proteases of the present invention provide advantages over many currently used enzymes, due to their stability under various conditions.
Indeed, there are a variety of wash conditions including varying detergent formulations, wash water volumes, wash water temperatures, and lengths of wash time, to which proteases involved in washing are exposed. In addition, detergent formulations used in different geographical areas have different concentrations of their relevant components present in the wash water. For example, a European detergent typically has about 4500-5000 ppm of detergent components in the wash water, while a Japanese detergent typically has approximately 667 ppm of detergent components in the wash water. In North America, particularly the United States, detergents typically have about 975 ppm of detergent components present in the wash water.
A low detergent concentration system includes detergents where less than about 800 ppm of detergent components are present in the wash water. Japanese detergents are typically considered low detergent concentration system as they have approximately 667 ppm of detergent components present in the wash water.
A medium detergent concentration includes detergents where between about 800 ppm and about 2000 ppm of detergent components are present in the wash water. North American detergents are generally considered to be medium detergent concentration systems as they have approximately 975 ppm of detergent components present in the wash water. Brazil typically has approximately 1500 ppm of detergent components present in the wash water.
A high detergent concentration system includes detergents where greater than about 2000 ppm of detergent components are present in the wash water. European detergents are generally considered to be high detergent concentration systems as they have approximately 4500-5000 ppm of detergent components in the wash water.
Latin American detergents are generally high suds phosphate builder detergents and the range of detergents used in Latin America can fall in both the medium and high detergent concentrations as they range from 1500 ppm to 6000 ppm of detergent components in the wash water. As mentioned above, Brazil typically has approximately 1500 ppm of detergent components present in the wash water. However, other high suds phosphate builder detergent geographies, not limited to other Latin American countries, may have high detergent concentration systems up to about 6000 ppm of detergent components present in the wash water.
In light of the foregoing, it is evident that concentrations of detergent compositions in typical wash solutions throughout the world varies from less than about 800 ppm of detergent composition (“low detergent concentration geographies”), for example about 667 ppm in Japan, to between about 800 ppm to about 2000 ppm (“medium detergent concentration geographies”), for example about 975 ppm in U.S. and about 1500 ppm in Brazil, to greater than about 2000 ppm (“high detergent concentration geographies”), for example about 4500 ppm to about 5000 ppm in Europe and about 6000 ppm in high suds phosphate builder geographies.
The concentrations of the typical wash solutions are determined empirically. For example, in the U.S., a typical washing machine holds a volume of about 64.4 L of wash solution. Accordingly, in order to obtain a concentration of about 975 ppm of detergent within the wash solution about 62.79 g of detergent composition must be added to the 64.4 L of wash solution. This amount is the typical amount measured into the wash water by the consumer using the measuring cup provided with the detergent.
As a further example, different geographies use different wash temperatures. The temperature of the wash water in Japan is typically less than that used in Europe. For example, the temperature of the wash water in North America and Japan can be between 10 and 30° C. (e.g., about 20° C.), whereas the temperature of wash water in Europe is typically between 30 and 60° C. (e.g., about 40° C.).
As a further example, different geographies typically have different water hardness. Water hardness is usually described in terms of the grains per gallon mixed Ca2+/Mg2+. Hardness is a measure of the amount of calcium (Ca2+) and magnesium (Mg2+) in the water. Most water in the United States is hard, but the degree of hardness varies. Moderately hard (60-120 ppm) to hard (121-181 ppm) water has 60 to 181 parts per million (parts per million converted to grains per U.S. gallon is ppm # divided by 17.1 equals grains per gallon) of hardness minerals.












Water
Grains per gallon 
Parts per million







Soft
less than 1.0 
less than 17


Slightly hard
1.0 to 3.5 
 17 to 60 


Moderately hard
3.5 to 7.0 
 60 to 120


Hard
7.0 to 10.5
120 to 180


Very hard
greater than 10.5
greater than 180









European water hardness is typically greater than 10.5 (for example 10.5-20.0) grains per gallon mixed Ca2+/Mg2+ (e.g., about 15 grains per gallon mixed Ca2+/Mg2+). North American water hardness is typically greater than Japanese water hardness; but less than European water hardness. For examples North American water hardness can be between 3 to 10 grains, 3-8 grains or about 6 grains. Japanese water hardness is typically lower than North American water hardness, usually less than 4, for example 3 grains-per gallon mixed Ca2+/Mg2+.
Accordingly, in some embodiments, the present invention provides proteases that show surprising wash performance in at least one set of wash conditions (e.g., water temperature, water hardness, and/or detergent concentration). In some embodiments, the proteases of the present invention are comparable in wash performance to subtilisin proteases. In some embodiments, the proteases of the present invention exhibit enhanced wash performance as compared to subtilisin proteases. Thus, in some preferred embodiments of the present invention, the proteases provided herein exhibit enhanced oxidative stability, enhanced thermal stability, and/or enhanced chelator stability.
In some preferred embodiments, the present invention provides the ASP protease, as well as homologues and variants fo the protease. These proteases find use in any applications in which it is desired to clean protein based stains from textiles or fabrics.
In some embodiments, the cleaning compositions of the present invention are formulated as hand and machine laundry detergent compositions including laundry additive compositions, and compositions suitable for use in the pretreatment of stained fabrics, rinse-added fabric softener compositions, and compositions for use in general household hard surface cleaning operations, as well as dishwashing operations. Those in the art are familiar with different formulations which can be used as cleaning compositions. In preferred embodiments, the proteases of the present invention comprise comparative or enhanced performance in detergent compositions (i.e., as compared to other proteases). In some embodiments, cleaning performance is evaluated by comparing the proteases of the present invention with subtilisin proteases in various cleaning assays that utilize enzyme-sensitive stains such as egg, grass, blood, milk, etc., in standard methods. Indeed, those in the art are familiar with the spectrophotometric and other analytical methodologies used to assess detergent performance under standard wash cycle conditions.
Assays that find use in the present invention include, but are not limited to those described in WO 99/34011, and U.S. Pat. No. 6,605,458 (See e.g., Example 3). In U.S. Pat. No. 6,605,458, at Example 3, a detergent dose of 3.0 g/l at pH10.5, wash time 15 minutes, at 15 C, water hardness of 6° dH, 10 nM enzyme concentration in 150 ml glass beakers with stirring rod, 5 textile pieces (phi 2.5 cm) in 50 ml, EMPA 117 test material from Center for Test Materials Holland are used. The measurement of reflectance “R” on the test material was done at 460 nm using a Macbeth ColorEye 7000 photometer. Additional methods are provided in the Examples herein. Thus, these methods also find use in the present invention.
The addition of proteases of the invention to conventional cleaning compositions does not create any special use limitation. In other words, any temperature and pH suitable for the detergent is also suitable for the present compositions, as long as the pH is within the range set forth herein, and the temperature is below the described protease's denaturing temperature. In addition, proteases of the present invention find use in cleaning compositions that do not include detergents, again either alone or in combination with builders and stabilizers.
When used in cleaning compositions or detergents, oxidative stability is a further consideration. Thus, in some applications, the stability is enhanced, diminished, or comparable to subtilisin proteases as desired for various uses. In some preferred embodiments, enhanced oxidative stability is desired. Some of the proteases of the present invention find particular use in such applications.
When used in cleaning compositions or detergents, thermal stability is a further ao consideration. Thus, in some applications, the stability is enhanced, diminished, or comparable to subtilisin proteases as desired for various uses. In some preferred embodiments, enhanced thermostability is desired. Some of the proteases of the present invention find particular use in such applications.
When used in cleaning compositions or detergents, chelator stability is a further consideration. Thus, in some applications, the stability is enhanced, diminished, or comparable to subtilisin proteases as desired for various uses. In some preferred embodiments, enhanced chelator stability is desired. Some of the proteases of the present invention find particular use in such applications.
In some embodiments of the present invention, naturally occurring proteases are provided which exhibit modified enzymatic activity at different pHs when compared to subtilisin proteases. A pH-activity profile is a plot of pH against enzyme activity and may be constructed as described in the Examples and/or by methods known in the art. In some embodiments, it is desired to obtain naturally occurring proteases with broader profiles (i.e., those having greater activity at range of pHs than a comparable subtilisin protease). In other embodiments, the enzymes have no significantly greater activity at any pH, or naturally occurring homologues with sharper profiles (i.e., those having enhanced activity when compared to subtilisin proteases at a given pH, and lesser activity elsewhere). Thus, in various embodiments, the proteases of the present invention have differing pH optima and/or ranges. It is not intended that the present invention be limited to any specific pH or pH range.
In some embodiments of the present invention, the cleaning compositions comprise, proteases of the present invention at a level from 0.00001% to 10% of 69B4 and/or other protease of the present invention by weight of the composition and the balance (e.g., 99.999% to 90.0%) comprising cleaning adjunct materials by weight of composition. In other aspects of the present invention, the cleaning compositions of the present invention comprise, the 69B4 and/or other proteases at a level of 0.0001% to 10%, 0.001% to 5%, 0.001% to 2%, 0.005% to 0.5% 69B4 or other protease of the present invention by weight of the composition and the balance of the cleaning composition (e.g., 99.9999% to 90.0%, 99.999% to 98%, 99.995% to 99.5% by weight) comprising cleaning adjunct materials.
In some embodiments, preferred cleaning compositions, in addition to the protease preparation of the invention, comprise one or more additional enzymes or enzyme derivatives which provide cleaning performance and/or fabric care benefits. Such enzymes include, but are not limited to other proteases, lipases, cutinases, amylases, cellulases, peroxidases, oxidases (e.g. laccases), and/or mannanases.
Any other protease suitable for use in alkaline solutions finds use in the compositions of the present invention. Suitable proteases include those of animal, vegetable or microbial origin. In particularly preferred embodiments, microbial proteases are used. In some embodiments, chemically or genetically modified mutants are included. In some embodiments, the protease is a serine protease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases include subtilisins, especially those derived from Bacillus (e.g., subtilisin, lentus, amyloliquefaciens, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168). Additional examples include those mutant proteases described in U.S. Pat. Nos. RE 34,606, 5,955,340, 5,700,676, 6,312,936, and 6,482,628, all of which are incorporated herein by reference. Additional protease examples include, but are not limited to trypsin (e.g., of porcine or bovine origin), and the Fusarium protease described in WO 89/06270. Preferred commercially available protease enzymes include those sold under the trade names MAXATASE®, MAXACALTM, MAXAPEMTM, OPTICLEAN®, OPTIMASE®, PROPERASE®, PURAFECT® and PURAFECT® OXP (Genencor), those sold under the trade names ALCALASE®, SAVINASE®, PRIMASE®, DURAZYMTM, RELASE® and ESPERASE® (Novozymes); and those sold under the trade name BLAPTM (Henkel Kommanditgesellschaft auf Aktien, Duesseldorf, Germany. Various proteases are described in WO95/23221, WO 92/21760, and U.S. Pat. Nos. 5,801,039, 5,340,735, 5,500,364, 5,855,625. An additional BPN′ variant (“BPN′-var 1” and “BPN-variant 1”; as referred to herein) is described in U.S. Pat. No. RE 34,606. An additional GG36-variant (“GG36-var.1” and “GG36-variant 1”; as referred to herein) is described in U.S. Pat. Nos. 5,955,340 and 5,700,676. A further GG36-variant is described in U.S. Pat. Nos. 6,312,936 and 6,482,628. In one aspect of the present invention, the cleaning compositions of the present invention comprise additional protease enzymes at a level from 0.00001% to 10% of additional protease by weight of the composition and 99.999% to 90.0% of cleaning adjunct materials by weight of composition. In other embodiments of the present invention, the cleaning compositions of the present invention also comprise, proteases at a level of 0.0001% to 10%, 0.001% to 5%, 0.001% to 2%, 0.005% to 0.5% 69B4 protease (or its homologues or variants) by weight of the composition and the balance of the cleaning composition (e.g., 99.9999% to 90.0%, 99.999% to 98%, 99.995% to 99.5% by weight) comprising cleaning adjunct materials.
In addition, any lipase suitable for use in alkaline solutions finds use in the present invention. Suitable lipases include, but are not limited to those of bacterial or fungal origin. Chemically or genetically modified mutants are encompassed by the present invention. Examples of useful lipases include Humicola lanuginosa lipase (See e.g., EP 258 068, and so EP 305 216), Rhizomucor miehei lipase (See e.g., EP 238 023), Candida lipase, such as C. antarctica lipase (e.g., the C. antarctica lipase A or B; See e.g., EP 214 761), a Pseudomonas lipase such as P. alcaligenes and P. pseudoalcaligenes lipase (See e.g., EP 218 272), P. cepacia lipase (See e.g., EP 331 376), P. stutzeri lipase (See e.g., GB 1,372,034), P. fluorescens lipase, Bacillus lipase (e.g., B. subtilis lipase [Dartois et al., Biochem. Biophys. Acta 1131:253-260 [1993]); B. stearothermophilus lipase [See e.g., JP 64/744992]; and B. pumilus lipase [See e.g., WO 91/16422]).
Furthermore, a number of cloned lipases find use in some embodiments of the present invention, including but not limited to Penicillium camembertii lipase (See, Yamaguchi et al., Gene 103:61-67 [1991]), Geotricum candidum lipase (See, Schimada et al., J. Biochem., 106:383-388 [1989]), and various Rhizopus lipases such as R. delemar lipase (See, Hass et al., Gene 109:117-113 [1991]), a R. niveus lipase (Kugimiya et al., Biosci. Biotech. Biochem. 56:716-719 [1992]) and R. oryzae lipase.
Other types of lipolytic enzymes such as cutinases also find use in some embodiments of the present invention, including but not limited to the cutinase derived from Pseudomonas mendocina (See, WO 88/09367), or cutinase derived from Fusarium solani pisi (See, WO 90/09446).
Additional suitable lipases include commercially available lipases such as M1 LIPASETM, LUMA FASTTM, and LIPOMAXTM (Genencor); LIPOLASE® and LIPOLASE® ULTRA (Novozymes); and LIPASE PTM “Amano” (Amano Pharmaceutical Co. Ltd., Japan).
In some embodiments of the present invention, the cleaning compositions of the present invention further comprise lipases at a level from 0.00001% to 10% of additional lipase by weight of the composition and the balance of cleaning adjunct materials by weight of composition. In other aspects of the present invention, the cleaning compositions of the present invention also comprise, lipases at a level of 0.0001% to 10%, 0.001% to 5%, 0.001% to 2%, 0.005% to 0.5% lipase by weight of the composition.
Any amylase (alpha and/or beta) suitable for use in alkaline solutions also find use in some embodiments of the present invention. Suitable amylases include, but are not limited to those of bacterial or fungal origin. Chemically or genetically modified mutants are included in some embodiments. Amylases that find use in the present invention, include, but are not limited to α-amylases obtained from B. licheniformis (See e.g., GB 1,296,839). Commercially available amylases that find use in the present invention include, but are not limited to DURAMYL®, TERMAMYL®, FUNGAMYL® and BANTM (Novozymes) and RAPIDASE® and MAXAMYL® P (Genencor International).
In some embodiments of the present invention, the cleaning compositions of the so present invention further comprise amylases at a level from 0.00001% to 10% of additional amylase by weight of the composition and the balance of cleaning adjunct materials by weight of composition. In other aspects of the present invention, the cleaning compositions of the present invention also comprise, amylases at a level of 0.0001% to 10%, 0.001% to 5%, 0.001% to 2%, 0.005% to 0.5% amylase by weight of the composition.
Any cellulase suitable for use in alkaline solutions find use in embodiments of the present invention. Suitable cellulases include, but are not limited to those of bacterial or fungal origin. Chemically or genetically modified mutants are included in some embodiments. Suitable cellulases include, but are not limited to Humicola insolens cellulases (See e.g., U.S. Pat. No. 4,435,307). Especially suitable cellulases are the cellulases having color care benefits (See e.g., EP 0 495 257).
Commercially available cellulases that find use in the present include, but are not limited to CELLUZYME® (Novozymes), and KAC-500(B)TM (Kao Corporation). In some embodiments, cellulases are incorporated as portions or fragments of mature wild-type or variant cellulases, wherein a portion of the N-terminus is deleted (See e.g., U.S. Pat. No. 5,874,276).
In some embodiments, the cleaning compositions of the present invention can further comprise cellulases at a level from 0.00001% to 10% of additional cellulase by weight of the composition and the balance of cleaning adjunct materials by weight of composition. In other aspects of the present invention, the cleaning compositions of the present invention also comprise cellulases at a level of 0.0001% to 10%, 0.001% to 5%, 0.001% to 2%, 0.005% to 0.5% cellulase by weight of the composition.
Any mannanase suitable for use in detergent compositions and or alkaline solutions find use in the present invention. Suitable mannanases include, but are not limited to those of bacterial or fungal origin. Chemically or genetically modified mutants are included in some embodiments. Various mannanases are known which find use in the present invention (See e.g., U.S. Pat. No. 6,566,114, U.S. Pat. No. 6,602,842, and U.S. Pat. No. 6,440,991, all of which are incorporated herein by reference).
In some embodiments, the cleaning compositions of the present invention can further comprise mannanases at a level from 0.00001% to 10% of additional mannanase by weight of the composition and the balance of cleaning adjunct materials by weight of composition. In other aspects of the present invention, the cleaning compositions of the present invention also comprise, mannanases at a level of 0.0001% to 10%, 0.001% to 5%, 0.001% to 2%, 0.005% to 0.5% mannanase by weight of the composition.
In some embodiments, peroxidases are used in combination with hydrogen peroxide or a source thereof (e.g., a percarbonate, perborate or persulfate). In alternative embodiments, oxidases are used in combination with oxygen. Both types of enzymes are used for “solution bleaching” (i.e., to prevent transfer of a textile dye from a dyed fabric to another fabric when the fabrics are washed together in a wash liquor), preferably together with an enhancing agent (See e.g., WO 94/12621 and WO 95/01426). Suitable peroxidases/oxidases include, but are not limited to those of plant, bacterial or fungal origin. Chemically or genetically modified mutants are included in some embodiments.
In some embodiments, the cleaning compositions of the present invention can further comprise peroxidase and/or oxidase enzymes at a level from 0.00001% to 10% of additional peroxidase and/or oxidase by weight of the composition and the balance of cleaning adjunct materials by weight of composition. In other aspects of the present invention, the cleaning compositions of the present invention also comprise, peroxidase and/or oxidase enzymes at a level of 0.0001% to 10%, 0.001% to 5%, 0.001% to 2%, 0.005% to 0.5% peroxidase and/or oxidase enzymes by weight of the composition.
Mixtures of the above mentioned enzymes are encompassed herein, in particular a mixture of a the 69B4 enzyme, one or more additional proteases, at least one amylase, at least one lipase, at least one mannanase, and/or at least one cellulase. Indeed, it is contemplated that various mixtures of these enzymes will find use in the present invention.
It is contemplated that the varying levels of the protease and one or more additional enzymes may both independently range to 10%, the balance of the cleaning composition being cleaning adjunct materials. The specific selection of cleaning adjunct materials are readily made by considering the surface, item, or fabric to be cleaned, and the desired form of the composition for the cleaning conditions during use (e.g., through the wash detergent use).
Examples of suitable cleaning adjunct materials include, but are not limited to, surfactants, builders, bleaches, bleach activators, bleach catalysts, other enzymes, enzyme stabilizing systems, chelants, optical brighteners, soil release polymers, dye transfer agents, dispersants, suds suppressors, dyes, perfumes, colorants, filler salts, hydrotropes, photoactivators, fluorescers, fabric conditioners, hydrolyzable surfactants, preservatives, anti-oxidants, anti-shrinkage agents, anti-wrinkle agents, germicides, fungicides, color speckles, silvercare, anti-tarnish and/or anti-corrosion agents, alkalinity sources, solubilizing agents, carriers, processing aids, pigments, and pH control agents (See e.g., U.S. Pat. Nos. 6,610,642, 6,605,458, 5,705,464, 5,710,115, 5,698,504, 5,695,679, 5,686,014 and 5,646,101, all of which are incorporated herein by reference). Embodiments of specific cleaning composition materials are exemplified in detail below.
If the cleaning adjunct materials are not compatible with the proteases of the present invention in the cleaning compositions, then suitable methods of keeping the cleaning adjunct materials and the protease(s) separated (i.e., not in contact with each other) until combination of the two components is appropriate are used. Such separation methods include any suitable method known in the art (e.g., gelcaps, encapulation, tablets, physical separation, etc.).
Preferably an effective amount of one or more protease(s) provided herein are included in compositions useful for cleaning a variety of surfaces in need of proteinaceous stain removal. Such cleaning compositions include cleaning compositions for such applications as cleaning hard surfaces, fabrics, and dishes. Indeed, in some embodiments, the present invention provides fabric cleaning compositions, while in other embodiments, the present invention provides non-fabric cleaning compositions. Notably, the present invention also provides cleaning compositions suitable for personal care, including oral care (including dentrifices, toothpastes, mouthwashes, etc., as well as denture cleaning compositions), skin, and hair cleaning compositions. It is intended that the present invention encompass detergent compositions in any form (i.e., liquid, granular, bar, semi-solid, gels, emulsions, tablets, capsules, etc.).
By way of example, several cleaning compositions wherein the protease of the present invention find use are described in greater detail below. In embodiments in which the cleaning compositions of the present invention are formulated as compositions suitable for use in laundry machine washing method(s), the compositions of the present invention preferably contain at least one surfactant and at least one builder compound, as well as one or more cleaning adjunct materials preferably selected from organic polymeric compounds, bleaching agents, additional enzymes, suds suppressors, dispersants, lime-soap dispersants, soil suspension and anti-redeposition agents and corrosion inhibitors. In some embodiments, laundry compositions also contain softening agents (i.e., as additional cleaning adjunct materials).
The compositions of the present invention also find use detergent additive products in solid or liquid form. Such additive products are intended to supplement and/or boost the performance of conventional detergent compositions and can be added at any stage of the cleaning process.
In embodiments formulated as compositions for use in manual dishwashing methods, the compositions of the invention preferably contain at least one surfactant and preferably at least one additional cleaning adjunct material selected from organic polymeric compounds, suds enhancing agents, group II metal ions, solvents, hydrotropes and additional enzymes.
In some embodiments, the density of the laundry detergent compositions herein ranges from 400 to 1200 g/liter, while in other embodiments, it ranges from 500 to 950 g/liter of composition measured at 20° C.
In some embodiments, various cleaning compositions such as those provided in U.S. Pat. No. 6,605,458 find use with the proteases of the present invention. Thus, in some embodiments, the compositions comprising at least one protease of the present invention is a compact granular fabric cleaning composition, while in other embodiments, the composition is a granular fabric cleaning composition useful in the laundering of colored fabrics, in further embodiments, the composition is a granular-fabric cleaning composition which provides softening through the wash capacity, in additional embodiments, the composition is a heavy duty liquid fabric cleaning composition.
In some embodiments, the compositions comprising at least one protease of the present invention are fabric cleaning compositions such as those described in U.S. Pat. Nos. 6,610,642 and 6,376,450. In addition, the proteases of the present invention find use in granular laundry detergent compositions of particular utility under European or Japanese washing conditions (See e.g., U.S. Pat. No. 6,610,642).
In alternative embodiments, the present invention provides hard surface cleaning compositions comprising at least one protease provided herein. Thus, in some embodiments, the compositions comprising at least one protease of the present invention is a hard surface cleaning composition such as those described in U.S. Pat. Nos. 6,610,642, 6,376,450, and 6,376,450.
In yet further embodiments, the present invention provides dishwashing compositions comprising at least one protease provided herein. Thus, in some embodiments, the compositions comprising at least one protease of the present invention is a hard surface cleaning composition such as those in U.S. Pat. Nos. 6,610,642 and 6,376,450.
In still further embodiments, the present invention provides dishwashing compositions comprising at least one protease provided herein. Thus, in some embodiments, the compositions comprising at least one protease of the present invention comprise oral care compositions such as those in U.S. Pat. Nos. 6,376,450, and 6,376,450.
The formulations and descriptions of the compounds and cleaning adjunct materials contained in the aforementioned U.S. Pat. Nos. 6,376,450, 6,605,458, 6,605,458, and 6,610,642, all of which are expressly incorporated by reference herein. Still further examples are set forth in the Examples below.
I) Processes of Making and Using the Cleaning Composition of the Present Invention
The cleaning compositions of the present invention can be formulated into any suitable form and prepared by any process chosen by the formulator, non-limiting examples of which are described in U.S. Pat. Nos. 5,879,584, 5,691,297, 5,574,005, 5,569,645, 5,565,422, 5,516,448, 5,489,392, and 5,486,303, all of which are incorporated herein by reference. When a low pH cleaning composition is desired, the pH of such composition may be adjusted via the addition of a material such as monoethanolamine or an acidic material such as HCl.
II) Adjunct Materials in Addition to the Serine Proteases of the Present Invention
While not essential for the purposes of the present invention, the non-limiting list of adjuncts illustrated hereinafter are suitable for use in the instant cleaning compositions and may be desirably incorporated in certain embodiments of the invention, for example to assist or enhance cleaning performance, for treatment of the substrate to be cleaned, or to modify the aesthetics of the cleaning composition as is the case with perfumes, colorants, dyes or the like. It is understood that such adjuncts are in addition to the serine proteases of the present invention. The precise nature of these additional components, and levels of incorporation thereof, will depend on the physical form of the composition and the nature of the cleaning operation for which it is to be used. Suitable adjunct materials include, but are not limited to, surfactants, builders, chelating agents, dye transfer inhibiting agents, deposition aids, dispersants, additional enzymes, and enzyme stabilizers, catalytic materials, bleach activators, bleach boosters, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersing agents, clay soil removal/anti-redeposition agents, brighteners, suds suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, processing aids and/or pigments. In addition to the disclosure below, suitable examples of such other adjuncts and levels of use are found in U.S. Pat. Nos. 5,576,282, 6,306,812, and 6,326,348, that are incorporated by reference. The aforementioned adjunct ingredients may constitute the balance of the cleaning compositions of the present invention.
Surfactants
The cleaning compositions according to the present invention may comprise a surfactant or surfactant system wherein the surfactant can be selected from nonionic surfactants, anionic surfactants, cationic surfactants, ampholytic surfactants, zwitterionic surfactants, semi-polar nonionic surfactants and mixtures thereof. When a low pH cleaning composition, such as composition having a neat pH of from about 3 to about 5, is desired, such composition typically does not contain alkyl ethoxylated sulfate as it is believed that such surfactant may be hydrolyzed by such compositions the acidic contents.
The surfactant is typically present at a level of from about 0.1% to about 60%, from about 1% to about 50% or even from about 5% to about 40% by weight of the subject cleaning composition.
Builders
The cleaning compositions of the present invention may comprise one or more detergent builders or builder systems. When a builder is used, the subject cleaning composition will typically comprise at least about 1%, from about 3% to about 60% or even from about 5% to about 40% builder by weight of the subject cleaning composition.
Builders include, but are not limited to, the alkali metal, ammonium and alkanolammonium salts of polyphosphates, alkali metal silicates, alkaline earth and alkali metal carbonates, aluminosilicate builders polycarboxylate compounds ether hydroxypolycarboxylates, copolymers of maleic anhydride with ethylene or vinyl methyl ether, 1,3,5-trihydroxy benzene-2,4,6-trisulphonic acid, and carboxymethyloxysuccinic acid, the various alkali metal, ammonium and substituted ammonium salts of polyacetic acids such as ethylenediamine tetraacetic acid and nitrilotriacetic acid, as well as polycarboxylates such as mellitic acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid, benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid; and soluble salts thereof.
Chelating Agents
The cleaning compositions herein may contain a chelating agent, Suitable chelating agents include copper, iron and/or manganese chelating agents and mixtures thereof.
When a chelating agent is used, the cleaning composition may comprise from about 0.1% to about 15% or even from about 3.0% to about 10% chelating agent by weight of the subject cleaning composition.
Deposition Aid
The cleaning compositions herein may contain a deposition aid. Suitable deposition aids include, polyethylene glycol, polypropylene glycol, polycarboxylate, soil release polymers such as polytelephthalic acid, clays such as Kaolinite, montmorillonite, atapulgite, illite, bentonite, halloysite, and mixtures thereof.
Dve Transfer Inhibiting Agents
The cleaning compositions of the present invention may also include one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof.
When present in a subject cleaning composition, the dye transfer inhibiting agents may be present at levels from about 0.0001% to about 10%, from about 0.01% to about 5% or even from about 0.1% to about 3% by weight of the cleaning composition.
Dispersants
The cleaning compositions of the present invention can also contain dispersants. Suitable water-soluble organic materials include the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms.
Enzymes
The cleaning compositions can comprise one or more detergent enzymes which provide cleaning performance and/or fabric care benefits. Examples of suitable enzymes include, but are not limited to, hemicellulases, peroxidases, proteases, cellulases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, keratinases, reductases, oxidases, phenol oxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, β-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase, and amylases, or mixtures thereof. A typical combination is cocktail of conventional applicable enzymes like protease, lipase, cutinase and/or cellulase in conjunction with amylase.
Enzyme Stabilizers
Enzymes for use in detergents can be stabilized by various techniques. The enzymes employed herein can be stabilized by the presence of water-soluble sources of calcium and/or magnesium ions in the finished compositions that provide such ions to the enzymes.
Catalytic Metal Complexes
The cleaning compositions of the present invention may include catalytic metal complexes. One type of metal-containing bleach catalyst is a catalyst system comprising a transition metal cation of defined bleach catalytic activity, such as copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cations, an auxiliary metal cation having little or no bleach catalytic activity, such as zinc or aluminum cations, and a sequestrate having defined stability constants for the catalytic and auxiliary metal cations, particularly ethylenediaminetetraacetic acid, ethylenediaminetetra (methylenephosphonic acid) and water-soluble salts thereof. Such catalysts are disclosed in U.S. Pat. No. 4,430,243.
If desired, the compositions herein can be catalyzed by means of a manganese compound. Such compounds and levels of use are well known in the art and include, for example, the manganese-based catalysts disclosed in U.S. Pat. No. 5,576,282.
Cobalt bleach catalysts useful herein are known, and are described, for example, in U.S. Pat. Nos. 5,597,936, and 5,595,967. Such cobalt catalysts are readily prepared by known procedures, such as taught for example in U.S. Pat. Nos. 5,597,936, and 5,595,967.
Compositions herein may also suitably include a transition metal complex of a macropolycyclic rigid ligand—abbreviated as “MRL”. As a practical matter, and not by way of limitation, the compositions and cleaning processes herein can be adjusted to provide on the order of at least one part per hundred million of the active MRL species in the aqueous washing medium, and will preferably provide from about 0.005 ppm to about 25 ppm, more preferably from about 0.05 ppm to about 10 ppm, and most preferably from about 0.1 ppm to about 5 ppm, of the MRL in the wash liquor.
Preferred transition-metals in the instant transition-metal bleach catalyst include manganese, iron and chromium. Preferred MRL's herein are a special type of ultra-rigid ligand that is cross-bridged such as 5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane.
Suitable transition metal MRLs are readily prepared by known procedures, such as taught for example in WO 00/332601, and U.S. Pat. No. 6,225,464.
III) Processes of Making and Using Cleaning Compositions
The cleaning compositions of the present invention can be formulated into any suitable form and prepared by any process chosen by the formulator, non-limiting examples of which are described in U.S. Pat. Nos. 5,879,584, 5,691,297, 5,574,005, 5,569,645, 5,516,448, 5,489,392, and 5,486,303, all of which are incorporated herein by reference.
IV) Method of Use
The cleaning compositions disclosed herein of can be used to clean a situs inter alia a surface or fabric. Typically at least a portion of the situs is contacted with an embodiment of the present cleaning composition, in neat form or diluted in a wash liquor, and then the situs is optionally washed and/or rinsed. For purposes of the present invention, washing includes but is not limited to, scrubbing, and mechanical agitation. The fabric may comprise most any fabric capable of being laundered in normal consumer use conditions. The disclosed cleaning compositions are typically employed at concentrations of from about 500 ppm to about 15,000 ppm in solution. When the wash solvent is water, the water temperature typically ranges from about 5° C. to about 90° C. and, when the situs comprises a fabric, the water to fabric mass ratio is typically from about 1:1 to about 30:1.
B. Animal Feed
Still further, the present invention provides compositions and methods for the production of a food or animal feed, characterized in that protease according to the invention is mixed with food or animal feed. In some embodiments, the protease is added as a dry product before processing, while in other embodiments it is added as a liquid before or after processing. In some embodiments, in which a dry powder is used, the enzyme is diluted as a liquid onto a dry carrier such as milled grain. The proteases of the present invention find use as components of animal feeds and/or additives such as those described U.S. Pat. No. 5,612,055, U.S. Pat. No. 5,314,692. and U.S. Pat. No. 5,147,642, all of which are hereby incorporated by reference.
The enzyme feed additive according to the present invention is suitable for preparation in a number of methods. For example, in some embodiments, it is prepared simply by mixing different enzymes having the appropriate activities to produce an enzyme mix. In some embodiments; this enzyme mix is mixed directly with a feed, while in other embodiments, it is impregnated onto a cereal-based carrier material such as milled wheat, maize or soya flour. The present invention also encompasses these impregnated carriers, as they find use as enzyme feed additives.
In some alternative embodiments, a cereal-based carrier (e.g., milled wheat or maize) is impregnated either simultaneously or sequentially with enzymes having the appropriate activities. For example, in some embodiments, a milled wheat carrier is first sprayed with a xylanase, secondly with a protease, and optionally with a β-glucanase. The present invention also encompasses these impregnated carriers, as they find use as enzyme feed additives. In preferred embodiments, these impregnated carriers comprise at least one protease of the present invention.
In some embodiments, the feed additive of the present invention is directly mixed with the animal feed, while in alternative embodiments, it is mixed with one or more other feed additives such as a vitamin feed additive, a mineral feed additive, and/or an amino acid feed additive. The resulting feed additive including several different types of components is then mixed in an appropriate amount with the feed.
In some preferred embodiments, the feed additive of the present invention, including cereal-based carriers is normally mixed in amounts of 0.01-50 g per kilogram of feed, more preferably 0.1-10 g/kilogram, and most preferably about 1 g/kilogram.
In alternative embodiments, the enzyme feed additive of the present invention involves construction of recombinant microorganisms that produces the desired enzyme(s) in the desired relative amounts. In some embodiments, this is accomplished by increasing the copy number of the gene encoding at least one protease of the present invention, and/or by using a suitably strong promoter operatively linked to the polynucleotide encoding the protease(s). In further embodiments, the recombinant microorganism strain has certain enzyme activities deleted (e.g., cellulases, endoglucanases, etc.), as desired.
In additional embodiments, the enzyme feed additives provided by the present invention also include other enzymes, including but not limited to at least one xylanase, α-amylase, glucoamylase, pectinase, mannanase, α-galactosidase, phytase, and/or lipase. In some embodiments, the enzymes having the desired activities are mixed with the xylanase and protease either before impregnating these on a cereal-based carrier or alternatively such enzymes are impregnated simultaneously or sequentially on such a cereal-based carrier. The carrier is then in turn mixed with a cereal-based feed to prepare the final feed. In alternative embodiments, the enzyme feed additive is formulated as a solution of the individual enzyme activities and then mixed with a feed material pre-formed as pellets or as a mash.
In still further embodiments, the enzyme feed additive is included in animals' diets by incorporating it into a second (i.e., different) feed or the animals' drinking water. Accordingly, it is not essential that the enzyme mix provided by the present invention be incorporated into the cereal-based feed itself, although such incorporation forms a particularly preferred embodiment of the present invention. The ratio of the units of xylanase activity per g of the feed additive to the units of protease activity per g of the feed additive is preferably 1:0.001-1,000, more preferably 1:0.01-100, and most preferably 1:0.1-10. As indicated above, the enzyme mix provided by the present invention is preferably finds use as a feed additive in the preparation of a cereal-based feed.
In some embodiments, the cereal-based feed comprises at least 25% by weight, or more preferably at least 35% by weight, wheat or maize or a combination of both of these cereals. The feed further comprises a protease (i.e., at least one protease of the present invention) in such an amount that the feed includes a protease in such an amount that the feed includes 100-100,000 units of protease activity per kg.
Cereal-based feeds provided the present invention according to the present invention find use as feed for a variety of non-human animals, including poultry (e.g., turkeys, geese, ducks, chickens, etc.), livestock (e.g., pigs, sheep, cattle, goats, etc.), and companion animals (e.g., horses, dogs, cats, rabbits, mice, etc.). The feeds are particularly suitable for poultry and pigs, and in particular broiler chickens.
C. Textile and Leather Treatment
The present invention also provides compositions for the treatment of textiles that include at least one of the proteases of the present invention. In some embodiments, at least one protease of the present invention is a component of compositions suitable for the treatment of silk or wool (See e.g., U.S. RE Pat. No. 216,034, EP 134,267, U.S. Pat. No. 4,533,359, and EP 344,259).
In addition, the proteases of the present invention find use in a variety of applications where it is desirable to separate phosphorous from phytate. Accordingly, the present invention also provides methods producing wool or animal hair material with improved properties. In some preferred embodiments, these methods comprise the steps of pretreating wool, wool fibres or animal hair material in a process selected from the group consisting of plasma treatment processes and the Delhey process; and subjecting the pretreated wool or animal hair material to a treatment with a proteolytic enzyme (e.g., at least one protease of the present invention) in an amount effective for improving the properties. In some embodiments, the proteolytic enzyme treatment occurs prior to the plasma treatment, while in other embodiments, it occurs after the plasma treatment. In some further embodiments, it is conducted as a separate step, while in other embodiments, it is conducted in combination with the scouring or the dyeing of the wool or animal hair material. In additional embodiments, at least one surfactant and/or at least one softener is present during the enzyme treatment step, while in other embodiments, the surfactant(s) and/or softener(s) are incorporated in a separate step wherein the wool or animal hair material is subjected to a softening treatment.
In some embodiments, the compositions of the present invention find us in methods for shrink-proofing wool fibers (See e.g., JP 4-327274). In some embodiments, the compositions are used in methods for shrink-proofing treatment of wool fibers by subjecting the fibers to a low-temperature plasma treatment, followed by treatment with a shrink-proofing resin such as a block-urethane resin, polyamide epochlorohydrin resin, glyoxalic resin, ethylene-urea resin or acrylate resin, and then treatment with a weight reducing proteolytic enzyme for obtaining a softening effect). In some embodiments, the plasma treatment step is a low-temperature treatment, preferably a corona discharge treatment or a glow discharge treatment.
In some embodiments, the low-temperature plasma treatment is carried out by using a gas, preferably a gas selected from the group consisting of air, oxygen, nitrogen, ammonia, helium, or argon. Conventionally, air is used but it may be advantageous to use any of the other indicated gasses.
Preferably, the low-temperature plasma treatment is carried out at a pressure between about 0.1 torr and 5 torr for from about 2 seconds to about 300 seconds, preferably for about 5 seconds to about 100 seconds, more preferably from about 5 seconds to about 30 seconds.
As indicated above, the present invention finds use in conjunction with methods such as the Delhey process (See e.g., DE-A-43 32 692). In this process, the wool is treated in an aqueous solution of hydrogen peroxide in the presence of soluble wolframate, optionally followed by treatment in a solution or dispersion of synthetic polymers, for improving the anti-felting properties of the wool. In this method, the wool is treated in an aqueous solution of hydrogen peroxide (0.1-35% (w/w), preferably 2-10% (w/w)), in the presence of a 2-60% (w/w), preferably 8-20% (w/w) of a catalyst (preferably Na2 WO4), and in the presence of a nonionic wetting agent. Preferably, the treatment is carried out at pH 8-11, and room temperature. The treatment time depends on the concentrations of hydrogen peroxide and catalyst, but is preferably 2 minutes or less. After the oxidative treatment, the wool is rinsed with water. For removal of residual hydrogen peroxide, and optionally for additional bleaching, the wool is further treated in acidic solutions of reducing agents (e.g., sulfites, phosphites etc.).
In some embodiments, the enzyme treatment step carried out for between about 1 minute and about 120 minutes. This step is preferably carried out at a temperature of between about 20° C. and about 60° C., more preferably between about 30° C. and about 50° C. Alternatively, the wool is soaked in or padded with an aqueous enzyme solution and then subjected to steaming at a conventional temperature and pressure, typically for about 30 seconds to about 3 minutes. In some preferred embodiments, the proteolytic enzyme treatment is carried out in an acidic or neutral or alkaline medium which may include a buffer.
In alternative embodiments, the enzyme treatment step is conducted in the presence of one or more conventional anionic, non-ionic (e.g.; Dobanol; Henkel AG) or cationic surfactants. An example of a useful nonionic surfactant is Dobanol (from Henkel AG). In further embodiments, the wool or animal hair material is subjected to an ultrasound treatment, either prior to or simultaneous with the treatment with a proteolytic enzyme. In some preferred embodiments, the ultrasound treatment is carried out at a temperature of about 50° C. for about 5 minutes. In some preferred embodiments, the amount of proteolytic enzyme used in the enzyme treatment step is between about 0.2 w/w % and about 10 w/w %, based on the weight of the wool or animal hair material. In some embodiments, in order to the number of treatment steps, the enzyme treatment is carried out during dyeing and/or scouring of the wool or animal hair material, simply by adding the protease to the dyeing, rinsing and/or scouring bath. In some embodiments, enzyme treatment is carried out after the plasma treatment but in other embodiments, the two treatment steps are carried out in the opposite order.
Softeners conventionally used on wool are usually cationic softeners, either organic cationic softeners or silicone based products, but anionic or non-ionic softeners are also so useful. Examples of useful softeners include, but are not limited to polyethylene softeners and silicone softeners (i.e., dimethyl polysiloxanes (silicone oils)), H-polysiloxanes, silicone elastomers, aminofunctional dimethyl polysiloxanes, aminofunctional silicone elastomers, and epoxyfunctional dimethyl polysiloxanes, and organic cationic softeners (e.g. alkyl quarternary ammonium derivatives).
In additional embodiments, the present invention provides compositions for the treatment of an animal hide that includes at least one protease of the present invention. In some embodiments, the proteases of the present invention find use in compositions for treatment of animal hide, such as those described in WO 03/00865 (Insect Biotech Co., Taejeon-Si, Korea). In additional embodiments, the present invention provides methods for processing hides and/or skins into leather comprising enzymatic treatment of the hide or skin with the protease of the present invention (See e.g., WO 96/11285). In additional embodiments, the present invention provides compositions for the treatment of an animal skin or hide into leather that includes at least one protease of the present invention.
Hides and skins are usually received in the tanneries in the form of salted or dried raw hides or skins. The processing of hides or skins into leather comprises several different process steps including the steps of soaking, unhairing and bating. These steps constitute the wet processing and are performed in the beamhouse. Enzymatic treatment utilizing the proteases of the present invention are applicable at any time during the process involved in the processing of leather. However, proteases are usually employed during the wet processing (i.e., during soaking, unhairing and/or bating). Thus, in some preferred embodiments, the enzymatic treatment with at least one of the proteases of the present invention occurs during the wet processing stage.
In some embodiments, the soaking processes of the present invention are performed under conventional soaking conditions (e.g., at a pH in the range pH 6.0-11).
In some preferred embodiments, the range is pH 7.0-10.0. In alternative embodiments, the temperature is in the range of 20-30° C., while in other embodiments it is preferably in the range 24-28° C. In yet further embodiments, the reaction time is in the range 2-24 hours, while preferred range is 4-16 hours. In additional embodiments, tensides and/or preservatives are provided as desired.
The second phase of the bating step usually commences with the addition of the bate itself. In some embodiments, the enzymatic treatment takes place during bating. In some preferred embodiments, the enzymatic treatment takes place during bating, after the deliming phase. In some embodiments, the bating process of the presents invention is performed using conventional conditions (e.g., at a pH in the range pH 6.0-9.0). In some preferred embodiments, the pH range is 6.0 to 8.5. In further embodiments, the temperature is in the range of 20-30° C., while in preferred embodiments, the temperature is in the range of 25-28° C. In some embodiments, the reaction time is in the range of 20-90 minutes, while in other embodiments, it is in the range 40-80 minutes. Processes for the manufacture of leather are well known to those skilled in the art (See e.g., WO 94/069429 WO 90/1121189, U.S. Pat. No. 3,840,433, EP 505920, GB 2233665, and U.S. Pat. No. 3,986,926, all of which are herein incorporated by reference).
In further embodiments, the present invention provides bates comprising at least one protease of the present invention. A bate is an agent or an enzyme-containing preparation comprising the chemically active ingredients for use in beamhouse processes, in particular in the bating step of a process for the manufacture of leather. In some embodiments, the present invention provides bates comprising protease and suitable excipients. In some embodiments, agents including, but not limited to chemicals known and used in the art, e.g. diluents, emulgators, delimers and carriers. In some embodiments, the bate comprising at least one protease of the present invention is formulated as known in the art (See. e.g., GB-A2250289, WO 96/11285, and EP 0784703).
In some embodiments, the bate of the present invention contains from 0.00005 to 0.01 g of active protease per g of bate, while in other embodiments, the bate contains from 0.0002 to 0.004 g of active protease per g of bate.
Thus, the proteases of the present invention find use in numerous applications and settings.
EXPERIMENTAL
The present invention is described in further detail in the following Examples which are not in any way intended to limit the scope of the invention as claimed. The attached Figures are meant to be considered as integral parts of the specification and description of the invention. All references cited are herein specifically incorporated by reference for all that is described therein. The following Examples are offered to illustrate, but not to limit the claimed invention
In the experimental disclosure which follows, the following abbreviations apply: PI (proteinase inhibitor), ppm (parts per million); M (molar); mM (millimolar); μM (micromolar); nM (nanomolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); gm (grams); mg (milligrams); μg (micrograms); pg (picograms); L (liters); ml and mL (milliliters); μl and μL (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); U (units); V (volts); MW (molecular weight); sec (seconds); min(s) (minute/minutes); h(s) and hr(s) (hour/hours); ° C. (degrees Centigrade); QS (quantity sufficient); ND (not done); NA (not applicable); rpm (revolutions per minute); H2O (water); dH2O (deionized water); (HCl (hydrochloric acid); aa (amino acid); by (base pair); kb (kilobase pair); kD (kilodaltons); cDNA (copy or complementary DNA); DNA (deoxyribonucleic acid); ssDNA (single stranded DNA); dsDNA (double stranded DNA); dNTP (deoxyribonucleotide triphosphate); RNA (ribonucleic acid); MgCl2 (magnesium chloride); NaCl (sodium chloride); w/v (weight to volume); v/v (volume to volume); g (gravity); OD (optical density); Dulbecco's phosphate buffered solution (DPBS); SOC (2% Bacto-Tryptone, 0.5% Bacto Yeast Extract, 10 mM NaCl, 2.5 mM KCl); Terrific Broth (TB; 12 g/l Bacto Tryptone, 24 g/l glycerol, 2.31 g/l KH2PO4, and 12.54 g/l K2HPO4); OD280 (optical density at 280 nm); OD600 (optical density at 600 nm); A405 (absorbance at 405 nm); Vmax (the maximum initial velocity of an enzyme catalyzed reaction); PAGE (polyacrylamide gel electrophoresis); PBS (phosphate buffered saline [150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2]); PBST (PBS+0.25% TWEEN® 20); PEG (polyethylene glycol); PCR (polymerase chain reaction); RT-PCR (reverse transcription PCR); SDS (sodium dodecyl sulfate); Tris (tris(hydroxymethyl)aminomethane); HEPES (N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]); HBS (HEPES buffered saline); SDS (sodium dodecylsulfate); Tris-HCl (tris[Hydroxymethyl]aminomethane-hydrochloride); Tricine (N-[tris-(hydroxymethyl)-methyl]-glycine); CHES (2-(N-cyclo-hexylamino) ethane-sulfonic acid); TAPS (3-{[tris-(hydroxymethyl)-methyl]-amino}-propanesulfonic acid); CAPS (3-(cyclo-hexylamino)-propane-sulfonic acid; DMSO (dimethyl sulfoxide); DTT (1,4-dithio-DL-threitol); SA (sinapinic acid (s,5-dimethoxy-4-hydroxy cinnamic acid); TCA (trichloroacetic acid); Glut and GSH (reduced glutathione); GSSG (oxidized glutathione); TCEP (Tris[2-carboxyethyl] phosphine); Ci (Curies); mCi (milliCuries); μCi (microCuries); HPLC (high pressure liquid chromatography); RP-HPLC (reverse phase high pressure liquid chromatography); TLC (thin layer chromatography); MALDI-TOF (matrix-assisted laser desorption/ionization—time of flight); Ts (tosyl); Bn (benzyl); Ph (phenyl); Ms (mesyl); Et (ethyl), Me (methyl); Taq (Thermus aquaticus DNA polymerase); Klenow (DNA polymerase I large (Klenow) fragment); rpm (revolutions per minute); EGTA (ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid); EDTA (ethylenediaminetetracetic acid); bla (β-lactamase or ampicillin-resistance gene); HDL (heavy duty liquid detergent, i.e., laundry detergent); MJ Research (MJ Research, Reno, Nev.); Baseclear (Baseclear BV, Inc., Leiden, the Netherlands); PerSeptive (PerSeptive Biosystems, Framingham, Mass.); ThermoFinnigan (ThermoFinnigan, San Jose, Calif.); Argo (Argo BioAnalytica, Morris Plains, N.J.); Seitz EKS (SeitzSchenk Filtersystems GmbH, Bad Kreuznach, Germany); Pall (Pall Corp., East Hills, N.Y.); Spectrum (Spectrum Laboratories, Dominguez Rancho, Calif.); Molecular Structure (Molecular Structure Corp., Woodlands, Tex.); Accelrys (Accelrys, Inc., San Diego, Calif.); Chemical Computing (Chemical Computing Corp., Montreal, Canada); New Brunswick (New Brunswick Scientific, Co., Edison, N.J.); CFT (Center for Test Materials, Vlaardingeng, the Netherlands); Procter & Gamble (Procter & Gamble, Inc., Cincinnati, Ohio); GE Healthcare (GE Healthcare, Chalfont St. Giles, United Kingdom); DNA2.0 (DNA2.0, Menlo Park, Calif.); OXOID (Oxoid, Basingstoke, Hampshire, UK); Megazyme (Megazyme International Ireland Ltd., Bray Business Park, Bray, Co., Wicklow, Ireland); Finnzymes (Finnzymes Oy, Espoo, Finland); Kelco (CP Kelco, Wilmington, Del.); Corning (Corning Life Sciences, Corning, N.Y.); (NEN (NEN Life Science Products, Boston, Mass.); Pharma AS (Pharma AS, Oslo, Norway); Dynal (Dynal, Oslo, Norway); Bio-Synthesis (Bio-Synthesis, Lewisville, Tex.); ATCC (American Type Culture Collection, Rockville, Md.); Gibco/BRL (Gibco/BRL, Grand Island, N.Y.); Sigma (Sigma Chemical Co., St. Louis, Mo.); Pharmacia (Pharmacia Biotech, Piscataway, N.J.); NCBI (National Center for Biotechnology Information); Applied Biosystems (Applied Biosystems, Foster City, Calif.); BD Biosciences and/or Clontech (BD Biosciences CLONTECH Laboratories, Palo Alto, Calif.); Operon Technologies (Operon Technologies, Inc., Alameda, Calif.); MWG Biotech (MWG Biotech, High Point, N.C.); Oligos Etc (Oligos Etc. Inc, Wilsonville, Oreg.); Bachem (Bachem Bioscience, Inc., King of Prussia, Pa.); Difco (Difco 15. Laboratories, Detroit, Mich.); Mediatech (Mediatech, Herndon, Va.; Santa Cruz (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.); Oxoid (Oxoid Inc., Ogdensburg, N.Y.); Worthington (Worthington Biochemical Corp., Freehold, N.J.); GIBCO BRL or Gibco BRL (Life Technologies, Inc., Gaithersburg, Md.); Millipore (Millipore, Billerica, Mass.); Bio-Rad (Bio-Rad, Hercules, Calif.); Invitrogen (Invitrogen Corp., San Diego, Calif.); NEB (New England Biolabs, Beverly, Mass.); Sigma (Sigma Chemical Co., St. Louis, Mo.); Pierce (Pierce Biotechnology, Rockford, Ill.); Takara (Takara Bio Inc., Otsu, Japan); Roche (Hoffmann-La Roche, Basel, Switzerland); EM Science (EM Science, Gibbstown, N.J.); Qiagen (Qiagen, Inc., Valencia, Calif.); Biodesign (Biodesign Intl., Saco, Me.); Aptagen (Aptagen, Inc., Herndon, Va.); Sorvall (Sorvall brand, from Kendro Laboratory Products, Asheville, N.C.); Molecular Devices (Molecular Devices, Corp., Sunnyvale, Calif.); R&D Systems (R&D Systems, Minneapolis, Minn.); Stratagene (Stratagene Cloning Systems, La Jolla, Calif.); Marsh (Marsh Biosciences, Rochester, N.Y.); Bio-Tek (Bio-Tek Instruments, Winooski, Vt.); (Biacore (Biacore, Inc., Piscataway, N.J.); PeproTech (PeproTech, Rocky Hill, N.J.); SynPep (SynPep, Dublin, Calif.); New Objective (New Objective brand; Scientific Instrument Services, Inc., Ringoes, N.J.); Waters (Waters, Inc., Milford, Mass.); Matrix Science (Matrix Science, Boston, Mass.); Dionex (Dionex, Corp., Sunnyvale, Calif.); Monsanto (Monsanto Co., St. Louis, Mo.); Wintershall (Wintershall AG, Kassel, Germany); BASF (BASF Co., Florham Park, N.J.); Huntsman (Huntsman Petrochemical Corp., Salt Lake City, Utah); Enichem (Enichem Iberica, Barcelona, Spain); Fluka Chemie AG (Fluka Chemie AG, Buchs, Switzerland); Gist-Brocades (Gist-Brocades, NV, Delft, the Netherlands); Dow Corning (Dow Corning Corp., Midland, Mich.); and Microsoft (Microsoft, Inc., Redmond, Wash.).
Example 1
Assays
In the following Examples, various assays were used, such as protein determinations, application-based tests, and stability-based tests. For ease in reading, the following assays are set forth below and referred to in the respective Examples. Any deviations from the protocols provided below in any of the experiments performed during the development of the present invention are indicated in the Examples.
Some of the detergents used in the following Examples had the following compositions. In Compositions I and II, the balance (to 100%) is perfume/dye and/or water. The pH of these compositions was from about 5 to about 7 for Composition I, and about 7.5 to about 8.5 Composition II. In Composition III, the balance (to 100%) comprised of water and/or the minors perfume, dye, brightener/SRPI/sodium carboxymethylcellulose/photobleach/MgSo4/PVPVI/suds suppressor/high molecular PEG/clay.










DETERGENT COMPOSITIONS










Composition I
Composition II












LAS
24.0
8.0


C12-C15 AE1.8S
—
11.0


C8-C10 propyl dimethyl amine
2.0
2.0


C12-C14 alkyl dimethyl amine oxide
—
—


C12-C15 AS
—
7.0


CFAA
—
4.0


C12-C14 Fatty alcohol ethoxylate
12.0
1.0


C12-C18 Fatty acid
3.0
4.0


Citric acid (anhydrous)
6.0
3.0


DETPMP
—
1.0


Monoethanolamińe
5.0
5.0


Sodium hydroxide
—
1.0


1 N HCl aqueous solution
#1
—


Propanediol
12.7
10.


Ethanol
1.8
5.4


DTPA
0.5
0.4


Pectin Lyase
—
0.005


Lipase
0.1
—


Amylase
0.001
—


Cellulase
—
0.0002


Protease A
—
—


Aldose Oxidase
—
—


DETBCHD
—
0.01


SRP1
0.5
0.3


Boric acid
2.4
2.8


Sodium xylene sulfonate
—
—


DC 3225C
1.0
1.0


2-butyl-octanol
0.03
0.03


Brightener 1
0.12
0.08









Composition III

















C14-C15AS or sodium tallow alkyl
3.0



sulfate




LAS
8.0



C12-C15AE3S
1.0



C12-C15E5 or E3
5.0



QAS
—



Zeolite A
11.0



SKS-6 (dry add)
9.0



MA/AA
2.0



AA
—



3Na Citrate 2H2O
—



Citric Acid (Anhydrous)
1.5



DTPA
—



EDDS
0.5



HEDP
0.2



PB1
—










Composition III

















Percarbonate
3.8



NOBS
—



NACA OBS
2.0



TAED
2.0



BB1
0.34



BB2
—



Anhydrous Na Carbonate
8.0



Sulfate
2.0



Silicate
—



Protease B
—



Protease C
—



Lipase
—



Amylase
—



Cellulase
—



Pectin Lyase
0.001



Aldose Oxidase
0.05



PAAC
—










A. TCA Assay for Protein Content Determination in 96-Well Microtiter Plates

This assay was started using filtered culture supernatant from microtiter plates grown 4 days at 33° C. with shaking at 230 RPM and humidified aeration. A fresh 96-well flat bottom plate was used for the assay. First, 100 μL/well of 0.25 N HCl were placed in the wells. Then, 50 μL filtered culture broth were added to the wells. The light scattering/absorbance at 405 nm (use 5 sec mixing mode in the plate reader) was then determined, in order to provide the “blank” reading.
For the test, 100 μL/well 15% (w/v) TCA was placed in the plates and incubated between 5 and 30 min at room temperature. The light scattering/absorbance at 405 nm (use 5 sec mixing mode in the plate reader) was then determined.
The calculations were performed by subtracting the blank (i.e., no TCA) from the test reading with TCA. If desired, a standard curve can be created by calibrating the TCA readings with AAPF assays of clones with known conversion factors. However, the TCA results are linear with respect to protein concentration from 50 to 0.500 ppm and can thus be plotted directly against enzyme performance for the purpose of choosing good-performing variants.
B. suc-AAPF-pNA Assay of Proteases in 96-Well Microtiter Plates
In this assay system, the reagent solutions used were:
1. 100 mM Tris/HCl, pH 8.6, containing 0.005% TWEEN®-80 (Tris buffer)
2. 100 mM Tris buffer, pH 8.6, containing 10 mM CaCl2 and 0.005% TWEEN®-80 (Tris buffer)
3. 160 mM suc-AAPF-pNA in DMSO (suc-AAPF-pNA stock solution) (Sigma: S-7388)
To prepare suc-AAPF-pNA working solution, 1 ml AAPF stock was added to 100 ml Tris/Ca buffer and mixed well for at least 10 seconds.
The assay was performed by adding 10 μl of diluted protease solution to each well, followed by the addition (quickly) of 190 μl 1 mg/ml AAPF-working solution. The solutions were mixed for 5 sec., and the absorbance change was read at 410 nm in an MTP reader, at 25° C. The protease activity was expressed as AU (activity=δOD·min−1·ml−1).

C. Keratin Hydrolysis Assay

In this assay system, the chemical and reagent solutions used were:
















Keratin
ICN 902111



Detergent
Detergent Composition II




1.6 g. detergent is dissolved in 1000 ml 




water (pH = 8.2) 0.6 ml. CaCl2/MgCl2 of 10,000 




gpg is added as well as 1190 mg




HEPES, giving a hardness 




and buffer strength of 6 gpg and 5 mM




respectively. The pH is adjusted to 8.2 with NaOH.



Picrylsulfonic 
Sigma P-2297 (5% solution in water)



acid (TNBS)




Reagent A
45.4 g Na2B4O7•10 H2O (Merck 6308) 




and 15 ml of 4N NaOH are dissolved 




together to a final volume




of 1000 ml (by heating if needed)



Reagent B
35.2 g NaH2PO4•1H2O (Merck 6346) 




and 0.6 g Na2SO3 (Merck 6657)




are dissolved together to a final volume of 1000 ml.










Method:

Prior to the incubations, keratin was sieved on a 100 μm sieve in small portions at a time. Then, 10 g of the <100 μm keratin was stirred in detergent solution for at least 20 minutes at room temperature with regular adjustment of the pH to 8.2. Finally, the suspension was centrifuged for 20 minutes at room temperature (Sorvall, GSA rotor, 13,000 rpm). This procedure was then repeated. Finally, the wet sediment was suspended in detergent to a total volume of 200 ml., and the suspension was kept stirred during pipetting.
Prior to incubation, microtiter plates (MTPs) were filled with 200 μl substrate per well with a Biohit multichannel pipette and 1200 μl tip (6 dispenses of 200 μl and dispensed as fast as possible to avoid settling of keratin in the tips). Then, 10 μl of the filtered culture was added to the substrate containing MTPs. The plates were covered with tape, placed in an incubator and incubated at 20° C. for 3 hours at 350 rpm (Innova 4330 [New Brunswick]). Following incubation, the plates were centrifuged for 3 minutes at 3000 rpm (iSigma 6K 15 centrifuge). About 15 minutes before removal of the 1st plate from the incubator, the TNBS reagent was prepared by mixing 1 ml TNBS solution per 50 ml of reagent A.
MTPs were filled with 60 μl TNBS reagent A per well. From the incubated plates, 10 μl was transferred to the MTPs with TNBS reagent A. The plates were covered with tape and shaken for 20 minutes in a bench shaker (BMG Thermostar) at room temperature and 500 rpm. Finally, 200 μl of reagent B was added to the wells, mixed for 1 minute on a shaker, and the absorbance at 405 nm was measured with the MTP-reader.
Calculation of the Keratin Hydrolyzing Activity:
The obtained absorbance value was corrected for the blank value (substrate without enzyme). The resulting absorbance provides a measure for the hydrolytic activity. For each sample (variant) the performance index was calculated. The performance index compares the performance of the variant (actual value) and the standard enzyme (theoretical value) at the same protein concentration. In addition, the theoretical values can be calculated, using the parameters of the Langmuir equation of the standard enzyme. A performance index (PI) that is greater than 1 (PI>1) identifies a better variant (as compared to the standard [e.g., wild-type]), while a PI of 1 (PI=1) identifies a variant that performs the same as the standard, and a PI that is less than 1 (PI<1) identifies a variant that performs worse than the standard. Thus, the PI identifies winners, as well as variants that are less desirable for use under certain circumstances.
D. Microswatch Assay for Testing Protease Performance
All of the detergents used in these assays did not contain enzymes.
Detergent Preparations:
1. European Detergent Solution:
Milli-Q water was adjusted to 15 gpg water hardness (Ca/Mg=4/1), add 7.6 g/l ARIEL® Regular detergent and stir the detergent solution vigorously for at least 30 minutes. The detergent was filtered before use in the assay through a 0.22 μm filter (e.g. Nalgene top bottle filter).
2. Japanese Detergent Solution
Milli-Q water was adjusted to 3 gpg water hardness (Ca/Mg=3/1), add 0.66 g/l Detergent Composition III, the detergent solution was stirred vigorously for at least 30 minutes. The detergent was filtered before use in the assay through a 0.22 μm filter (e.g. Nalgene top bottle filter).
3. Cold Water Liquid Detergent (US Conditions):
Milli-Q water was adjusted to 6 gpg water hardness (Ca/Mg=3/1), add 1.60 g/l TIDE® LVJ-1 detergent and stir the detergent solution vigorously for at least 15 minutes. Add 5 mM Hepes buffer and set pH at 8.2. The detergent was filtered before use in the assay through a 0.22 μm filter (e.g. Nalgene top bottle filter).
4. Low pH Liquid Detergent (US Conditions):
Milli-Q water was adjusted to 6 gpg water hardness (Ca/Mg=3/1), 1.60 g/l Detergent Composition I, was added and the detergent solution stirred vigorously for at least 15 minutes. The pH was set at 6.0 using 1N NaOH solution. The detergent was filtered before use in the assay through a 0.22 μm filter (e.g. Nalgene top bottle filter).
Microswatches:
Microswatches of 1⁄4′′ circular diameter were ordered and delivered by CFT Vlaardingen. The microswatches were pretreated using the fixation method described below. Single microswatches were placed in each well of a 96-well microtiter plate vertically to expose the whole surface area (i.e., not flat on the bottom of the well).
Bleach Fixation (“Superfixed”):
In a 10 L stainless steel beaker containing 10 L of water, the water was heated to 60° C. for fixation of swatches used in European conditions (=Super fixed). For Japanese condition(s) and other conditions, the swatches were fixed at room temperature (=3K). Then, 10 ml of 30% hydrogen peroxide (1 ml/L of H2O2, final conc. of H2O2 is 300 ppm) were added. Then, 100 swatches (10 swatches/L) were added to the solution. The solution was allowed to sit for 30 minutes with occasional stirring and monitoring of the temperature. The swatches were rinsed 7-8 times with cold water and placed on bench to dry. A towel was placed on top of swatches, as this prevents the swatches from curling up. For the 3K swatches, the procedure is repeated (except the water was not heated and 10× the amount of hydrogen peroxide was added).
Alternative Fixation (“3K” Swatch Fixation):
This particular swatch fixation was done at room temperature, however the amount of 30% H2O2 added is 10× more than in the Superfixed Swatch Fixation. Bubble formation (frothing) will be visible and therefore it is necessary to use a bigger beaker to account for this. First, 8 liters of distilled water are placed in a 10 L beaker, and 80 ml of 30% hydrogen peroxide are added. The water and peroxide are mixed well with a ladle. Then, 40 pieces of EMPA 116 swatches were spread into a fan before adding into the solution to ensure uniform fixation. The swatches were swirled in the solution (using the ladle) for 30 minutes, continuously for the first five minutes and occasionally for the remaining 25 minutes. The solution was discarded and the swatches were rinsed 6 times with approximately 6 liters of distilled water each time. The swatches were placed on top of paper towels to dry. The air-dried swatches were punched using a 1⁄4′′ circular die on an expulsion press. A single microswatch was placed vertically into each well of a 96-well microtiter plate to expose the whole surface area (i.e. not flat on the bottom of the well).
Enzyme Samples:
The enzyme samples were tested at appropriate concentrations for the respective geography, and diluted in 10 mM NaCl, 0.005% TWEEN®-80 solution.
Test Method:
The incubator was set at the desired temperature: 20° C. for cold water liquid conditions; 30° C. for low-pH liquid conditions; 40° C. for European conditions; 20° C. for Japanese and North American conditions. The pretreated and precut swatches were placed into the wells of a 96-well MTP, as described above. The enzyme samples were diluted, if needed, in 10 mM NaCl, 0.005% TWEEN®-80 to 20× the desired concentration. The desired detergent solutions were prepared as described above. Then, 190 μl of detergent solution were added to each well of the MTP. To this mixture, 10 μl of enzyme solution were added to each well (to provide a total volume to 200 μl/well). The MTP was sealed with a plate sealer and placed in an incubator for 60 minutes, with agitation at 350 rpm. Following incubation under the appropriate conditions, 100 μl of solution from each well were removed and placed into a fresh MTP. The new MTP containing 100 μl of solution/well was read at 405 nm in a MTP reader. Blank controls, as well as a control containing a microswatch and detergent but no enzyme were also included.







TABLE 1-1







Detergent Composition and Incubation Conditions in the μSwatch Assay.














Ref-

Water
Enzyme





erence

Hard-
Dosage




Geography
Enzyme
Detergent
ness
[ppm]
Temp.
Swatch





European
ASP
7.6 g/l
15 gpg -
0.5-4
40°
Super-



GG36
ARIEL ®
Ca/Mg:


fix




Regular
4/1





Japanese
ASP
0.66 g/l
3 gpg -
0.5-4
20°
3K



GG36
Detergent
Ca/Mg:







Comp. III
3/1





Cold Water
ASP
1.6 g/l 
6 gpg - 
0.5-4
20°
3K




Tide ®
Ca/Mg:





Liquid

LVJ-1
3/1





Liquid
ASP
1.6 g/l
6 gpg -
0.5-4
30°
3K


Detergent

Detergent
Ca/Mg:





Comp. I

Comp. I
3/1








**The stock solution was used at a concentration of 15,000 gpg


stock #1 = Ca/Mg 3:1


(1.92 M Ca2+ = 282.3 g/L CaCl2•2H2O; 0.64 M Mg2+ = 30.1 g/L MgCl2•6H2O)


stock #2 = Ca/Mg 4:1


(2.05 M Ca2+ = 301.4 g/L CaC12•2H2O; 0.51 M Mg2+ = 103.7 g/L MgCl2•6H2O)






Calculation of the BMI Performance:

The obtained absorbance value was corrected for the blank value (obtained after incubation of microswatches in the absence of enzyme). The resulting absorbance was a measure for the hydrolytic activity. For each sample (variant) the performance index was calculated. The performance index compares the performance of the variant (actual value) and the standard enzyme (theoretical value) at the same protein concentration. In addition, the theoretical values can be calculated, using the parameters of the Langmuir equation of the standard enzyme. A performance index (PI) that is greater than 1 (PI>1) identifies a better variant (as compared to the standard [e.g., wild-type]), while a PI of 1 (PI=1) identifies a variant that performs the same as the standard, and a PI that is less than 1 (PI<1) identifies a variant that performs worse than the standard. Thus, the PI identifies winners, as well as variants that are less desirable for use under certain circumstances.
D. Dimethylcasein Hydrolysis Assay (96 Wells)
In this assay system, the chemical and reagent solutions used were:













Dimethylcasein 
Sigma C-9801


(DMC):



TWEEN ®-80:
Sigma P-8074


PIPES buffer 
Sigma P-1851; 15.1 g is dissolved in 


(free acid):
about 960 ml water; pH is adjusted: to 7.0 with 



4N NaOH, 1 ml 5% TWEEN ®-80 is added and 



the volume brought up to 1000 ml. The final



concentration of PIPES and TWEEN ®-80 is 



50 mM and 0.005% respectively.


Picrylsulfonic acid 
Sigma P-2297 (5% solution in water)


(TNBS):



Reagent A:
45.4 g Na2B4O7•10 H2O (Merck 6308) 



and 15 ml of 4N NaOH are dissolved together 



to a final volume of 1000 ml (by heating if 


Reagent B:
needed) 35.2 g NaH2PO4•1H2O (Merck 6346) 



and 0.6 g Na2SO3 (Merck 6657) are 



dissolved together to a final volume of 1000 ml.









Method:

To prepare the substrate, 4 g DMC were dissolved in 400 ml PIPES buffer. The filtered culture supernatants were diluted with PIPES buffer; the final concentration of the controls in the growth plate was 20 ppm. Then, 10 μl of each diluted supernatant were added to 200 μl substrate in the wells of a MTP. The MTP plate was covered with tape, shaken for a few seconds and placed in an oven at 37° C. for 2 hours without agitation.
About 15 minutes before removal of the 1st plate from the oven, the TNBS reagent was prepared by mixing 1 ml TNBS solution per 50 ml of reagent A. MTPs were filled with 60 μl TNBS reagent A per well. The incubated plates were shaken for a few seconds, after which 10 μl were transferred to the MTPs with TNBS reagent A. The plates were covered with tape and shaken for 20 minutes in a bench shaker (BMG Thermostar) at room temperature and 500 rpm. Finally, 200 μl reagent B were added to the wells, mixed for 1 minute on a shaker, and the absorbance at 405 nm was determined using an MTP-reader.
Calculation of Dimethylcasein Hydrolyzing Activity:
The obtained absorbance value was corrected for the blank value (substrate without enzyme). The resulting absorbance is a measure for the hydrolytic activity. The (arbitrary) specific activity of a sample was calculated by dividing the absorbance and the determined protein concentration.
E. Thermostability Assay
This assay is based on the dimethylcasein hydrolysis, before and after heating of the buffered culture supernatant. The same chemical and reagent solutions were used as described in the dimethylcasein hydrolysis assay.
Method:
The filtered culture supernatants were diluted to 20 ppm in PIPES buffer (based on the concentration of the controls in the growth plates). Then, 50 μl of each diluted supernatant were placed in the empty wells of a MTP. The MTP plate was incubated in an iEMS incubator/shaker HT (Thermo Labsystems) for 90 minutes at 60° C. and 400 rpm. The plates were cooled on ice for 5 minutes. Then, 10 μl of the solution was added to a fresh MTP containing 200 μl dimethylcasein substrate/well. This MTP was covered with tape, shaken for a few seconds and placed in an oven at 37° C. for 2 hours without agitation. The same detection method as used for the DMC hydrolysis assay was used.
Calculation of Thermostability:
The residual activity of a sample was expressed as the ratio of the final absorbance and the initial absorbance, both corrected for blanks.
F. LAS Stability Assay
LAS stability was measured after incubation of the test protease in the presence of 0.06% LAS (dodecylbenzenesulfonate sodium), and the residual activity was determined using the AAPF assay.
Reagents:


    
    
        Dodecylbenzenesulfonate, Sodium salt (=LAS): Sigma D-2525
        TWEEN®-80: Sigma P-8074
        TRIS buffer (free acid): Sigma T-1378); 6.35 g is dissolved in about 960 ml water; pH is adjusted to 8.2 with 4N HCl. Final concentration of TRIS is 52.5 mM.
        LAS stock solution: Prepare a 10.5% LAS solution in MQ water (=10.5 g per 100 ml MQ)
        TRIS buffer-100 mM/pH 8.6 (100 mM Tris/0.005% Tween80)
        TRIS-Ca buffer, pH 8.6 (100 mM Tris/10 mM CaCl2/0.005% Tween80)

Hardware:

        Flat bottom MTPs: Costar (#9017)
        Biomek FX
        ASYS Multipipettor
        Spectramax MTP Reader
        iEMS Incubator/Shaker
        Innova 4330 Incubator/Shaker
        Biohit multichannel pipette
        BMG Thermostar Shaker

Method:

    
    


A 10 μl 0.063% LAS solution was prepared in 52.5 mM Tris buffer pH 8.2. The AAPF working solution was prepared by adding 1 ml of 100 mg/ml AAPF stock solution (in DMSO) to 100 ml (100 mM) TRIS buffer; pH 8.6. To dilute the supernatants, flat-bottomed plates were filled with dilution buffer and an aliquot of the supernatant was added and mixed well. The dilution ratio depended on the concentration of the ASP-controls in the growth plates (AAPF activity). The desired protein concentration was 80 ppm.
Ten μl of the diluted supernatant was added to 190 μl 0.063% LAS buffer/well. The MTP was covered with tape, shaken for a few seconds and placed in an incubator (Innova 4230) at 25° C., for 60 minutes at 200 rpm agitation. The initial activity (t=10 minutes) was determined after 10 minutes of incubation by transferring 10 μl of the mixture in each well to a fresh MTP containing 190 μl AAPF work solution. These solutions were mixed well and the AAPF activity was measured using a MTP Reader (20 readings in 5 minutes and 25° C.).
The final activity (t=60 minutes) was determined by removing another 10 μl of solution from the incubating plate after 60 minutes of incubation. The AAPF activity was then determined as described above. The calculations were performed as follows: the % Residual Activity was [t−60 value]*100/[t−10 value].
G. Scrambled Egg Hydrolysis Assay
Proteases release insoluble particles from scrambled egg, which was baked into the wells of 96-well microtiter plates. The scrambled egg coated wells were treated with a mixture of protease containing culture filtrate and ADW (automatic dishwash detergent) to determine the enzyme performance in scrambled egg removal. The rate of turbidity is a measure of the enzyme activity.
Materials:


    
    
        Water bath
        Oven with mechanical air circulation (Memmert ULE 400)
        Incubator/shaker with amplitude of 0.25 cm (Multitron), equipped with MTP-holders and aluminum covers and bottoms
        Biomek FX liquid-handling system (Beckman)
        Micro plate reader (Molecular Devices Spectramax 340, SOFTmax Pro Software)
        Nichiryo 8800 multi channel syringe dispenser+syringes
        Micro titer plate tape
        Single and multi channel pipettes with tips
        Grade A medium eggs
        CaCl2.2H2O (Merck 102382); MgCl2.6H2O (Merck105833); Na2CO3 (Merck 6392)
        ADW product:
        
            LH-powder (=Light House)

Procedure:

        
        
    
    


Three eggs were stirred with a fork in a glass beaker and 100 ml milk (at 4° C. or room temperature) was added. The beaker was placed in an 85° C. water bath, and the mixture was stirred constantly with a spoon. As the mixture became thicker, care was taken to scrape the solidifying material continuously from the walls and bottom of the beaker. When the mixture was slightly runny (after about 25 minutes) the beaker was removed from the bath. Another 40 ml milk was added to the mixture and blended with a hand mixer or blender for 2 minutes. The mixture was cooled to room temperature (an ice bath can be used). The substrate was then stirred with an additional amount of 5 to 15% water (usually 7.5%).
Test Method:
First, 50 μl of scrambled egg substrate were dispensed into each well of a MTP. The plates were allowed to dry at room temperature overnight (about 17 hours), baked in oven at 80° C. for 2 hours, then cooled to room temperature.
ADW product solution was prepared by dissolving 2.85 g of LH-powder into 1 L water. Only about 15 minutes dissolution time was needed and filtration of the solution was not needed. Then, 1.16 mL artificial hardness solution was added and 2120 mg Na2CO3 was dissolved in the solution.
Hardness solution was prepared by mixing 188.57 g CaCl2.2H2O and 86.92 g MgCl2.6H2O in 1 L demi water (equal to 1.28 M Ca+0.43 M Mg and totally 10000 gpg). The above-mentioned amounts of ADW, CaCl2 and MgCl2 were already proportionally increased values (200/190x) because of the addition of 10 μl supernatant to 190 μl ADW solution.
ADW solution (190 μl) was added to each well of the substrate plate. The MTPs were processed by adding 10 μl of supernatant to each well and sealing the'plate with tape. The plate was placed in a pre-warmed incubator/shaker and secured with a metal cover and clamp. The plate was then washed for 30 minutes at the appropriate temperature (50° C. for US) at 700 rpm. The plate was removed from the incubator/shaker. With gentle up and down movements of the liquid, about 125 μl of the warm supernatant were transferred to an empty flat bottom plate. After cooling, exactly 100 μl of the dispersion was dispensed into the wells of an empty flat bottom plate. The absorbance at 405 nm was determined using a microtiter plate reader.
Calculation of the Scrambled Egg Hydrolyzing Activity:
The obtained absorbance value was corrected for the blank value (substrate without enzyme). The resulting absorbance is a measure for the hydrolytic activity. For each sample (variant) the performance index was calculated. The performance index compares the performance of the variant (actual value) and the standard enzyme (theoretical value) at the same protein concentration. In addition, the theoretical values can be calculated, using the parameters of the Langmuir equation of the standard enzyme. A performance index (PI) that is greater than 1 (PI>1) identifies a better variant (as compared to the standard [e.g., wild-type]), while a PI of 1 (PI=1) identifies a variant that performs the same as the standard, and a PI that is less than 1 (PI<1) identifies a variant that performs worse than the standard. Thus, the PI identifies winners, as well as variants that are less desirable for use under certain circumstances.
Example 2
Production of 69B4 Protease from the Gram-Positive Alkaliphilic Bacterium 69B4
This Example provides a description of the Cellulomonas strain 69B4 used to initially isolate the novel protease 69B4 provided by the present invention. The alkaliphilic micro-organism Cellulomonas strain 69B.4, (DSM 16035) was isolated at 37° C. on an alkaline casein medium containing (g L−1) (See e.g., Duckworth et al., FEMS Microbiol. Ecol., 19:181-191 [1996]).
















Glucose (Merck 1.08342)
10



Peptone (Difco 0118)
5



Yeast extract (Difco 0127)
5



K2HPO4
1



MgSO4•7H2O
0.2



NaCl
40



Na2CO3
10



Casein
20



Agar
20










An additional alkaline cultivation medium (Grant Alkaliphile Medium) was also used to cultivate Cellulomonas strain 69B.4, as provided below:
Grant Alkaliphile Medium (“GAM”) solution A (g L−1)
















Glucose (Merck 1.08342)
10



Peptone (Difco 0118)
5



Yeast extract (Difco 0127)
5



K2HPO4
1



MgSO4•7H2O
0.2










Dissolved in 800 ml distilled water and sterilized by autoclaving

GAM solution B (g L1)

















NaCl
40



Na2CO3
10










Dissolved in 200 ml distilled water and sterilized by autoclaving.

Complete GAM medium was prepared by mixing Solution A (800 ml) with Solution B (200 ml). Solid medium is prepared by the addition of agar (2% w/v).
Growth Conditions
From a freshly thawed glycerol vial of culture (stored as a frozen glycerol (20% v/v, stock stored at −80° C.), the micro-organisms were inoculated using an inoculation loop on Grant Alkaliphile Medium (GAM) described above in agar plates and grown for at least 2 days at 37° C. One colony was then used to inoculate a 500 ml shake flask containing 100 ml of GAM at pH 10. This flask was then incubated at 37° C. in a rotary shaker at 280 rpm for 1-2 days until good growth (according to visual observation) was obtained. Then, 100 ml of broth culture was subsequently used to inoculate a 7 L fermentor containing 5 liters of GAM. The fermentations were run at 37° C. for 2-3 days in order to obtain maximal production of protease. Fully aerobic conditions were maintained throughout by injecting air, at a rate of 5 L/min, into the region of the impeller, which was rotating at about 500 rpm. The pH was set at pH 10 at the start, but was not controlled during the fermentation.
Preparation of 69B4 Crude Enzyme Samples
Culture broth was collected from the fermentor, and cells were removed by centrifugation for 30 min at 5000×g at 10° C. The resulting supernatant was clarified by depth filtration over Seitz EKS (SeitzSchenk Filtersystems). The resulting sterile culture supernatant was further concentrated approximately 10 times by ultra filtration using an ultra filtration cassette with a 10 kDa cut-off (Pall Omega 10 kDa Minisette; Pall). The resulting concentrated crude 69B4 samples were frozen and stored at −20° C. until further use.
Purification
The cell separated culture broth was dialyzed against 20 mM (2-(4-morpholino)-ethane sulfonic acid (“MES”), pH 5.4, 1 mM CaCl2 using 8K Molecular Weight Cut Off (MWCO) Spectra-Por7 (Spectrum) dialysis tubing. The dialysis was performed overnight or until the conductivity of the sample was less than or equal to the conductivity of the MES buffer. The dialyzed enzyme sample was purified using a BioCad VISION (Applied Biosystems) with a 10×100 mm (7.845 mL) POROS High Density Sulfo-propyl (HS) 20 (20 micron) cation-exchange column (PerSeptive Biosystems). After loading the enzyme on the previously equilibrated column at 5 mL/min, the column was washed at 40 mL/min with a pH gradient from 25 mM MES, pH 6.2, 1 mM CaCl2 to 25 mM (N-[2-hydroxyethyl]piperazine-N′-[2-ethane]sulfonic acid [C8H18N2O4S, CAS #7365-45-9]) (“HEPES”) pH 8.0, 1 mM CaCl2 in 25 column volumes. Fractions (8 mL) were collected across the run. The pH 8.0 wash step was held for 5 column volumes and then the enzyme was eluted using a gradient (0-100 mM NaCl in the same buffer in 35 column volumes). Protease activity in the fractions was monitored using the pNA assay (sAAPF-pNA assay; DelMar, et al., supra). Protease activity which eluted at 40 mM NaCl was concentrated and buffer exchanged (using a 5K MWCO VIVA Science 20 mL concentrator) into 20 mM MES, pH 5.8, 1 mM CaCl2. This material was used for further characterization of the enzyme.
Example 3
PCR Amplification of a Serine Protease Gene Fragment
In this Example, PCR amplification of a serine protease gene fragment is described.
Degenerate Primer Design
Based on alignments of published serine protease amino acid sequences, a range of degenerate primers were designed against conserved structural and catalytic regions. Such regions included those that were highly conserved among the serine proteases, as well as those known to be important for enzyme structure and function.
During the development of the present invention, protein sequences of nine published serine proteases (Streptogrisin C homologues) were aligned, as shown in below. The sequences were Streptomyces griseus Streptogrisin C (accession no. P52320); alkaline serine protease precursor fromThermobifida fusca (accession no. AAC23545); alkaline proteinase (EC 3.4.21.-) from Streptomyces sp. (accession no. PC2053); alkaline serine proteinase I from Streptomyces sp. (accession no. S34672); serine protease from Streptomyces lividans (accession no. CAD4208); putative serine protease from Streptomyces coelicolor A3(2) (accession no. NP—625129); putative serine protease from Streptomyces avermitilis MA-4680 (accession no. NP—822175); serine protease from Streptomyces lividans (accession no. CAD42809); putative serine protease precursor from Streptomyces coelicolor A3(2) (accession no. NP—628830). All of these sequences are publicly available from GenBank. These alignments are provided below. In this alignment, two conserved boxes are underlined and shown in bold.










AAC23545
  (1)
 --MNHSSR--RTTSLLFTAALAATALVAATTPAS----------------



PC2053
  (1)
 --MRHTGR-NAIGAAIAASALAFALVPSQAAAN------DTLTERAEAAV


S34672
  (1)
 --MRLKGRTVAIGSALAASALALSLVPANASSELP----SAETAKADALV


CAD42808
  (1)
 MVGRHAAR-SRRAALTALGALVLTALPSAASAAPPPVPGPRPAVARTPDA


NP_625129
  (1)
 MVGRHAAR-SRRAALTALGALVLTALPSAASAAPPPVPGPRPAVARTPDA


NP_822175
  (1)
 MVHRHVG--AGCAGLSVLATLVLTGLPAAAAIEPP-GPAPAPSAVQPLGA


CAD42809
  (1)
 MPHRHRHH-RAVGAAVAATAALLVAGLSGSASAGTAPAGSAPTAAETLRT


NP_628830
  (1)
 MPHRHRHH-RAVGAAVAATAALLVAGLSGSASAGTAPAGSAPTAAETLRT


P52320
  (1)
 ---MERTT-LRRRALVAGTATVAVGALALAGLTGVASADPAATAAPPVSA


 




51                                             100


AAC23545
 (31)
 -----AQELALKRDLGLSDAEVAELRAAEAEAVELEEELRDSLGSDFGGV


PC2053
 (42)
 ADLPAGVLDAMERDLGLSEQEAGLKLVAEHDAALLGETLSADLDAFAGSW


S34672
 (45)
 EQLPAGMVDAMERDLGVPAAEVGNQLVAEHEAAVLEESLSEDLSGYAGSW


CAD42808
 (50)
 ATAPARMLSAMERDLRLAPGQAAARPVNEAEAGTRAGMLRNTLGDRFAGA


NP_625129 
 (50)
 ATAPARMLSAMERDLRLAPGQAAARLVNEAEAGTRAGMLRNTLGDRFAGA


NP_822175 
 (48)
 GNPSTAVLGALQRDLHLTDTQAKTRLVNEMEAGTRAGRLQNALGKHFAGA


CAD42809
 (50)
 DAAPPALLKAMQRDLGIDRRQAERRLVNEAEAGATAGRLRAALGGDFAGA


NP_628830
 (50)
 DAAPPALLKAMQRDLGLDRRQAERRLVNEAEAGATAGRLRAALGGDFAGA


P52320
 (47)
 DSLSPGMLAALERDLGLDEDAARSRIANEYRAAAVAAGLEKSLGARYAGA


 




 101                                            150


AAC23545
 (76)
 YLDADT-TEITVAVTDPAAVSRVDADDVTVDVVDFGETALNDFVASLNAI


PC2053
 (92)
 LAEGT---ELVVATTSEAEAAEITEAGATAEVVDHTLAELDSVKDALDTA


S34672
 (95)
 IVEGTS--EHVVATTDRAEAAEITAAGATATVVEHSLAELEAVKDILDEA


CAD42808
(100)
 WVSGATSAELTVATTDAADTAAIEAQGAKAAVVGRNLAELRAVKEKLDAA


NP_625129
(100)
 WVSGATSAELTVATTDAADTAAIEAQGAKAAVVGRNLAELRAVKEKLDAA


NP_822175
 (98)
 WVHGAASADLTVATTHATDIPAITAGGATAVVVKTGLDDLKGAKKKLDSA


CAD42809
(100)
 WVRGAESGTLTVATTDAGDVAAVEARGAEAKVVRHSLADLDAAKARLDTA


NP_628830
(100)
 WVRGAESGTLTVATTDAGDVAAIEARGAEAKVVRHSLADLDAAKARLDTA


P52320
 (97)
 RVSGAK-ATLTVATTDASEAARITEAGARAEVVGHSLDRFEGVKKSLDKA


 




 151                                            200


AAC23545
(125)
 ADT--ADPKVTGWYTDLESDAVVITTLRGGTPAAEELAERAGLDERAVRI


PC2053
(139)
 AES-YDTTDAPVWYVDVTTNGVVLLTSD--VTEAEGFVEAAGVNAAAVDI


S34672
(143)
 ATA-NPEDAAPVWYVDVTTNEVVVLASD--VPAAEAFVAASGADASTVRV


CAD42808
(150)
 AVR-TRTRQTPVWYVDVKTNRVTVQATG--ASAAAAFVEAAGVPAADVGV


NP_625129
(150)
 AVR-TRTRQTPVWYVDVKTNRVTVQATG--ASAAAAFVEAAGVPAADVGV


NP_822175
(148)
 VAHGGTAVNTPVRYVDVRTNRVTLQARS--RAAADALIAAAGVDSGLVDV


CAD42809
(150)
 AAG-LNTADAPVWYVDTRTNTVVVEAIR--PAAARSLLTAAGVDGSLAHV


NP_628830
(150)
 AAG-LNTADAPVWYVDTRTNTVVVEAIR--PAAARSLLTAAGVDGSLAHV


P52320
(146)
 ALD-KAPKNVPVWYVDVAANRVVVNAAS--PAAGQAFLKVAGVDRGLVTV


 




 201                                            250


AAC23545
(173)
 VEEDEEPQSLAAIIGGNPYYFGN-YRCSIGFSVRQGSQTGFATAGHCGST


PC2053
(186)
 QTSDEQPQAFYDLVGGDAYYMGG-GRCSVGFSVTQGSTPGFATAGHCGTV


S34672
(190)
 ERSDESPQPFYDLVGGDAYYIGN-GRCSIGFSVRQGSTPGFVTAGHCGSV


CAD42808
(197)
 RVSPDQPRVLEDLVGGDAYYIDDQARCSIGFSVTKDDQEGFATAGHCGDP


NP_625129
(197)
 RVSPDQPRVLEDLVGGDAYYIDDQARCSIGFSVTKDDQEGFATAGHCGDP


NP_822175
(196)
 KVSEDRPRALFDIRGGDAYYIDNTARCSVGFSVTKGNQQGFATAGHCGRA


CAD42809
(197)
 KNRTERPRTFYDLRGGEAYYINNSSRCSIGFPITKGTQQGFATAGHCDRA


NP_628830
(197)
 KNRTERPRTFYDLRGGEAYYINNSSRCSIGFPITKGTQQGFATAGHCGRA


P52320
(193)
 ARSAEQPRALADIRGGDAYYMNGSGRCSVGFSVTRGTQNGFATAGRCGRV


 




 251                                            300


AAC23545
(222)
 GTRVS----SPSGTVAGSYFPGRDMGWVRITSADTVTPLVNRYNGGTVTV


PC2053
(235)
 GTSTTGYNQAAQGTFEESSFPGDDMAWVSVNSDWNTTPTVNE--GE-VTV


S34672
(239)
 GNATTGFNRVSQGTFRGSWFPGRDMAWVAVNSNWTPTSLVRNS-GSGVRV


CAD42808
(247)
 GATTTGYNEADQGTFQASTFPGKDMAWVGVNSDWTATPDVKAEGGEKIQL


NP_625129
(247)
 GATTTGYNEADQGTFQASTFPGKDMAWVGVNSDWTATPDVKAEGGEKIQL


NP_822175
(246)
 GAPTAGFNEVAQGTVQASVFPGHDMAWVGVNSDWTATPDVAGAAGQNVSI


CAD42809
(247)
 GSSTTGANRVAQGTFQGSIFPGRDMAWVATNSSWTATPYVLGAGGQNVQV


NP_628830
(247)
 GSSTTGANRVAQGTFQGSIFPGRDMAWVATNSSWTATPYVLGAGGQNVQV


P52320
(243)
 GTTTNGVNQQAQGTFQGSTFPGRDIAWVATNANWTPRPLVNGYGRGDVTV


 




 301                                            350


AAC23545
(268)
 TGSQEAATGSSVCRSGATTGWRCGTIQSKNQTVRYAEGTVTGLTRTTACA


PC2053
(282)
 SGSTEAAVGASICRSGSTTGWHCGTIQQHNTSVTYPEGTITGVTRTSVCA


S34672
(288)
 TGSTQATVGSSICRSGSTTGWRCGTIQQHNTSVTYPQGTITGVTRTSACA


CAD42808
(297)
 AGSVEALVGASVCRSGSTTGWHCGTIQQHDTSVTYPEGTVDGLTGTTVCA


NP_625129
(297)
 AGSVEALVGASVCRSGSTTGWHCGTIQQHDTSVTYPEGTVDGLTETTVCA


NP_822175
(296)
 AGSVQAIVGAAICRSGSTTGWHCGTVEEHDTSVTYEEGTVDGLTRTTVCA


CAD42809
(297)
 TGSTASPVGASVCRSGSTTGWHCGTVTQLNTSVTYQEGTISPVTRTTVCA


NP_628830
(297)
 TGSTASPVGASVCRSGSTTGWHCGTVTQLNTSVTYQEGTISPVTRTTVCA


P52320
(293)
 AGSTASVVGASVCRSGSTTGWHCGTIQQLNTSVTYPEGTISGVTRTSVCA


 




 351                                            400


AAC23545
(318)
 EGGDSGGPWLTGSQAQGVTSGGTGDCRSGGITFFQPINPLLSYFGLQLVT


PC2053
(332)
 EPGDSGGSYISGSQAQGVTSGGSGNCTSGGTTYHQPINPLLSAYGLDLVT


S34672
(338)
 QPGDSGGSFISGTQAQGVTSGGSGNCSIGGTTFHQPVNPILSQYGLTLVR


CAD42808
(347)
 EPGDSGGPFVSGVQAQGTTSGGSGDCTNGGTTFYQPVNPLLSDFGLTLKT


NP_625129
(347)
 EPGDSGGPFVSGVQAQGTTSGGSGDCTNGGTTFYQPVNPLLSDFGLTLKT


NP_822175
(346)
 EPGDSGGSFVSGSQAQGVTSGGSGDCTRGGTTYYQPVNPILSTYGLTLKT


CAD42809
(347)
 EPGDSGGSFISGSQAQGVTSGGSGDCRTGGGTFFQPINALLQNYGLTLKT


NP_628830
(347)
 EPGDSGGSFISGSQAQGVTSGGSGDCRTGGETFFQPINALLQNYGLTLKT


P52320
(343)
 EPGDSGGSYISGSQAQGVTSGGSGNCSSGGTTYFQPINPLLQAYGLTLVT


 




 401                                            450


AAC23545
(368)
 G-------------------------------------------------


PC2053
(382)
 G-------------------------------------------------


S34672
(388)
 S-------------------------------------------------


CAD42808
(397)
 TSAATQTPAPQDNAAA------DAWTAGRVYEVGTTVSYDGVRYRCLQSH


NP_625129
(397)
 TSAATQTPAPQDNAAA------DAWTAGRVYEVGTTVSYDGVRYRCLQSH


NP_822175
(396)
 STAPTDTPSDPVDQSG-------VWAAGRVYEVGAQVTYAGVTYQCLQSH


CAD42809
(397)
 TGGDDGGGDDGG-----EEPGG-TWAAGTVYQPGDTVTYGGATFRCLQGH


NP_628830
(397)
 TGGDDGGGDDGGGDDGGEEPGG-TWAAGTVYQPGDTVTYGGATFRCLQGH 


P52320
(393)
 SGGGTPTDPPTTPPTDSP---GGTWAVGTAYAAGATVTYGGATYRCLQAH


 




 451            468


AAC23545
(369)
 ------------------ (SEQ ID NO: 648)


PC2053
(383)
 ------------------ (SEQ ID NO: 649)


S34672
(389)
 ------------------ (SEQ ID NO: 650)


CAD42808
(441)
 QAQGVGSPASVPALWQRV (SEQ ID NO: 651)


NP_625129
(441)
 QAQGVGSPASVPALWQRV (SEQ ID NO: 652)


NP_822175
(439)
 QAQGVWQPAATPALWQRL (SEQ ID NO: 653)


CAD42809
(441)
 QAYAGWEPPNVPALWQRV (SEQ ID NO: 654)


NP_628830
(446)
 QAYAGWEPPNVPALWQRV (SEQ ID NO: 655)


P52320
(440)
 TAQPGWTPADVPALWQRV (SEQ ID NO: 656)






Two particular regions were chosen to meet the criteria above, and a forward and a reverse primer were designed based on these amino acid regions. The specific amino acid regions used to design the primers are highlighted in black in the sequences shown in the alignments directly above. Using the genetic code for codon usage, degenerate nucleotide PCR primers were synthesized by MWG-Biotech. The degenerate primer sequences produced were:









forward primer TTGWXCGT_FW:



(SEQ ID NO: 10)



5′ ACNACSGGSTGGCRGTGCGGCAC 3′



 



reverse primer GDSGGX_RV:



(SEQ ID NO: 11)



5′-ANGNGCCGCCGGAGTCNCC-3′






As all primers were synthesized in the 5′-3′ direction and standard IUB code for mixed base sites was used (e.g., to designate “N” for A/C/T/G). Degenerate primers TTGWXCGT_FW and GDSGGX_RV successfully amplified a 177 bp region from Cellulomonas sp. isolate 69B4 by PCR, as described below.
PCR Amplification of a Serine Protease Gene Fragment
Cellulomonas sp. isolate 69B4 genomic DNA was used as a template for PCR amplification of putative serine protease gene fragments using the above-described primers. PCR was carried out using High Fidelity Platinum Taq polymerase (Catalog number 11304-102; Invitrogen). Conditions were determined by individual experiments, but typically thirty cycles were run in a thermal cycler (MJ Research). Successful amplification was verified by electrophoresis of the PCR reaction on a 1% agarose TBE gel. A PCR product that was amplified from Cellulomonas sp. 69B4 with the primers TTGWXCGT_FW and GDSGGX_RV was purified by gel extraction using the Qiaquick Spin Gel Extraction kit (Catalogue 28704; Qiagen) according to the manufacturer's instructions. The purified PCR product was cloned into the commercially available pCR2.1TOPO vector System (Invitrogen) according to the manufacturer's instructions, and transformed into competent E. coli TOP10 cells. Colonies containing recombinant plasmids were visualized using blue/white selection. For rapid screening of recombinant transformants, plasmid DNA was prepared from cultures of putative positive (i.e., white) colonies. DNA was isolated using the Qiagen plasmid purification kit, and was sequenced by Baseclear. One of the clones contained a DNA insert of 177 bp that showed some homology with several streptogrisin-like protease genes of various Streptomyces species and also with serine protease genes from other bacterial species. The DNA and protein coding sequence of this 177 bp fragment is provided in FIG. 13.
Sequence Analysis
The sequences were analyzed by BLAST and other protein translation sequence tools. BLAST comparison at the nucleotide level showed various levels of identity to published serine protease sequences. Initially, nucleotide sequences were submitted to BLAST (Basic BLAST version 2.0). The program chosen was “BlastX”, and the database chosen was “nr.” Standard/default parameter values were employed. Sequence data for putative Cellulomonas 69B4 protease gene fragment was entered in FASTA format and the query submitted to BLAST to compare the sequences of the present invention to those already in the database. The results returned for the 177 bp fragment a high number of hits for protease genes from various Streptomyces spp., including S. griseus, S. fividans, S. coelicolor, S. albogriseolus, S. platensis, S. fradiae; and Streptomyces sp. It was concluded that further analysis of the 177 bp fragment cloned from Cellulomonas sp. isolate 69B4 was desired.
Example 4
Isolation of a Polynucleotide Sequence from the Genome of Cellulomonas 69B4 Encoding a Serine Protease by Inverse PCR
In this Example, experiments conducted to isolate a polynucleotide sequence encoding a serine protease produced by Cellulomonas sp. 69B4 are described.
Inverse PCR of Cellulomonas sp. 69B4 Genomic DNA to Isolate the Gene Encoding Cellulomonas strain 69B4 Protease
Inverse PCR was used to isolate and clone the full-length serine protease gene from Cellulomonas sp. 6984. Based on the DNA sequence of the 177 bp fragment of the Cellulomonas protease gene described in Example 3, novel DNA primers were designed:









69B4int_RV1



(SEQ ID NO: 14)



5′-CGGGGTAGGTGACCGAGGAGTTGAGCGCAGTG-3′



 



69B4int_FW2



(SEQ ID NO: 15)



5′-GCTCGCCGGCAACCAGGCCCAGGGCGTCACGTC-3′






Chromosomal DNA of Cellulomonas sp. 69B4 was digested with the restriction enzymes ApaI, BamHI, BssHII, KpnI, NarI, NcoI, NheI, PvuI, SalI or SstII, purified using the Qiagen PCR purification kit (Qiagen, Catalogue #28106) and self-ligated with T4 DNA ligase (Invitrogen) according to the manufacturers' instructions. Ligation mixtures were purified using the Qiagen PCR purification kit, and PCR was performed with primers 69B4int_RV1 and 69B4int_FW2. PCR on DNA fragments that were digested with NcoI, and then self-ligated, resulting in a PCR product of approximately 1.3 kb. DNA sequence analysis (BaseClear) revealed that this DNA fragment covers the main part of a streptogrisin-like protease gene from Cellulomonas. This protease was designated as “69B4 protease,” and the gene encoding Cellulomonas 69B4 protease was designated as the “asp gene.” The entire sequence of the asp gene was derived by additional inverse PCR reactions with primer 69B40int_FW2 and an another primer: 69B4-for 4 (5′ MC GGC GGG TTC ATC ACC GCC GGC CAC TGC GGC C 3′ (SEQ ID NO:16). Inverse PCR with these primers on NcoI, BssHII, ApaI and PvuI digested and self-ligated DNA fragments of genomic DNA of Cellulomonas sp. 69B4 resulted in the identification of the entire sequence of the asp gene.
Nucleotide and Amino Acid Sequences
For convenience, various sequences are included below. First, the DNA sequence of the asp gene (SEQ ID NO:1) provided below encodes the signal peptide (SEQ ID NO:9) and the precursor serine protease (SEQ ID NO:7) derived from Cellulomonas strain 69B4 (DSM 16035). The initiating polynucleotide encoding the signal peptide of the Cellulomonas strain 69B4 protease is in bold (ATG).










(SEQ ID NO: 1)



   1
GCGCGCTGCG CCCACGACGA CGCCGTCCGC CGTTCGCCGG CGTACCTGCG TTGGCTCACC




CGCGCGACGC GGGTGCTGCT GCGGCAGGCG GCAAGCGGCC GCATGGACGC AACCGAGTGG


 


  61
ACCCACCAGA TCGACCTCCA TAACGAGGCC GTATGACCAG AAAGGGATCT GCCACCGCCC



TGGGTGGTCT AGCTGGAGGT ATTGCTCCGG CATACTGGTC TTTCCCTAGA CGGTGGCGGG


 


 121
ACCAGCACGC TCCTAACCTC CGAGCACCGG CGACCGCCGG GTGCGATGAA AGGGACGAAC



TGGTCGTGCG AGGATTGGAG GCTCGTGGCC GCTGGCGGCC CACGCTACTT TCCCTGCTTG


 


 181
CGAGATGACA CCACGCACAG TCACGCGGGC CCTGGCCGTG GCCACCGCAG CCGCCACACT



GCTCTACTGT GGTGCGTGTC AGTGCGCCCG GGACCGGCAC CGGTGGCGTC GGCGGTGTGA


 


 241
CCTGGCAGGC GGCATGGCCG CCCAGGCCAA CGAGCCCGCA CCACCCGGGA GCGCGAGCGC



GGACCGTCCG CCGTACCGGC GGGTCCGGTT GCTCGGGCGT GGTGGGCCCT CGCGCTCGCG


 


 301
ACCGCCACGC CTGGCCGAGA AGCTCGACCC CGACCTCCTC GAGGCCATGG AGCGCGACCT



TGGCGGTGCG GACCGGCTCT TCGAGCTGGG GCTGGAGGAG CTCCGGTACC TCGCGCTGGA


 


 361
GGGCCTCGAC GOGGAGGAAG CCGCCGCCAC CCTGGCGTTC CAGCACGACG CAGCCGAGAC



CCCGGAGCTG CGCCTCCTTC GGCGGCGGTG GGACCGCAAG GTCGTGCTGC GTCGGCTCTG


 


 421
CGGCGAGGCC CTCGCCGAAG AGCTCGACGA GGACTTCGCC GGCACCTGGG TCGAGGACGA



GCCGCTCCGG GAGCGGCTTC TCGAGCTGCT CCTGAAGCGG CCGTGGACCC AGCTCCTGCT


 


 481
CGTCCTGTAC GTCGCCACCA CCGACGAGGA CGCCGTCGAG GAGGTCGAGG GCGAAGGCGC



GCAGGACATG CAGCGGTGGT GGCTGCTCCT GCGGCAGCTC CTCCAGCTCC CGCTTCCGCG


 


 541
CACGGCCGTC ACCGTCGAGC ACTCCCTGGC CGACCTCGAG GCCTGGAAGA CCGTCCTCGA



GTGCCGGCAG TGGCAGCTCG TGAGGGACCG GCTGGAGCTC CGGACCTTCT GGCAGGAGCT


 


 601
CGCCGCCCTC GAGGGCCACG ACGACGTGCC CACCTGGTAC GTCGACGTCC CGACCAACAG



GCGGCGGGAG CTCCCGGTGC TGCTGCACGG GTGGACCATG CAGCTGCAGG GCTGGTTGTC


 


 661
CGTCGTCGTC GCCGTCAAGG CCGGAGCCCA GGACGTCGCC GCCGGCCTCG TCGAAGGTGC



GCAGCAGCAG CGGCAGTTCC GGCCTCGGGT CCTGCAGCGG CGGCCGGAGC AGCTTCCACG


 


 721
CGACGTCCCG TCCGACGCCG TGACCTTCGT CGAGACCGAC GAGACCCCGC GGACCATGTT



GCTGCAGGGC AGGCTGCGGC ACTGGAAGCA GCTCTGGCTG CTCTGGGGCG CCTGGTACAA


 


 781
CGACGTGATC GGCGGCAACG CCTACACCAT CGGGGGGCGC AGCCGCTGCT CGATCGGGTT



GCTGCACTAG CCGCCGTTGC GGATGTGGTA GCCCCCCGCG TCGGCGACGA GCTAGCCCAA


 


 841
CGCGGTCAAC GGCGGGTTCA TCACCGCCGG CCACTGCGGC CGCACCGGCG CCACCACCGC



GCGCCAGTTG CCGCCCAAGT AGTGGCGGCC GGTGACGCCG GCGTGGCCGC GGTGGTGGCG


 


 901
CAACCCCACC GGGACCTTCG CCGGGTCCAG CTTCCCGGGC AACGACTACG CGTTCGTCCG



GTTGGGGTGG CCCTGGAAGC GGCCCAGGTC GAAGGGCCCG TTGCTGATGC GCAAGCAGGC


 


 961
TACCGGGGCC GGCGTGAACC TGCTGGCCCA GGTCAACAAC TACTCCGGTG GCCGCGTCCA



ATGGCCCCGG CCGCACTTGG ACGACCGGGT CCAGTTGTTG ATGAGGCCAC CGGCGCAGGT


 


1021
GGTCGCCGGG CACACCGCGG CCCCCGTCGG CTCGGCCGTG TGCCGGTCCG GGTCGACCAC



CCAGCGGCCC GTGTGGCGCC GGGGGCAGCC GAGCCGGCAC ACGGCCAGGC CCAGCTGGTG


 


1081
CGGGTGGCAC TGCGGCACCA TCACTGCGCT CAACTCCTCG GTCACCTACC CCGAGGGCAC



GCCCACCGTG ACGCCGTGGT AGTGACGCGA GTTGAGGAGC CAGTGGATGG GGCTCCCGTG


 


1141
CGTCCGCGGC CTGATCCGCA CCACCGTCTG CGCCGAGCCC GGCGACTCCG GTGGCTCGCT



GCAGGCGCCG GACTAGGCGT GGTGGCAGAC GCGGCTCGGG CCGCTGAGGC CACCGAGCGA


 


1201
GCTCGCCGGC AACCAGGCCC AGGGCGTCAC GTCCGGCGGC TCCGGCAACT GCCGCACCGG



CGAGCGGCCG TTGGTCCGGG TCCCGCAGTG CAGGCCGCCG AGGCCGTTGA CGGCGTGGCC


 


1261
TGGCACCACG TTCTTCCAGC CGGTCAACCC CATCCTCCAG GCGTACGGCC TGAGGATGAT



ACCGTGGTGC AAGAAGGTCG GCCAGTTGGG GTAGGAGGTC CGCATGCCGG ACTCCTACTA


 


1321
CACCACGGAC TCGGGCAGCA GCCCGGCCCC TGCACCGACC TCCTGCACCG GCTACGCCCG



GTGGTGCCTG AGCCCGTCGT CGGGCCGGGG ACGTGGCTGG AGGACGTGGC CGATGCGGGC


 


1381
CACCTTCACC GGGACCCTCG CGGCCGGCCG GGCCGCCGCC CAGCCCAACG GGTCCTACGT



GTGGAAGTGG CCCTGGGAGC GCCGGCCGGC CCGGCGGCGG GTCGGGTTGC CCAGGATGCA


 


1441
GCAGGTCAAC CGGTCCGGGA CCCACAGCGT GTGCCTCAAC GGGCCCTCCG GTGCGGACTT



CGTCCAGTTG GCCAGGCCCT GGGTGTCGCA CACGGAGTTG CCCGGGAGGC CACGCCTGAA


 


1501
CGACCTCTAC GTGCAGCGCT GGAACGGCAG CTCCTGGGTG ACCGTCGCCC AGAGCACCTC



GCTGGAGATG CACGTCGCGA CCTTGCCGTC GAGGACCCAC TGGCAGCGGG TCTCGTGGAG


 


1561
CCCCGGCTCC AACGAGACCA TCACCTACCG CGGCAACGCC GGCTACTACC GCTACGTGGT



GGGGCCGAGG TTGCTCTGGT AGTGGATGGC GCCGTTGCGG CCGATGATGG CGATGCACCA


 


1621
CAACGCCGCG TCCGGCTCCG GTGCCTACAC CATGGGGCTC ACCCTCCCCT GACGTAGCGC



GTTGCGGCGC AGGCCGAGGC CACGGATGTG GTACCCCGAG TGGGAGGGGA CTGCATCGCG






The following DNA sequence (SEQ ID NO:2) encodes the signal peptide (SEQ ID NO:9) that is operatively linked to the precursor protease (SEQ ID NO:7) derived from Cellulomonas strain 69B4 (DSM 16035). The initiating polynucleotide encoding the signal peptide of the Cellulomonas strain 69B4 protease is in bold (ATG). The asterisk indicates the termination codon (TGA), beginning with residue 1486. Residues 85, 595, and 1162, relate to the initial residues of the N terminal prosequence, mature sequence and Carboxyl terminal prosequence, respectively, are bolded and underlined.










(SEQ ID NO: 2)



   1
ATGACACCAC GCACAGTCAC GCGGGCCCTG GCCGTGGCCA CCGCAGCCGC CACACTCCTG




TACTGTGGTG CGTGTCAGTG CGCCCGGGAC CGGCACCGGT GGCGTCGGCG GTGTGAGGAC


 



                         .85


  61
GCAGGCGGCA TGGCCGCCCA GGCCAACGAG CCCGCACCAC CCGGGAGCGC GAGCGCACCG



CGTCCGCCGT ACCGGCGGGT CCGGTTGCTC GGGCGTGGTG GGCCCTCGCG CTCGCGTGGC


 


 121
CCACGCCTGG CCGAGAAGCT CGACCCCGAC CTCCTCGAGG CCATGGAGCG CGACCTGGGC



GGTGCGGACC GGCTCTTCGA GCTGGGGCTG GAGGAGCTCC GGTACCTCGC GCTGGACCCG


 


 181
CTCGACGCGG AGGAAGCCGC CGCCACCCTG GCGTTCCAGC ACGACGCAGC CGAGACCGGC



GAGCTGCGCC TCCTTCGGCG GCGGTGGGAC CGCAAGGTCG TGCTGCGTCG GCTCTGGCCG


 


 241
GAGGCCCTCG CCGAAGAGCT CGACGAGGAC TTCGCCGGCA CCTGGGTCGA GGACGACGTC



CTCCGGGAGC GGCTTCTCGA GCTGCTCCTG AAGCGGCCGT GGACCCAGCT CCTGCTGCAG


 


 301
CTGTACGTCG CCACCACCGA CGAGGACGCC GTCGAGGAGG TCGAGGGCGA AGGCGCCACG



GACATGCAGC GGTGGTGGCT GCTCCTGCGG CAGCTCCTCC AGCTCCCGCT TCCGCGGTGC


 


 361
GCCGTCACCG TCGAGCACTC CCTGGCCGAC CTCGAGGCCT GGAAGACCGT CCTCGACGCC



CGGCAGTGGC AGCTCGTGAG GGACCGGCTG GAGCTCCGGA CCTTCTGGCA GGAGCTGCGG


 


 421
GCCCTCGAGG GCCACGACGA CGTGCCCACC TGGTACGTCG ACGTCCCGAC CAACAGCGTC



CGGGAGCTCC CGGTGCTGCT GCACGGGTGG ACCATGCAGC TGCAGGGCTG GTTGTCGCAG


 


 481
GTCGTCGCCG TCAAGGCCGG AGCCCAGGAC GTCGCCGCCG GCCTCGTCGA AGGTGCCGAC



CAGCAGCGGC AGTTCCGGCC TCGGGTCCTG CAGCGGCGGC CGGAGCAGCT TCCACGGCTG


 



                                                          595


 541
GTCCCGTCCG ACGCCGTGAC CTTCGTCGAG ACCGACGAGA CCCCGCGGAC CATGTTCGAC



CAGGGCAGGC TGCGGCACTG GAAGCAGCTC TGGCTGCTCT GGGGCGCCTG GTACAAGCTG


 


 601
GTGATCGGCG GCAACGCCTA CACCATCGGG GGGCGCAGCC GCTGCTCGAT CGGGTTCGCG



CACTAGCCGC CGTTGCGGAT GTGGTAGCCC CCCGCGTCGG CGACGAGCTA GCCCAAGCGC


 


 661
GTCAACGGCG GGTTCATCAC CGCCGGCCAC TGCGGCCGCA CCGGCGCCAC CACCGCCAAC



CAGTTGCCGC CCAAGTAGTG GCGGCCGGTG ACGCCGGCGT GGCCGCGGTG GTGGCGGTTG


 


 721
CCCACCGGGA CCTTCGCCGG GTCCAGCTTC CCGGGCAACG ACTACGCGTT CGTCCGTACC



GGGTGGCCCT GGAAGCGGCC CAGGTCGAAG GGCCCGTTGC TGATGCGCAA GCAGGCATGG


 


 781
GGGGCCGGCG TGAACCTGCT GGCCCAGGTC AACAACTACT CCGGTGGCCG CGTCCAGGTC



CCCCGGCCGC ACTTGGACGA CCGGGTCCAG TTGTTGATGA GGCCACCGGC GCAGGTCCAG


 


 841
GCCGGGCACA CCGCGGCCCC CGTCGGCTCG GCCGTGTGCC GGTCCGGGTC GACCACCGGG



CGGCCCGTGT GGCGCCGGGG GCAGCCGAGC CGGCACACGG CCAGGCCCAG CTGGTGGCCC


 


0901
TGGCACTGCG GCACCATCAC TGCGCTCAAC TCCTCGGTCA CCTACCCCGA GGGCACCGTC



ACCGTGACGC CGTGGTAGTG ACGCGAGTTG AGGAGCCAGT GGATGGGGCT CCCGTGGCAG


 


0961
CGCGGCCTGA TCCGCACCAC CGTCTGCGCC GAGCCCGGCG ACTCCGGTGG CTCGCTGCTC



GCGCCGGACT AGGCGTGGTG GCAGACGCGG CTCGGGCCGC TGAGGCCACC GAGCGACGAG


 


1021
GCCGGCAACC AGGCCCAGGG CGTCACGTCC GGCGGCTCCG GCAACTGCCG CACCGGTGGC



CGGCCGTTGG TCCGGGTCCC GCAGTGCAGG CCGCCGAGGC CGTTGACGGC GTGGCCACCG


 


1081
ACCACGTTCT TCCAGCCGGT CAACCCCATC CTCCAGGCGT ACGGCCTGAG GATGATCACC



TGGTGCAAGA AGGTCGGCCA GTTGGGGTAG GAGGTCCGCA TGCCGGACTC CTACTAGTGG


 



                     1162


1141
ACGGACTCGG GCAGCAGCCC GGCCCCTGCA CCGACCTCCT GCACCGGCTA CGCCCGCACC



TGCCTGAGCC CGTCGTCGGG CCGGGGACGT GGCTGGAGGA CGTGGCCGAT GCGGGCGTGG


 


1201
TTCACCGGGA CCCTCGCGGC CGGCCGGGCC GCCGCCCAGC CCAACGGGTC CTACGTGCAG



AAGTGGCCCT GGGAGCGCCG GCCGGCCCGG CGGCGGGTCG GGTTGCCCAG GATGCACGTC


 


1261
GTCAACCGGT CCGGGACCCA CAGCGTGTGC CTCAACGGGC CCTCCGGTGC GGACTTCGAC



CAGTTGGCCA GGCCCTGGGT GTCGCACACG GAGTTGCCCG GGAGGCCACG CCTGAAGCTG


 


1321
CTCTACGTGC AGCGCTGGAA CGGCAGCTCC TGGGTGACCG TCGCCCAGAG CACCTCCCCC



GAGATGCACG TCGCGACCTT GCCGTCGAGG ACCCACTGGC AGCGGGTCTC GTGGAGGGGG


 


1381
GGCTCCAACG AGACCATCAC CTACCGCGGC AACGCCGGCT ACTACCGCTA CGTGGTCAAC



CCGAGGTTGC TCTGGTAGTG GATGGCGCCG TTGCGGCCGA TGATGGCGAT GCACCAGTTG


 



                                              1486*


1441
GCCGCGTCCG GCTCCGGTGC CTACACCATG GGGCTCACCC TCCCCTGA 



CGGCGCAGGC CGAGGCCACG GATGTGGTAC CCCGAGTGGG AGGGGACT






The following DNA sequence (SEQ ID NO:3) encodes the precursor protease derived from Cellulomonas strain 69B4 (DSM 16035).










(SEQ ID NO: 3)



   1
AACGAGCCCG CACCACCCGG GAGCGCGAGC GCACCGCCAC GCCTGGCCGA GAAGCTCGAC




TTGCTCGGGC GTGGTGGGCC CTCGCGCTCG CGTGGCGGTG CGGACCGGCT CTTCGAGCTG


 


  61
CCCGACCTCC TCGAGGCCAT GGAGCGCGAC CTGGGCCTCG ACGCGGAGGA AGCCGCCGCC



GGGCTGGAGG AGCTCCGGTA CCTCGCGCTG GACCCGGAGC TGCGCCTCCT TCGGCGGCGG


 


 121
ACCCTGGCGT TCCAGCACGA CGCAGCCGAG ACCGGCGAGG CCCTCGCCGA AGAGCTCGAC



TGGGACCGCA AGGTCGTGCT GCGTCGGCTC TGGCCGCTCC GGGAGCGGCT TCTCGAGCTG


 


 181
GAGGACTTCG CCGGCACCTG GGTCGAGGAC GACGTCCTGT ACGTCGCCAC CACCGACGAG



CTCCTGAAGC GGCCGTGGAC CCAGCTCCTG CTGCAGGACA TGCAGCGGTG GTGGCTGCTC


 


 241
GACGCCGTCG AGGAGGTCGA GGGCGAAGGC GCCACGGCCG TCACCGTCGA GCACTCCCTG



CTGCGGCAGC TCCTCCAGCT CCCGCTTCCG CGGTGCCGGC AGTGGCAGCT CGTGAGGGAC


 


 301
GCCGACCTCG AGGCCTGGAA GACCGTCCTC GACGCCGCCC TCGAGGGCCA CGACGACGTG



CGGCTGGAGC TCCGGACCTT CTGGCAGGAG CTGCGGCGGG AGCTCCCGGT GCTGCTGCAC


 


 361
CCCACCTGGT ACGTCGACGT CCCGACCAAC AGCGTCGTCG TCGCCGTCAA GGCCGGAGCC



GGGTGGACCA TGCAGCTGCA GGGCTGGTTG TCGCAGCAGC AGCGGCAGTT CCGGCCTCGG


 


 421
CAGGACGTCG CCGCCGGCCT CGTCGAAGGT GCCGACGTCC CGTCCGACGC CGTGACCTTC



GTCCTGCAGC GGCGGCCGGA GCAGCTTCCA CGGCTGCAGG GCAGGCTGCG GCACTGGAAG


 


 481
GTCGAGACCG ACGAGACCCC GCGGACCATG TTCGACGTGA TCGGCGGCAA CGCCTACACC



CAGCTCTGGC TGCTCTGGGG CGCCTGGTAC AAGCTGCACT AGCCGCCGTT GCGGATGTGG


 


 541
ATCGGGGGGC GCAGCCGCTG CTCGATCGGG TTCGCGGTCA ACGGCGGGTT CATCACCGCC



TAGCCCCCCG CGTCGGCGAC GAGCTAGCCC AAGCGCCAGT TGCCGCCCAA GTAGTGGCGG


 


 601
GGCCACTGCG GCCGCACCGG CGCCACCACC GCCAACCCCA CCGGGACCTT CGCCGGGTCC



CCGGTGACGC CGGCGTGGCC GCGGTGGTGG CGGTTGGGGT GGCCCTGGAA GCGGCCCAGG


 


 661
AGCTTCCCGG GCAACGACTA CGCGTTCGTC CGTACCGGGG CCGGCGTGAA CCTGCTGGCC



TCGAAGGGCC CGTTGCTGAT GCGCAAGCAG GCATGGCCCC GGCCGCACTT GGACGACCGG


 


 721
CAGGTCAACA ACTACTCCGG TGGCCGCGTC CAGGTCGCCG GGCACACCGC GGCCCCCGTC



GTCCAGTTGT TGATGAGGCC ACCGGCGCAG GTCCAGCGGC CCGTGTGGCG CCGGGGGCAG


 


 781
GGCTCGGCCG TGTGCCGGTC CGGGTCGACC ACCGGGTGGC ACTGCGGCAC CATCACTGCG



CCGAGCCGGC ACACGGCCAG GCCCAGCTGG TGGCCCACCG TGACGCCGTG GTAGTGACGC


 


 841
CTCAACTCCT CGGTCACCTA CCCCGAGGGC ACCGTCCGCG GCCTGATCCG CACCACCGTC



GAGTTGAGGA GCCAGTGGAT GGGGCTCCCG TGGCAGGCGC CGGACTAGGC GTGGTGGCAG


 


 901
TGCGCCGAGC CCGGCGACTC CGGTGGCTCG CTGCTCGCCG GCAACCAGGC CCAGGGCGTC



ACGCGGCTCG GGCCGCTGAG GCCACCGAGC GACGAGCGGC CGTTGGTCCG GGTCCCGCAG


 


 961
ACGTCCGGCG GCTCCGGCAA CTGCCGCACC GGTGGCACCA CGTTCTTCCA GCCGGTCAAC



TGCAGGCCGC CGAGGCCGTT GACGGCGTGG CCACCGTGGT GCAAGAAGGT CGGCCAGTTG


 


1021
CCCATCCTCC AGGCGTACGG CCTGAGGATG ATCACCACGG ACTCGGGCAG CAGCCCGGCC



GGGTAGGAGG TCCGCATGCC GGACTCCTAC TAGTGGTGCC TGAGCCCGTC GTCGGGCCGG


 


1081
CCTGCACCGA CCTCCTGCAC CGGCTACGCC CGCACCTTCA CCGGGACCCT CGCGGCCGGC



GGACGTGGCT GGAGGACGTG GCCGATGCGG GCGTGGAAGT GGCCCTGGGA GCGCCGGCCG


 


1141
CGGGCCGCCG CCCAGCCCAA CGGGTCCTAC GTGCAGGTCA ACCGGTCCGG GACCCACAGC



GCCCGGCGGC GGGTCGGGTT GCCCAGGATG CACGTCCAGT TGGCCAGGCC CTGGGTGTCG


 


1201
GTGTGCCTCA ACGGGCCCTC CGGTGCGGAC TTCGACCTCT ACGTGCAGCG CTGGAACGGC



CACACGGAGT TGCCCGGGAG GCCACGCCTG AAGCTGGAGA TGCACGTCGC GACCTTGCCG


 


1261
AGCTCCTGGG TGACCGTCGC CCAGAGCACC TCCCCCGGCT CCAACGAGAC CATCACCTAC



TCGAGGACCC ACTGGCAGCG GGTCTCGTGG AGGGGGCCGA GGTTGCTCTG GTAGTGGATG


 


1321
CGCGGCAACG CCGGCTACTA CCGCTACGTG GTCAACGCCG CGTCCGGCTC CGGTGCCTAC



GCGCCGTTGC GGCCGATGAT GGCGATGCAC CAGTTGCGGC GCAGGCCGAG GCCACGGATG


 


1381
ACCATGGGGC TCACCCTCCC CTGA



TGGTACCCCG AGTGGGAGGG GACT






The following DNA sequence (SEQ ID NO:4) encodes the mature protease derived from Cellulomonas strain 69B4 (DSM 16035).










(SEQ ID NO: 4)



  1
TTCGACGTGA TCGGCGGCAA CGCCTACACC ATCGGGGGGC GCAGCCGCTG CTCGATCGGG




AAGCTGCACT AGCCGCCGTT GCGGATGTGG TAGCCCCCCG CGTCGGCGAC GAGCTAGCCC


 


 61
TTCGCGGTCA ACGGCGGGTT CATCACCGCC GGCCACTGCG GCCGCACCGG CGCCACCACC



AAGCGCCAGT TGCCGCCCAA GTAGTGGCGG CCGGTGACGC CGGCGTGGCC GCGGTGGTGG


 


121
GCCAACCCCA CCGGGACCTT CGCCGGGTCC AGCTTCCCGG GCAACGACTA CGCGTTCGTC



CGGTTGGGGT GGCCCTGGAA GCGGCCCAGG TCGAAGGGCC CGTTGCTGAT GCGCAAGCAG


 


181
CGTACCGGGG CCGGCGTGAA CCTGCTGGCC CAGGTCAACA ACTACTCCGG TGGCCGCGTC



GCATGGCCCC GGCCGCACTT GGACGACCGG GTCCAGTTGT TGATGAGGCC ACCGGCGCAG


 


241
CAGGTCGCCG GGCACACCGC GGCCCCCGTC GGCTCGGCCG TGTGCCGGTC CGGGTCGACC



GTCCAGCGGC CCGTGTGGCG CCGGGGGCAG CCGAGCCGGC ACACGGCCAG GCCCAGCTGG


 


301
ACCGGGTGGC ACTGCGGCAC CATCACTGCG CTCAACTCCT CGGTCACCTA CCCCGAGGGC



TGGCCCACCG TGACGCCGTG GTAGTGACGC GAGTTGAGGA GCCAGTGGAT GGGGCTCCCG


 


361
ACCGTCCGCG GCCTGATCCG CACCACCGTC TGCGCCGAGC CCGGCGACTC CGGTGGCTCG



TGGCAGGCGC CGGACTAGGC GTGGTGGCAG ACGCGGCTCG GGCCGCTGAG GCCACCGAGC


 


421
CTGCTCGCCG GCAACCAGGC CCAGGGCGTC ACGTCCGGCG GCTCCGGCAA CTGCCGCACC



GACGAGCGGC CGTTGGTCCG GGTCCCGCAG TGCAGGCCGC CGAGGCCGTT GACGGCGTGG


 


481
GGTGGCACCA CGTTCTTCCA GCCGGTCAAC CCCATCCTCC AGGCGTACGG CCTGAGGATG



CCACCGTGGT GCAAGAAGGT CGGCCAGTTG GGGTAGGAGG TCCGCATGCC GGACTCCTAC


 


561
ATCACCACGG ACTCGGGCAG CAGCCCG



TAGTGGTGCC TGAGCCCGTC GTCGGGC






The following DNA sequence (SEQ ID NO:5) encodes the signal peptide derived from Cellulomonas strain 69B4 (DSM 16035)








(SEQ ID NO: 5)










 1
ATGACACCAC CACAGTCAC GCGGGCCCTG GCCGTGGCCA CCGCAGCCGC CACACTCCTG




TACTGTGGTG CGTGTCAGTG CGCCCGGGAC CGGCACCGGT GGCGTCGGCG GTGTGAGGAC


 


61
GCAGGCGGCA TGGCCGCCCA GGCC



CGTCCGCCGT ACCGGCGGGT CCGG






The following sequence is the amino acid sequence (SEQ ID NO:6) of the signal sequence and precursor protease derived from Cellulomonas strain 69B4 (DSM 16035), including the signal sequence [segments 1a-c] (residues 1-28 [-198 to −171]), an N-terminal prosequence [segments 2a-r] (residues 29-198 [-170 to -1]), a mature protease [segments 3a-t] (residues 199-387 [1-189]), and a C-terminal prosequence [segments 4a-l] (residues 388-495 [190-398]) encoded by the DNA sequences set forth in SEQ ID NOS:1, 2, 3 and 4. The N-terminal sequence of the mature protease amino acid sequence is in bold.








(SEQ ID NO: 6)










  1
MTPRTVTRAL AVATAAATLL AGGMAAQA NE PAPPGSASAP PRLAEKLDPD




    1a         1b         1c      2a 2b        2c


 


 51
LLEAMERDLG LDAEEAAATL AFQHDAAETG EALAEELDED FAGTWVEDDV



    2d         2e         2f        2g              2h


 


101
LYVATTDEDA VEEVEGEGAT AVTVEHSLAD LEAWKTVLDA ALEGHDDVPT



    2i        2j               2k         2l         2m


 


151
WYVDVPTNSV VVAVKAGAQD VAAGLVEGAD VPSDAVTFVE TDETPRTM FD



   2n         2o         2p               2q         2r


 



3a


201
VIGGNAYTIG GRSRCSIGFA VNGGFITAGH CGRTGATTAN PTGTFAGSSF



    3b         3c        3d         3e               3f


 


251
PGNDYAFVRT GAGVNLLAQV NNYSGGRVQV AGHTAAPVGS AVCRSGSTTG



    3g        3h         3i         3j               3k


 


301
WHCGTITALN SSVTYPEGTV RGLIRTTVCA EPGDSGGSLL AGNQAQGVTS



   3l         3m         3n               3o         3p


 


351
GGSGNCRTGG TTFFQPVNPI LQAYGLRMIT TDSGSSP APA PTSCTGYART



   3q          3r        3s         3t       4a 4b


 


401
FTGTLAAGRA AAQPNGSYVQ VNRSGTHSVC LNGPSGADFD LYVQRWNGSS



    4c         4d        4e               4f         4g


 


451
WVTVAQSTSP GSNETITYRG NAGYYRYVVN AASGSGAYTM GLTLP



         4h         4i         4j         4k         4l






The following sequence (SEQ ID NO:7) is the amino acid sequence of the precursor protease derived from Cellulomonas strain 69B4 (DSM 16035) (SEQ ID NO:7).








(SEQ ID NO: 7)










  1
NEPAPPGSAS APPRLAEKLD PDLLEAMERD.LGLDAEEAAA.TLAFQHDAAE



 


 51
TGEALAEELD EDFAGTWVED DVLYVATTDE DAVEEVEGEG ATAVTVEHSL


 


101
ADLEAWKTVL DAALEGHDDV PTWYVDVPTN SVVVAVKAGA QDVAAGLVEG


 


151
ADVPSDAVTF VETDETPRTM FDVIGGNAYT IGGRSRCSIG FAVNGGFITA


 


201
GHCGRTGATT ANPTGTFAGS SFPGNDYAFV RTGAGVNLLA QVNNYSGGRV


 


251
QVAGHTAAPV GSAVCRSGST TGWHCGTITA LNSSVTYPEG TVRGLIRTTV


 


301
CAEPGDSGGS LLAGNQAQGV TSGGSGNCRT GGTTFFQPVN PILQAYGLRM


 


351
ITTDSGSSPA PAPTSCTGYA RTFTGTLAAG RAAAQPNGSY VQVNRSGTHS


 


401
VCLNGPSGAD FDLYVQRWNG SSWVTVAQST SPGSNETITY RGNAGYYRYV


 


451
VNAASGSGAY TMGLTLP






The following sequence (SEQ ID NO:8) is the amino acid sequence of the mature protease derived from Cellulomonas strain 69B4 (DSM 16035). The catalytic triad residues H32, D56 and S132 are bolded and underlined.








(SEQ ID NO: 8)










  1
FDVIGGNAYT IGGRSRCSIG FAVNGGFITA GHCGRTGATT ANPTGTFAGS



 


 51
SFPGNDYAFV RTGAGVNLLA QVNNYSGGRV QVAGHTAAPV GSAVCRSGST


 


101
TGWHCGTITA LNSSVTYPEG TVRGLIRTTV CAEPGDSGGS LLAGNQAQGV


 


151
TSGGSGNCRT GGTTFFQPVN PILQAYGLRM ITTDSGSSP






The following sequence (SEQ ID NO:9) is the amino acid sequence of the signal peptide of the protease derived from Cellulomonas strain 6984 (DSM 16035).









1
MTPRTVTRAL AVATAAATLL AGGMAAQA
(SEQ ID NO: 9)






The following sequence (SEQ ID NO:10) is the degenerate primer used to identify a 177 bp fragment of the protease of Cellulomonas strain 69B4.









(SEQ ID NO: 10)



TTGWXCGT_FW: 5′ ACNACSGGSTGGCRGTGCGGCAC 3′






The following sequence (SEQ ID NO:11) is the reverse primer used to identity a 177 bp fragment of the protease derived from Cellulomonas strain 69B4.









(SEQ ID NO: 11)



GDSGGX_RV: 5′-ANGNGCCGCCGGAGTCNCC-3′






The following DNA (SEQ ID NO:13) and amino acid sequence of the 177 bp fragment (SEQ ID NO:12) encoding part of the protease gene derived from Cellulomonas strain 69B4. The sequences of the degenerate primers (SEQ ID NOS:10 and 11) are underlined and in bold.










   D  G  W   D  C  G   T  I  T  A   L  N  S   S  V  T   Y  P  E  G .



  1
ACGACGGCTG GGACTGCGGC ACCATCACTG CGCTCAACTC CTCGGTCACC TACCCCGAGG



TGCTGCCGAC CCTGACGCCG TGGTAGTGAC GCGAGTTGAG GAGCCAGTGG ATGGGGCTCC


 



.  T  V  R   G  L  I   R  T  T  V   C  A  E   P  G  D   S  G  G  S .


 61
GCACCGTCCG CGGCCTGATC CGCACCACCG TCTGCGCCGA GCCCGGCGAC TCCGGTGGCT



CGTGGCAGGC GCCGGACTAG GCGTGGTGGC AGACGCGGCT CGGGCCGCTG AGGCCACCGA


 



.  L  L  A   G  N  Q   A  Q  G  V   T  S  G   D  S  G   G  S


121
CGCTGCTCGC CGGCAACCAG GCCCAGGGCG TCACGTCCGG CGACTCCGGC GGCTCAT



GCGACGAGCG GCCGTTGGTC CGGGTCCCGC AGTGCAGGCC GCTGAGGCCG CCGAGTA






Analysis of the Sequence of Cellulomonas sp. 69B4 Protease

A saturated sinapinic acid (3,5-dimethoxy-4-hydroxy cinnamic acid) (“SA”) solution in a 1:1 v/v acetonitrile (“ACN”)/0.1% formic acid solution was prepared. The resulting mixture was vortexed for 60 seconds and then centrifuged for 20 seconds at 14,000 rpm. Then, 5 μl of the matrix supernatant was transferred to a 0.5 ml Eppendorf tube and 1 μl of a 10 pmole/μl protease 69B4 sample was added to the SA matrix supernatant and vortexed for 5 seconds. Then, 1 μl of the analyte/matrix solution was transferred onto a sample plate and, after being completely dry, analyzed by a Voyager DE-STR (PerSeptive), matrix assisted laser desorption/ionization—time of flight (MALDI-TOF) mass spectrophotometer, with the following settings: Mode of operation: Linear; Extraction mode: Delayed; Polarity: Positive; Accelerating voltage: 25000 V; Extraction delay time: 350 nsec; Acquisition mass range: 4000-20000 Da; Number of laser shots: 100/spectrum; and Laser intensity: 2351. The resulting spectrum is provided in FIG. 4.
A tryptic map was produced using methods known in the art (Christianson et al., Anal. Biochem. 223:119-29 [1994]), modified as described herein. The protease solution, containing 10-50 μg protease was diluted 1:1 with chilled water in a 1.5 ml microtube. 1.0 N HCl was added to a final concentration of 0.1 N HCl, mixed thoroughly and incubated for 10 minutes on ice. Then, 50% trichloro-acetic acid (“TCA”) was added to a final concentration of 10% TCA and mixed. The sample was incubated for 10 minutes on ice, centrifuged for two minutes and the supernatant discarded. Then, 1 ml of cold 90% acetone was added to resuspend the pellet. The resulting sample was then centrifuged for one minute, the supernatant quickly decanted and remaining liquid was removed by vacuum aspiration. The dry pellet was dissolved in 12 μl of 8.0 M urea solution (480 mg urea [Roche, catalog #1685899]) in 0.65 ml of ammonium bicarbonate solution (final concentration of bicarbonate: 0.5 M) and incubated for 3-5 minutes at 37° C. The solution was slowing diluted with 48 μl of a n-octyl-beta-D-glucopyranoside solution (“o-water”) (200 mg of n-octyl-beta-D-glucopyranoside [C14H28O6, f.w. 292.4] in 200 ml of water). Then, 2.0 μl of trypsin (2.5 mg/ml in 1 mM HCl) was added and the mixture was incubated for 15 minutes at 37° C. The proteolytic reaction was quenched with 6 μl of 10% trifluoroacetic acid (“TFA”). Insoluble material and bubbles were removed from the sample by centrifugation for one minute. The tryptic digest was separate by RP-HPLC on 2.1×150 mm C-18 column (5 μl particle size, 300 angstroms pore size). The elution gradient was formed from 0.1% (v/v) TFA in water and 0.08% (v/v) TFA in acetonitrile at a flow rate of 0.2 ml-min. The column compartment was heated to 50° C. Peptide elution was monitored at 215 nm and data were collected at 215 nm and 280 nm. The samples were then analyzed on a LCQ Advantage mass spectrometer with a Surveyor HPLC (both from Thermo Finnigan). The LCQ mass spectrophotometer was run with the following settings: Spray voltage: 4.5 kV; Capillary temperature: 225° C. Data processing was performed using TurboSEQUEST and Xcalibur (ThermoFinnigan). Sequencing of the tryptic digest portions was also performed in part by Argo BioAnalytica.
Analysis of the full sequence of the asp gene revealed that it encodes a prosequence protease of 495 amino acids (SEQ ID NO:6). The first 28 amino acids were predicted to form a signal peptide. The mass of the mature chain of 69B4 protease as produced by Cellulomonas strain 69B4 has a molecular weight of 18764 (determined by MALDI-TOF). The sequence of the N-terminus of the mature chain was also determined by MALDI-TOF analysis and starts with the sequence FDVIGGNAYTIGGR (SEQ ID NO:17). It is believed that the 69B4 protease has a unique precursor structure with NH2— and COOH terminal pro-sequences, as is known to occur with some other enzymes (e.g., T. aquaticus aqualysin I; See e.g., Lee et al., FEMS Microbiol. Lett., 1:69-74 [1994]; Sakamoto et al., Biosci. Biotechnol. Biochem., 59:1438-1443 [1995]; Sakamoto et al., Appl. Microbiol. Biotechnol., 45:94-101 [1996]; Kim et al., Biochem. Biophys. Res. Commun., 231:535-539 [1997]; and Oledzka et al., Protein Expr. Purific., 29:223-229 [2003]). The predicted molecular weight of mature 69B4 protease as provided in SEQ ID NO:8, was 18776.42, which corresponds well with the molecular weight of the purified enzyme with proteolytic activity isolated from Cellulomonas sp. 69B4 18764). The prediction of the COON terminal pro-sequence in 69B4 protease was also based on an alignment of the 69B4 protease with T. aquaticus aqualysin I, provided below. In this alignment, the amino acid sequence of the Cellulomonas 69B4 signal sequence and precursor protease are aligned with the signal sequence and precursor protease Aqualysin I of Thermus aquaticus (COOH-terminal pro-sequence of Aqualysin I is underlined and in bold).










Aqualysin I
  (1)
----MRKTYWLMALFAVLVLGGCQMASRSDPTPTLAEAFWPKEAPVYGLD



69B4
  (1)
MTPRTVTRALAVATAAATLLAGGMAAQANEPAPPGSASAPPRLAEKLDPD


Consensus
  (1)
           MA  A  LLAG   A   DP P  A A  PK A     D


 




51                                             100


Aqualysin I
 (47)
DPEAIPGRYIVVFKKGKGQSLLQGGITTLQARLAPQGVVVTQAYTGALQG


69B4
 (51)
LLEAMERDLGLDAEEAAATLAFQHDAAETGEALAEE---LDEDFAGTWVE


Consensus
 (51)
  EAI     L    A A    Q         LA     L   F G


 




101                                            150


Aqualysin I
 (97)
FAAEMAPQALEAFRQSPDVEFIEADKVVRAWATQSPAPWGLDRIDQRDLP


69B4
 (98)
DDVLYVATTDEDAVEEVEGEGATAVTVEHSLADLEANKTVLDAALEGHDD


Consensus
(101)
          E      D E   A  V  A A        LD


 




151                                            200


Aqualysin I
(147)
LSNSYTYTATGRGVNVYVIDTGIRTTHREFGGRARVGYDALGGNGQDCNG


69B4
(148)
VPTWYVDVPTNS--VVVAVKAGAQDVAAGLVEGADVPSDAVT--FVETDE


Consensus
(151)
L   Y    T     V  I  G           A V  DAL     D


 




201                                            250


Aqualysin I
(197)
HGTHVAGTIGGVTYGVAKAVNLYAVRVLDCNGSGSTSGVIAGVDWVTRNH


69B4
(194)
TPRTMFDVIGGNAYTIGGRS--------RCSIGFAVNGGFITAGHCGRTG


Consensus
(201)    
    M   IGG  Y IA            C    A  G         R


 




251                                            300


Aqualysin I
(247)
RRPAVANMSLGGGVSTALDNAVKNSIAAGVVYAVAAGNDNANACNYSPAR


69B4
(236)
ATTANPTGTFAGSSFPGNDYAFVRTGAG--------VNLLAQVNNYSGGR


Consensus
(251)
   A    S AG    A D A   S AA         N  AN  NYS AR


 




301                                            350


Aqualysin I
(297)
VAEALTVGATTSSDARASFSNYGSCVDLFAPGASIPSAWYTSDTATQTLN


69B4
(278)
VQVAGHTAAPVGSAVCRSGSTTGWHCGTIT--ALNSSVTYPEGTVRGLIR


Consensus
(301)
V  A   AA   S    S S  G         A   S  Y   T    I


 




351                                            400


Aqualysin I
(347)
GTSMATPHVAGVAALYLEQNPSATPASVASAILNGATTGRLSGIGSGSPN


69B4
(326)
TTVCAEPGDSGGSLLAGNQAQGVTSGGSGNCRTGGTTFFQPVNPILQAYG


Consensus
(351)
 T  A P  AG A L   Q    T A  A     G T          A


 




401                                            450


Aqualysin I
(397)
ALLYSLLSSGSGSTAPCTSCSYYTGSLSG---PGDYNFQPNGTYYYSP-A


69B4
(376)
LRMITTDS-GSSPAPAPTSCTGYARTFTGTLAAGRAAAQPNGSYVQVNRS


Consensus
(401)
  L S  S GS      TSCS Y  S SG    G    QPNGSY     A


 




451                                            500


Aqualysin I
(443)
GTHRAWLRGPAGTDFDLYLWREDGSRELTVGSSTGPTSEESLSYSGTAGY


69B4
(425)
GTHSVCLNGPSGADFDLYVQRWNGSSWVTVAQSTSPGSNETITYRGNAGY


Consensus
(451)
GTH   L GPAG DFDLYL RW GS WLTVA ST P S ESISY G AGY


 




501 521


Aqualysin I
(493)
YLWRIYAYSGSGMYEFWLQRP (SEQ ID NO: 644)


69B4
(475)
YRYVVNAASGSGAYTMGLTLP (SEQ ID NO: 645)


Consensus
(501)
Y W I A SGSG Y   L  P (SEQ ID NO: 646)






The sequences of three internal peptides of the purified enzyme from Cellulomonas sp. 69B4 having proteolytic activity were determined by MALDI-TOF analysis. All three peptides were also identified in the translation product of the isolated asp gene, confirming the identification of the correct protease gene (See, SEQ ID NO:1, above).
Percentage Identity Comparison Between Asp and Streptogrisin
The deduced polypeptide product of the asp gene (mature chain) was used in homology analysis with other serine proteases using the BLAST program and settings as described in Example 3. The preliminary analyses showed identities of from about 44-48% (See, Table 4-1, below). Together with analysis of the translated sequence, these results provided evidence that the asp gene encodes a protease having less than 50% sequence identity with the mature chains of Streptogrisin-like serine proteases. An alignment of Asp with Streptogrisin A, Streptogrisin B, Streptogrisin C, Streptogrisin D of Streptomyces griseus is provided below. In this alignment, the amino acid sequences of Cellulomonas 69B4 mature protease (“69B4 mature”) are aligned with mature proteases amino acid sequences of Streptogrisin C (“Sq-streptogrisinC_mature”), Streptogrisin B (“Sq—streptogrisinBmature”), Streptogrisin A (“Sq-streptogrisinAmature”), Streptogrisin D (“Sq—streptogrisinDmature”) and consensus residues.












1                                               50



69B4 mature
  (1)
 FDVIGGNAYTIGGRSRCSIGFAVN----GGFITAGHCGRTGATT------


Sg-StreptogrisinC mature
  (1)
 ADIRGGDAYYNNGSGRCSVGFSVTRGTQNGFATAGHCGRVGTTTNG--VN


Sg-StreptogrisinBmature
  (1)
 --ISGGDAIYSST-GRCSLGFNVRSGSTYYFLTAGHCTDGATTWWANSAR


Sg-StreptogrisinAmature
  (1)
 --IAGGEAITTGG-SRCSLGFNVSVNGVAHALTAGHCTNISASWS-----


Sg-StreptogrisinDmature
  (1)
 --IAGGDAIWGSG-SRCSLGFNVVKGGEPYFLTAGHCTESVTSWSD-TQG


Consensus
  (1)
   IAGGDAIY  G SRCSLGFNV  G   YFLTAGHCT  GTTW


 




 51                                             100


Asp mature
 (41)
 ANPTGTFAGSSFPGNDYAFVRTGAGVNLLAQVNNYSGGRVQVAGHTAAPV


Sg-StreptogrisinC mature
 (49)
 QQAQGTFQGSTFPGRDIAWVATNANWTPRPLVNGYGRGDVTVAGSTASVV


Sg-StreptogrisinBmature
 (48)
 TTVLGTTSGSSFPNNDYGIVRYTNTTIPKDGTVGG----QDITSAANATV


Sg-StreptogrisinAmature
 (43)
 ---IGTRTGTSFPNNDYGIIRHSNPAAADGRVYLYNGSYQDITTAGNAFV


Sg-StreptogrisinDmature
 (47)
 GSEIGANEGSSFPENDYGLVKYTSDTAHPSEVNLYDGSTQAITQAGDATV


Consensus
 (51)
    IGT  GSSFP NDYGIVRYTA       VN Y G  Q IT AG A V


 




 101                                            150


Asp mature
 (91)
 GSAVCRSGSTTGWHCGTITALNSSVTYPEG-TVRGLIRTTVCAEPGDSGG


Sg-StreptogrisinC mature
 (99)
 GASVCRSGSTTGWHCGTIQQLNTSVTYPEG-TISGVTRTSVCAEPGDSGQ


Sg-StreptogrisinBmature
 (94)
 GMAVTRRGSTTGTHSGSVTALNATVNYGGGDVVYGNIRTNVCAEPGDSGG


Sg-StreptogrisinAmature
 (90)
 GQAVQRSGSTTGLRSGSVTGLNATVNYGSSGIVYGMIQTNVCAEPGDSGG


Sg-StreptogrisinDmature
 (97)
 GQAVTRSGSTTQVHDGEVTALDATVNYGNGDIVNGLIQTTVCAEPGDSGG


Consensus
(101)
 G AV RSGSTTG H GSVTALNATVNYG G IV GLIRTTVCAEPGDSGG


 




 151                                            200


Asp mature
(140)
 SLLAGNQAQGVTSGGSGNCRTGGTTFFQPVNPILQAYGLRMITTDSGSSP


Sg-StreptogrisinC mature
(148)
 SYISGSQAQGVTSGGSGNCSSGGTTYFQPINPLLQAYGLTLVTSGGGTPT


Sg-StreptogrisinBmature
(144)
 PLYSGTRAIGLTSGGSGNCSSGGTTFFQPVTEALSAYGVSVY--------


Sg-StreptogrisinAmature
(140)
 SLFAGSTALGLTSGGSGNCRTGGTTFYQPVTEALSAYGATVL--------


Sg-StreptogrisinDmature
(147)
 ALFAGDTALGLTSGGSGDCSSGGTTFFQPVPEALAAYGAEIG--------


Consensus
(151)
 SLFAGS ALGLTSGGSGNCSSGGTTFFQPV EALSAYGLTVI


 




 201                                            250


Asp mature
(190)
 --------------------------------------------------


Sg-StreptogrisinC mature
(198)
 DPPTTPPTDSPGGTWAVGTAYAAGATVTYGGATYRCLQAHTAQPGWTPAD


Sg-StreptogrisinBmature
(186)
 --------------------------------------------------


Sg-StreptogrisinAmature
(182)
 --------------------------------------------------


Sg-StreptogrisinDmature
(189)
 --------------------------------------------------


Consensus
(201)



 




 251


Asp mature
(190)
 -------- (SEQ ID NO: 8)


Sg-StreptogrisinC mature
(248)
 VPALWQRV (SEQ ID NO: 639)


Sg-StreptogrisinBmature
(186)
 -------- (SEQ ID NO: 640)


Sg-StreptogrisinAmature
(182)
 -------- (SEQ ID NO: 641)


Sg-StreptogrisinDmature
(189)
 -------- (SEQ ID NO: 642)


Consensus
(251)
 -------- (SEQ ID NO: 643)













TABLE 4-1







Percentage Identity: Comparison between Cellulomonas sp. 69B4 Protease


Encoded by asp and Other Serine Proteases (identity between the mature chains)

















Alphalytic







endopeptidase



Streptogrisin A
Streptogrisin B
Streptogrisin C
Streptogrisin D
Lysobacter



S. griseus
S. griseus
S. griseus
S. griseus
enzymogenes





Asp protease
48%
45%
47%
46%
44%


Cellulomonas sp.







Isolate 69B4









Additional protease sequences were also investigated. In these analyses, proteases homologous in protein sequence to the mature domain of ASP were searched for using BLAST. Those identified were then aligned using the multiple sequence alignment program clustalW. The numbers on the top of the alignment below refer to the amino-acid sequence of the mature ASP protease. The numbers at the side of the alignment are sequence identifiers, as described at the bottom of the alignment.









Sequence
1        10        20             30        40



ASP
FDVIGGNAYTIGGRSRCSIGFAVN-----GGFITAGHCGRTGATTANPTG--------TF


 


2
TPLIAGGEAITTGGSRCSLGFNV-SVNGVAHALTAGHCTNISASWS----------IGTR



 


3
--IAGGEAIYAAGGGRCSLGFNVRSSSGATYALTAGHCTEIASTWYTNSGQTSL--LGTR


 


4
NKLIQGGDAIYASSWRCSLGFNVRTSSGAEYFLTAGHCTDGAGAWRASSGGTV---IGQT


 


5
NKLIQGGDAIYASSWRCSLGFNVRTSSGAEYFLTAGHCTDGAGAWRASSGGTV---IGQT


 


6
TKLIQGGDAIYASSWRCSLGFNVRSSSGVDYFLTAGHCTDGAGTWYSNSARTTA--IGST


 


7
TKLISGGDAIYSSTGRCSLGFNVRSGS-TYYFLTAGHCTDGATTWWANSARTTV--LGTT


 


8
---VLGGGAIYGGGSRCSAAFNV-TKGGARYFVTAGHCTNISANWSASSGGSV---VGVR


 


9
QREVAGGDAIYGGGSRCSAAFNV-TKNGVRYFLTAGHCTNLSSTWSSTSGGTS---IGVR


 


10
KPFIAGGDAITGNGGRCSLGFNVTKG-GEPHFLTAGHCTEGISTWSDSSG--QV--IGEN


 


11
KPFVAGGDAITGGGGRCSLGFNVTKG-GEPYFITAGHCTESISTWSDSSG--NV--IGEN


 


12
TPLIAGGDAIWGSGSRCSLGFNVVKG-GEPYFLTAGHCTESVTSWSDTQGG-SE--IGAN


 


13
KTFASGGDAIFGGGARCSLGFNVTAGDGSAAFLTRGHCGGGATMWSDAQGGQPI--ATVD


 


14
KTFASGGDAIFGGGARCSLGFNVTAGDGSPAFLTAGHCGVAADQWSDAQGGQPI--ATVD


 


15
------------------------------------------------------------


 


16
TTRLNGAEPILSTAGRCSAGFNVTDG-TSDFILTAGHCGPTGSVWFGDRPGDGQ--VGRT


 


17
ATVQGGDVYYINRSSRCSIGFAVT-----TGFVSAGHCGGSGASATTSSGEAL----GTF


 


18
ADIRGGDAYYMNGSGRCSVGFSVTRG-TQNGFATAGHCGRVGTTTNGVNQQAQ----GTF


 


19
YDLRGGEAYYINNSSRCSIGFPITKG-TQQGFATAGHCGRAGSSTTGANRVAQ----GTF


 


20
YDLVGGDAYYIGN-GRCSIGFSVRQG-STPGFVTAGHCGSVGNATTGFNRVSQ----GTF


 


21
YDLVGGDAYYMGG-GRCSVGFSVTQG-STPGFATAGHCGTVGTSTTGYNQAAQ----GTF


 


22
EDLVGGDAYYIDDQARCSIGFSVTKD-DQEGFATAGHCGDPGATTTGYNEADQ----GTF


 


23
LAAIIGGNPYYFGNYRCSIGFSVRQG-SQTGFATAGHCGSTGTRVSSPSG--------TV


 


24
ANIVGGIEYSINNASLCSVGFSVTRG-ATKGFVTAGHCGTVNATARIGGAVV-----GTF


 


25
AAGTVGGDPYYTGNVRCSIGFSVH-----GGFVTAGHCGRAGAGVSGWDRSYI----GTF


 


26
VIVPVRDYWGGDALSGCTLAFPVYGG-----FLTAGHCAVEGKGHILKTEMTGGQ-IGTV


 


27
DPPLRSGLAIYGTNVRCSSAFMAYSG-SSYYMMTAGHCAEDSSYWEVPTYSYGYQGVGHV


 



  50         60        70         80        90        100


ASP
AGSSFPGN-DYAFVRTGAGVNLLAQVNNYSGGR-VQVAGHTAAPVGSAVCRSGSTTGWHC



 


2
TGTSFPNNDYGIIRHSNPAAA--DGRVYLYNGSYQDITTAGNAFVGQAVQRSGSTTGLRS



 


3
AGTSFPGNDYGLIRHSNASAA--DGRVYLYNGSYRDITGAGNAYVGQTVQRSGSTTGLHS


 


4
AGSSFPGNDYGIVQYTGS-------VSRPGTANGVDITRAATPSVGTTVIRDGSTTGTHS


 


5
AGSSFPGNDYGIVQYTGS-------VSRPGTANGVDITRAATPSVGTTVIRDGSTTGTHS


 


6
AGSSFPGNDYGIVRYTGS-------VSRPGTANGVDITRAATPSVGTTVIRDGSTTGTHS


 


7
SGSSFPNNDYGIVRYTNTT------IPKDGTVGGQDITSAANATVGMAVTRRGSTTGTHS


 


8
EGTSFPTNDYGIVRYTDGSSP--AGTVDLYNGSTQDISSAANAVVGQAIKKSGSTTKVTS


 


9
EGTSFPTNDYGIVRYTTTTNV--DGRVNLYNGGYQDIASAADAVVGQAIKKSGSTTKVTS


 


10
AASSFPGDDYGLVKYTADVAH--PSQVNLYDGSSQSISGAAEAAVGMQVTRSGSTTQVHS


 


11
AASSFPDNDYGLVKYTADVDH--PSEVNLYNGSSQAISGAAEATVGMQVTRSGSTTQVHD


 


12
EGSSFPENDYGLVKYTSDTAH--PSEVNLYDGSTQAITQAGDATVGQAVTRSGSTTQVHD


 


13
QAVFPPEGDFGLVRYDGPSTE--APSEVDLGDQTLPISGAAEASVGQEVFRMGSTTGLAD


 


14
QAVFPGEGDFALVRYDDPATE--APSEVDLGDQTLPISGAAEAAVGQEVFRMGSTTGLAD


 


15
------------------------------------------------------------


 


16
VAGSFFGDDFSLVEYANGKAGDGADVVAVGDGKGVRITGAGEPAVGQRVFRSGSTSGLRD


 


17
SGSVFPGSADMAYVRTVSGTVLRGYINGYGQGS-FPVSGSSEAAVGASICRSGSTTQVHC


 


18
QGSTFPGR-DIAWVATNANWTPRPLVNGYGRGD-VTVAGSTASVVGASVCRSGSTTGWHC


 


19
QGSIFPGR-DMAWVATNSSWTATPYVLGAGGQN-VQVTGSTASPVGASVCRSGSTTGWHC


 


20
RGSWFPGR-DMAWVAVNSNWTPTSLVRNSGSG--VRVTGSTQATVGSSICRSGSTTGWRC


 


21
EESSFPGD-DMAWVSVNSDWNTTPTVNEGE----VTVSGSTEAAVGASICRSGSTTGWHC


 


22
QASTFPGK-DMAWVGVNSDWTATPDVKAEGGEK-IQLAGSVEALVGASVCRSGSTTGWHC


 


23
AGSYFPGR-DMGWVRITSADTVTPLVNRYNGGT-VTVTGSQEAATGSSVCRSGATTGWRC


 


24
AARVFPGN-DRAWVSLTSAQTLLPRVANGSSF--VTVRGSTEAAVGAAVCRSGRTTGYQC


 


25
QGSSFPDN-DYAWVSVGSGWWTVPVVLGWGTVSDQLVRGSNVAPVGASICRSGSTTHWHC


 


26
EASQFGDGIDAAWAKNYGDWNGRGRVTHWNGGGGVDIKGSNEAAVGAHMCKSGRTTKWTC


 


27
ADYTFGYYGDSAIVRVDDPGF---WQPRGWVYPSTRITNWDYDYVGQYVCKQGSTTGYTC


 



    110       120        130       140        150



ASP
GTITALNSSVTYPEGTV-RGLIRTTVCAEPGDSGGSLLAGN-QAQGVTSGGS--------


 


2
GSVTGLNATVNYGSSGIVYGMIQTNVCAEPGDSGGSLF-AGSTALGLTSGGS--------



 


3
GRVTGLNATVNYGGGDIVSGLIQTNVCAEPGDSGGALF-AGSTALGLTSGGS--------


 


4
GRVTALNATVNYGGGDVVGGLIQTTVCAEPGDSGGSLYGSNGTAYGLTSGGS--------


 


5
GRVTALNATVNYGGGDVVGGLIQTTVCAEPGDSGGSLYGSNGTAYGLTSGGS--------


 


6
GRVTALNATVNYGGGDIVSGLIQTTVCAEPGDSGGPLYGSNGTAYGLTSGGS--------


 


7
GSVTALNATVNYGGGDVVYGMIRTNVCAEPGDSGGPLY-SGTRAIGLTSGGS--------


 


8
GTVTAVNVTVNYGDGP-VYNMGRTTACSAGGDSGGAHF-AGSVALGIHSGSS--------


 


9
GTVSAVNVTVNYSDGP-VYGMVRTTACSAGGDSGGAHF-AGSVALGIHSGSS--------


 


10
GTVTGLDATVNYGNGDIVNGLIQTDVCAEPGDSGGSLFSGDK-AVGLTSGGS--------


 


11
GTVTGLDATVNYGNGDIVNGLIQTDVCAEPGDSGGSLFSGDQ-AIGLTSGGS--------


 


12
GEVTALDATVNYGNGDIVNGLIQTTVCAEPGDSGGALFAGDT-ALGLTSGGS--------


 


13
GQVLGLDVTVNYPEG-TVTGLIQTDVCAEPGDSGGSLFTRDGLAIRLTSGGT--------


 


14
GQVLGLDATVNYPEG-MVTGLIQTDVCAEPGDSGGSLFTRDGLAIGLTSGGS--------


 


15
-----------------VDGLIQTDVCAEPGDSGGALFDGDA-AIGLTSGGS--------


 


16
GRVTALDATVNYPEG-TVTGLIETDVCAEPGDSGGPMFSEGV-ALGVTSGGS--------


 


17
GTIGAKGATVNYPQGAV-SGLTRTSVCAEPGDSGGSFYSGS-QAQGVTSGGS--------


 


18
GTIQQLNTSVTYPEGTI-SGVTRTSVCAEPGDSGGSYISGS-QAQGVTSGGS--------


 


19
GTVTQLNTSVTYQEGTI-SPVTRTTVCAEPGDSGGSFISGS-QAQGVTSGGS--------


 


20
GTIQQHNTSVTYPQGTI-TGVTRTSACAQPGDSGGSFISGT-QAQGVTSGGS--------


 


21
GTIQQHNTSVTYPEGTI-TGVTRTSVCAEPGDSGGSYISGS-QAQGVTSGGS--------


 


22
GTIQQHDTSVTYPEGTV-DGLTETTVCAEPGDSGGPFVSGV-QAQGTTSGGS--------


 


23
GTIQSKNQTVRYAEGTV-TGLTRTTACAEGGDSGGPWLTGS-QAQGVTSGGT--------


 


24
GTITAKNVTANYAEGAV-RGLTQGNACMGRGDSGGSWITSAGQAQGVMSGGNVQSNGNNC


 


25
GTVLAHNETVNYSDGSVVHQLTKTSVCAEGGDSGGSFISGD-QAQGVTSGGW--------


 


26
GYLLRKDVSVNYGNGHI-VTLNETSACALGGDSGGAYVWND-QAQGITSGSN--------


 


27
GQITETNATVSYPGRTL-TGMTWSTACDAPGDSGSGVYDGSTAHGILSGGPN--------


 



    160       170       180      189



ASP
GNCRTGGTTFFQPVNPILQAYGLRMITTDSGSSP (SEQ ID NO: 18)


 


2
GNCRTGGTTFYQPVTEALSAYGATVL-------- (SEQ ID NO: 19)



 


3
GNCRTGGTT------------------------- (SEQ ID NO: 20)


 


4
GNCSSGGTTFFQPVTEALSAYGVSVY-------- (SEQ ID NO: 21)


 


5
GNCSSGGTTFFQPVTEALSAYGVSVY-------- (SEQ ID NO: 22)


 


6
GNCSSGGTTFFQPVTEALSAYGVSVY-------- (SEQ ID NO: 23)


 


7
GNCSSGGTTFFQPVTEALSAYGVSVY-------- (SEQ ID NO: 24)


 


8
GCSGTAGSAIHQPVTKALSAYGVTVYL------- (SEQ ID NO: 25)


 


9
GCTGTNGSAIHQPVREALSAYGVNVY-------- (SEQ ID NO: 26)


 


10
GDCTSGGTTFFQPVTEALSATGTQIG-------- (SEQ ID NO: 27)


 


11
GDCTSGGETFFQPVTEALSATGTQIG-------- (SEQ ID NO: 28)


 


12
GDCSSGGTTFFQPVPEALAAYGAEIG-------- (SEQ ID NO: 29)


 


13
RDCTSGGETFFQPVTTALAAVGGTLGGEDGGDG- (SEQ ID NO: 30)


 


14
GDCTVGGETFFQPVTTALAAVGATLGGEDGGAGA (SEQ ID NO: 31)


 


15
GDCSQGGETFFQPVTEALKAYGAQIGGGQGEPPE (SEQ ID NO: 32)


 


16
GDCAKGGTTFFQPLPEAMASLGVRLIVPGREGAA (SEQ ID NO: 33)


 


17
GDCSRGGTTYFQPVNRILQTYGLTLVTA------ (SEQ ID NO: 34)


 


18
GNCSSGGTTYFQPINPLLQAYGLTLVTSGG--GT (SEQ ID NO: 35)


 


19
GDCRTGGETFFQPINALLQNYGLTLKTTGGDDGG (SEQ ID NO: 36)


 


20
GNCSIGGTTFHQPVNPILSQYGLTLVRS------ (SEQ ID NO: 37)


 


21
GNCTSGGTTYHQPINPLLSAYGLDLVTG------ (SEQ ID NO: 38)


 


22
GDCTNGGTTFYQPVNPLLSDFGLTLKTTSA---- (SEQ ID NO: 39)


 


23
GDCRSGGITFFQPINPLLSYFGLQLVTG------ (SEQ ID NO: 40)


 


24
GIPASQRSSLFERLQPILSQYGLSLVTG------ (SEQ ID NO: 41)


 


25
GNCSSGGETWFQPVNEILNRYGLTLHTA------ (SEQ ID NO: 42)


 


26
-MDTNNCRSFYQPVNTVLNKWKLSLVTSTDVTTS (SEQ ID NO: 43)


 


27
----SGCGMIHEPISRALADRGVTLLAG------ (SEQ ID NO: 44)






In the above listing, the numbers correspond as follows:














1
ASP Protease


2
Streptogrisin A (Streptomyces griseus)


3
Glutamyl endopeptidase (Streptomyces fradiae)


4
Streptogrisin B (Streptomyces lividans)


5
SAM-P20 (Streptomyces coelicolor)


6
SAM-P20 (Streptomyces albogriseolus)


7
Streptogrisin B (Streptomyces griseus)


8
Glutamyl endopeptidase II (Streptomyces griseus)


9
Glutamyl endopeptidase II (Streptomyces fradiae)


10
Streptogrisin D (Streptomyces albogriseolus)


11
Streptogrisin D (Streptomyces coelicolor)


12
Streptogrisin D (Streptomyces griseus)


13
Subfamily S1E unassigned peptidase 



(SalO protein) (Streptomyces lividans)


14
Subfamily S1E unassigned peptidase 



(SALO protein) (Streptomyces coelicolor)


15
Streptogrisin D (Streptomyces platensis)


16
Subfamily S1E unassigned peptidase 



(3SC5B7.10 protein)(Streptomyces coelicolor)


17
CHY1 protease (Metarhizium anisopliae)


18
Streptogrisin C (Streptomyces griseus)


19
Streptogrisin C (SCD40A.16c protein) 



(Streptomyces coelicolor)


20
Subfamily S1E unassigned peptidase 



(I) (Streptomyces sp.)


21
Subfamily S1E unassigned peptidase 



(II) (Streptomyces sp.)


22
Subfamily S1E unassigned peptidase 



(SCF43A.19 protein)(Streptomyces coelicolor)


23
Subfamily S1E unassigned peptidase 



(Thermobifida fusca; basonym Thermomonospora fusca)


24
Alpha-lytic endopeptidase (Lysobacter enzymogenes)


25
Subfamily S1E unassigned peptidase 



(SC10G8.13C protein) (Streptomyces coelicolor)


26
Yeast-lytic endopeptidase (Rarobacter faecitabidus)


27
Subfamily S1E unassigned peptidase 



(SC10A5.18 protein) (Streptomyces coelicolor)









Example 5
Screening for Novel Homologues of 69B4 Protease by PCR
In this Example, methods used to screen for novel homologues of 69B4 protease are described. Bacterial strains of the suborder Micrococcineae, and in particular from the family Cellulomonadaceae and Promicromonosporaceae were ordered from the German culture collection, DSMZ (Braunschweig) and received as freeze dried cultures. Additional strains were received from the Belgian Coordinated Collections of Microorganisms, BCCMTTM/LMG (University of Ghent). The freeze-dried ampoules were opened according to DSMZ instructions and the material rehydrated with sterile physiological saline (1.5 ml) for 1 h. Well-mixed, rehydrated cell suspensions (300 μL) were transferred to sterile Eppendorf tubes for subsequent PCR.
PCR Methods
i) Pretreatment of the Samples
The rehydrated microbial cell suspensions were placed in boiling water bath for 10 min. The suspensions were then centrifuged at 16000 rpm for 5 min. (Sigma 1-15 centrifuge) to remove cell debris and remaining cells, the clear supernatant fraction serving as template for the PCR reaction.
(ii) PCR Test Conditions
The DNA from these types of bacteria (Actinobacteria) is characteristically highly GC rich (typically >55 mol %), so addition of DMSO is a necessity. The chosen concentration based on earlier work with the Cellulomonas sp. strain 69B4 was 4% v/v DMSO.
(iii) PCR Primers (Chosen from the Following Pairs)









(SEQ ID NO: 45)


Prot-int_FW1
5′-TGCGCCGAGCCCGGCGACTC-3′


 



(SEQ ID NO: 46)


Prot-int_RV1
5′-GAGTCGCCGGGCTCGGCGCA-3′


 



(SEQ ID NO: 47)


Prot-int_FW2
5′-TTCCCCGGCAACGACTACGCGTGGGT-3′


 



(SEQ ID NO: 48)


Prot-int_RV2
5′-ACCCACGCGTAGTCGTTGCCGGGGAA-3′


 



(SEQ ID NO: 49)


Cellu-FW1
5′-GCCGCTGCTCGATCGGGTTC-3′


 



(SEQ ID NO: 50)


Cellu-RV1
5′-GCAGTTGCCGGAGCCGCCGGACGT-3′






(Iv) PCR Mixture (all Materials Supplied by Invitrogen)
















Template DNA
4 μ1



10x PCR buffer
5 μ1



50 mM MgSO4
2 μ1



10 mM dNTP's
1 μ1



Primers (10 μM soln.)
1 μ1 each



Platinum Taq hifi polymerase
0.5 μ1



DMSO
2 μ1



MilliQ water
33.5 μ1










(v) PCR Protocol
















1) 94° C.
 5 min



2) 94° C.
30 sec



3) 55° C.
30 sec



4) 68° C.
 3 min









5) Repeat steps 2-4 repeat for 29 cycles










6) 68° C.
10 min



7) 15° C.
 1 min










The amplified PCR products were examined by agarose gel electrophoresis. Distinct bands for each organism were excised from the gel, purified using the Qiagen gel extraction kit, and sequenced by BaseClear, using the same primer combinations.
(vi) Sequence Analysis
Nucleotide sequence data were analyzed and the DNA sequences were translated into amino acid sequences to review the homology to 69B4-mature protein. Sequence alignments were performed using AlignX, a component of Vector NTI suite 9.0.0. The results are compiled in Table 5-1. The numbering is that used in SEQ ID NO:8.







TABLE 5-1







Percent Identity of (translated) Amino Acid Sequences found


in Natural Isolate Strains Compared to 69B4 Mature Protease











No. of





Amino
Overlap
%


Microorganism
Acids
Position
Identity





Cellulomonas flavigena DSM 20109
101
34-134
62


Cellulomonas biazotea DSM 20112
114
26-139
68


Cellulomonas fimi DSM20113
109
32-140
72


Cellulomonas gelida DSM 20111
 48
142-189 
69


Cellulomonas iranensis DSM 14785
 85
52-123
66


Cellulomonas cellasea DSM 20109
102
32-133
63


Cellulomonas xylanilytica LMG 21723
143
16-158
73


Oerskovia turbata DSM 20577
111
34-144
74


Oerskovia jenensis DSM 46000
129
22-150
70


Cellulosimicrobium cellulans DSM 20424
134
35-168
53


Promicromonospora citrea DSM 43110
 85
52-136
75


Promicromonospora sukumoe DSM
 85
52-136
73


44121





Xylanibacterium ulmi LMG 21721
141
16-156
64








Streptomyces griseus ATCC 27001
No PCR product 


Streptomyces griseus ATCC 10137
detected 


Streptomyces griseus ATCC 23345
homologous


Streptomyces fradiae ATCC 14544
to 69B4 protease


Streptomyces coelicolor ATCC 10147



Streptomyces lividans TK23









These results show that PCR primers based on polynucleotide sequences of the 69B4 protease gene (mature chain), SEQ ID NO:4 are successful in detecting homologous genes in bacterial strains of the suborder Micrococcineae, and in particular from the family Cellulomonadaceae and Promicromonosporaceae.
FIG. 2 provides a phylogeny tree of ASP protease. The phylogeny of this protease was examined by a variety of approaches from mature sequences of similar members of the chymotrypsin superfamily of proteins and ASP homologues for which significant mature sequence has been deduced. Using protein distance methods known in the art (See e.g., Kimura, The Neutral Theory of Molecular Evolution, Cambridge University Press, Cambridge, UK [1983]) similar trees were obtained either including or excluding gaps. The phylogenetic tree of FIG. 2 was constructed from aligned sequences (positions 16-181 of SEQ ID NO:8) using TREECONW v.1.3b (Van de Peer and De Wachter, Comput. Appl. Biosci., 10:569-570 [1994]) and with tree topology inferred by the Neighbor-Joining algorithm (Saitou and Nei, Mol. Biol. Evol., 4:406-425 [1987]). As indicated by this tree, the data indicate that the ASP series of homologous proteases (“cellulomonadins”) forms a separate subfamily of proteins. In FIG. 2, the numbers provided in brackets correspond to the sequences provided herein.
The following is an alignment between the Cellulomonas 69B4 ASP protease and homologous proteases of related genera described herein.












1                                               50



69B4(ASP)complete
  (1)
MTPRTVTRALAVATAAATLLAGGMAAQANEPAPPGSASAPPRLAEKLDPD


 


Cellulomonas gelida
  (1)
--------------------------------------------------


 


Cellulomonas flavigena
  (1)
--------------------------------------------------


 


Cellulomonas biazotea
  (1)
--------------------------------------------------


 


Cellulomonas fimi
  (1)
--------------------------------------------------


 


Cellulomonas iranensis
  (1)
--------------------------------------------------


 


Cellulomonas cellasea
  (1)
--------------------------------------------------


 


C. xylanilytica
  (1)
--------------------------------------------------


 


Oerskovia turbata
  (1)
MARSFWATLATACAATALVAGPAALTANAATPTPDTPTVSPQTSSKVSPE


 


Oerskovia jenensis
  (1)
--------------------------------------------------


 


Cm. cellulans
  (1)
--------------------------------------------------


 


Pm. citrea
  (1)
--------------------------------------------------


 


Pm. sukumoe
  (1)
--------------------------------------------------


 


69B4 (ASP) mature
  (1)
--------------------------------------------------


 


Consensus
  (1)



 




51                                             100


69B4(ASP)complete
 (51)
LLEAMERDLGLDAEEAAATLAFQHDAAETGEALAEELDEDF-AGTWVEDD


 


Cellulomonas gelida
  (1)
--------------------------------------------------


 


Cellulomonas flavigena
  (1)
--------------------------------------------------


 


Cellulomonas biazotea
  (1)
--------------------------------------------------


 


Cellulomonas fimi
  (1)
--------------------------------------------------


 


Cellulomonas iranensis
  (1)
--------------------------------------------------


 


Cellulomonas cellasea
  (1)
-------------------------------------------------V


 


C. xylanilytica
  (1)
--------------------------------------------------


 


Oerskovia turbata
 (51)
VLRALQRDLGLSAKDATKRLAFQSDAASTEDALADSLDAYAGAWVDPARN


 


Oerskovia jenensis
  (1)
--------------------------------------------------


 


Cm. cellulans
  (1)
--------------------------------PRAAGRAARSSGSRASAS


 


Pm. citrea
  (1)
--------------------------------------------------


 


Pm. sukumoe
  (1)
--------------------------------------------------


 


69B4 (ASP) mature
  (1)
--------------------------------------------------


 


Consensus
 (51)



 




101                                            150


69B4(ASP)complete
(100)
VLYVATTDEDAVEEVEGEGATAVTVEHSLADLEAWKTVLDAALEGHDDVP


 


Cellulomonas gelida
  (1)
--------------------------------------------------


 


Cellulomonas flavigena
  (1)
--------------------------------------------------


 


Cellulomonas biazotea
  (1)
---------------KQTASEFVIRLTIGELNLAAANSPLPIGHAWSTAL


 


Cellulomonas fimi
  (1)
--------------------------------------------------


 


Cellulomonas iranensis
  (1)
--------------------------------------------------


 


Cellulomonas cellasea
  (2)
GRVRQLPLRGHDVLPARERDPAGLASASRPGLTRSRRARLDAAGPSARVA


 


C. xylanilytica
  (1)
--------------------------------------------------


 


Oerskovia turbata
(101)
TLYVGVADRAEAKEVRSAGATPVVVDHTLAELDTWKAALDGELNDPAGVP


 


Oerskovia jenensis
  (1)
--------------------------------------------------


 


Cm. cellulans
 (19)
TSPGPTSVTASASSCGRATGRRQRWTFEADGTVRAGGKCMDVAWAPRPTA


 


Pm. citrea
  (1)
--------------------------------------------------


 


Pm. sukumoe
  (1)
--------------------------------------------------


 


69B4 (ASP) mature
  (1)
--------------------------------------------------


 


Consensus 
(101)



 




151                                            200


69B4(ASP)complete
(150)
TWYVDVPTNSVVVAVKAGAQDVAAGLVEGADVPSDAVTFVETDETPRTMF


 


Cellulomonas gelida
  (1)
--------------------------------------------------


 


Cellulomonas flavigena
  (1)
-------------------------------------------------V


 


Cellulomonas biazotea
 (36)
GWYVDVTTNTVVVNATALAVAQATEIVAAATVPADAVRVVETTEAPRTFI


 


Cellulomonas fimi
  (1)
-------------------------------------------------V


 


Cellulomonas iranensis
  (1)
--------------------------------------------------


 


Cellulomonas cellasea
 (52)
AWYVDVPTNKLVVESVG--DTAAAADAVAAAGLPADAVTLATTEAPRTFV


 


C. xylanilytica
  (1)
--------------------------------------------------


 


Oerskovia turbata
(151)
SWFVDVTTNQVVVNVHDGGRALAELAAASAGVPADAITYVTTTEAPRPLV


 


O. jenenensis revi
  (1)
--------------------------------------------------


 


Cm. cellulans
 (69)
RASSSRTARQRGPEVRAQRRGRPRVGAGEQSASTPPGAHRGTRGAVRAHG


 


Pm. citrea
  (1)
--------------------------------------------------


 


Pm. sukumoe
  (1)
--------------------------------------------------


 


69B4 (ASP) mature
  (1)
-------------------------------------------------F


 


Consensus 
(151)



 




201                                            250


69B4(ASP)complete
(200)
DVIGGNAYTIGGRSR-----CSIGFAVNGGFITAGHCGRTGA-----TTA


 


Cellulomonas gelida
  (1)
--------------------------------------------------


 


Cellulomonas flavigena
  (2) 
DVIGGNAYYIGSRSR-----CSIGFAVEGGFVTAGHCGRAGA-----STS


 


Cellulomonas biazotea
 (86) 
DVIGGNRYRINNTSR-----CSVGFAVSGGFVTAGHCGTTGA-----TTT


 


C. fimi. revi
  (2) 
DVIGGDAYYIGGRSR-----CSIGFAVTGGFVTAGHCGRTGA-----ATT


 


C. iranensis revi
  (1)
--------------------------------------------------


 


Cellulomonas cellasea
(100)
DVIGGNAYYINASSR-----CSVGFAVEGGFVTAGHCGRAGA-----STS


 


C. xylanilytica
  (1)
--------------R-----CSIGFAVTGGFVTAGHCGRSGA-----TTT


 


Oerskovia turbata
(201)
DVVGGNAYTMGSGGR-----CSVGFAVNGGFITAGHCGSVGT-----RTS


 


Oerskovia jenensis
  (1)
--------------R-----CSVGFAVNGGFVTAGHCGTVGT-----RTS


 


Cm. cellulans
(119)
DVRGGDRYITRDPGASSGSACSIGYAVQGGFVTAGHCGRGGTRRVLTASW


 


Pm. citrea
  (1)
--------------------------------------------------


 


Pm. sukumoe
  (1)
--------------------------------------------------


 


69B4 (ASP) mature
  (2)
DVIGGNAYTIGGRSR-----CSIGFAVNGGFITAGHCGRTGA-----TTA


 


Consensus
(201)
DVIGG  Y I    R     CSIGFAV GGFVTAGHCGR GA      TS


 




251                                            300


69B4(ASP)complete
(240)
NPTGTFAGSSFPGNDYAFVRTGAGVNLLAQVNNYSGGRVQVAGHTAAPVG


 


Cellulomonas gelida
  (1)
--------------------------------------------------


 


Cellulomonas flavigena
 (42)
SPSGTFRGSSFPGNDYAWVQVASGNTPRGLVNNHSGGTVRVTGSQQAAVG


 


Cellulomonas biazotea
(126)
KPSGTFAGSSFPGNDYAWVRVASGNTPVGAVNNYSGGTVAVAGSTQATVG


 


Cellulomonas fimi
 (42)
SPSGTFAGSSFPGNDYAWVRVASGNTPVGAVNNYSGGTVAVAGSTQAAVG


 


Cellulomonas iranensis
  (1)
----------FPGNDYAWVQVGSGDTPRGLVNNYAGGTVRVTGSQQAAVG


 


Cellulomonas cellasea
(140)
SPSGTFRGSSFPGNDYAWVQVASGMTPRGLVNNHSGGTVRVTGSQQAAVG


 


C. xylanilytica
 (27)
SPSGTFAGSSFPGNDYAWVRAASGNTPVGAVNRYDGSRVTVAGSTDAAVG


 


Oerskovia turbata
(241)
GPGGTFRGSNFPGNDYAWVQVDAGNTPVGAVNNYSGGRVAVAGSTAAPVG


 


Oerskovia jenensis
 (27)
GPGGTFRGSSFPGNDYAWVQVDAGNTPVGAVNNYSGGRVAVAGSTAAPVG


 


Cm. cellulans
(169)
ARMGTVQAASFPGHDYAWVRVDAGFSPVPRVNNYAGGTVDVAGSAEAPVG


 


Pm. citrea
  (1)
----------FPGNDYAWVNTGTDDTLVGAVNNYSGGTVNVAGSTRAAVG


 


Pm. sukumoe
  (1)
----------FPGNDYAWVNVGSDDTPIGAVNNYSGGTVNVAGSTQAAVG


 


69B4 (ASP) mature
 (42)
NPTGTFAGSSFPGNDYAFVRTGAGVNLLAQVNNYSGGRVQVAGHTAAPVG


 


Consensus
(251)
 P GTF GSSFPGNDYAWVQVASGNTPVGAVNNYSGGTV VAGST AAVG


 




301                                            350


69B4(ASP)complete
(290)
SAVCRSGSTTGWHCGTITALNSSVTYPEGTVRGLIRTTVCAEPGDSGGSL


 


Cellulomonas gelida
  (1)
--------------------------------------------------


 


Cellulomonas flavigena
 (92)
SYVCRSGSTTGWRCGYVRAYNTTVRYAEGSVSGLIRTSVCAEPGDSGGSL


 


Cellulomonas biazotea
(176)
ASVCRSGSTTGWRCGTIQAFNSTVNYAQGSVSGLIRTNVCAEPGDSGGSL


 


Cellulomonas fimi
 (92) 
ATVCRSGSTTGWRCGTIQAFNATVNYAEGSVSGLIRTNVCAEPGDSGGSL


 


Cellulomonas iranensis
 (41)
AYVCRSGSTTGWRCGTVQAYNASVRYAEGTVSGLIRTNVCAEPGD-----


 


Cellulomonas cellasea
(190)
SYVCRSGSTTGWRCGYVRAYNTTVRYAEGSVSGLIRTSVCAEPGDSGGSL


 


C. xylanilytica
 (77)
AAVCRSGSTTAWGCGTIQSRGASVTYAQGTVSGLIRTNVCAEPGDSGGSL


 


Oerskovia turbata
(291)
ASVCRSGSTTGWHCGTIGAYNTSVTYPQGTVSGLIRTNVCAEPGDSGGSL


 


Oerskovia jenensis
 (77)
SSVCRSGSTTGWRCGTIAAYNSSVTYPQGTVSGLIRTNVCAEPGDSGGSL


 


Cm. cellulans
(219)
ASVCRSGATTGWRCGVIEQKNITVNYGNGDVPGLVRGSACAEGGDSGGSV


 


Pm. citrea
 (41)
ATVCRSGSTTGWHCGTIQALNASVTYAEGTVSGLIRTNVCAEPGD-----


 


Pm. sukumoe
 (41)
STVCRSGSTTGWHCGTIQAFNASVTYAEGTVSGLIRTNVCAEPGD-----


 


69B4 (ASP) mature
 (92)
SAVCRSGSTTGWHCGTITALNSSVTYPEGTVRGLIRTTVCAEPGDSGGSL


 


Consensus
(301)
ASVCRSGSTTGWRCGTI AYNASV YAEGTVSGLIRTNVCAEPGDSGGSL


 




351                                            400


69B4(ASP)complete
(340)
LAGNQAQGVTSGGSGNCRTGGTTFFQPVNPILQAYGLRMITT-DSGSSPA


 


Cellulomonas gelida
  (1)
LAGNQAQGVTSGGSGNCSSGGTTYFQPVNEALRVYGLTLVTS-DGGGTE-


 


Cellulomonas flavigena
(142)
VAGTQAQGVTSGGSGNCRYGGTTYFQPVNEILQDQPGPSTTR-AL-----


 


Cellulomonas biazotea
(226)
IAGNQAQGLTSGGSGNCTTGGTTYFQPVNEALSAYGLTLVTSSGGGGGGG


 


Cellulomonas fimi
(142)
VAG-----------------------------------------------


 


Cellulomonas iranensis
 (86)
--------------------------------------------------


 


Cellulomonas cellasea
(240)
VAGTQAQGVTSGGSGNCRYGGTTYFQPVNEILQAYGLRLVLG-HARGGPS


 


C. xylanilytica
(127)
IAGTQARGVTSGGSGNC---------------------------------


 


Oerskovia turbata
(341)
LAGNQAQGVTSGGSGNCSSGGTTYFQPVNEALGGYGLTLVTSDGGGPSRR


 


Oerskovia jenensis
(127)
LAGNQAQGLTSGGSGNCSSGGTTYFQPVNEALSAYGLTLVTSGGRGNC--


 


Cm. cellulans
(269)
ISGNQAQGVTSGRINDCSNGGKFLYQPDRRPVARDHGRRVGQRARRARGQ


 


Pm. citrea
 (86)
--------------------------------------------------


 


Pm. sukumoe
 (86)
--------------------------------------------------


 


69B4 (ASP) mature
(142)
LAGNQAQGVTSGGSGNCRTGGTTFFQPVNPILQAYGLRMITTDSGSSP--


 


Consensus
(351)
LAGNQAQGVTSGGSGNC  GGTTYFQPVN  L  YGL LV


 


69B4(ASP)complete
(389)
-PAPTSCTGYARTFTGTLAAGRAAAQPNGSYVQVNRSGTHSVCLNGPSGA


 


Cellulomonas gelida
 (49)
-PPPTGCQGYARTYQGSVSAGTSVAQPNGSYVTTG-GGTHRVCLSGPAGT


 


Cellulomonas flavigena
(186)
--------------------------------------------------


 


Cellulomonas biazotea
(276)
----TTCTGYARTYTGSLASRQSAVQPSGSYVTVGSSGTIRVCLDGPSGT


 


Cellulomonas fimi
(145)
--------------------------------------------------


 


Cellulomonas iranensis
 (86)
--------------------------------------------------


 


Cellulomonas cellasea
(289)
-PARRAPAPPARA-------------------------------------


 


C. xylanilytica
(144)
--------------------------------------------------


 


Oerskovia turbata
(391)
RPGARAMRGPTRAASRPGRRSRSERFVRHDRGRATGCA------------


 


Oerskovia jenensis
(175)
--------------------------------------------------


 


Cm. cellulans
(319)
VHRRPRVRLQ----------------------------------------


 


Pm. citrea
 (86)
--------------------------------------------------


 


Pm. sukumoe
 (86)
--------------------------------------------------


 


69B4 (ASP) mature
(190)
--------------------------------------------------


 


Consensus
(401)



 




451                                            500


69B4(ASP)complete
(438)
DFDLYVQRWNGSSWVTVAQSTSPGSNETITYRGNAGYYRYVVNAASGSGA


 


Cellulomonas gelida
 (97)
DLDLYLQKWNGYSWASVAQSTSPGATEAVTYTGTAGYYRYVVHAYAGSGA


 


Cellulomonas flavigena
(186)
--------------------------------------------------


 


Cellulomonas biazotea
(322)
DFDLYLQKWNGSAW------------------------------------


 


Cellulomonas fimi
(145)
--------------------------------------------------


 


Cellulomonas iranensis
 (86)
--------------------------------------------------


 


Cellulomonas cellasea
(301)
--------------------------------------------------


 


C. xylanilytica
(144)
--------------------------------------------------


 


Oerskovia turbata
(429)
--------------------------------------------------


 


Oerskovia jenensis
(175)
--------------------------------------------------


 


Cm. cellulans
(329)
--------------------------------------------------


 


Pm. citrea
 (86)
--------------------------------------------------


 


Pm. sukumoe
 (86)
--------------------------------------------------


 


69B4 (ASP) mature
(190)
--------------------------------------------------


 


Consensus
(451)



 




501


69B4(ASP)complete
(488)
YTMGLTLP (SEQ ID NO: 6)


 


Cellulomonas gelida
(147)
YTLGATTP (SEQ ID NO: 60)


 


Cellulomonas flavigena
(186)
-------- (SEQ ID NO: 54)


 


Cellulomonas biazotea
(336)
-------- (SEQ ID NO: 56)


 


Cellulomonas fimi
(145)
-------- (SEQ ID NO: 58)


 


Cellulomonas iranensis
 (86)
-------- (SEQ ID NO: 62)


 


Cellulomonas cellasea
(301)
-------- (SEQ ID NO: 64)


 


C. xylanilytica
(144)
-------- (SEQ ID NO: 66)


 


Oerskovia turbata
(429)
-------- (SEQ ID NO: 68)


 


Oerskovia jenensis
(175)
-------- (SEQ ID NO: 70)


 


Cm. cellulans
(329)
-------- (SEQ ID NO: 72)


 


Pm. citrea
 (86)
-------- (SEQ ID NO: 74)


 


Pm. sukumoe
 (86)
-------- (SEQ ID NO: 76)


 


69B4 (ASP) mature
(190)
-------- (SEQ ID NO: 8)


 


Consensus
(501)
         (SEQ ID NO: 647)






Example 6
Detection of Novel Homologues of 69B4 Protease by Immunoblotting
In this Example, immunoblotting experiments used to detect homologues of 6984 are described. The following organisms were used in these experiments:
1. Cellulomonas biazotea DSM 20112
2. Cellulomonas flavigena DSM 20109
3. Cellulomonas fimi DSM 20113
4. Cellulomonas cellasea DSM 20118
5. Cellulomonas uda DSM 20107
6. Cellulomonas gelida DSM 20111
7. Cellulomonas xylanilytica LMG 21723
8. Cellulomonas iranensis DSM 14785
9. Oerskovia jenensis DSM 46000
10. Oerskovia turbata DSM 20577
11. Cellulosimicrobium cellulans DSM 20424
12. Xylanibacterium ulmi LMG21721
13. Isoptericola variabilis DSM 10177
14. Xylanimicrobium pachnodae DSM 12657
15. Promicromonospora citrea DSM 43110
16. Promicromonospora sukumoe DSM 44121
17. Agromyces ramosus DSM 43045
The strains were first grown on Heart Infusion/skim milk agar plates (72 h, 30° C.) to confirm strain purity, protease reaction by clearing of the skim milk and to serve as inoculum. Bacterial strains were cultivated on Brain Heart Infusion broth supplemented with casein (0.8% w/v) in 100/500 Erlenmeyer flasks with baffles at 230 rpm, 30° C. for 5 days. Microbial growth was checked by microscopy. Supernatants were separated from cells by centrifugation for 30 min at 4766×g. Further solids were removed by centrifugation at 9500 rpm. Supernatants were concentrated using Vivaspin 20 ml concentrator (Vivascience), cutoff 10 kDa, by centrifugation at 4000×g. Concentrates were stored in aliquots of 0.5 mL at −20° C.
Primary Antibody
The primary antibody (EP034323) for the immunoblotting reaction, prepared by Eurogentec (Liege Science Park, Seraing, Belgium) was raised against 2 peptides consisting of amino acids 151-164 and 178-189 in the 69B4 mature protease (SEQ ID NO:8), namely:
TSGGSGNCRTGGTT (epitope 1; SEQ ID NO:51) and LRMITTDSGSSP (epitope 2; SEQ ID NO:52) as shown below in the amino acid sequence of 69B4 mature protease:




Electrophoresis and Immunoblotting

Sample Preparation
1. Concentrated culture supernatant (50 μL)
2. PMSF (1 μL; 20 mg/ml)
3. 1M HCl (25 μL)
4. Nu PAGE LDS sample buffer (25 μL) (Invitrogen, Carlsbad, Calif., USA) Mixed and heated at 90° C. for 10 min.
Electrophoresis
SDS-PAGE was performed in duplicate using NuPAGE 10% Bis-Tris gels (Invitrogen) with MES-SDS running buffer at 100 v for 5 min. and 200 v constant. Where possible, 25 μL sample were loaded in each slot. One gel of each pair was stained with Coomassie Blue and the other gel was used for immunoblotting using the Boehringer Mannheim chromogenic Western blotting protocol (Roche).
Immunoblotting
The transfer buffer used was Transfer buffer: Tris (0.25M)-glycine (1.92M)-methanol (20% v/v). The PVDF membrane was pre-wetted by successive moistening in methanol, deionized water, and finally transfer buffer.
The PAGE gel was briefly washed in deionized water and transferred to blotting pads soaked in transfer buffer, covered with pre-wetted PVDF membrane and pre-soaked blotting pads. Blotting was performed in transfer buffer at 400 mA constant for 2.5-3 h. The membrane was briefly washed (2×) in Tris buffered saline (TBS) (0.5M Tris, 0.15M NaCl, pH7.5). Non-specific antibody binding was prevented by incubating the membrane in 1% v/v mouse/rabbit Blocking Reagent (Roche) in maleic acid solution (100 mM maleic acid, 150 mM NaCl, pH7.5) overnight at 4° C.
The primary antibody used in these reactions was EP034323 diluted 1:1000. The reaction was performed with the Ab diluted in 1% Blocking Solution with a 30 min. action time. The membrane was washed 4×10 min. in TBST (TSB+0.1% v/v Tween 20).
The secondary antibody consisted of anti-mouse/anti-rabbit IgG (Roche) 73 μL in 20 ml in 1% Blocking Solution with a reaction time of 30 min. The membrane was washed 4×15 min. in TBST and the substrate reaction (alkaline phosphatase) performed with BM Chromogenic Western Blotting Reagent (Roche) until staining occurred.
The results of the cross-reactivity with primary polyclonal antibody are shown in Table 6-1.







TABLE 6-1







Immunoblotting Results















Protease




Estimated
% Sequence
Activity




Molecular
Identity to
On HI-



Immuno-
Mass
69B4 Mature
Skim Milk


Strain
Blot Result
kDa
Protease
Agar














C. flavigena DSM
positive
21
66
positive


20109






C. biazotea DSM
negative

65
positive


20112






C. fimi DSM 20112
negative

72
weak +


C. gelida DSM 20111
positive
20
69
weak +


C. uda DSM 20107
negative


weak +


C. iranensis DSM
negative

33
weak +


14785






C. cellasea DSM
positive
27
61 
positive


20118






C. xylanilytica LMG
negative

69
positive


21723






O. turbata DSM
positive
18
73
positive


20577






O. jenensis DSM
positive
35
78
positive


46000






C. cellulans DSM
negative

48
positive


20424






P. citrea DSM 43110
negative

28
positive


P. sukumoe DSM
negative

69
positive


44121






X. ulmi LMG21721
negative

72
negative


I. variabilis DSM
negative


positive 


10177






X. pachnodae DSM
negative


weak +


12657






A. ramosus DSM
negative


weak +


43045









Based on these results, it is clear that the antibody used in these experiments is highly specific at detecting homologues with avery high percentage of amino acid sequence identity to 69B4 protease. Furthermore, these results indicate that the C-terminal portion of the 69B4 mature protease chain is fairly variable especially in the region of the 2-peptide epitopes. In these experiments, it was determined that in cases where there were more than 2 amino acid differences in this region a negative Western blotting reaction resulted.
Example 7
Inverse PCR and Genome Walking
In this Example, experiments conducted to elucidate polynucleotide sequences of ASP are described. The microorganisms utilized in these experiments were:
1. Cellulomonas biazotea DSM 20112
2. Cellulomonas flavigena DSM 20109
3. Cellulomonas fimi DSM 20113
4. Cellulomonas cellasea DSM 20118
5. Cellulomonas gefida DSM 20111
6. Cellulomonas iranensis (DSM 14785)
7. Oerskovia jenensis DSM 46000
8. Oerskovia turbata DSM 20577
9. Cellulosimicrobium cellulans DSM 20424
10. Promicromonospora citrea DSM 43110
11. Promicromonospora sukumoe DSM 44121
These bacterial strains were cultivated on Brain Heart Infusion broth or Tryptone Soya broth in 100/500 Erlenmeyer flasks with baffles at 230 rpm, 30° C. for 2 days: Cells were separated from the culture broth by centrifugation for 30 min at 4766×g.
Chromosomal DNA was obtained by standard phenol/chloroform extraction method known in the art from cells digested by lysozyme/EDTA (See e.g., Sambrook et al., supra). Chromosomal DNA was digested with the restriction enzymes selected from the following list: ApaI, BamHI, BssHII, KpnI, NarI, NcoI, NheI, PvuI, SalI or SstII.
The nucleotide and amino acid sequences of these organisms are provided below. In these listings, the mature protease is indicated in bold and the signal sequence is underlined.








C. flavigena (DSM 20109)



(SEQ ID NO: 53)










   1
GTCGACGTCA TCGGGGGCAA CGCGTACTAC ATCGGGTCGC GCTCGCGGTG




CAGCTGCAGT AGCCCCCGTT GCGCATGATG TAGCCCAGCG CGAGCGCCAC


 


  51
CTCGATCGGG TTCGCGGTCG AGGGCGGGTT CGTCACCGCG GGGCACTGCG



GAGCTAGCCC AAGCGCCAGC TCCCGCCCAA GCAGTGGCGC CCCGTGACGC


 


 101
GGCGCGCGGG CGCGAGCACG TCGTCACCGT CGGGGACCTT CCGCGGCTCG



CCGCGCGCCC GCGCTCGTGC AGCAGTGGCA GCCCCTGGAA GGCGCCGAGC


 


 151
TCGTTCCCCG GCAACGACTA CGCGTGGGTC CAGGTCGCCT CGGGCAACAC



AGCAAGGGGC CGTTGCTGAT GCGCACCCAG GTCCAGCGGA GCCCGTTGTG


 


 201
GCCGCGCGGG CTGGTGAACA ACCACTCGGG CGGCACGGTG CGCGTCACCG



CGGCGCGCCC GACCACTTGT TGGTGAGCCC GCCGTGCCAC GCGCAGTGGC


 


 251
GCTCGCAGCA GGCCGCGGTC GGCTCGTACG TGTGCCGATC GGGCAGCACG



CGAGCGTCGT CCGGCGCCAG CCGAGCATGC ACACGGCTAG CCCGTCGTGC


 


 301
ACGGGATGGC GGTGCGGCTA CGTCCGGGCG TACAACACGA CCGTGCGGTA



TGCCCTACCG CCACGCCGAT GCAGGCCCGC ATGTTGTGCT GGCACGCCAT


 


 351
CGCGGAGGGC TCGGTCTCGG GCCTCATCCG CACGAGCGTG TGCGCCGAGC



GCGCCTCCCG AGCCAGAGCC CGGAGTAGGC GTGCTCGCAC ACGCGGCTCG


 


 401
CGGGCGACTC CGGCGGCTCG CTGGTCGCCG GCACGCAGGC CCAGGGCGTC



GCCCGCTGAG GCCGCCGAGC GACCAGCGGC CGTGCGTCCG GGTCCCGCAG


 


 451
ACGTCGGGCG GGTCCGGCAA CTGCCGCTAC GGGGGCACGA CGTACTTCCA



TGCAGCCCGC CCAGGCCGTT GACGGCGATG CCCCCGTGCT GCATGAAGGT


 


 501
GCCCGTGAAC GAGATCCTGC AGGACCAGCC CGGGCCGTCG ACCACGCGTG



CGGGCACTTG CTCTAGGACG TCCTGGTCGG GCCCGGCAGC TGGTGCGCAC


 


 551
CCCTA



GGGAT


 








Cellulomonas flavigena (DSM 20109)



(SEQ ID NO: 54)










   1
VDVIGGNAYY IGSRSRCSIG FAVEGGFVTA GHCGRAGAST SSPSGTFRGS



 


  51
SFPGNDYAWV QVASGNTPRG LVNNHSGGTV RVTGSQQAAV GSYVCRSGST


 


 101
TGWRCGYVRA YNTTVRYAEG SVSGLIRTSV CAEPGDSGGS LVAGTQAQGV


 


 151
TSGGSGNCRY GGTTYFQPVN EILQDQPGPS TTRAL


 








Cellulomonas biazotea (DSM 20112)



(SEQ ID NO: 55)










   1
TAAAACAGAC GGCCAGTGAA TTTGTAATAC GACTCACTAT AGGCGAATTG




ATTTTGTCTG CCGGTCACTT AAACATTATG CTGAGTGATA TCCGCTTAAC


 


  51
AATTTAGCGG CCGCGAATTC GCCCTTACCT ATAGGGCACG CGTGGTCGAC



TTAAATCGCC GGCGCTTAAG CGGGAATGGA TATCCCGTGC GCACCAGCTG


 


 101
GGCCCTGGGC TGGTACGTCG ACGTCACTAC CAACACGGTC GTCGTCAACG



CCGGGACCCG ACCATGCAGC TGCAGTGATG GTTGTGCCAG CAGCAGTTGC


 


 151
CCACCGCCCT CGCCGTGGCC CAGGCGACCG AGATCGTCGC CGCCGCAACG



GGTGGCGGGA GCGGCACCGG GTCCGCTGGC TCTAGCAGCG GCGGCGTTGC


 


 201
GTGCCCGCCG ACGCCGTCCG GGTCGTCGAG ACCACCGAGG CGCCCCGCAC



CACGGGCGGC TGCGGCAGGC CCAGCAGCTC TGGTGGCTCC GCGGGGCGTG


 


 251
GTTCATCGAC GTCATCGGCG GCAACCGTTA CCGGATCAAC AACACCTCGC



CAAGTAGCTG CAGTAGCCGC CGTTGGCAAT GGCCTAGTTG TTGTGGAGCG


 


 301
GCTGCTCGGT CGGCTTCGCC GTCAGCGGCG GCTTCGTCAC CGCCGGGCAC



CGACGAGCCA GCCGAAGCGG CAGTCGCCGC CGAAGCAGTG GCGGCCCGTG


 


 351
TGCGGCACGA CCGGCGCGAC CACGACGAAA CCGTCCGGCA CGTTCGCCGG



ACGCCGTGCT GGCCGCGCTG GTGCTGCTTT GGCAGGCCGT GCAAGCGGCC


 


 401
CTCGTCGTTC CCCGGCAACG ACTACGCGTG GGTGCGCGTC GCGTCCGGCA



GAGCAGCAAG GGGCCGTTGC TGATGCGCAC CCACGCGCAG CGCAGGCCGT


 


 451
ACACCCCGGT CGGCGCCGTG AACAACTACA GCGGCGGCAC CGTGGCCGTC



TGTGGGGCCA GCCGCGGCAC TTGTTGATGT CGCCGCCGTG GCACCGGCAG


 


 501
GCCGGCTCGA CGCAGGCGAC CGTCGGTGCG TCCGTCTGCC GCTCCGGCTC



CGGCCGAGCT GCGTCCGCTG GCAGCCACGC AGGCAGACGG CGAGGCCGAG


 


 551
CACCACGGGG TGGCGCTGCG GGACGATCCA GGCGTTCAAC TCCACCGTCA



GTGGTGCCCC ACCGCGACGC CCTGCTAGGT CCGCAAGTTG AGGTGGCAGT


 


 601
ACTACGCGCA GGGCAGCGTC TCCGGCCTCA TCCGCACGAA CGTGTGCGCC



TGATGCGCGT CCCGTCGCAG AGGCCGGAGT AGGCGTGCTT GCACACGCGG


 


 651
GAGCCCGGCG ACTCCGGCGG CTCGCTCATC GCCGGCAACC AGGCCCAGGG



CTCGGGCCGC TGAGGCCGCC GAGCGAGTAG CGGCCGTTGG TCCGGGTCCC


 


 701
CCTGACGTCC GGCGGGTCGG GCAACTGCAC CACCGGCGGG ACGACGTACT



GGACTGCAGG CCGCCCAGCC CGTTGACGTG GTGGCCGCCC TGCTGCATGA


 


 751
TCCAGCCCGT CAACGAGGCG CTCTCCGCCT ACGGCCTGAC GCTCGTCACG



AGGTCGGGCA GTTGCTCCGC GAGAGGCGGA TGCCGGACTG CGAGCAGTGC


 


 801
TCGTCCGGCG GCGGCGGTGG CGGCGGCACG ACCTGCACCG GGTACGCGCG



AGCAGGCCGC CGCCGCCACC GCCGCCGTGC TGGACGTGGC CCATGCGCGC


 


 851
GACCTACACC GGCTCGCTCG CCTCGCGGCA GTCCGCCGTC CAGCCGTCCG



CTGGATGTGG CCGAGCGAGC GGAGCGCCGT CAGGCGGCAG GTCGGCAGGC


 


 901
GCAGCTATGT GACCGTCGGG TCCAGCGGCA CCATCCGCGT CTGCCTCGAC



CGTCGATACA CTGGCAGCCC AGGTCGCCGT GGTAGGCGCA GACGGAGCTG


 


 951
GGCCCGAGCG GGACGGACTT CGACCTGTAC CTGCAGAAGT GGAACGGGTC



CCGGGCTCGC CCTGCCTGAA GCTGGACATG GACGTCTTCA CCTTGCCCAG


 


1001
CGCGTGGGC



GCGCACCCG


 








Cellulomonas biazotea (DSM 20112)



(SEQ ID NO: 56)










   1
KQTASEFVIR LTIGELNLAA ANSPLPIGHA WSTALGWYVD VTTNTVVVNA



 


  51
TALAVAQATE IVAAATVPAD AVRVVETTEA PRTFIDVIGG NRYRINNTSR


 


 101
CSVGFAVSGG FVTAGHCGTT GATTTKPSGT FAGSSFPGND YAWVRVASGN


 


 151
TPVGAVNNYS GGTVAVAGST QATVGASVCR SGSTTGWRCG TIQAFNSTVN


 


 201
YAQGSVSGLI RTNVCAEPGD SGGSLIAGNQ AQGLTSGGSG NCTTGGTTYF


 


 251
QPVNEALSAY GLTLVTSSGG GGGGGTTCTG YARTYTGSLA SRQSAVQPSG


 


 301
SYVTVGSSGT IRVCLDGPSG TDFDLYLQKW NGSAW


 








Cellulomonas fimi (DSM 20113)



(SEQ ID NO: 57)










   1
GTGGACGTGA TCGGCGGCGA CGCCTACTAC ATCGGCGGCC GCAGCCGCTG




CACCTGCACT AGCCGCCGCT GCGGATGATG TAGCCGCCGG CGTCGGCGAC


 


  51
TTCGATCGGG TTCGCCGTCA CCGGGGGCTT CGTGACCGCC GGGCACTGCG



AAGCTAGCCC AAGCGGCAGT GGCCCCCGAA GCACTGGCGG CCCGTGACGC


 


 101
GCCGCACCGG CGCGGCCACG ACGAGCCCGT CGGGCACGTT CGCCGGCTCG



CGGCGTGGCC GCGCCGGTGC TGCTCGGGCA GCCCGTGCAA GCGGCCGAGC


 


 151
AGCTTCCCGG GCAACGACTA CGCGTGGGTG CGGGTCGCGT CGGGCAACAC



TCGAAGGGCC CGTTGCTGAT GCGCACCCAC GCCCAGCGCA GCCCGTTGTG


 


 201
GCCCGTCGGC GCGGTGAACA ACTACAGCGG CGGCACGGTC GCCGTCGCCG



CGGGCAGCCG CGCCACTTGT TGATGTCGCC GCCGTGCCAG CGGCAGCGGC


 


 251
GCTCGACCCA GGCCGCCGTC GGTGCGACCG TGTGCCGCTC GGGCTCCACC



CGAGCTGGGT CCGGCGGCAG CCACGCTGGC ACACGGCGAG CCCGAGGTGG


 


 301
ACCGGCTGGC GGTGCGGCAC CATCCAGGCG TTCAACGCGA CCGTCAACTA



TGGCCGACCG CCACGCCGTG GTAGGTCCGC AAGTTGCGCT GGCAGTTGAT


 


 351
CGCCGAGGGC AGCGTCTCCG GCCTCATCCG CACGAACGTG TGCGCCGAGC



GCGGCTCCCG TCGCAGAGGC CGGAGTAGGC GTGCTTGCAC ACGCGGCTCG


 


 401
CCGGCGACTC GGGCGGCTCG CTCGTCGCCG GCAACCAGGC GCAGGGCATG



GGCCGCTGAG CCCGCCGAGC GAGCAGCGGC CGTTGGTCCG CGTCCCGTAC


 


 451
ACGTCCGGCG GCTCCGACAA CTGC



TGCAGGCCGC CGAGGCTGTT GACG


 








Cellulomonas fimi (DSM 20113)



(SEQ ID NO: 58)










   1
VDVIGGDAYY IGGRSRCSIG FAVTGGFVTA GHCGRTGAAT TSPSGTFAGS



 


  51
SFPGNDYAWV RVASGNTPVG AVNNYSGGTV AVAGSTQAAV GATVCRSGST


 


 101
TGWRCGTIQA FNATVNYAEG SVSGLIRTNV CAEPGDSGGS LVAG


 








Cellulomonas gelida (DSM 20111)



(SEQ ID NO: 59)










   1
CTCGCGGGCA ACCAGGCGCA GGGCGTGACG TCGGGCGGGT CGGGCAACTG




GAGCGCCCGT TGGTCCGCGT CCCGCACTGC AGCCCGCCCA GCCCGTTGAC


 


  51
CTCGTCGGGC GGGACGACGT ACTTCCAGCC CGTCAACGAG GCCCTCCGGG



GAGCAGCCCG CCCTGCTGCA TGAAGGTCGG GCAGTTGCTC CGGGAGGCCC


 


 101
TGTACGGGCT CACGCTCGTG ACCTCTGACG GTGGGGGCAC CGAGCCGCCG



ACATGCCCGA GTGCGAGCAC TGGAGACTGC CACCCCCGTG GCTCGGCGGC


 


 151
CCGACCGGGT GCCAGGGCTA TGCGCGGACC TACCAGGGCA GCGTCTCGGC



GGCTGGCCCA CGGTCCCGAT ACGCGCCTGG ATGGTCCCGT CGCAGAGCCG


 


 201
CGGGACGTCG GTCGCGCAGC CGAACGGTTC GTACGTCACG ACCGGGGGCG



GCCCTGCAGC CAGCGCGTCG GCTTGCCAAG CATGCAGTGC TGGCCCCCGC


 


 251
GGACGCACCG GGTGTGCCTG AGCGGACCGG CGGGCACGGA CCTGGACCTG



CCTGCGTGGC CCACACGGAC TCGCCTGGCC GCCCGTGCCT GGACCTGGAC


 


 301
TACCTGCAGA AGTGGAACGG GTACTCGTGG GCCAGCGTCG CGCAGTCGAC



ATGGACGTCT TCACCTTGCC CATGAGCACC CGGTCGCAGC GCGTCAGCTG


 


 351
GTCGCCTGGT GCCACGGAGG CGGTCACGTA CACCGGGACC GCCGGCTACT



CAGCGGACCA CGGTGCCTCC GCCAGTGCAT GTGGCCCTGG CGGCCGATGA


 


 401
ACCGCTACGT GGTCCACGCG TACGCGGGTT CGGGGGCGTA CACCCTGGGG



TGGCGATGCA CCAGGTGCGC ATGCGCCCAA GCCCCCGCAT GTGGGACCCC


 


 451
GCGACGACCC CG



CGCTGCTGGG GC


 








Cellulomonas gelida (DSM 20111)



(SEQ ID NO: 60)










   1
LAGNQAQGVT SGGSGNCSSG GTTYFQPVNE ALRVYGLTLV TSDGGGTEPP



 


  51
PTGCQGYART YQGSVSAGTS VAQPNGSYVT TGGGTHRVCL SGPAGTDLDL


 


 101
YLQKWNGYSW ASVAQSTSPG ATEAVTYTGT AGYYRYVVHA YAGSGAYTLG


 


 151
ATTP


 








Cellulomonas iranensis (DSM 14785)



(SEQ ID NO: 61)










   1
TTCCCCGGCA ACGACTACGC GTGGGTCCAG GTCGGGTCGG GCGACACCCC




AAGGGGCCGT TGCTGATGCG CACCCAGGTC CAGCCCAGCC CGCTGTGGGG


 


  51
CCGCGGCCTG GTCAACAACT ACGCGGGCGG CACCGTGCGG GTCACCGGGT



GGCGCCGGAC CAGTTGTTGA TGCGCCCGCC GTGGCACGCC CAGTGGCCCA


 


 101
CGCAGCAGGC CGCGGTCGGC GCGTACGTCT GCCGGTCGGG CAGCACGACG



GCGTCGTCCG GCGCCAGCCG CGCATGCAGA CGGCCAGCCC GTCGTGCTGC


 


 151
GGCTGGCGCT GCGGCACCGT GCAGGCCTAC AACGCGTCGG TCCGCTACGC



CCGACCGCGA CGCCGTGGCA CGTCCGGATG TTGCGCAGCC AGGCGATGCG


 


 201
CGAGGGCACC GTCTCGGGCC TCATCCGCAC CAACGTCTGC GCCGAGCCCG



GCTCCCGTGG CAGAGCCCGG AGTAGGCGTG GTTGCAGACG CGGCTCGGGC


 


 251
GCGACTC



CGCTGAG


 








Cellulomonas iranensis (DSM 14785)



(SEQ ID NO: 62)










   1
FPGNDYAWVQ VGSGDTPRGL VNNYAGGTVR VTGSQQAAVG AYVCRSGSTT



 


  51
GWRCGTVQAY NASVRYAEGT VSGLIRTNVC AEPGD


 








Cellulomonas cellasea (DSM 20118)



(SEQ ID NO: 63)










   1
GTCGGGCGGG TCCGGCAACT GCCGCTACGG GGGCACGACG TACTTCCAGC




CAGCCCGCCC AGGCCGTTGA CGGCGATGCC CCCGTGCTGC ATGAAGGTCG


 


  51
CCGTGAACGA GATCCTGCAG GCCTACGGTC TGCGTCTCGT CCTGGGCTGA



GGCACTTGCT CTAGGACGTC CGGATGCCAG ACGCAGAGCA GGACCCGACT


 


 101
CACGCTCGCG GCGGGCCCGG CTCGACGCGG CCGGCCCGTC GGCCCGGGTC



GTGCGAGCGC CGCCCGGGCC GAGCTGCGCC GGCCGGGCAG CCGGGCCCAG


 


 151
GCCGCCTGGT ACGTCGACGT GCCGACCAAC AAGCTCGTCG TCGAGTCGGT



CGGCGGACCA TGCAGCTGCA CGGCTGGTTG TTCGAGCAGC AGCTCAGCCA


 


 201
CGGCGACACC GCGGCGGCCG CCGACGCCGT CGCCGCCGCG GGCCTGCCTG



GCCGCTGTGG CGCCGCCGGC GGCTGCGGCA GCGGCGGCGC CCGGACGGAC


 


 251
CCGACGCCGT GACGCTCGCG ACCACCGAGG CGCCACGGAC GTTCGTCGAC



GGCTGCGGCA CTGCGAGCGC TGGTGGCTCC GCGGTGCCTG CAAGCAGCTG


 


 301
GTCATCGGCG GCAACGCGTA CTACATCAAC GCGAGCAGCC GCTGCTCGGT



CAGTAGCCGC CGTTGCGCAT GATGTAGTTG CGCTCGTCGG CGACGAGCCA


 


 351
CGGCTTCGCG GTCGAGGGCG GGTTCGTCAC CGCGGGCCAC TGCGGGCGCG



GCCGAAGCGC CAGCTCCCGC CCAAGCAGTG GCGCCCGGTG ACGCCCGCGC


 


 401
CGGGCGCGAG CACGTCGTCA CCGTCGGGGA CCTTCCGCGG CTCGTCGTTC



GCCCGCGCTC GTGCAGCAGT GGCAGCCCCT GGAAGGCGCC GAGCAGCAAG


 


 451
CCCGGCAACG ACTACGCGTG GGTCCAGGTC GCCTCGGGCA ACACGCCGCG



GGGCCGTTGC TGATGCGCAC CCAGGTCCAG CGGAGCCCGT TGTGCGGCGC


 


 501
CGGGCTGGTG AACAACCACT CGGGCGGCAC GGTGCGCGTC ACCGGCTCGC



GCCCGACCAC TTGTTGGTGA GCCCGCCGTG CCACGCGCAG TGGCCGAGCG


 


 551
AGCAGGCCGC GGTCGGCTCG TACGTGTGCC GATCGGGCAG CACGACGGGA



TCGTCCGGCG CCAGCCGAGC ATGCACACGG CTAGCCCGTC GTGCTGCCCT


 


 601
TGGCGGTGCG GCTACGTCCG GGCGTACAAC ACGACCGTGC GGTACGCGGA



ACCGCCACGC CGATGCAGGC CCGCATGTTG TGCTGGCACG CCATGCGCCT


 


 651
GGGCTCGGTC TCGGGCCTCA TCCGCACGAG CGTGTGCGCC GAGCCGGGCG



CCCGAGCCAG AGCCCGGAGT AGGCGTGCTC GCACACGCGG CTCGGCCCGC


 


 701
ACTCCGGCGG CTCGCTGGTC GCCGGCACGC AGGCCCAGGG CGTCACGTCG



TGAGGCCGCC GAGCGACCAG CGGCCGTGCG TCCGGGTCCC GCAGTGCAGC


 


 751
GGCGGGTCCG GCAACTGCCG CTACGGGGGC ACGACGTACT TCCAGCCCGT



CCGCCCAGGC CGTTGACGGC GATGCCCCCG TGCTGCATGA AGGTCGGGCA


 


 801
GAACGAGATC CTGCAGGCCT ACGGTCTGCG TCTCGTCCTG GGCTGACACG



CTTGCTCTAG GACGTCCGGA TGCCAGACGC AGAGCAGGAC CCGACTGTGC


 


 851
CTCGCGGCGG GCCCTCCCCT GCCCGTCGCG CGCCGGCCCC ACCAGCCCGG



GAGCGCCGCC CGGGAGGGGA CGGGCAGCGC GCGGCCGGGG TGGTCGGGCC


 


 901
GCCG



CGGC


 








Cellulomonas cellasea (DSM 20118)



(SEQ ID NO: 64)










   1
VGRVRQLPLR GHDVLPARER DPAGLRSASR PGLTRSRRAR LDAAGPSARV



 


  51 
AAWYVDVPTN KLVVESVGDT AAAADAVAAA GLPADAVTLA TTEAPRTFVD


 


 101
VIGGNAYYIN ASSRCSVGFA VEGGFVTAGH CGRAGASTSS PSGTFRGSSF


 


 151
PGNDYAWVQV ASGNTPRGLV NNHSGGTVRV TGSQQAAVGS YVCRSGSTTG


 


 201
WRCGYVRAYN TTVRYAEGSV SGLIRTSVCA EPGDSGGSLV AGTQAQGVTS


 


 251
GGSGNCRYGG TTYFQPVNEI LQAYGLRLVL G*HARGGPSP ARRAPAPPAR


 


 301
A


 








Cellulomonas xylanilytica (LMG 21723)



(SEQ ID NO: 65)










   1
CGCTGCTCGA TCGGGTTCGC CGTGACGGGC GGCTTCGTGA CCGCCGGCCA




CTGCGGACGG TCCGGCGCGA CGACGACGTC GCCGAGCGGC ACGTTCGCCG


 



GCGACGAGCT AGCCCAAGCG GCACTGCCCG CCGAAGCACT GGCGGCCGGT



GACGCCTGCC AGGCCGCGCT GCTGCTGCAG CGGCTCGCCG TGCAAGCGGC


 


 101
GGTCCAGCTT TCCCGGCAAC GACTACGCCT GGGTCCGCGC GGCCTCGGGC



AACACGCCGG TCGGTGCGGT GAACCGCTAC GACGGCAGCC GGGTGACCGT


 



CCAGGTCGAA AGGGCCGTTG CTGATGCGGA CCCAGGCGCG CCGGAGCCCG



TTGTGCGGCC AGCCACGCCA CTTGGCGATG CTGCCGTCGG CCCACTGGCA


 


 201
GGCAGGGTCC ACCGACGCGG CCGTCGGTGC CGCGGTCTGC CGGTCGGGGT



CGACGACCGC GTGGGGCTGC GGCACGATCC AGTCCCGCGG CGCGAGCGTC


 



CCGGCCCAGG TGGCTGCGCC GGCAGCCACG GCGCCAGACG GCCAGCCCCA



GCTGCTGGCG CACCCCGACG CCGTGCTAGG TCAGGGCGCC GCGCTCGCAG


 


 301
ACGTACGCCC AGGGCACCGT CAGCGGGCTC ATCCGCACCA ACGTGTGCGC



CGAGCCGGGT GACTCCGGGG GGTCGCTGAT CGCGGGCACC CAGGCGCGGG


 



TGCATGCGGG TCCCGTGGCA GTCGCCCGAG TAGGCGTGGT TGCACACGCG



GCTCGGCCCA CTGAGGCCCC CCAGCGACTA GCGCCCGTGG GTCCGCGCCC


 


 401
GCGTGACGTC CGGCGGCTCC GGCAACTGC



CGCACTGCAG GCCGCCGAGG CCGTTGACG


 








Cellulomonas xylanilytica (LMG 21723)



(SEQ ID NO: 66)










   1
RCSIGFAVTG GFVTAGHCGR SGATTTSPSG TFAGSSFPGN DYAWVRAASG



 


  51
NTPVGAVNRY DGSRVTVAGS TDAAVGAAVC RSGSTTAWGC GTIQSRGASV


 


 101
TYAQGTVSGL IRTNVCAEPG DSGGSLIAGT QARGVTSGGS GNC


 








Oerskovia turbata (DSM 20577)



(SEQ ID NO: 67)










   1
ATGGCACGAT CATTCTGGAG GACGCTCGCC ACGGCGTGCG CCGCGACGGC




TACCGTGCTA GTAAGACCTC CTGCGAGCGG TGCCGCACGC GGCGCTGCCG


 


  51
ACTGGTTGCC GGCCCCGCAG CGCTCACCGC GAACGCCGCG ACGCCCACCC



TGACCAACGG CCGGGGCGTC GCGAGTGGCG CTTGCGGCGC TGCGGGTGGG


 


 101
CCGACACCCC GACCGTTTCA CCCCAGACCT CCTCGAAGGT CTCGCCCGAG



GGCTGTGGGG CTGGCAAAGT GGGGTCTGGA GGAGCTTCCA GAGCGGGCTC


 


 151
GTGCTCCGCG CCCTCCAGCG GGACCTGGGG CTGAGCGCCA AGGACGCGAC



CACGAGGCGC GGGAGGTCGC CCTGGACCCC GACTCGCGGT TCCTGCGCTG


 


 201
GAAGCGTCTG GCGTTCCAGT CCGACGCGGC GAGCACCGAG GACGCTCTCG



CTTCGCAGAC CGCAAGGTCA GGCTGCGCCG CTCGTGGCTC CTGCGAGAGC


 


 251
CCGACAGCCT GGACGCCTAC GCGGGCGCCT GGGTCGACCC TGCGAGGAAC



GGCTGTCGGA CCTGCGGATG CGCCCGCGGA CCCAGCTGGG ACGCTCCTTG


 


 301
ACCCTGTACG TCGGCGTCGC CGACAGGGCC GAGGCCAAGG AGGTCCGTTC



TGGGACATGC AGCCGCAGCG GCTGTCCCGG CTCCGGTTCC TCCAGGCAAG


 


 351
GGCCGGAGCG ACCCCCGTGG TCGTCGACCA CACGCTCGCC GAGCTCGACA



CCGGCCTCGC TGGGGGCACC AGCAGCTGGT GTGCGAGCGG CTCGAGCTGT


 


 401
CGTGGAAGGC GGCGCTCGAC GGTGAGCTCA ACGACCCCGC GGGCGTCCCG



GCACCTTCCG CCGCGAGCTG CCACTCGAGT TGCTGGGGCG CCCGCAGGGC


 


 451
AGCTGGTTCG TCGACGTCAC GACCAACCAG GTCGTCGTCA ACGTGCACGA



TCGACCAAGC AGCTGCAGTG CTGGTTGGTC CAGCAGCAGT TGCACGTGCT


 


 501
CGGCGGACGC GCCCTCGCGG AGCTGGCTGC CGCGAGCGCG GGCGTGCCCG



GCCGCCTGCG CGGGAGCGCC TCGACCGACG GCGCTCGCGC CCGCACGGGC


 


 551
CCGACGCCAT CACCTACGTG ACGACGACCG AGGCTCCTCG TCCCCTCGTC



GGCTGCGGTA GTGGATGCAC TGCTGCTGGC TCCGAGGAGC AGGGGAGCAG


 


 601
GACGTGGTGG GCGGCAACGC GTACACCATG GGTTCGGGCG GGCGCTGCTC



CTGCACCACC CGCCGTTGCG CATGTGGTAC CCAAGCCCGC CCGCGACGAG


 


 651
GGTCGGCTTC GCGGTGAACG GGGGCTTCAT CACGGCCGGG CACTGCGGCT



CCAGCCGAAG CGCCACTTGC CCCCGAAGTA GTGCCGGCCC GTGACGCCGA


 


 701
CGGTCGGCAC CCGCACCTCG GGGCCGGGCG GCACGTTCCG GGGGTCGAAC



GCCAGCCGTG GGCGTGGAGC CCCGGCCCGC CGTGCAAGGC CCCCAGCTTG


 


 751
TTCCCCGGCA ACGACTACGC CTGGGTGCAG GTCGACGCGG GTAACACCCC



AAGGGGCCGT TGCTGATGCG GACCCACGTC CAGCTGCGCC CATTGTGGGG


 


 801
GGTCGGCGCG GTCAACAACT ACAGCGGTGG GCGCGTCGCG GTCGCAGGGT



CCAGCCGCGC CAGTTGTTGA TGTCGCCACC CGCGCAGCGC CAGCGTCCCA


 


 851
CGACGGCCGC GCCCGTGGGG GCCTCGGTCT GCCGGTCCGG TTCCACGACG



GCTGCCGGCG CGGGCACCCC CGGAGCCAGA CGGCCAGGCC AAGGTGCTGC


 


 901
GGCTGGCACT GCGGCACCAT CGGCGCGTAC AACACCTCGG TGACGTACCC



CCGACCGTGA CGCCGTGGTA GCCGCGCATG TTGTGGAGCC ACTGCATGGG


 


 951
GCAGGGCACC GTCTCGGGGC TCATCCGCAC GAACGTGTGC GCCGAGCCCG



CGTCCCGTGG CAGAGCCCCG AGTAGGCGTG CTTGCACACG CGGCTCGGGC


 


1001
GCGACTCGGG CGGCTCGCTC CTCGCGGGCA ACCAGGCGCA GGGCGTGACC



CGCTGAGCCC GCCGAGCGAG GAGCGCCCGT TGGTCCGCGT CCCGCACTGG


 


1051
TCGGGCGGGT CGGGCAACTG CTCGTCGGGC GGGACGACGT ACTTCCAGCC



AGCCCGCCCA GCCCGTTGAC GAGCAGCCCG CCCTGCTGCA TGAAGGTCGG


 


1101
CGTCAACGAG GCCCTCGGGG GGTACGGGCT CACGCTCGTG ACCTCTGACG



GCAGTTGCTC CGGGAGCCCC CCATGCCCGA GTGCGAGCAC TGGAGACTGC


 


1151
GTGGGGGCCC GAGCCGCCGC CGACCGGGTG CCAGGGCTAT GCGCGGACCT



CACCCCCGGG CTCGGCGGCG GCTGGCCCAC GGTCCCGATA CGCGCCTGGA


 


1201
ACCAGGGCAG CGTCTCGGCC GGGACGTCGG TCGCGCAGCG AACGGTTCGT



TGGTCCCGTC GCAGAGCCGG CCCTGCAGCC AGCGCGTCGC TTGCCAAGCA


 


1251
ACGTCACGAC CGGGGGCGGG CGACCGGGTG TGCC



TGCAGTGCTG GCCCCCGCCC GCTGGCCCAC ACGG


 








Oerskovia turbata (DSM 20577)



(SEQ ID NO: 68)










   1
MARSFWRTLA TACAATALVA GPAALTANAA TPTPDTPTVS PQTSSKVSPE



 


  51
VLRALQRDLG LSAKDATKRL AFQSDAASTE DALADSLDAY AGAWVDPARN


 


 101
TLYVGVADRA EAKEVRSAGA TPVVVDHTLA ELDTWKAALD GELNDPAGVP


 


 151
SWFVDVTTNQ VVVNVHDGGR ALAELAAASA GVPADAITYV TTTEAPRPLV


 


 201
DVVGGNAYTM GSGGRCSVGF AVNGGFITAG HCGSVGTRTS GPGGTFRGSN


 


 251
FPGNDYAWVQ VDAGNTPVGA VNNYSGGRVA VAGSTAAPVG ASVCRSGSTT


 


 301
GWHCGTIGAY NTSVTYPQGT VSGLIRTNVC AEPGDSGGSL LAGNQAQGVT


 


 351
SGGSGNCSSG GTTYFQPVNE ALGGYGLTLV TSDGGGPSRR RPGARAMRGP


 


 401
TRAASRPGRR SRSERFVRHD RGRATGCA


 








Oerskovia jenensis (DSM 46000)



(SEQ ID NO: 69)










   1
GCCGCTGCTC GGTCGGCTTC GCGGTGAACG GCGGCTTCGT CACCGCAGGC




CGGCGACGAG CCAGCCGAAG CGCCACTTGC CGCCGAAGCA GTGGCGTCCG


 


  51
CACTGCGGGA CGGTGGGCAC CCGCACCTCG GGGCCGGGCG GCACGTTCCG



GTGACGCCCT GCCACCCGTG GGCGTGGAGC CCCGGCCCGC CGTGCAAGGC


 


 101
CGGGTCGAGC TTCCCCGGCA ACGACTACGC CTGGGTGCAG GTCGACGCGG



GCCCAGCTCG AAGGGGCCGT TGCTGATGCG GACCCACGTC CAGCTGCGCC


 


 151
GGAACACCCC GGTCGGGGCC GTCAACAACT ACAGCGGTGG ACGCGTCGCG



CCTTGTGGGG CCAGCCCCGG CAGTTGTTGA TGTCGCCACC TGCGCAGCGC


 


 201
GTCGCGGGCT CGACGGCCGC ACCCGTGGGT TCCTCGGTCT GCCGGTCCGG



CAGCGCCCGA GCTGCCGGCG TGGGCACCCA AGGAGCCAGA CGGCCAGGCC


 


 251
TTCCACGACG GGCTGGCGCT GCGGCACGAT CGCGGCCTAC AACAGCTCGG



AAGGTGCTGC CCGACCGCGA CGCCGTGCTA GCGCCGGATG TTGTCGAGCC


 


 301
TGACGTACCC GCAGGGGACC GTCTCCGGGC TCATCCGCAC CAACGTGTGC



ACTGCATGGG CGTCCCCTGG CAGAGGCCCG AGTAGGCGTG GTTGCACACG


 


 351
GCCGAGCCGG GCGACTCGGG CGGCTCGCTC CTCGCGGGCA ACCAGGCACA



CGGCTCGGCC CGCTGAGCCC GCCGAGCGAG GAGCGCCCGT TGGTCCGTGT


 


 401
GGGCCTGACG TCGGGCGGGT CGGGCAACTG CTCGTCGGGC GGCACGACGT



CCCGGACTGC AGCCCGCCCA GCCCGTTGAC GAGCAGCCCG CCGTGCTGCA


 


 451
ACTTCCAGCC CGTCAACGAG GCGCTCTCGG CCTACGGCCT CACGCTCGTG



TGAAGGTCGG GCAGTTGCTC CGCGAGAGCC GGATGCCGGA GTGCGAGCAC


 


 501
ACCTCCGGCG GCAGGGGCAA CTGC



TGGAGGCCGC CGTCCCCGTT GACG


 








Oerskovia jenensis (DSM 46000)



(SEQ ID NO: 70)










   1
RCSVGFAVNG GFVTAGHCGT VGTRTSGPGG TFRGSSFPGN DYAWVQVDAG



 


  51
NTPVGAVNNY SGGRVAVAGS TAAPVGSSVC RSGSTTGWRC GTIAAYNSSV


 


 101
TYPQGTVSGL IRTNVCAEPG DSGGSLLAGN QAQGLTSGGS GNCSSGGTTY


 


 151
FQPVNEALSA YGLTLVTSGG RGNC


 








Cellulosimicrobium cellulans (DSM 20424)



(SEQ ID NO: 71)










   1
CCACGGGCGG CGGGTCGGGC AGCGCGCTCG TCGGGCTCGC GGGCAAGTGC




GGTGCCCGCC GCCCAGCCCG TCGCGCGAGC AGCCCGAGCG CCCGTTCACG


 


  51
ATCGACGTCC CCGGGTCCGA CTTCAGTGAC GGCAAGCGCC TCCAGCTGTG



TAGCTGCAGG GGCCCAGGCT GAAGTCACTG CCGTTCGCGG AGGTCGACAC


 


 101
GACGTGCAAC GGGTCGCAGG CAGCGCTGGA CGTTCGAAGC CGACGGCACC



CTGCACGTTG CCCAGCGTCC GTCGCGACCT GCAAGCTTCG GCTGCCGTGG


 


 151
GTACGCGCGG GCGGCAAGTG CATGGACGTC GCGTGGGCGC CGCGGCCGAC



CATGCGCGCC CGCCGTTCAC GTACCTGCAG CGCACCCGCG GCGCCGGCTG


 


 201
GGCACGGCGC TCCAGCTCGC GAACTGCACG GCAACGCGGC CCAGAAGTTC



CCGTGCCGCG AGGTCGAGCG CTTGACGTGC CGTTGCGCCG GGTCTTCAAG


 


 251
GTGCTCAACG GCGCGGGCGA CCTCGTGTCG GTGCTGGCGA ACAAAGTGCG



CACGAGTTGC CGCGCCCGCT GGAGCACAGC CACGACCGCT TGTTTCACGC


 


 301
TCGACGCCGC CGGGTGCGCA CCGAGGTACT CGCGGCGCCG TACGAGCTCA



AGCTGCGGCG GCCCACGCGT GGCTCCATGA GCGCCGCGGC ATGCTCGAGT


 


 351
CGGCGACGTG CGCGGCGGCG ACCGCTACAT CACACGGGAC CCGGGCGCGT



GCCGCTGCAC GCGCCGCCGC TGGCGATGTA GTGTGCCCTG GGCCCGCGCA


 


 401
CGTCGGGCTC GGCCTGCTCG ATCGGGTACG CCGTCCAGGG CGGCTTCGTC



GCAGCCCGAG CCGGACGAGC TAGCCCATGC GGCAGGTCCC GCCGAAGCAG


 


 451
ACGGCGGGGC ACTGCGGACG CGGCGGGACA AGGAGAGTGC TCACCGCGAG



TGCCGCCCCG TGACGCCTGC GCCGCCCTGT TCCTCTCACG AGTGGCGCTC


 


 501
CTGGGCGCGC ATGGGGACGG TCCAGGCGGC GTCGTTCCCC GGCCACGACT



GACCCGCGCG TACCCCTGCC AGGTCCGCCG CAGCAAGGGG CCGGTGCTGA


 


 551
ACGCGTGGGT GCGCGTCGAC GCCGGGTTCT CCCCCGTCCC GCGGGTGAAC



TGCGCACCCA CGCGCAGCTG CGGCCCAAGA GGGGGCAGGG CGCCCACTTG


 


 601
AACTACGCCG GCGGCACCGT CGACGTCGCC GGCTCGGCCG AGGCGCCCGT



TTGATGCGGC CGCCGTGGCA GCTGCAGCGG CCGAGCCGGC TCCGCGGGCA


 


 651
GGGTGCGTCG GTGTGCCGCT CGGGCGCCAC GACCGGCTGG CGCTGCGGCG



CCCACGCAGC CACACGGCGA GCCCGCGGTG CTGGCCGACC GCGACGCCGC


 


 701
TCATCGAGCA GAAGAACATC ACCGTCAACT ACGGCAACGG CGACGTTCCC



AGTAGCTCGT CTTCTTGTAG TGGCAGTTGA TGCCGTTGCC GCTGCAAGGG


 


 751
GGCCTCGTGC GCGGCAGCGC GTGCGCGGAG GGCGGCGACT CGGGCGGGTC



CCGGAGCACG CGCCGTCGCG CACGCGCCTC CCGCCGCTGA GCCCGCCCAG


 


 801
GGTGATCTCC GGCAACCAGG CGCAGGGCGT CACGTCGGGC AGGATCAACG



CCACTAGAGG CCGTTGGTCC GCGTCCCGCA GTGCAGCCCG TCCTAGTTGC


 


 851
ACTGCTCGAA CGGCGGCAAG TTCCTCTACC AGCCCGATCG ACGGCCTGTC



TGACGAGCTT GCCGCCGTTC AAGGAGATGG TCGGGCTAGC TGCCGGACAG


 


 901
GCTCGTGACC ACGGGCGGCG GGTCGGGCAG CGCGCTCGTC GGGCTCGCGG



CGAGCACTGG TGCCCGCCGC CCAGCCCGTC GCGCGAGCAG CCCGAGCGCC


 


 951
GCAAGTGCAT CGACGTCCCC GGGTCCGACT TCAG



CGTTCACGTA GCTGCAGGGG CCCAGGCTGA AGTC


 








Cellulosimicrobium cellulans (DSM 20424)



(SEQ ID NO: 72)










   1
PRAAGRAARS SGSRASASTS PGPTSVTASA SSCGRATGRR QRWTFEADGT



 


  51
VRAGGKCMDV AWAPRPTARR SSSRTARQRG PEVRAQRRGR PRVGAGEQSA


 


 101
STPPGAHRGT RGAVRAHGDV RGGDRYITRD PGASSGSACS IGYAVQGGFV


 


 151
TAGHCGRGGT RRVLTASWAR MGTVQAASFP GHDYAWVRVD AGFSPVPRVN


 


 201
NYAGGTVDVA GSAEAPVGAS VCRSGATTGW RCGVIEQKNI TVNYGNGDVP


 


 251
GLVRGSACAE GGDSGGSVIS GNQAQGVTSG RINDCSNGGK FLYQPDRRPV


 


 301
ARDHGRRVGQ RARRARGQVH RRPRVRLQ


 








Promicromonospora citrea (DSM 43110)



(SEQ ID NO: 73)










   1
TTCCCCGGCA ACGACTACGC GTGGGTGAAC ACGGGCACGG ACGACACCCT




AAGGGGCCGT TGCTGATGCG CACCCACTTG TGCCCGTGCC TGCTGTGGGA


 


  51
CGTCGGCGCC GTGAACAACT ACAGCGGCGG CACGGTCAAC GTCGCGGGCT



GCAGCCGCGG CACTTGTTGA TGTCGCCGCC GTGCCAGTTG CAGCGCCCGA


 


 101
CGACCCGTGC CGCCGTCGGC GCGACGGTCT GCCGCTCGGG CTCCACGACC



GCTGGGCACG GCGGCAGCCG CGCTGCCAGA CGGCGAGCCC GAGGTGCTGG


 


 151
GGCTGGCACT GCGGCACCAT CCAGGCGCTG AACGCGTCGG TCACCTACGC



CCGACCGTGA CGCCGTGGTA GGTCCGCGAC TTGCGCAGCC AGTGGATGCG


 


 201
CGAGGGCACC GTGAGCGGCC TCATCCGCAC CAACGTGTGC GCCGAGCCCG



GCTCCCGTGG CACTCGCCGG AGTAGGCGTG GTTGCACACG CGGCTCGGGC


 


 251
GCGACTC



CGCTGAG


 








Promicromonospora citrea (DSM 43110)



(SEQ ID NO: 74)










   1
FPGNDYAWVN TGTDDTLVGA VNNYSGGTVN VAGSTRAAVG ATVCRSGSTT



 


  51
GWHCGTIQAL NASVTYAEGT VSGLIRTNVC AEPGD


 








Promicromonospora sukumoe (DSM 44121)



(SEQ ID NO: 75)










   1
TTCCCCGGCA ACGACTACGC GTGGGTGAAC GTCGGCTCCG ACGACACCCC




AAGGGGCCGT TGCTGATGCG CACCCACTTG CAGCCGAGGC TGCTGTGGGG


 


  51
GATCGGTGCG GTCAACAACT ACAGCGGCGG CACCGTGAAC GTCGCGGGCT



CTAGCCACGC CAGTTGTTGA TGTCGCCGCC GTGGCACTTG CAGCGCCCGA


 


 101
CGACCCAGGC CGCCGTCGGC TCCACCGTCT GCCGCTCCGG TTCCACGACC



GCTGGGTCCG GCGGCAGCCG AGGTGGCAGA CGGCGAGGCC AAGGTGCTGG


 


 151
GGCTGGCACT GCGGCACCAT CCAGGCCTTC AACGCGTCGG TCACCTACGC



CCGACCGTGA CGCCGTGGTA GGTCCGGAAG TTGCGCAGCC AGTGGATGCG


 


 201
CGAGGGCACC GTGTCCGGCC TGATCCGCAC CAACGTCTGC GCCGAGCCCG



GCTCCCGTGG CACAGGCCGG ACTAGGCGTG GTTGCAGACG CGGCTCGGGC


 


 251
GCGACTC



CGCTGAG


 








Promicromonospora sukumoe (DSM 44121)



(SEQ ID NO: 76)










   1
FPGNDYAWVN VGSDDTPIGA VNNYSGGTVN VAGSTQAAVG STVCRSGSTT



 


  51
GWHCGTIQAF NASVTYAEGT VSGLIRTNVC AEPGD


 








Xylanibacterium ulmi (LMG 21721)



(SEQ ID NO: 77)










   1
GCCGCTGCTC GATCGGGTTC GCCGTGACGG GCGGCTTCGT GACCGCCGGC




CGGCGACGAG CTAGCCCAAG CGGCACTGCC CGCCGAAGCA CTGGCGGCCG


 


  51
CACTGCGGAC GGTCCGGCGC GACGACGACG TCCGCGAGCG GCACGTTCGC



GTGACGCCTG CCAGGCCGCG CTGCTGCTGC AGGCGCTCGC CGTGCAAGCG


 


 101
CGGGTCCAGC TTTCCCGGCA ACGACTACGC CTGGGTCCGC GCGGCCTCGG



GCCCAGGTCG AAAGGGCCGT TGCTGATGCG GACCCAGGCG CGCCGGAGCC


 


 151
GAACACGCCG GTCGGTGCGG TGAACCGCTA CGACGGCAGC CGGGTGACCG



CTTGTGCGGC CAGCCACGCC ACTTGGCGAT GCTGCCGTCG GCCCACTGGC


 


 201
TGGCCGGGTC CACCGACGCG GCCGTCGGTG CCGCGGTCTG CCGGTCGGGG



ACCGGCCCAG GTGGCTGCGC CGGCAGCCAC GGCGCCAGAC GGCCAGCCCC


 


 251
TCGACGACCG CGTGGCGCTG CGGCACGATC CAGTCCCGCG GCGCGACGGT



AGCTGCTGGC GCACCGCGAC GCCGTGCTAG GTCAGGGCGC CGCGCTGCCA


 


 301
CACGTACGCC CAGGGCACCG TCAGCGGGCT CATCCGCACC AACGTGTGCG



GTGCATGCGG GTCCCGTGGC AGTCGCCCGA GTAGGCGTGG TTGCACACGC


 


 351
CCGAGCCGGG TGACTCCGGG GGGTCGCTGA TCGCGGGCAC CCAGGCGCAG



GGCTCGGCCC ACTGAGGCCC CCCAGCGACT AGCGCCCGTG GGTCCGCGTC


 


 401
GGCGTGACGT CCGGCGGCTC CGGCAACTGC



CCGCACTGCA GGCCGCCGAG GCCGTTGACG


 








Xylanibacterium ulmi: (LMG 21721)



(SEQ ID NO: 78)










   1
RCSIGFAVTG GFVTAGHCGR SGATTTSASG TFAGSSFPGN DYAWVRAASG



 


  51
NTPVGAVNRY DGSRVTVAGS TDAAVGAAVC RSGSTTAWRC GTIQSRGATV


 


 101
TYAQGTVSGL IRTNVCAEPG DSGGSLIAGT QAQGVTSGGS G






Inverse PCR

Inverse PCR was used to determine the full-length serine protease genes from chromosomal DNA of bacterial strains of the suborder Micrococcineae shown by PCR or immunoblotting to be novel homologues of the new Cellulomonas sp. 69B4 protease described herein.
Digested DNA was purified using the PCR purification kit (Qiagen, Catalogue #28106), and self-ligated with T4 DNA ligase (Invitrogen) according to the manufacturers' instructions. Ligation mixtures were purified with the PCR purification kit (Qiagen) and a PCR was performed with primers selected from the following list;










RV-1 Rest
5′ - ACCCACGCGTAGTCGTTGCC - 3′ 
(SEQ ID NO: 79)



 


RV-1 Cellul
5′ - ACCCACGCGTAGTCGTKGCCGGGG - 3′ 
(SEQ ID NO: 80)


 


RV-2 biaz-fimi
5′ - TCGTCGTGGTCGCGCCGG - 3′ 
(SEQ ID NO: 81)


 


RV-2 cella-flavi
5′ - CGACGTGCTCGCGCCCG - 3′ 
(SEQ ID NO: 82)


 


RV-2 cellul
5′ - CGCGCCCAGCTCGCGGTG - 3′ 
(SEQ ID NO: 83)


 


RV-2 turb
5′ - CGGCCCCGAGGTGCGGGTGCCG - 3′ 
(SEQ ID NO: 84)


 


Fw-1 biaz-fimi
5′ - CAGCGTCTCCGGCCTCATCCGC - 3′ 
(SEQ ID NO: 85)


 


Fw-1 cella-flavi
5′ - CTCGGTCTCGGGCCTCATCCGC - 3′ 
(SEQ ID NO: 86)


 


Fw-1 cellul
5′ - CGACGTTCCCGGCCTCGTGCGC - 3′ 
(SEQ ID NO: 87)


 


Fw-1 turb
5′ - CACCGTCTCGGGGCTCATCCGC - 3′ 
(SEQ ID NO: 88)


 


Fw-2 rest
5′ - AGCARCGTGTGCGCCGAGCC - 3′ 
(SEQ ID NO: 89)


 


Fw-2 cellul
5′ - GGCAGCGCGTGCGCGGAGGG - 3′ 
(SEQ ID NO: 90)


 


Fw-1 gelida
5′ - GCCGCTGCTCGATCGGGTTC - 3′  
(SEQ ID NO: 91)


 


Rv-1 gelida
5′ - GCAGTTGCCGGAGCCGCCGGACGT - 3'. 
(SEQ ID NO: 92)






The amplified PCR products were examined by agarose gel electrophoresis (0.8% agarose in TBE buffer (Invitrogen)). Distinct bands in the range 1.3-2.2 kbp for each organism were excised from the gel, purified using the Qiagen gel extraction kit and the sequence analyzed by BaseClear. Sequence analysis revealed that these DNA fragments covered some additional parts of protease gene homologues to the Cellulomonas 69B4 protease gene.
Genome Walking Using Rapid Amplification of Genomic Ends (RAGE)
A genome walking methodology (RAGE) known in the art was used to determine the full-length serine protease genes from chromosomal DNA of bacterial strains of the suborder Micrococcineae shown by PCR or immunoblotting to be novel homologues of the new Cellulomonas sp. 69B4 protease. RAGE was performed using the Universal GenomeWalkerTM Kit (BD Biosciences Clontech), some with modifications to the manufacturer's protocol (BD Biosciences user manual PT3042-1, Version # PR03300). Modifications to the manufacturer's protocol included addition of DMSO (3 μL) to the reaction mixture in 50 μL total volume due to the high GC content of the template DNA and use of AdvantageTM—GC Genomic Polymerase Mix (BD Biosciences Clontech) for the PCR reactions which were performed as follows;
















PCR 1
PCR 2









99° C. - 0.05 sec





94° C. - 0.25 sec/72° C. - 3.00 min
 7 cycles
 4 cycles



94° C. - 0.25 sec/67° C. - 4.00 min
39 cycles
24 cycles



67° C. - 7.00 min





15° C. - 1.00 min










PCR was performed with primers (Invitrogen, Paisley, UK) selected from the following list (listed in 5′ to 3′ orientation);











RV-1 Rest
ACCCACGCGTAGTCGTTGCC 
(SEQ ID NO: 79)



 


RV-1 Cellul
ACCCACGCGTAGTCGTKGCCGGGG 
(SEQ ID NO: 80)


 


RV-2 biaz-fimi
TCGTCGTGGTCGCGCCGG 
(SEQ ID NO: 81)


 


RV-2 cella-flavi
CGACGTGCTCGCGCCCG 
(SEQ ID NO: 82)


 


RV-2 cellul
CGCGCCCAGCTCGCGGTG 
(SEQ ID NO: 83)


 


RV-2 turb
CGGCCCCGAGGTGCGGGTGCCG 
(SEQ ID NO: 84)


 


Fw-1 biaz-fimi
CAGCGTCTCCGGCCTCATCCGC 
(SEQ ID NO: 85)


 


Fw-1 cella-flavi
CTCGGTCTCGGGCCTCATCCGC 
(SEQ ID NO: 86)


 


Fw-1 cellul
CGACGTTCCCGGCCTCGTGCGC 
(SEQ ID NO: 87)


 


Fvv-1 turb
CACCGTCTCGGGGCTCATCCGC 
(SEQ ID NO: 88)


 


Fw-2 rest
AGCARCGTGTGCGCCGAGCC 
(SEQ ID NO: 89)


 


Fw-2 cellul
GGCAGCGCGTGCGCGGAGGG 
(SEQ ID NO: 90)


 


Fw-1 gelida
GCCGCTGCTCGATCGGGTTC 
(SEQ ID NO: 91)


 


Rv-1 gelida
GCAGTTGCCGGAGCCGCCGGACGT 
(SEQ ID NO: 92)


 


Flavi FW1
TGCGCCGAGCCCGGCGACTCCGGC 
(SEQ ID NO: 93)


 


Flavi FW2
GGCACGACGTACTTCCAGCCCGTGAAC 
(SEQ ID NO: 94)


 


Flavi RV1
GACCCACGCGTAGTCGTTGCCGGGGAACGACGA 
(SEQ ID NO: 95)


 


Flavi RV2
GAAGGTCCCCGACGGTGACGACGTGCTCGCGCC 
(SEQ ID NO: 96)


 


Turb FW1
CAGGCGCAGGGCGTGACCTCGGGCGGGTCG 
(SEQ ID NO: 97)


 


Turb FW2
GGCGGGACGACGTACTTCCAGCCCGTCAA 
(SEQ ID NO: 98)


 


Cellu RV1
CACCCACGCGTAGTCGTGGCCGGGGAACGA 
(SEQ ID NO: 99)


 


Cellu RV2
GAAGCCGCCCTGGACGGCGTACCCGATCGAGCA 
(SEQ ID NO: 100)


 


Cellu FW1
TGCGCGGAGGGCGGCGACTCGGGCGGGTCG 
(SEQ ID NO: 101)


 


Cellu FW2
TTCCTCTACCAGCCCGTCAACCCGATCCTA 
(SEQ ID NO: 102)


 


Cella RV2
CGCCGCGGGGACGAACCCGCCCTCGACCGCGAA 
(SEQ ID NO: 103)


 


Cella RV1
CGCGTAGTCGTTGCCGGGGAACGACGAGCC 
(SEQ ID NO: 104)


 


Cella FW1
GGCCTCATCCGCACGAGCGTGTGCGCCGAG 
(SEQ ID NO: 105)


 


Cella FW2
ACGTCGGGCGGGTCCGGCAACTGCCGCTACGGGGGC 
(SEQ ID NO: 106)


 


Gelida RV1
GAGCCCGTACACCCGGAGGGCCTCGTTGACGGGCTGGAA 
(SEQ ID NO: 107)


 


Gelida RV2
CGTCACGCCCTGCGCCTGGTTGCCCGCGAG 
(SEQ ID NO: 108)


 


Gelida FW1
TCCAGCCCGTCAACGAGGCCCTCCGGGTGTACGGGCTC 
(SEQ ID NO: 109)


 


Gelida FW2
ACGTCGGTCGCGCAGCCGAACGGTTCGTACGTC 
(SEQ ID NO: 110)


 


Biazot RV1
CGTGGTCGCGCCGGTCGTGCCGCAGTGCCC 
(SEQ ID NO: 111)


 


Biazot RV2
GACGACGACCGTGTTGGTAGTGACGTCGACGTACCA 
(SEQ ID NO: 112)


 


Biazot FW1
TCCACCACGGGGTGGCGCTGCGGGACGATC 
(SEQ ID NO: 113)


 


Biazot FW2
GTGTGCGCCGAGCCCGGCGACTCCGGCGGC 
(SEQ ID NO: 114)


 


Turb RV C-mature
GCTCGGGCCCCCACCGTCAGAGGTCACGAGCGTGAG 
(SEQ ID NO: 115)


 


Turb FW signal
ATGGCACGATCATTCTGGAGGACGCTCGCCACGGCG 
(SEQ ID NO: 116)


 


Cellu internal FW
TGCTCGATCGGGTACGCCGTCCAGGGCGGCTTC 
(SEQ ID NO: 117)


 


Cellu internal RV
TAGGATCGGGTTGACGGGCTGGTAGAGGAA 
(SEQ ID NO: 118)


 


Biazot Int Fw
TGGTACGTCGACGTCACTACCAACACGGTCGTCGTC 
(SEQ ID NO: 119)


 


Biazot Int Rv
5′ - GCCGCCGGAGTCGCCGGGCTCGGCGCACAC 
(SEQ ID NO: 120)


 


flavi Nterm
5′ - GTSGACGTSATCGGSGGSAACGCSTACTAC 
(SEQ ID NO: 121)


 


flavi Cterm
5′ - SGCSGTSGCSGGNGANGA 
(SEQ ID NO: 122)


 


fimi Nterm
5′ - GTSGAYGTSATCGGCGGCGAYGCSTAC 
(SEQ ID NO: 123)


 


fimi Cterm
5′ - SGASGCGTANCCCTGNCC 
(SEQ ID NO: 124)






The PCR products were subcloned in the pCR4-TOPO TA cloning vector (Invitrogen) and transformed to E. coli Top10 one-shot electrocompetent cells (Invitrogen). The transformants were incubated (37° C., 260 rpm, 16 hours) in 2×TY medium with 100 μg/ml ampicillin. The isolated plasmid DNA (isolated using the Qiagen Qiaprep pDNA isolation kit) was sequenced by BaseClear.
Sequence Analysis
Full length polynucleotide sequences were assembled from PCR fragment sequences using the ContigExpress and AlignX programs in Vector NTI suite v. 9.0.0 (Invitrogen) using the original polynucleotide sequence obtained in Example 4 as template and the ASP mature protease and ASP full-length sequence for alignment. The results for the polynucleotide sequences are displayed in Table 7-1 and the translated amino acid sequences are displayed in Table 7-2. For each of the natural bacterial strains the polynucleotide sequences and translated amino acid sequences for each of the homologous proteases are provided above.
Table 7-1 provides comparison information between ASP protease and various other sequences obtained from other bacterial strains. Amino acid sequence information for Asp-mature-protease homologues is available from 13 species:
1. Cellulomonas biazotea DSM 20112
2. Cellulomonas flavigena DSM 20109
3. Cellulomonas fimi DSM 20113
4. Cellulomonas cellasea DSM 20118
5. Cellulomonas gelida DSM 20111
6. Cellulomonas iranensis DSM 14784
7. Cellulomonas xylanilytica LMG 21723
8. Oerskovia jenensis DSM 46000
9. Oerskovia turbata DSM 20577
9. Oerskovia turbata DSM 20577
10. Cellulosimicrobium cellulans DSM 20424
11. Promicromonospora citrea DSM 43110
12. Promicromonospora sukumoe DSM 44121
13. Xylanibacterium ulmi LMG 21721
Notably, the sequence from Cellulomonas gelida at 48 amino acids is too short for useful consensus alignment. Sequence alignment against Asp-mature for the remaining 12 species are provided herein. To date, complete mature sequence has been determined for Oerskovia turbata, Cellulomonas cellasea, Cellulomonas biazotea and Cellulosimicrobium cellulans. However, there are some problems and sequence fidelity is not guaranteed for the sequence information known to the public, Cellulomonas cellasea protease is clearly homologous to Asp (61.4% identity). However, the sequencing of 10 independent PCR fragments of the C-terminal region all gives a stop codon at position 184, suggesting that there is no C-terminal prosequence. In addition, Cellulosimicrobium cellulans is a close relative of Cellulomonas and clearly has an Asp homologous protease. However, the sequence identity is low, only 47.7%. It contains an insertion of 4 amino acids at position 43-44 and it is uncertain where the N-terminus of the protein begins. Nonetheless, the data provided here clearly show that there are enzymes homologous to the ASP protease described herein. Thus, it is intended that the present invention encompass the ASP protease isolated from Cellulomonas strain 69B4, as well as other homologous proteases.
In this Table, the nucleotide numbering is based on full-length gene of 69B4 protease (SEQ ID NO:2), where nt 1-84 encode the signal peptide, nt 85-594 encode the N-terminal prosequence, nt 595-1161 encode the mature 69B4 protease, and nt 1162-1485 encode the C-terminal prosequence.







TABLE 7-1







Percent Identity of Homologous Polynucleotide 


Sequences from Natural Isolate Strains Compared 


with ASP Mature Protease Gene Sequence













% Identity



Total

Overlap


Strain
Base Pairs
Overlap*
Mature Protease













69B4 (ASP) Protease
1485
 1-1485



Cellulomonas flavigena
555
595-1156
72.3


DSM20109





Cellulomonas
627
332-1355
73.7


biazotea DSM 20112





Cellulomonas
474
595-1068
78.7


fimi DSM 20113





Cellulomonas
462
1018-1485 
72.2


gelida DSM 20118





Cellulomonas
257
748-1004
75.2


iranensis DSM14784





Cellulomonas
904
294-1201
72.7


cellasea DSM 20118





Cellulomonas
429
640-1068
75.1


xylanilytica LMG 21723





Oerskovia
1284
 1-1291
73.1


turbata DSM 20577





Oerskovia
387
638-1158
72.7


jenensis DSM 46000





Cellulosimicrobium
984
251-1199
63.1


cellulans DSM20424





Promicromonospora
257
748-1004 
75.9


citrea DSM 43110





Promicromonospora
257
748-1004 
77.4


sukumoe DSM 44121





Xylanibacterium
430
638-1068 
77.0


ulmi LMG21721









The following Table (Table 7-2) provides information regarding the translated amino acid sequence data in natural isolate strains compared with full-length ASP.







TABLE 7-2







Translated Amino Acid Sequence Data Comparisons














Signal






Total
peptide
N-terminal pro
Mature protease
C-terminal pro



amino
overlap:
overlap:
overlap:
overlap:


Strain
acids
position
position
position
position















69B4 (ASP)
495
28 (1-28)
170 (29-198)
189 (199-387)
108 (388-


Protease




495)


Cellulomonas
185


185 (199-383)



flavigena



id 68.6%



DSM20109







Cellulomonas
335

84 (104-198)
189 (199-387)
62 (388-451)


biazotea DSM


id 35.8%
id 70.4%
id 64.1%


20112



complete



Cellulomonas
144 


144 (199-342)



fimi DSM 20113



id 74.3%



Cellulomonas
154


48 (340-387)
106 (388-495)


gelida DSM 20118



id 68.8%
id 63.9%







complete


Cellulomonas
85


85 (250-334)



iranensis



id 65.9%



DSM14784







Cellulomonas
301

98 (99-198)
189 (199-387)
13 (388-400)


cellasea DSM


id 31.0%
id 68.3%
id 30.8%


20118



complete



Cellulomonas
143


143(214-356)



xylanilytica LMG



id 73.4%



21723







Oerskovia
428
29 (2-30)
171 (31-198)
188 (201-389)
40 (390-429)


turbata DSM 20577

id 43.3%
id 44.4%
id 73.0%
id 10.0%






complete



Oerskovia
174


174 (214-334)



jenensis DSM



id 73.6%



46000







Cellulosimicrobium
328

117 (82-198)
199 (199-387)
12 (388-399)


cellulans


id 6%
id 47.7%



DSM20424



complete



Promicromonospora
85


85 (250-334)



citrea DSM 43110



id 75.3%



Promicromonospora
85


85 (250-334)



sukumoe DSM



id 64.7%



44121







Xylanibacterium
141


141 (214-354)



ulmi LMG21721



id 72.3%









These results clearly show that bacterial strains of the suborder Micrococcineae, including the families Cellulomonadaceae and Promicromonosporaceae posess genes that are homologous with the 69B4 protease. Over the region of the mature 69B protease, the gene sequence identities range from about 60%-80%. The amino acid sequences of these homologous sequences exhibit about 45%-80% identity with the mature 69B4 protease protein. In contrast to the majority of streptogrisin proteases derived from members of the suborder Streptomycineae, these 69B4 (Asp) protease homologues from the suborder Micrococcineae possess six cysteine residues, which form three disulfide bridges in the mature 69B4 protease protein.
Indeed, in spite of the incomplete sequences provided herein and questions regarding fidelity, the present invention provides essential elements of the Asp group of proteases and comparisons with streptogrisins. Asp is uniquely Asp is characterized, along with Streptogrisin C, as having 3 disulfide bridges. In the following sequence, the Asp amino acids are printed in bold and the fully conserved residues are underlined. The active site residues are marked with # and double underlined. The cysteine residues are marked with * and underlined. The disulfide bonds are located between C17 and C38, C95 and C105, and C131 and C158.








(SEQ ID NO: 125)



1           5           8  17          20              25            30   32



X D V [I, V] G G [N, D] [X9] C* S [I, V] G [F, Y ] A V X G G F [I, V] T A G H#


 


33    35        40     45        50        55             60


C* G [X2] G [X2] TN [X4] G T F X G S S F P G N D# Y A [F, W] V [X4]


 


65                    72          75                           80


[G, D] [X2] [L, P] [X3] V N [N, R] [Y, H] [S, D] G [G, S] [R, T] V X V [A, T] G


 


85                 90             95             100                 105


[H, S] [T, Q] X A X V G [S, A] X V C* R S G [S, A] T T [G, A] W [H, R] C* G


 


                 112            115                  120                    125


[T, Y] [I, V] [X3] [N, G] X [S, T] V X Y [P, A] [E, Q] G [T, S, D] V [R, S] G L


 


                          130   131          135  137    140


[I, V] R [T, G] [T, N, S] [V, A] C* A E [P, G] G D S# G G S [L, V] [L, V, I] [A. S]


 


   145                 150                155                 158


G [N, T] Q A [Q, R] G [V, L] T S G [G, R] [S, I] [G, N] [N, D] C* [X2] G


 


162  167 169  189


G [X4] Q P [X21]






Table 7-3 (below) indicates the positions where ASP and Streptogrisin C differ:







TABLE 7-3







Positions At Which ASP and Streptogrisin C Differ












ASP
ASP
ASP
Streptogrisin C



Position
Amino Acid
Homologs
Amino Acid







22
A
 R?
S



25
G
G
N



28
I
V
A



51
S
 N?
T



55
N
 H?
R



57
Y
Y
I



65
G
D
N



74
N
R
G



76
S
D
G



77
G
G
R



79
R
T
D



88
A
A
S



122
V
V
I



125
L
L
V



126
I
V
T



141
L
V
Y



145
N
T
S










Example 8
Mass Spectrometric Sequencing of ASP Homologues
In this Example, experiments conducted to confirm the DNA-derived sequence as well as verify/establish the N-terminal and C-terminal sequences of the mature ASP homologues are described. The microorganisms utilized in these experiments were the following:
1. Cellulomonas biazotea DSM 20112
2. Cellulomonas flavigena DSM 20109
3. Cellulomonas fimi DSM 20113
4. Cellulomonas cellasea DSM 20118
7. Oerskovia jenensis DSM 46000
8. Oerskovia turbata DSM 20577
9. Cellulosimicrobium cellulans DSM 20424
The micropurified ASP homologues were subjected to mass spectrometry-based protein sequencing procedures which consisted of these major steps: micropurification, gel electrophoresis, in-gel proteolytic digestion, capillary liquid chromatography electrospray tandem mass spectrometry (nanoLC-ESI-MS/MS), database searching of the mass spectrometric data, and de novo sequencing. Details of these steps are described what follows. As described previously in Example 6, concentrated culture sample (about 200 ml) was added to 500 ml 1M CaCl2 and centrifuged at 14,000 rpm (model 5415C Eppendorf) for min. The supernatant was cooled on ice and acidified with 200 ml 1N HCl. After 5 min, 200 ml 50% trichloroacetic acid were added and the sample was centrifuged for 4 min at 14,000 rpm (model 5415C Eppendorf). The supernatant was discarded and the pellet was washed first with water and then with 90% acetone. The pellet, after being dried in the speed vac, was dissolved in 2× Protein Preparation (Tris-Glycine Sample Buffer; Novex) buffer and diluted 1+1 with water before being applied to the SDS-PAGE gel. SDS-PAGE was run with NuPAGE MES SDS Running Buffer. SDS-PAGE gel (1 mm NuPAGE 10% Bis-Tris; Novex) was developed and stained using standard protocols known in the art. Following SDS-PAGE, bands corresponding to ASP homologues were excised and processed for mass spectrometric peptide sequencing using standard protocols in the art.
Peptide mapping and sequencing was performed using capillary liquid chromatography electrospray tandem mass spectrometry (nanoLC-ESI-MS/MS). This analysis systems consisted of capillary HPLC system (model COLO; Waters) and mass spectrometer (model Qtof Ultima API; Waters). Peptides were loaded on a pre-column (PepMap100 C18, 5 um, 100 A, 300 um ID×1 mm; Dionex) and chromatographed on capillary columns (Biobasic C18 75 um×10 cm; New Objectives) using a gradient from 0 to 100% solvent B in 45 min at a flow rate of 200nL/min (generated using a static split from a pump flow rate of 5uL/min). Solvent A consisted of 0.1% formic acid in water; and solvent B was 0.1% formic acid in acetonitrile. The mass spectrometer was operated with the following parameters: spray voltage of 3.1 kV, desolavation zone at 15° C., mass spectra acquired from 400 to 1900 m/z, resolution of 6000 in v-mode. Tandem MS spectra were acquired in data dependent mode with two most intense peaks selected and fragmented with mass dependent collision energy (as specified by vendor) and collision gas (argon) at 2.5×10-5 torr.
The identities of the peptides were determined using a database search program (Mascot, Matrix Science) using a database containing ASP homologue DNA-obtained sequences. Database searches were performed with the following parameters: no enzyme selected, peptide error of 2.5Da, MS/MS ions error of 0.1Da, and variable modification of carboxyaminomethyl cysteine). For unmatched MS/MS spectra, manual de novo sequence assignments were performed. For example, FIG. 4 shows the sequence of N-terminal most tryptic peptide from C. flavigena determined from this tandem mass spectrum. In Table 8-1, the percentage of the sequence verified on the protein level for various homologues are reported along with N-terminal and C-terminal peptide sequences.







TABLE 8-1







Mass Spec. Sequencing of ASP Homologues










Sequence




Verified
N-terminal



%
and



Trypsin,
C-terminal


ASP
Chymotrypsin
Sequences


Homologue
Digests
(Peptide Mass in Da)





Cellulomonas
81, 81
[IY]AWDAFAENVVDWSSR (SEQ ID


cellasea

NO:126)(2026.7)




YGGTTYFQPVNEILQAY (SEQ ID




NO:127)(1961.8)


Cellulomonas
70, 50
VDVI\LGGNAYYI/L[ . . . ]R (SEQ ID


flavigena

NO:128)(1697.7)


Cellulomonas
21, ND
VDVI/LGGDAY[ . . . ]R (SEQ ID 


fimi

NO:129)(1697.6)





Notes:


ND: not determined


sequence not determined indicated in [..] 


sequence order not determined indicated by [ ] 


isobaric residues not distinguished indicated by I\L






Example 9
Protease Production in Streptomyces lividans 
This Example describes experiments conducted to develop methods for production of protease by S. lividans. Thus, a plasmid comprising a polypeptide encoding a polypeptide having proteolytic activity was constructed and used such vector to transform Streptomyces lividans host cells The methods used for this transformation are more fully described in U.S. Pat. No. 6,287,839 and WO 02/50245, both of which are herein expressly incorporated by reference.
One plasmid developed during these experiments was designated as “pSEG69B4T.” The construction of this plasmid made use of one pSEGCT plasmid vector (See, WO 02/50245). A glucose isomerase (“GI”) promoter operably linked to the structural gene encoding the 69B4 protease was used to drive the expression of the protease. A fusion between the GI-promoter and the 69B4 signal-sequence, N-terminal prosequence and mature sequence was constructed by fusion-PCR techniques as a XbaI-BamHI fragment. The fragment was ligated into plasmid pSEGCT digested with XbaI and BamHI, resulting in plasmid pSEG69B4T (See, FIG. 6). Although the present Specification provides specific expression vectors, it is contemplated that additional vectors utilizing different promoters and/or signal sequences combined with various prosequences of the 69B4 protease will find use in the present invention.
An additional plasmid developed during the experiments was designated as “pSEA469B4CT” (See, FIG. 7). As with the pSEG69B4T plasmid, one pSEGCT plasmid vector was used to construct this plasmid. To create the pSEA469B4CT, the Aspergillus niger (regulatory sequence) (“A4”) promoter was operably linked to the structural gene encoding the 69B4 protease, and used to drive the expression of the protease. A fusion between the A4-promoter and the Cel A (from Streptomyces coelicolor) signal-sequence, the asp-N-terminal prosequence and the asp mature sequence was constructed by fusion-PCR techniques, as a XbaI-BamHI fragment. The fragment was ligated into plasmid pSEA4GCT digested with XbaI and BamHI, resulting in plasmid pSEA469B4CT (See, FIG. 7). The sequence of the A4 (A. niger) promoter region is:










(SEQ ID NO: 130)



  1
         TCGAA CTTCAT GTTCGA GTTCTT GTTCAC GTAGAA GCCGGA GATGTG AGAGGT




         AGCTT GAAGTA CAAGCT CAAGAA CAAGTG CATCTT CGGCCT CTACAC TCTCCA


 


 61
 GATCTG GAACTG CTCACC CTCGTT GGTGGT GACCTG GAGGTA AAGCAA GTGACC CTTCTG



 CTAGAC CTTGAC GAGTGG GAGCAA CCACCA CTGGAC CTCCAT TTCGTT CACTGG GAAGAC


 


121
 GCGGAG GTGGTA AGGAAC GGGGTT CCACGG GGAGAG AGAGAT GGCCTT GACGGT CTTGGG



 CGCCTC CACCAT TCCTTG CCCCAA GGTGCC CCTCTC TCTCTA CCGGAA CTGCCA GAACCC


 


181
 AAGGGG AGCTTC NGCGCG GGGGAG GATGGT CTTGAG AGAGGG GGAGCT AGTAAT GTCGTA



 TTCCCC TCGAAG NCGCGC CCCCTC CTACCA GAACTC TCTCCC CCTCGA TCATTA CAGCAT


 


241
 CTTGGA CAGGGA GTGCTC CTTCTC CGACGC ATCAGC CACCTC AGCGGA GATGGC ATCGTG



 GAACCT GTCCCT CACGAG GAAGAG GCTGCG TAGTCG GTGGAG TCGCCT CTACCG TAGCAC


 


301
 CAGAGA CAGACC



 GTCTCT GTCTGG






In these experiments, the host Streptomyces lividans TK23 was transformed with either of the vectors described above using protoplast methods known in the art (See e.g., Hopwood, et al., Genetic Manipulation of Streptomyces, A Laboratory Manual, The John Innes Foundation, Norwich, United Kingdom [1985]).
The transformed culture was expanded to provide two fermentation cultures. At various time points, samples of the fermentation broths were removed for analysis. For the purposes of this experiment, a skimmed milk procedure was used to confirm successful cloning. In these methods, 30 μl of the shake flask supernatant was spotted in punched out holes in skim milk agar plates and incubated at 37° C. The incubated plates were visually reviewed after overnight incubation for the presence of halos. For purposes of this experiment, the same samples were also assayed for protease activity and for molecular weight (SDS-PAGE). At the end of the fermentation run, full length protease was observed by SDS-PAGE.
A sample of the fermentation broth was assayed as follows: 10 μl of the diluted 15 supernatant was taken and added to 190 μl AAPF substrate solution (conc. 1 mg/ml, in 0.1 M Tris/0.005% TWEEN, pH 8.6). The rate of increase in absorbance at 410 nm due to release of p-nitroaniline was monitored (25° C.). The assay results of the fermentation broth of 3 clones (X, Y, W) obtained using the pSEG69B4T and two clones using the pSEA469B4T indicated that Asp was expressed by both constructs. able XXI. Results for Two Clones (pSEA469B4T). Indeed, the results obtained in these experiments showed that the polynucleotide encoding a polypeptide having proteolytic activity was expressed in Streptomyces lividans, using both of these expression vectors. Although two vectors are described in this Example, it is contemplated that additional expression vectors using different promoters and/or signal sequences combined with different combinations of 69B4 protease: +/−N terminal and C terminal prosequence in the pSEA4CT backbone (vector), as well as other constructs will find use in the present invention.
Example 10
Protease Production in B. subtilis 
In this Example, experiments conducted to produce protease 69B4 (also referred to herein as “ASP,” “Asp,” and “ASP protease,” and “Asp protease”) in B. subtilis are described. In this Example, the transformation of plasmid pHPLT-ASP-C1-2 (See, Table 10-1; and FIG. 9), into B. subtilis is described. Transformation was performed as known in the art (See e.g., WO 02/14490, incorporated herein by reference. To optimize ASP expression in B. subtilis a synthetic DNA sequence was produced by DNA2.0, and utilized in these expression experiments. The DNA sequence (synthetic ASP DNA sequence) provided below, with codon usage adapted for Bacillus species, encodes the wild type ASP precursor protein:








(SEQ ID NO: 131)



ATGACACCACGAACTGTCACAAGAGCTCTGGCTGIGGCAACAGCAGCTGCTACACTCTTGGCTGGGGGTAT



 


GGCAGCACAAGCTAACGAACCGGCTCCTCCAGGATCTGCATCAGCCCCTCCACGATTAGCTGAAAAACTTGA


 


CCCTGACTTACTTGAAGCAATGGAACGCGATCTGGGGTTAGATGCAGAGGAAGCAGCTGCAACGTTAGCTTT


 


TCAGCATGACGCAGCTGAAACGGGAGAGGCTCTTGCTGAGGAACTCGACGAAGATTTCGCGGGCACGTGGG


 


TTGAAGATGATGTGCTGTATGTTGCAACCACTGATGAAGATGCTGTTGAAGAAGTCGAAGGCGAAGGAGCAA


 


CTGCTGTGACTGTTGAGCATTCTCTTGCTGATTTAGAGGCGTGGAAGACGGTTTTGGATGCTGCGCTGGAGG


 


GTCATGATGATGTGCCTACGTGGTACGTCGACGTGCCTACGAATTCGGTAGTCGTTGCTGTAAAGGCAGGAG


 


CGCAGGATGTAGCTGCAGGACTTGTGGAAGGCGCTGATGTGCCATCAGATGCGGTCACTTTTGTAGAAACG


 


GACGAAACGCCTAGAACGATGTTCGACGTAATTGGAGGCAACGCATATACTATTGGCGGCCGGTCTAGATG


 


TTCTATCGGATTCGCAGTAAACGGTGGCTTCATTACTGCCGGTCACTGCGGAAGAACAGGAGCCACTACTG


 


CCAATCCGACTGGCACATTTGCAGGTAGCTCGTTTCCGGGAAATGATTATGCATTCGTCCGAACAGGGGCA


 


GGAGTAAATTTGCTTGCCCAAGTCAATAACTACTCGGGCGGCAGAGTCCAAGTAGCAGGACATACGGCCG


 


CACCAGTTGGATCTGCTGTATGCCGCTCAGGTAGCACTACAGGTTGGCATTGCGGAACTATCACGGCGCT


 


GAATTCGTCTGTCACGTATCCAGAGGGAACAGTCCGAGGACTTATCCGCACGACGGTTTGTGCCGAACCA


 


GGTGATAGCGGAGGTAGCCTTTTAGCGGGAAATCAAGCCCAAGGTGTCACGTCAGGTGGTTCTGGAAATT


 


GTCGGACGGGGGGAACAACATTCTTTCAACCAGTCAACCCGATTTTGCAGGCTTACGGCCTGAGAATGATT


 


ACGACTGACTCTGGAAGTTCCCCTGCTCCAGCACCTACATCATGTACAGGCTACGCAAGAACGTTCACAGG


 


AACCCTCGCAGCAGGAAGAGCAGCAGCTCAACCGAACGGTAGCTATGTTCAGGTCAACCGGAGCGGTACAC


 


ATTCCGTCTGTCTCAATGGACCTAGCGGTGCGGACTTTGATTTGTATGTGCAGCGATGGAATGGCAGTAGCT


 


GGGTAACCGTCGCTCAATCGACATCGCCGGGAAGCAATGAAACCATTACGTACCGCGGAAATGCTGGATATT


 


ATCGCTACGTGGTTAACGCTGCGTCAGGATCAGGAGCTTACACAATGGGACTCACCCTCCCCTGA






In the above sequence, bold indicates the DNA that encodes the mature protease, standard font indicates the leader sequence, and the underline indicates the N-terminal and so C-terminal prosequences.
Expression of the Synthetic ASP Gene
Asp expression cassettes were constructed in the pXX-KpnI (See, FIG. 15) or p2JM103-DNNDPI (See, FIG. 16) vectors and subsequently cloned into the pHPLT vector ss (See, FIG. 17) for expression of ASP in B. subtilis. pXX-KpnI is a pUC based vector with the aprE promoter (B. subtilis) driving expression, a cat gene, and a duplicate aprE promoter for amplification of the copy number in B. subtilis. The bla gene allows selective growth in E. coli. The KpnI, introduced in the ribosomal binding site, downstream of the aprE promoter region, together with the HindIII site enables cloning of Asp expression cassettes in pXX-KpnI. The vector p2JM103-DNNDPI contains the aprE promoter (B. subtilis) to drive expression of the BCE103 cellulase core (endo-cellulase from an obligatory alkaliphilic Bacillus; See, Shaw et al., J. Mol. Biol., 320:303-309 [2002]), in frame with an acid labile linker (DDNDPI [SEQ ID NO:132]; See, Segalas et al., FEBS Lett., 371:171-175 [1995]). The ASP expression cassette (BamHI and HindIII) was fused to BCE103-DDNDPI fusion protein. When secreted, ASP is cleaved of the cellulase core to turn into the mature protease
pHPLT (See, FIG. 17; and Solingen et al., Extremophiles 5:333-341 [2001]) contains the thermostable amylase LAT promoter (PLAT) of Bacillus licheniformis, followed by XbaI and HpaI restriction sites for cloning ASP expression constructs. The following sequence is that of the BCE103 cellulase core with DNNDPI acid labile linker. In this sequence, the bold indicates the acid-labile linker, while the standard font indicates the BCE103 core.








(DNA; SEQ ID NO: 133) and (Amino Acid; SEQ ID NO: 134)











 V  R   S  K   K  L   W  I   S  L   L  F   A  L   T  L   I  F   T  M



  1
GTGAGA AGCAAA AAATTG TGGATC AGCTTG TTGTTT GCGTTA ACGTTA ATCTTT ACGATG



CACTCT TCGTTT TTTAAC ACCTAG TCGAAC AACAAA CGCAAT TGCAAT TAGAAA TGCTAC


 



 A  F   S  N   M  S   A  Q   A  D   D  Y   S  V   V  E   E  H   G  Q


 61
GCGTTC AGCAAC ATGAGC GCGCAG GCTGAT GATTAT TCAGTT GTAGAG GAACAT GGCCAA



CGCAAG TCGTTG TACTCG CGCGTC CGACTA CTAATA AGTCAA CATCTC CTTGTA CCCGTT


 



 L  S   I  S   N  G   E  L   V  N   E  R   G  E   Q  V   Q  L   K  G


121
CTAAGT ATTAGT AACGGT GAATTA GTCAAT GAACGA GGCGAA CAAGTT CAGTTA AAAGGG



GATTCA TAATCA TTGCCA CTTAAT CAGTTA CTTGCT CCGCTT GTTCAA GTCAAT TTTCCC


 



 M  S   S  H   G  L   Q  W   Y  G   Q  F   V  N   Y  E   S  M   K  W


181
ATGAGT TCCCAT GGTTTG CAATGG TACGGT CAATTT GTAAAC TATGAA AGCATG AAATGG



TACTCA AGGGTA CCAAAC GTTACC ATGCCA GTTAAA CATTTG ATACTT TCGTAC TTTACC


 



 L  R   D  D   W  G   I  T   V  F   R  A   A  M   Y  T   S  S   G  G


241
CTAAGA GATGAT TGGGGA ATAACT GTATTC CGAGCA GCAATG TATACC TCTTCA GGAGGA



GATTCT CTACTA ACCCCT TATTGA CATAAG GCTCGT CGTTAC ATATGG AGAAGT CCTCCT


 



 Y  I   D  D   P  S   V  K   E  K   V  K   E  T   V  E   A  A   I  D


301
TATATT GACGAT CCATCA GTAAAG GAAAAA GTAAAA GAGACT GTTGAG GCTGCG ATAGAC



ATATAA CTGCTA GGTAGT CATTTC CTTTTT CATTTT CTCTGA CAACTC CGACGC TATCTG


 



 L  G   I  Y   V  I   I  D   W  H   I  L   S  D   N  D   P  N   I  Y


361
CTTGGC ATATAT GTGATC ATTGAT TGGCAT ATCCTT TCAGAC AATGAC CCGAAT ATATAT



GAACCG TATATA CACTAG TAACTA ACCGTA TAGGAA AGTCTG TTACTG GGCTTA TATATA


 



 K  E   E  A   K  D   F  F   D  E   M  S   E  L   Y  G   D  Y   P  N


421
AAAGAA GAAGCG AAGGAT TTCTTT GATGAA ATGTCA GAGTTG TATGGA GACTAT CCGAAT



TTTCTT CTTCGC TICCTA AAGAAA CTACTT TACAGT CTCAAC ATACCT CTGATA GGCTTA


 



 V  I   Y  E   I  A   N  E   P  N   G  S   D  V   T  W   D  N   Q  I


481
GTGATA TACGAA ATTGCA AATGAA CCGAAT GGTAGT GATGTT ACGTGG GACAAT CAAATA



CACTAT ATGCTT TAACGT TTACTT GGCTTA CCATCA CTACAA TGCACC CTGTTA GTTTAT


 



 K  P   Y  A   E  E   V  I   P  V   I  R   D  N   D  P   N  N   I  V


541
AAACCG TATGCA GAAGAA GTGATT CCGGTT ATTCGT GACAAT GACCCT AATAAC ATTGTT



TTTGGC ATACGT CTTCTT CACTAA GGCCAA TAAGCA CTGTTA CTGGGA TTATTG TAACAA


 



 I  V   G  T   G  T   W  S   Q  D   V  H   H  A   A  D   N  Q   L  A


601
ATTGTA GGTACA GGTACA TGGAGT CAGGAT GTCCAT CATGCA GCCGAT AATCAG CTTGTA



TAACAT CCATGT CCATGT ACCTCA GTCCTA CAGGTA GTACGT CGGCTA TTAGTC GAACGT


 



 D  P   N  V   M  Y   A  F   H  F   Y  A   G  T   H  G   Q  N   L  R


661
GATCCT AACGTC ATGTAT GCATTT CATTTT TATGCA GGAACA CATGGA CAAAAT TTACGA



CTAGGA TTGCAG TACATA CGTAAA GTAAAA ATACGT CCTTGT GTACCT GTTTTA AATGCT


 



 D  Q   V  D   Y  A   L  D   Q  G   A  A   I  F   V  S   E  W   G  T


721
GACCAA GTAGAT TATGCA TTAGAT CAAGGA GCAGCG ATATTT GTTAGT GAATGG GGGACA



CTGGTT CATCTA ATACGT AATCTA GTTCCT CGTCGC TATAAA CAATCA CTTACC CCCTGT


 



 S  A   A  T   G  D   G  G   V  F   L  D   E  A   Q  V   W  I   D  F


781
AGTGCA GCTACA GGTGAT GGTGGT GTGTTT TTAGAT GAAGCA CAAGTG TGGATT GACTTT



TCACGT CGATGT CCACTA CCACCA CACAAA AATCTA CTTCGT GTTCAC ACCTAA CTGAAA


 



 M  D   E  R   N  L   S  W   A  N   W  S   L  T   H  K   D  E   S  S


841
ATGGAT GAAAGA AATTTA AGCTGG GCCAAC TGGTCT CTAACG CATAAG GATGAG TCATCT



TACCTA CTTTCT TTAAAT TCGACC CGGTTG ACCAGA GATTGC GTATTC CTACTC AGTAGA


 



 A  A   L  M   P  G   A  N   P  T   G  G   W  T   E  A   E  L   S  P


901
GCAGCG TTAATG CCAGGT GCAAAT CCAACT GGTGGT TGGACA GAGGCT GAACTA TCTCCA



CGTCGC AATTAC GGTCCA CGTTTA GGTTGA CCACCA ACCTGT CTCCGA CTTGAT AGAGGT


 



 S  G   T  F   V  R   E  K   I  R   E  S   A  S   D  N   N  D   P  I


961 
TCTGGT ACATTT GTGAGG GAAAGA ATAAGA GAATCA GCATCT GACAAC AATGAT CCCATA



AGACCA TGTAAA CACTCC CTTTTT TATTCT CTTAGT CGTAGA CTGTTG TTACTA GGGTAT






The Asp expression cassettes were cloned in the pXX-KpnI vector containing DNA encoding the wild type Asp signal peptide, or a hybrid signal peptide constructed of 5 subtilisin AprE N-terminal signal peptide amino acids fused to the 25 Asp C-terminal signal peptide amino acids (MRSKKRTVTRALAVATAAATLLAGGMAAQA (SEQ ID NO:135), or a hybrid signal peptide constructed of 11 subtilisin AprE N-terminal signal peptide amino acids fused to the 19 asp C-terminal signal peptide amino acids (MRSKKLWISLLLAVATAAATLLAGGMAAQA (SEQ ID NO:136). These expression cassettes were also constructed with the asp C-terminal prosequence encoding DNA in frame. Another expression cassette, for cloning in the p2JM103-DNNDPI vector, encodes the ASP N-terminal pro- and mature sequence.
The Asp expression cassettes cloned in the pXX-KpnI or p2JM103-DNNDPI vector were transformed into E. coli (Electromax DH10B, Invitrogen, Cat. No. 12033-015). The primers and cloning strategy used are provided in Table 10-1. Subsequently, the expression cassettes were cloned from these vectors and introduced in the pHPLT expression vector for transformation into a B. subtilis (ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32, ΔamyE::(xyIR,pxyIA-comK) strain. The primers and cloning strategy for ASP expression cassettes cloning in pHPLT are provided in Table 10-2. Transformation to B. subtilis was performed as described in WO 02/14490, incorporated herein by reference. FIGS. 12-21 provide plasmid maps for various plasmids described herein.







TABLE 10-1







ASP in pXX-KnpI and p2JM103-DNNDPI



















Restriction




ASP C-



Sites Used


Vector
Signal
Terminal

DNA
Host
for


Construct
Peptide
prosequence
Primers
Template
vector
Cloning





pXX-ASP-
ASP
In frame
pXX-ASP-III/IV-Fw
ASP
pXX-KnpI 
KnpI and


1


CTAGCTAGGTACCATGACA
synthetic

HindIII





CCACGAACTGTCACAAGAG
gene







CT (SEQ ID NO: 137)
G00222







ASP-syntc-ProC-RV








GTGTGCAAGCTTTCAGGG








GAGGGTGAGTCCCATTGT








GTAA (SEQ ID NO: 138)





 


pXX-
ASP
not
pXX-ASP-III/IV-Fw
ASP
pXX-KnpI
KnpI and


ASP-2

incorporated
CTAGCTAGGTACCATGACA
synthetic

HindIII





CCACGAACTGTCACAAGAG
gene







CT (SEQ ID NO: 139)
G00222







ASP-syntc-mature-RV








GTGTGCAAGCTTTCAAGGG








GAACTTCCAGAGTCAGTC








(SEQ ID NO: 140)





 


pXX-
MRSKK
In frame
ASP-PreCross-I-FW
ASP
pXX-KnpI
KnpI and


ASP-3
RTVTR

TCATGCAGGGTACCATGAG
synthetic

HindIII



ALAVA

AAGCAAGAAGCGAACTGTC
gene





TAAATL

ACAAGAGCTCTGGCT
G00222





LAGGM

(SEQ ID NO: 141)






AAQA

ASP-syntc-ProC-RV






(SEQ ID

GTGTGCAAGCTTTCAGGG






NO: 135)

GAGGGTGAGTCCCATTGT








GTAA (SEQ ID NO: 142)





 


pXX-
MRSKK
not
ASP-PreCross-I-FW
ASP
pXX-KnpI
KnpI and


ASP-4
RTVTR
incorporated
TCATGCAGGGTACCATGAG
synthetic

HindIII



ALAVA

AAGCAAGAAGCGAACTGTC
gene





TAAATL

ACAAGAGCTCTGGCT
G00222





LAGGM

(SEQ ID NO: 143)






AAQA

ASP-syntc-mature-RV






(SEQ ID

GTGTGCAAGCTTTCAAGGG






NO: 135)

GAACTTCCAGAGTCAGTC








(SEQ ID NO: 144)





 


pXX-
MRSKK
In frame
ASP-PreCross-II-FW
ASP
pXX-KnpI
KnpI and


ASP-5
LWISLL

TCATGCAGGGTACCATGAG
synthetic

HindIII



LAVAT

AAGCAAGAAGTTGTGGATC
gene





AAATLL

AGTTTGCTGCTGGCTGTGG
G00222





AGGMA

CAACAGCAGCTGCTACA






AQA

(SEQ ID NO: 145)






(SEQ ID

ASP-syntc-ProC-RV






NO: 136)

GTGTGCAAGCTTTCAGGG








GAGGGTGAGTCCCATTGT








GTAA (SEQ ID NO: 146)





 


pXX-
MRSKK
not
ASP-PreCross-II-FW
ASP
pXX-KnpI
KnpI and


ASP-6
LWISLL
incorporated
TCATGCAGGGTACCATGAG
synthetic

HindIII



LAVAT

AAGCAAGAAGTTGTGGATC
gene





AAATLL

AGTTTGCTGCTGGCTGTGG
G00222





AGGMA

CAACAGCAGCTGCTACA






AQA

(SEQ ID NO: 147)






(SEQ ID

ASP-syntc-mature-RV






NO: 136)

GTGTGCAAGCTTTCAAGGG








GAACTTCCAGAGTCAGTC








(SEQ ID NO: 148)





 


p2JM-103
BCE103
not
DPI-ASP-syntc-ProN-FW
ASP
p2JM103-
BamHI and


ASP
cellulase
incorporated
CCATACCGGATCCAAACGA
synthetic
DNNDPI
HindIII



core +

ACCGGCTCCTCCAGGATCT
gene





acid

(SEQ ID NO: 149)
G00222





labile

DPI-ASP-syntc-Mature-RV






linker

CTCGAGTTAAGCTTTTAAG








GGGAACTTCCAGAGTCAGT








C (SEQ ID NO: 150)
















TABLE 10-2







ASP Expression Cassettes in pHPLT















Restriction


Vector

DNA

sites used


construct
Primers
template
Host vector
for cloning





pHPLT-ASP-
ASP-III&IV-FW
pXX-ASP-1
pHPLT (XbaI x
NheI x SmaI


III
TGAGCTGCTAGCAAAAGGAGAGGGTA

HpaI)




AAGAATGACACCACGAACTGTC (SEQ






ID NO: 151)






pHPLT-ASPproC-RV






CGTACATCCCGGGTCAGGGGAGGGTG






AGTCCCATTG (SEQ ID NO: 152)





 


pHPLT-ASP-
ASP-III&IV-FW
pXX-ASP-2
pHPLT (XbaI x
NheI x SmaI


IV
TGAGCTGCTAGCAAAAGGAGAGGGTA

HpaI)




AAGAATGACACCACGAACTGTC (SEQ






ID NO: 153)






pHPLT-ASPmat-RV






CATGCATCCCGGGTTAAGGGGAACTT






CCAGAGTCAGTC (SEQ ID NO: 154)





 


pHPLT-ASP-
ASP-Cross-1&2-FW
pXX-ASP-3
pHPLT (XbaI x
NheI x SmaI


C1-1
TGAGCTGCTAGCAAAAGGAGAGGGTA

HpaI)




AAGAATGAGAAGCAAGAAG (SEQ ID






NO: 155)






pHPLT-ASPproC-RV






CGTACATCCCGGGTCAGGGGAGGGTG






AGTCCCATTG (SEQ ID NO: 156)





 


pHPLT-ASP-
ASP-Cross-1&2-FW
pXX-ASP-4
pHPLT (XbaI x
NheI x SmaI


C1-2
TGAGCTGCTAGCAAAAGGAGAGGGTA

HpaI)




AAGAATGAGAAGCAAGAAG (SEQ ID






NO: 157)






pHPLT-ASPmat-RV






CATGCATCCCGGGTTAAGGGGAACTT






CCAGAGTCAGTC (SEQ ID NO: 158)





 


pHPLT-ASP-
ASP-Cross-1&2-FW
pXX-ASP-5
pHPLT (XbaI x
NheI x SmaI


C2-1
TGAGCTGCTAGCAAAAGGAGAGGGTA

HpaI)




AAGAATGAGAAGCAAGAAG (SEQ ID






NO: 159)






pHPLT-ASPproC-RV






CGTACATCCCGGGTCAGGGGAGGGTG






AGTCCCATTG (SEQ ID NO: 160)





 


pHPLT-ASP-
ASP-Cross-1&2-FW
pXX-ASP-6
pHPLT (XbaI x
NheI x SmaI


C2-2
TGAGCTGCTAGCAAAAGGAGAGGGTA

HpaI)




AAGAATGAGAAGCAAGAAG (SEQ ID






NO: 161)






pHPLT-ASPmat-RV






CATGCATCCCGGGTTAAGGGGAACTT






CCAGAGTCAGTC (SEQ ID NO: 162)





 


pHPLT-ASP-
pHPLT-BCE/ASP-FW
p2JM103-
pHPLT
NheI x SmaI


VII
TGCAGTCTGCTAGCAAAAGGAGAGGG 
ASP





TAAAGAGTGAGAAG (SEQ ID NO: 163)






pHPLT-ASPmat-RV






CATGCATCCCGGGTTAAGGGGAACTT






CCAGAGTCAGTC (SEQ ID NO: 164)









Primers were obtained from MWG and Invitrogen. Invitrogen Platinum Taq DNA polymerase High Fidelity (Cat. No. 11304-029) was used for PCR amplification (0.2 μM primers, 25 up to 30 cycles) according to the Invitrogen's protocol. Ligase reactions of ASP expression cassettes and host vectors were completed by using Invitrogen T4 DNA Ligase (Cat. No. 15224-025), utilizing Invitrogen's protocol as recommended for general cloning of cohesive ends).
Selective growth of B. subtilis (ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32, ΔamyE::(xyIR,pxyIA-comK) transformants harboring the p2JM103-ASP vector or one of the pHPLT-ASP vectors was performed in shake flasks containing 25 ml Synthetic Maxatase Medium (SMM), with 0.97 g/l CaCl2.6H2O instead of 0.5 g/l CaCl2 (See, U.S. Pat. No. 5,324,653, herein incorporated by reference) with either 25 mg/L chloramphenicol or 20 mg/L neomycin. This growth resulted in the production of secreted ASP protease with proteolytic activity. However. Gel analysis was performed using NuPage Novex 10% Bis-Tris gels (Invitrogen, Cat. No. NP0301 BOX). To prepare samples for analysis, 2 volumes of supernatant were mixed with 1 volume 1M HCl, 1 volume 4xLDS sample buffer (Invitrogen, Cat. No. NP0007), and 1% PMSF (20 mg/ml) and subsequently heated for 10 minutes at 70° C. Then, 25 μL of each sample was loaded onto the gel, together with 10 μL of SeeBlue plus 2 pre-stained protein standards (Invitrogen, Cat. No. LC5925). The results' clearly demonstrated that all asp cloning strategies described in this Example yield suffident amounts of active Asp produced by B. subtilis. 
In addition, samples of the same fermentation broths were assayed as follows: 10 μl of the diluted supernatant was taken and added to 190 μl AAPF substrate solution (conc. 1 mg/ml, in 0.1 M Tris/0.005% TWEEN®, pH 8.6). The rate of increase in absorbance at 410 nm due to release of p-nitroaniline was monitored (25° C.), as it provides a measure of the ASP concentration produced. These results indicated that all of the constructs resulted in the production of measurable ASP protease.
The impact of the synthetic asp gene was investigated in Bacillus subtilis comparing the expression levels of the pHPLT-ASP-c-1-2 construct with the synthetic and native asp gene in a B. subtilis (ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32, ΔamyE::(xyIR,pxyIA-comK) strain. The native gene was amplified from plasmid containing the native asp gene, using platinum pfx polymerase (Invitrogen) with the following primers:







AK04-12.1: NheI thru RBS


(SEQ ID NO: 165)


TTATGCGAGGCTAGCAAAAGGAGAGGGTAAAGAGTGAGAAGCAAAAAACG


 


AK04-11: RBS thru


5 aa aprE for ASP native C1 fusion in pHPLT


(SEQ ID NO: 166)


taaagagtgagaagcaaaaaacgcacagtcacgcgggccctg


 


AK04-13: HpaI 3′ of native ASP mature


(SEQ ID NO: 167)


gtcctctgttaacttacgggctgctgcccgagtcc






The following conditions were used for these PCRs: 94° C. for 2 min.; followed by 25 cycles of 94° C. for 45 sec., 60° C. for 30 sec., and 68° C. for 2 min. for 30 sec.; followed by 68° C. for 5 min. The resulting PCR product was run on an E-gel (Invitrogen), excised, and purified with a gel extraction kit (Qiagen). Ligase reaction of this fragment containing the native ASP with the pHPLT vector was completed by using ligated (T4 DNA Ligase, NEB) and transformed directly into B. subtilis (ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32, ΔamyE::(xyIR,pxyIA-comK). Transformation to B. subtilis was performed as described in WO 02/14490 A2, herein incorporated by reference.

The Asp protein was produced by growth in shake flasks at 37° C. in medium containing the following ingredients; 0.03 g/L MgSO4, 0.22 g/L K2HPO4, 21.3 g/L NA2HPO4*7H2O, 6.1 g/L NaH2PO4*H2O, 3.6 g/L Urea, 7 g/L soymeal, 70 g/L Maltrin M150, and 42 g/L glucose, with a final pH7.5. In these experiments, the production level of the host carrying the synthetic gene cassette was found to be 3-fold higher than the host carrying the native gene cassette.
In additional experiments, expression of ASP was investigated in Bacillus subtilis using the sacB promoter and aprE signal peptide. The gene was amplified from plasmid containing the synthetic asp gene using TGO polymerase (Roche) and the primers:









CF 520 (+) Fuse ASP (pro) to aprE ss



(SEQ ID NO: 168)



GCAACATGTCTGCGCAGGCTAACGAACCGGCTCCTCCAGGA



 



CF 525 (−) End of Asp gene HindIII



(SEQ ID NO: 169)



GACATGACATAAGCTTAAGGGGAACTTCCAGAGTC






The sacB promoter (Bacillus subtilis), the start of the messenger RNA (+1) from aprE, and the aprE signal peptide were amplified from the plasmid pJHsacBJ2 using TGO polymerase (Roche) and the primers:









CF 161 (+) EcoRI at start of sacB promoter



(SEQ ID NO: 170)



GAGCCGAATTCATATACCTGCCGTT



 



CF 521 (−) Reverse complement of CF 520



(SEQ ID NO: 171)



TCCTGGAGGAGCCGGTTCGTTAGCCTGCGCAGACATGTTGC 






The following PCR conditions were used to amplify both pieces: 94° C. for 2 min.; followed by 30 cycles of 94° C. for 30 sec., 50° C. for 1 min., and 66° C. for 1 min.; followed by 72° C. for 7 min. The resulting PCR products were run on an E-gel (Invitrogen), excised, and purified with a gel extraction kit (Qiagen).
In addition, a PCR overlap extension fusion (Ho, Gene, 15:51-59 [1989]) was used to fuse the above gene fragment to the sacB promoter-aprE signal peptide fragment with PFX polymerase (Invitrogen) using the following primers:









CF 161 (+) EcoRI at start of sacB promoter



(SEQ ID NO: 170)



GAGCCGAATTCATATACCTGCCGTT



 



CF 525 (−) End of Asp gene HindIII



(SEQ ID NO: 169)



GACATGACATAAGCTTAAGGGGAACTTCCAGAGTC






The following conditions were used for these PCRs: 94° C. for 2 min.; followed by 25 cycles of 94° C. for 45 sec., 60° C. for 30 sec., and 68° C. for 2 min. 30 sec.; followed 68° C. for 5 min. The resulting PCR fusion products were run on an E-gel (Invitrogen), excised, and purified with a gel extraction kit (Qiagen). The purified fusions were cut (EcoRI/HindIII) and ligated (T4 DNA Ligase, NEB) into an EcoRI/HindIII pJH101 (Ferrari et al., J. Bacteriol., 152:809-814 [1983]) vector containing a strong transcriptional terminator. The ligation mixture was transformed into competent E. coli cells (Top 10 chemically competent cells, Invitrogen) and plasmid preps were done to retrieve the plasmid (Qiagen spin-prep).
The plasmid, pJHsacB-ASP (1-96 sacB promoter; 97-395 aprE+1 through end of aprE ss; and 396-1472 pro+mature asp; See, sequence provided below) was transformed to B. subtilis. Transformation to B. subtilis (ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32, ΔamyE::(xyIR,pxyIA-comK) strain was performed as described in WO 02/14490 A2, herein incorporated by reference. The chromosomal DNA was extracted from an overnight culture of the strain (grown in LB media) then transformed to strain BG 3594 and named “CF 202.” This strain produced a clear halo on the indicator plate (LA+1.6% skim milk).
pJHsacB-ASP Sequence:








(SEQ ID NO: 172)



CATCACATATACCTGCCGTTCACTATTATTTAGTGAAATGAGATATTATGATATTTTCTG



 


AATTGTGATTAAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGA


 


ATGAAAAGAAACAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCAAATCCTTTTAT


 


GATTTTCTATCAAACAAAAGAGGAAAATAGACCAGTTGCAATCCAAACGAGAGTCTAAT


 


AGAATGAGGTCacaGAATAGTCTTTTAAGTAAGTCTACTCTGAATTTTTTTAAAAGGAGA


 


GGGTAAAGAgtgAGAAGCAAAAAATTGTGGATCAGCTTGTTGTTTGCGTTAACGTTAATC


 


TTTACGATGGCGTTCAGCAACATGTCTGCGCAGGCTaacgaaccggctcctccaggatctgcatcag


 


cccctccacgattagctgaaaaacttgaccctgacttacttgaagcaatggaacgcgatctggggttagatgcagaggaagca


 


gctgcaacgttagcttttcagcatgacgcagctgaaacgggagaggctcttgctgaggaactcgacgaagatttcgcgggcac


 


gtgggttgaagatgatgtgctgtatgttgcaaccactgatgaagatgctgttgaagaagtcgaaggcgaaggagcaactgctgt


 


gactgttgagcattctcttgctgatttagaggcgtggaagacggttttggatgctgcgctggagggtcatgatgatgtgcctacgtg


 


gtacgtcgacgtgcctacgaattcggtagtcgttgctgtaaaggcaggagcgcaggatgtagctgcaggacttgtggaaggcg


 


ctgatgtgccatcagatgcggtcacttttgtagaaacggacgaaacgcctagaacgatgttcgacgtaattggaggcaacgcat


 


atactattggcggccggtctagatgttctatcggattcgcagtaaacggtggcttcattactgccggtcactgcggaagaacagg


 


agccactactgccaatccgactggcacatttgcaggtagctcgtttccgggaaatgattatgcattcgtccgaacaggggcagg


 


agtaaatttgcttgcccaagtcaataactactcgggcggcagagtccaagtagcaggacatacggccgcaccagttggatctg


 


ctgtatgccgctcaggtagcactacaggttggcattgcggaactatcacggcgctgaattcgtctgtcacgtatccagagggaac


 


agtccgaggacttatccgcacgacggtttgtgccgaaccaggtgatagcggaggtagccttttagcgggaaatcaagcccaag


 


gtgtcacgtcaggtggttctggaaattgtcggacggggggaacaacattctttcaaccagtcaacccgattttgcaggcttacggc


 


ctgagaatgattacgactgactctggaagttcccctTAAGCTTAAAAAACCGGCCTTGGCCCCGCCGGTT


 


TTTTATTATTTTTCTTCCTCCGCATGTTCAATCCGCTCCATAATCGACGGATGGCTCCCT


 


CTGAAAATTTTAACGAGAAACGGCGGGTTGACCCGGCTCAGTCCCGTAACGGCCAAGT


 


CCTGAAACGTCTCAATCGCCGCTTCCCGGTTTCCGGTCAGCTCAATGCCGTAACGGTC


 


GGCGGCGTTTTCCTGATACCGGGAGACGGCATTCGTAATCGGATCCCGGACGCATCG


 


TGGCCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCA


 


CCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGG


 


GTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCAC


 


CATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAAT


 


GCAGGAGTCGCATAAGGGAGAGCGTCGACCGATGCCCTTGAGAGCCTTCAACCCAGT


 


CAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCACTTATGACTGTCTTC


 


TTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCATTTTCGGCGAGG


 


ACCGCTTTCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAATCTT


 


GCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTTCGGCGAGAA


 


GCAGGCCATTATCGCCGGCATGGCGGCCGACGCGCTGGGCTACGTCTTGCTGGCGTT


 


CGCGACGCGAGGCTGGATGGCCTTCCCCATTATGATTCTTCTCGCTTCCGGCGGCATC


 


GGGATGCCCGCGTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAGGGA


 


CAGCTTCAAGGATCGCTCGCGGCTCTTACCAGCCTAACTTCGATCACTGGACCGCTGA


 


TCGTCACGGCGATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATTG


 


AGGCGCCGCCCTATACCTTATTTATGTTACAGTAATATTGACTTTTAAAAAAGGATTGAT


 


TCTAATGAAGAAAGCAGACAAGTAAGCCTCCTAAATTCACTTTAGATAAAAATTTAGGAG


 


GCATATCAAATGAACTTTAATAAAATTGATTTAGACAATTGGAAGAGAAAAGAGATATTT


 


AATCATTATTTGAACCAACAAACGACTTTTAGTATAACCACAGAAATTGATATTAGTGTTT


 


TATACCGAAACATAAAACAAGAAGGATATAAATTTTACCCTGCATTTATTTTCTTAGTGA


 


CAAGGGTGATAAACTCAAATACAGCTTTTAGAACTGGTTACAATAGCGACGGAGAGTTA


 


GGTTATTGGGATAAGTTAGAGCCACTTTATACAATTTTTGATGGTGTATCTAAAACATTC


 


TCTGGTATTTGGACTCCTGTAAAGAATGACTTCAAAGAGTTTTATGATTTATACCTTTCT


 


GATGTAGAGAAATATAATGGTTCGGGGAAATTGTTTCCCAAAACACCTATACCTGAAAA


 


TGCTTTTTCTCTTTCTATTATTCCATGGACTTCATTTACTGGGTTTAACTTAAATATCAAT


 


AATAATAGTAATTACCTTCTACCCATTATTACAGCAGGAAAATTCATTAATAAAGGTAATT


 


CAATATATTTACCGCTATCTTTACAGGTACATCATTCTGTTTGTGATGGTTATCATGCAG


 


GATTGTTTATGAACTCTATTCAGGAATTGTCAGATAGGCCTAATGACTGGCTTTTATAAT


 


ATGAGATAATGCCGACTGTACTTTTTACAGTCGGTTTTCTAATGTCACTAACCTGCCCC


 


GTTAGTTGAAGAAGGTTTTTATATTACAGCTCCAGATCCTGCCTCGCGCGTTTCGGTGA


 


TGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAA


 


GCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTG


 


TCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAAC


 


TATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGC


 


ACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGA


 


CTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGT


 


AATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGC


 


CAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCC


 


GCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGA


 


CAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGT


 


TCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCG


 


CTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCT


 


GGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTA


 


TCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGT


 


AACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGG


 


CCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAG


 


TTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAG


 


CGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAG


 


ATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGG


 


ATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGA


 


AGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTA


 


ATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACT


 


CCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCA


 


ATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAG


 


CCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTAT


 


TAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTG


 


TTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAG


 


CTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCG


 


GTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCAC


 


TCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTT


 


TCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGA


 


GTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAA


 


AGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTG


 


TTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTAC


 


TTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGA


 


ATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGC


 


ATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAA


 


CAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCA


 


TTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAA






Expression of the asp gene was investigated in a nine-protease delete Bacillus subtilis host. The plasmid pHPLT-ASP-C1-2 (See, Table 10-2, and FIG. 9), was transformed into B. subtilis (ΔaprE, ΔnprE, Δepr, LispA, Δbpr, Δvpr, ΔwprA, Δmpr-ybfJ, ΔnprB) and (degUHy32, oppA, AspoIIE3501, amyE:(xyIRPxyIAcomK-ermC). Transformation was performed as known in the art (See e.g., WO 02/14490, incorporated herein by reference). The Asp protein was produced by growth in shake flasks at 37° C. in MBD medium, a MOPS based defined medium. MBD medium was made essentially as known in the art (See, Neidhardt et al., J. Bacteriol., 119: 736-747 [1974]), except NH4Cl2, FeSO4, and CaCl2 were left out of the base medium, 3 mM K2HPO4 was used, and the base medium was supplemented with 60 mM urea, 75 g/L glucose, and 1% soytone. Also, the micronutrients were made up as a 100× stock containing in one liter, 400 mg FeSO4 0.7H2O, 100 mg MnSO4.H2O, 100 mg ZnSO4.7H2O, 50 mg CuCl2.2H2O, 100 mg CoCl2.6H2O, 100 mg NaMoO4.2H2O, 100 mg Na2B4O7.10H2O, 10 ml of 1M CaCl2, and 10 ml of 0.5 M sodium citrate. The expression levels obtained in these experiments were found to be fairly high.
In additional embodiments, “consensus” promoters such as those developed through site-saturation mutagenesis to create promoters that more perfectly conform to the established consensus sequences for the “−10” and “−35” regions of the vegetative “sigma A-type” promoters for B. subtilis (See, Voskuil et al., Mol. Microbiol., 17:271-279 [1995]) find use in the present invention. However, it is not intended that the present invention be limited to any particular consensus promoter, as it is contemplated that other promoters that function in Bacillus cells will find use in the present invention.
Example 11
Protease Production in Bacillus clausii 
In this Example, experiments conducted to produce protease 69B4 (also referred to as “Asp” herein) in B. clausii are described. In order to express the Asp protein in Bacillus clausii, it was necessary to use a promoter that works in this alkaliphilic microorganism due to its unique regulation systems. The production profile of the alkaline serine protease of B. clausii PB92 (MAXACAL® protease) has shown that it has to have a strong promoter (referred to as “MXL-prom.” herein; SEQ ID NOS:173, 174, and 175, See, FIG. 18) with a delicate regulation. Besides the promoter region, also signal sequences (leader sequences) are known to be very important for secreting proteins in B. clausii. Therefore, 3 constructs were designed With the MAXACAL® protease promoter region and separate fusions of the MAXACAL® protease leader sequence and the Asp leader sequence in front of the N-terminal Pro and the mature Asp protein with 3, 6 and 27 amino acids of the MAXACAL® protease leader fused to 25, 25 and 0 amino acids of the Asp leader, respectively.
To make these constructs, amplification of DNA fragments needed to be done in order to enable the fusion. Therefore, PCRs were performed on both MAXACAL® protease and Asp template DNA with Phusion high fidelity polymerase (Finnzymes) according to the manufacturer's instructions.
PCR reactions were executed with the following primers (bold indicates the MAXACAL® protease part of the primer) synthesized at MWG-Biotech AG:










1:
B. clau-3F: agggaaccgaatgaagaaacgaactgtcacaagagctctg 
(SEQ ID NO: 176)



 


2:
B. clau-3R: cagagctcttgtgacagttcgtttcttcattcggttccct 
(SEQ ID NO: 177)


 


3:
B. clau-6F: aatgaagaaaccgttggggcgaactgtcacaagagctctg 
(SEQ ID NO: 178)


 


4:
B. clau-6R: cagagctcttgtgacagttcgccccaacggtttcttcatt 
(SEQ ID NO: 179)


 


5:
B. clau-27F: agttcatcgatcgcatcggctaacgaaccggctcctccagga 
(SEQ ID NO: 180)


 


6:
B. clau-27R: tcctggaggagccggttcgttagccgatgcgatcgatgaact 
(SEQ ID NO: 181)


 


7:
B. clau-vector 5′: tcagggggatcctagattctgttaacttaacgtt. 
(SEQ ID NO: 182)



This primer contains the HpaI-site (GTTAAC) from the  




promoter region and a BamHI-site (GGATCC) for cloning  




reasons (both underlined).



 


8:
pHPLT-HindIII-R: gtgctgttttatcctttaccttgtctcc.  
(SEQ ID NO: 183)



The sequence of this primer lays just upstream of the        




HindIII-site of pHPLT-ASP-C1-2 (See, Table 10-2).














TABLE 11-1







PCR Setup to Create Fused MAXACAL ® 


Protease-Asp Leader Fragments










Template DNA
Primer 1
Primer 2
Fragment Name













pHPLT-ASP-C1-2
1
8
3F


pHPLT-ASP-C1-2
3
8
6F


pHPLT-ASP-C1-2
5
8
27F


pMAX4
2
7
3R


pMAX4
4
7
6R


pMAX4
6
7
27R


3F + 3R
7
8
3F3R


6F + 6R
7
8
6F6R


27F + 27R
7
8
27F27R









In Table 11-1, “pMAX4” refers to the template described in WO 88/06623, herein incorporated by reference. PCR fragments 3F3R, 6F6R, 27F27R were digested with both BamHI and HindIII. The digested PCR fragments were ligated with T4 ligase (Invitrogen) into BamHIH+HindIII-opened plasmid pHPLT-ASP-C1-2 (See, FIG. 18). The ligation product was transformed to competent B. subtilis cells ((ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32, ΔamyE::(xyIR,pxyIA-comk; See e.g., WO 02/14490, incorporated herein by reference) and selected on neomycin (20 mg/l). Heart Infusion-agar plates containing neomycin were used to identify neomycin resistant colonies. DNA of the B. subtilis transformants was isolated using Qiagen's plasmid isolation kit according to manufacture's instructions, and were tested on the appearance of the fused MAXACAL® protease-Asp fragment by their pattern after digestion with both NcoI+HpaI together in one tube. The restriction enzymes used in this Example (i.e., BamHI, HindIII, NcoI and HpaI) were all purchased from NEB, and used following the instructions of the supplier. DNA of B. subtilis transformants that showed 2 bands with restriction enzymes (NcoI+HpaI) was used to transform protease negative B. clausii strain PBT142 protoplast cells (these were derived from PB92).
The protoplast transformation of B. clausii strain PBT142 was performed according to the protocol mentioned for the protoplast transformation of B. alkalophilus (renamed B. clausii) strain PB92 in patent WO88/06623, herein incorporated by reference A modification to this protocol was the use of an alternative recipe for the regeneration plates, in that instead of 1.5% agar, 8.0 g/l Gelrite gellam gum (Kelco) was used. In addition, instead of 1000 mg/l neomycin, 20 mg/l neomycin was used as described by Van der Laan et al., (Van der Laan et al., Appl. Environ. Microbiol., 57:901-909 [1991]).
DNA from all 3 constructs isolated from B. subtilis (see above) was transformed into B. clausii PBT142 protoplasts using the same protocol as above. Transformants in B. clausii PBT142 were selected by replica-plating on Heart Infusion agar plates containing 20 mg/l neomycin. The B. clausii strains with the different construct were produced as indicated in Table 11-2.







TABLE 11-2







B.clausii Constructs










Construct (length




MAXACALI ® protease




leader)
B. clausii Strain







3 MXL/25ASP
PMAX-ASP3



6 MXL/25ASP
PMAX-ASP2



27 MXL/0ASP
PMAX-ASP1










These 3 strains were fermented in shake flasks containing 100 ml Synthetic Maxatase Medium (SMM) (See, U.S. Pat. No. 5,324,653, herein incorporated by reference). However, instead of 0.97 g/l CaCl2.6H2O, 0.5 g/l CaCl2 was used. Also, instead of 0.5 ml/l antifoam 5693, 0.25 ml/l Basildon was used. The 100 ml SSM shake flasks were inoculated with 0.2 ml of a pre-culture of the 3 B. clausii strains containing the leader constructs in 10 ml TSB (Tryptone Soya Broth) with 20 mg/l neomycin. The protease production values were measured via the AAPF-assay (as described above) after growth in the shake flasks for 3 days. The results indicated that these constructs were able to express protease with proteolytic activity.
In an additional experiment, integration of the leader construct with the entire MAXACAL® protease leader length (27 amino acids) was investigated. However, it is not intended that the present invention be limited to any particular mechanism.
Stable integration of heterologous DNA in the B. alcalophilus (now, B. clausii) chromosome is described in several publications (See e.g., WO 88/06623, and Van der Laan et al., supra). The procedure described in patent WO 88/06623 for integration of 1 or 2 copies of the MAXACAL® protease gene in the chromosome of B. alcalophilus (now, B. clausii) was used to integrate at least 1 copy of the asp gene in the chromosome of B. clausii PBT142. However, a derivative of pE194-neo: pENM#3 (See, FIG. 19) was used instead of the integration vector pE194-neo (to make pMAX4 containing the MAXACAL® protease gene). In the integration vector pENM#3, the Asp leader PCR product 27F27R was cloned in the unique blunt end site HpaI in between the 5′ and the 3′ flanking regions of the MAXACAL® protease gene. Therefore, 27F27R was made blunt-ended as follows: it was first digested with HpaI (5′ end), purified with the Qiagen PCR purification kit, and then digested with HindIII (3′ end). This treated PCR fragment 27F27R was purified again after HindIII digestion (using the same Qiagen kit) and filled in with dNTP's using T4 polymerase (Invitrogen) and purified again with Qiagen kit. The HpaI-opened pENM#3 and the blunt-ended PCR product 27F27R were ligated with T4 ligase (Invitrogen). The ligation product was transformed directly to B. clausii PBT142 protoplasts and selected after replica-plating on HI agar plates with 20 mg/l neomycin. Two transformants with the correct orientation of the asp gene in the integration vector were identified and taken into the integration procedure as described in patent WO 88/06623. Selections were done at 2 mg/l and 20 mg/l neomycin for integration in the MAXACAL® protease locus and at an illegitimate locus, respectively. These results indicated that B. clausii is also suitable as an expression host for the Asp protease.
Example 12
Protease Production in B. licheniformis 
In this Example, experiments conducted to produce protease 69B4 in B. licheniformis are described. During these experiments, various expression constructs were created to produce protease 69B4 protease (also referred to as “ASP protease”) in Bacillus licheniformis. Constructs were cloned into expression plasmid pHPLT (replicating in Bacillus) and/or into integration vector plCatH. Plasmid pHPLT (See, FIG. 17; and U.S. Pat. No. 6,562,612 [herein incorporated by reference) is a pUB110 derivative, has a neomycin resistance marker for selection, and contains the B. licheniformis α-amylase (LAT) promoter (PLAT), a sequence encoding the LAT signal peptide (preLAT), followed by PstI and HpaI restriction sites for cloning and the LAT transcription terminator. The plCatH vector (See, FIG. 20) contains a temperature sensitive origin of replication (ori pE194, for replication in Bacillus), ori pBR322 (for amplification in E. coli), a neomycin resistance gene for selection, and the native B. licheniformis chloramphenicol resistance gene (cat) with repeats for selection, chromosomal integration and cassette amplification.
Construct ASPc1 was created as a PstI-HpaI fragment by fusion PCR with High Fidelity Platinum Taq Polymerase (Invitrogen) according to the manufacturer's instructions, and with the following primers:







pHPLT-BgIII_FW


(SEQ ID NO: 184)


AGTTAAGCAATCAGATCTTCTTCAGGTTA


 


fusionC1_FW


(SEQ ID NO: 185)


CATTGAAAGGGGAGGAGAATCATGAGAAGCAAGAAGCGAACTGTCAC


 


fusionC1_RV


(SEQ ID NO: 186)


GTGACAGTTCGCTTCTTGCTTCTCATGATTCTCCTCCCCTTTCAATG


 


pHPLT-HindIII_RV


(SEQ ID NO: 187)


CTTTACCTTGTCTCCAAGCTTAAAATAAAAAAACGG






These primers were obtained from MWG Biotech. PCR reactions were typically performed on a thermocycler for 30 cycles with High Fidelity Platinum Taq polymerase (Invitrogen) according to the manufacturer's instructions, with annealing temperature of 55° C. PCR-I was performed with the primers pHPLT-BgIII_FW and fusionCl_RV on pHPLT as template DNA. PCR-II was performed with primers fusionC1_FW and pHPLTHindIII_RV on plasmid pHPLT-ASP-C1-2. The fragments from PCR-I and PCR-II were assembled in a fusion PCR with the primers pHPLT-BgIII_FW and pHPLT-HindIII—RV. This final PCR fragment was purified using the Qiagen PCR purification kit, digested with BglII and HindIII, and ligated with T4 DNA ligase according to the manufacturers' instructions into BglII and HindIII digested pHPLT. The ligation mixture was transformed into B. subtilis strain OS14 as known in the art (See, U.S. Pat. Appl. No. US20020182734 and WO 02/14490, both of which are incorporated herein by reference). Correct transformants produced a halo on a skimmed milk plate and one of them was selected to isolate plasmid pHPLT-ASPc1. This plasmid was introduced into B. licheniformis host BML780 (BRA7 derivative, cat-, amyL-, spo-, aprL-, endoGluC-) by protoplast transformation as known in the art (See, Pragai et al., Microbiol., 140:305-310 [1994]). Neomycin resistant transformants formed halos on skim plates, whereas the parent strain without pHPLT-ASPc1 did not. This result shows that B. licheniformis is capable of expressing and secreting ASP protease when expression is driven by the LAT promoter and when it is fused to a hybrid signal peptide (MRSKKRTVTRALAVATAAATLLAGGMAAQA; SEQ ID NO:135).
Construct ASPc3 was created as a PstI-HpaI fragment by fusion PCR (necessary to remove the internal PstI site in the synthetic asp gene) as described above with the following primers:









ASPdelPstI_FW



(SEQ ID NO: 188)



GCGCAGGATGTAGCAGCTGGACTTGTGG



 



ASPdelPstI_RV



(SEQ ID NO: 189)



CCACAAGTCCAGCTGCTACATCCTGCGC



 



AspPstI_FW



SEQ ID NO: 190)



GCCTCATTCTGCAGCTTCAGCAAACGAACCGGCTCCTCCAGG



 



AspHpaI_RV



(SEQ ID NO: 191)



CGTCCTCTGTTAACTCAGTCGTCACTTCCAGAGTCAGTCGTAATC






After purification, the PCR product was digested with PstI-HpaI and ligated into PstI and HpaI digested pHPLT and then transformed into B. subtilis strain OS14. Plasmid pHPLT-ASPc3 was isolated from a neomycin resistant that formed a relatively (compared to other transformants) large halo on a skim milk plate. Plasmid DNA was isolated using the Qiagen plasmid purification kit and sequenced by BaseClear.
Sequencing confirmed that the ASPc3 construct encodes mature ASP that has two aspartic acid residues at the extreme C-terminal end (S188D, P189D). These mutations were deliberately introduced by PCR to make the C-terminus of ASP less susceptible against proteolytic degradation (See, WO 02055717). It also appeared that two mutations were introduced into the coding region of the N-terminal pro region by the PCR methods. These mutations caused two amino acid changes in the N-terminal pro-region: L42I and Q141P. Since this particular clone with these two pro(N) mutations gives a somewhat larger halo than other clones without these mutations, it was contemplated that expression and/or secretion of ASP protease in Bacillus is positively affected by these N-terminal pro mutations. However, it is not intended that the present invention be limited to these specific mutations, as it is also contemplated that further mutations will find use in the present invention.
Next, pHPLT-ASPc3 was transformed into BML780 as described above. In contrast to the parental strain without the plasmid, BML780(pHLPT-ASPc3) produced a halo on a skim milk plate indicating that also this ASPc3 construct leads to ASP expression in B. licheniformis. To make an integrated, amplified strain containing the ASPc3 expression cassette, the C3 construct was amplified from pHPLT-ASPc3 with the following primeis:









EBS2XhoI_FW



(SEQ ID NO: 192)



ATCCTACTCGAGGCCTTTTGGAAGAAAATATAGGG



 



EBS2XhoI_RV



(SEQ ID NO: 193)



TGGAATCTCGAGGTTTTATCCTTTACCTTGTCTCC






The PCR product was digested with XhoI, ligated into XhoI-digested plCatH (See, FIG. 20) and transformed into B. subtilis OS14 as described above. The plasmid from an ASP expressing clone (judged by halo formation on skim milk plates) was isolated and designated plCatH-ASPc3. DNA sequencing by BaseClear confirmed that no further mutations were introduced in the ASPc3 cassette in plCatH-ASPC3. The plasmid was then transformed into BML780 at the permissive temperature (37° C.) and one neomycin resistant (neoR) and chloramphenicol resistant (capR) transformant were selected and designated BML780(plCatH-ASPc3). The plasmid in BML780(plCatH-ASPc3) was integrated into the cat region on the B. licheniformis genome by growing the strain at a non-permissive temperature (50° C.) in medium with chloramphenicol. One capR resistant clone was selected and designated BML780-plCatH-ASPc3. BML780-plCatH-ASPc3 was grown again at the permissive temperature for several generations without antibiotics to loop-out vector sequences and then one neomycin sensitive (neoS), capR clone was selected. In this clone, vector sequences of plCatH on the chromosome were excised (including the neomycin resistance gene) and only the ASPc3-cat cassette was left. Note that the cat gene is a native B. licheniformis gene and that the asp gene is the only heterologous piece of DNA introduced into the host. Next, the ASPc3-cat cassette on the chromosome was amplified by growing the strain in/on media with increasing concentrations of chloramphenicol. After various rounds of amplification, one clone (resistant against 75 μg/ml chloramphenicol) was selected and designated “BML780-ASPc3.” This clone produced a clear halo on a skim milk plate, whereas the parental strain BML780 did not, indicating that ASP protease is produced and secreted by the BML780-ASPc3 strain.
Construct ASPc4 is similar to ASPc3, but ASP protease expressed from ASPc4 does not have two aspartic acid residues at the C-terminal end of the mature chain. ASPc4 was created by amplification of the asp gene in pHPLT-ASPc3 with the following Hypur primers from MWG Biotech (Germany):







XhoPlatPRElat_FW


SEQ ID NO: 194)


acccccctcgaggcttttcttttggaagaaaatatagggaaaatggtact


 


tgttaaaaattcggaatatttatacaatatcatatgtttcacattgaaag


 


gggaggagaatcatgaaacaacaaaaacggctttac


 


ASPendTERMXhoI_RV


(SEQ ID NO: 195)


gtcgacctcgaggttttatcctttaccttgtctccaagcttaaaataaaa


 


aaacggatttccttcaggaaatccgtcctctgttaactcaaggggaactt


 


ccagagtcagtcgtaatc






The ASPc4 PCR product was purified and digested with XhoI, ligated into XhoI-digested plCatH, and transformed into B. subtilis OS14 as described above for ASPc3. Plasmid was isolated from a neoR, capR clone and designated plCatH-ASPc4. plCatH-ASPc4 was transformed into BML780, integrated in the genome, vector sequences were excised, and the cat-ASPc4 cassette was amplified as described above for the ASPc3 construct. Strains with the ASPc4 cassette did not produce smaller halos on skim milk plates than strains with the AspC3 cassette, suggesting that the polarity of the C-terminus of ASP mature is not a significant factor for ASP production, secretion and/or stability in Bacillus. However, it is not intended that the present invention be limited to any particular method.
To explore whether the native ASP signal peptide can drive export in Bacillus, ASPc5 was constructed. PCR was performed on the synthetic asp gene of DNA2.0 with primers ASPendTERMXhoI_RV (above) and XhoPlatPREasp_FW.







XhoPlatPREasp_FW


(SEQ ID NO: 196)


:acccccctcgaggcttttcttttggaagaaaatatagggaaaatggtac


 


ttgttaaaaattcggaatatttatacaatatcatatgtttcacattgaaa


 


ggggaggagaatcatgacaccacgaactgtcacaag






The ASPc5 PCR product was purified and digested with XhoI, ligated into XhoI digested plCatH, and transformed into B. subtilis OS14 as described above for ASPc3. Plasmid was isolated from a neoR, capR clone and designated “plCatH-ASPc5.” DNA sequencing confirmed that no unwanted mutations were introduced into the asp gene by the PCR. plCatH-ASPc5 was transformed into BML780, integrated in the genome, vector sequences were excised, and the cat-ASPc5 cassette was amplified as described above for the ASPc3 construct. It was observed that B. licheniformis strains with the ASPc5 construct also form halos on skim milk plates, confirming that the native signal peptide of ASP functions as a secretion signal in Bacillus species.
Finally, construct ASPc6 was created. It has the B. licheniformis subtilisin (aprL) promoter, RBS and signal peptide sequence fused in-frame to the DNA sequence encoding mature ASP from the optimized DNA2.0 gene. It was created by a fusion PCR with primer ASPendTERMXhoI_RV and the following primers:







AprLupXhoI_FW


(SEQ ID NO: 197)


attagtctcgaggatcgaccggaccgcaacctcc


 


AprLAsp_FW


(SEQ ID NO: 198)


cgatggcattcagcgattccgcttctgctaacgaaccggctcctccagga


 


tctgc


 


AprLAsp_RV


(SEQ ID NO: 199)


gcagatcctggaggagccggttcgttagcagaagcggaatcgctgaatgc


 


catcg






PCR-I was performed with the primers AprLupXhoI_FW and AprLAsp_RV on chromosomal DNA of BRAT as template DNA. PCR-II was performed with primers AprLAsp_FW and ASPendTERMXhoI_RV on the synthetic asp gene of DNA2.0. The fragments from PCR-I and PCR-II were assembled in a fusion PCR with the primers so ASPendTERMXhoI_RV and AprLupXhoI_FW. This final PCR fragment was purified using Qiagen's PCR purification kit (according to the manufacturers instructions), digested with XhoI, ligated into plCatH, and transformed into B. subtilis OS14, as described above for ASPc3. Plasmid was isolated from a neoR, capR clone and designated “plCatH-ASPc6.” DNA sequencing confirmed that no unwanted mutations were introduced into the asp gene or aprL region by the PCRs. plCatH-ASPc6 was transformed into BML780, integrated in the genome, vector sequences were excised, and the cat-ASPc6 cassette was amplified as described above for the ASPc3 construct. B. licheniformis strains with the ASPc6 construct also formed halos on skim milk plates, indicating that the aprL promoter in combination with the AprL signal peptide drives expression/secretion of ASP protease in B. licheniformis. 
Example 13
Protease Production in T. reesei 
In this Example, experiments conducted to produce protease 69B4 in T. reesei are described. In these experiments, three different fungal constructs (fungal expression vectors comprising cbhl fusions) were developed. One contained the ASP 5′ pro region, mature gene, and 3′ pro region; the second contained the ASP 5′ pro region and the mature gene; and the third contained only the ASP mature gene.
The following primer pairs were used to PCR (in the presence of 10% DMSO), the different fragments from the chromosomal DNA K25.10, carrying the ASP gene and introduced SpeI-AscI sites to clone the fragments into the vector pTREX4 (See, FIG. 21) digested with SpeI and AscI restriction enzymes.
1. CBHI fusion with the ASP 5′ pro region, mature gene, and 3′ pro region:







AspproF


forward primer (SpeI-Kexin site-ATG-pro sequence):


(SEQ ID NO: 200)


5′-ACTAGTAAGCGGATGAACGAGCCCGCACCACCCGGGAGCGCGAGC


 


AspproR reverse primer


(AscI site; C-term pro region


from the TAA stop codon to the end of the gene):


(SEQ ID NO: 201)


5′- GGCGCGCC TTA GGGGAGGGTGAGCCCCATGGTGTAGGCACCG






2. The ASP 5′ pro region and mature gene:








AspproF


forward primer (SpeI-Kexin site-ATG-pro sequence):


(SEQ ID NO: 202)


5′-ACTAGTAAGCGGATGAACGAGCCCGCACCACCCGGGAGCGCGAGC


 


AspmatR reverse primer (AscI site: TAA


stop to the end of the mature sequence)


(SEQ ID NO: 203)


5′- GGCGCGCC TTA CGGGCTGCTGCCCGAGTCCGTGGTGATCA-3′






3. The ASP mature gene only:








AspmatF forward primer SpeI-Kexin site-ATG-mature: 


(SEQ ID NO: 204)


5′-ACTAGT AAGCGG ATG TTCGACGTGATCGGCGGCAACGCCTACAC


 


CAT


 


AspmatR Reverse Primer


(AscI site: TAA stop to end of mature sequence)


(SEQ ID NO: 205)


5′-GGCGCGCC TTA CGGGCTGCTGCCCGAGTCCGTGGTGATCA-3′






After construction, the different plasmids were transformed into a Trichoderma reesei strain with disruptions in the cbh1, cbh2, egl1, and egl2 genes, using biolistic transformation methods known in the art. Stable transformants were screened, based on morphology. Ten stable transformants for each construct were screened in shake flasks. The initial inoculum media used contained 30 g/L α-lactose, 6.5 g/L (NH4)2SO4, 2 g/L KH2PO4, 0.3 g/L MgSO4*7H2O, 0.2 g/L CACl2, 1 ml/L 1000× T. reesei Trace Salts, 2 mL/L 10% TWEEN®-80, 22.5 g/L Proflo, and 0.72 g/L CaCO3, in which the transformants were grown for approximately 48 hr. After this incubation period, 10% of the culture was transferred into flasks containing minimal medium known in the art (See, Foreman et al., J. Biol. Chem., 278:31988-31997 [2003]), with 16 g/L of lactose to induce expression. The flasks were placed in a 28° C. shaker. Four-day samples were run on NuPAGE 4-12% gels, and stained with Coomassie Blue. After five-days the protease activity was measured by adding 10 μl of the supernatant to 190 μl AAPF substrate solution (conc. 1 mg/ml, in 0.1 M Tris/0.005% TWEEN, pH 8.6). The rate of increase in absorbance at 410 nm due to release of p-nitroaniline was monitored (25° C.)
The activity data showed that there was a 5× higher production over the control strain (i.e., the parent strain), indicating that T. reesei is suitable for the expression of ASP protease.
Example 14
Protease Production in A. niger 
In this Example, experiments conducted to produce protease 69B4 in Aspergillus niger var. awamori (PCT WO90/00192) are described. In these experiments, four different fungal constructs (fungal expression vectors comprising glaA fusions) were developed. One contained the ASP pre-region, 5′ pro-region, mature gene, and the 3′ pro-region: the second contained the ASP pre-region, 5′ pro-region, and the mature gene; the third contained the ASP 5′ pro-region, mature gene, and the 3′ pro-region; the fourth contained the ASP 5′ pro-region, and the mature gene.
Selected from the following primer pairs, primers were used to PCR (in the presence of 10% DMSO) the different fragments from the chromosomal DNA 69B4 carrying the asp gene and introduced the Nhe 1-BstEII sites to clone the fragments into the vector pSLGAMpR2 (See, FIG. 22) digested with Nhe1 and BstEII restriction enzymes.
Primers Anforward 01 and Anforward 02 contained attB1 Gateway cloning sequences (Invitrogen) at the 5′ end of the primer. Primers Anreversed 01 and Anreversed 02 contained attB2 Gateway cloning sequences (Invitrogen) at the 5′ end of the primer. These primers were used to PCR (in the presence of 10% DMSO) the different fragments from the chromosomal DNA 69B4 carrying the ASP genes.
The different constructs were transferred to a A. niger Gateway compatible destination vector pRAXdes2 (See, FIG. 23; See also, U.S. patent application Ser. No. 10/804,785, and PCT Appln. No. US04/08520, both of which are incorporated herein by reference).







Anforward 01 (without the attB1 sequence)


(SEQ ID NO: 206)


5′-ATGACACCACGAACTGTCACAAGAGCTCTG-3′


 


Anforward 02 (without the attB1 sequence)


(SEQ ID NO: 207)


5′-AACGAACCGGCTCCTCCAGGATCTGCATCA-3′


 


Anreversed 01(without the attB2 sequence)


(SEQ ID NO: 208)


5′-AGGGGAACTTCCAGAGTCAGTCGTAATCATTCTCAGGCC-3′


 


Anreversed 02 (without the attB1 sequence)


(SEQ ID NO: 209)


5′-GGGGAGGGTGAGTCCCATTGTGTAAGCTCCTGA-3′


 


pSLGAM-NT_FW


(SEQ ID NO: 210)


5′-ACCGCGACTGCTAGCAACGTCATCTCCAAGCGCGGCGGTGGCAACGA


 


ACCGGCTCCTCCAGGATCt-3′


 


pSLGAM-MAT_FW


(SEQ ID NO: 211)


5′-ACCGCGACTGCTAGCAACGTCATCTCCAAGCGCGGCGGTGGCAACGA


 


ACCGGCTCCTCCAGGATCT-3′


 


pSLGAM-MAT_RV


(SEQ ID NO: 212)


5′-CCGCCAGGTGTCGGTCACCTAAGGGGAACTTCCAGAGTCAGTCGTAA


 


TCATTCT-3′






PCR conditions were as follows: 5 μL of 10×PCR reaction buffer (Invitrogen); 20 mM MgSO4; 0.2 mM each of dATP, dTTP, dGTP, dCTP (final concentration), 1 μL of 10 ng/μL genomic DNA, 1 μL of High Fidelity Taq polymerase (Invitrogen) at 1 unit per μL, 0.2 μM of each primer (final concentration), 5 μl DMSO and water to 50 μL. The PCR protocol was: 94° C. for 5 min.; followed by 30 cycles of 94° C. for 30 sec., 55° C. for 30 sec., and 68° C. for 3 min; followed by 68° C. for 10 min., and 15° C. for 1 min.
After construction, the different plasmids and a helper plasmid (HM 396 pAPDI) were transformed into Aspergillus niger var awamori (Delta Ap4 strain), using protoplast transformation methods known in the art. Stable transformants were screened, based on morphology. Ten stable transformants for each construct were screened in shake flasks. After this period, a piece of agar containing the strain was transferred into flasks containing RoboSoy medium or the formula 12 g/l Tryptone, 8 g/l Soytone, 15 g/l Ammonium sulfate, 12.1 g/l NaH2PO4.H2O, 2.19 g/l Na2HPO4, 5 ml 20% MgSO4.7H2O, 10 ml 10% Tween 80, 500 ml 30% Maltose and 50 ml 1M phosphate buffer pH 5.8 and 2 g/l uridine to induce expression. The flasks were placed in a 28° C. shaker. Four-day samples were run on NuPAGE 10% Bis Tris protein gels, and stained with Coomassie Blue. Five-day samples were assayed for protease activity using the AAPF method.
The amount of ASP expressed was found to be low, such that it could not be detected in the Coomassie stained gel. Colonies on plates however showed a clear halo formation on skim milk plate agar plates that were significantly larger than the control strain. Thus, although the expression was low, these results clearly indicate that A. niger is suitable for the expression of ASP protease.
Example 15
Generation of Asp Site-Saturated Mutagenesis (SSM) Libraries
In this Example, experiments conducted to develop site-saturation mutagenesis libraries of asp are described. Site saturated Asp libraries each contained 96 B. subtilis (ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32, ΔamyE::(xyIR,pxyIA-comK) clones harboring the pHPLT-ASP-c1-2 expression vector. This vector, containing the Asp expression cassette composed of the synthetic DNA sequence (See, Example 10) encoding the Asp hybrid Signal peptide and the Asp N-terminal pro and mature protein were found to enable expression of the protein indicated below (the signal peptide and precursor protease) and secretion of the mature Asp protease.
DNA Sequence encoding synthetic Asp hybrid signal peptide:







(SEQ ID NO: 213)


ATGAGAAGCAAGAAGCGAACTGTCACAAGAGCTCTGGCTGTGGCAACAGC


 


AGCTGCTACACTCTTGGCTGGGGGTATGGCAGCACAAGCT






The signal peptide and precursor protease are provided in the following sequence (SEQ ID NO:214) (in this sequence, bold indicates the mature protease, underlining indicates the N-terminal prosequence, and the standard font indicates the signal peptide):








(SEQ ID NO: 214)


MRSKKRTVTRALAVATAAATLLAGGMAAQANEPAPPGSASAPPRLAEKLD


 


PDLLEAMERDLGLDAEEAAATLAFQHDAAETGEALAEELDEDFAGTWVED


 


DVLYVATTDEDAVEEVEGEGATAVTVEHSLADLEAWKTVLDAALEGHDDV


 


PTWYVDVPTNSVVVAVKAGAQDVAAGLVEGADVPSDAVTFVETDETPRTM


 


FDVIGGNAYTIGGRSRCSIGFAVNGGFITAGHCGRTGATTANPTGTFAGS


 


SFPGNDYAFVRTGAGVNLLAQVNNYSGGRVQVAGHTAAPVGSAVCRSGST


 


TGWHCGTITALNSSVTYPEGTVRGLIRTTVCAEPGDSGGSLLAGNQAQGV


 


TSGGSGNCRTGGTTFFQPVNPILQAYGLRMITTDSGSSP 






Construction of the 189 asp site saturated mutagenesis libraries was completed by using the pHPLT-ASP-C1-2 expression vector as template and primers listed in Table 15-1. The mutagenesis primers used in these experiments all contain the triple DNA sequence code NNS (N=A, C, T or G and S=C or G) at the position that corresponds with the codon of the Asp mature sequence to be mutated and guaranteed random incorporation of nucleotides at that position. Construction of each SSM library started with two PCR amplifications using pHPLT-BgIII-FW primer and a specific Reverse mutagenesis primer, and pHPLT-BgIII-RV primer and a specific Forward mutagenesis primer (equal positions for the mutagenesis primers). Platinum Taq DNA polymerase High Fidelity (Cat. No. 11304-029; Invitrogen) was used for PCR amplification (0.2 μM primers, 20 up to 30 cycles) according to protocol provided by Invitrogen. Briefly, 1 μL amplified DNA fragment of both specific PCR mixes, both targeted the same codon, was added to 48 μL of fresh PCR reaction solution together with primers pHPLT-BgIII-FW and pHPLT-BgIII-RV. This fusion PCR amplification (22 cycles) resulted in a linear pHPLT-ASP-c1-2 DNA fragment with a specific Asp mature codon randomly mutated and a unique BgIII restriction site on both ends. Purification of this DNA fragment (Qiagen PCR purification kit, Cat. No. 28106), digesting it with BgIII, performing an additional purification step and a ligation reaction (Invitrogen T4 DNA Ligase (Cat. No. 15224-025) generated circular and multimeric DNA that was subsequently transformed into B. subtilis (ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32, ΔamyE:(xyIR,pxyIA-comK). For each library, after overnight incubation at 37° C., 96 single colonies were picked from Heart Infusion agar plates with 20 mg/L neomycin and grown for 4 days at 37° C. in MOPS media with 20 mg/ml neomycin and 1.25 g/L yeast extract (See, WO 03/062380, incorporated herein by reference, for the exact medium formulation used herein) for sequence analysis (BaseClear) and protease expression for screening purposes. The library numbers ranged from 1 up to 189, with each number representing the codon of the mature asp sequence that is randomly mutated. After selection, each library included a maximum of 20 Asp protease variants.







TABLE 15-1





Primers Used to Generate Synthetic ASP SSM Libraries
















pHPLT-BgIII-FW
GCAATCAGATCTTCCTTCAGGTTATGACC (SEQ ID NO: 215)


 


pHPLT-BgIII-RV
GCATCGAAGATCTGATTGCTTAACTGCTTC (SEQ ID NO: 216)





Forward



Mutagenesis



Primer
DNA sequence, 5′ to 3′





asp1F
GAAACGCCTAGAACGATGNNSGACGTAATTGGAGGCAAC



(SEQ ID NO: 217)


 


asp2F
ACGCCTAGAACGATGTTCNNSGTAATTGGAGGCAACGCA



(SEQ ID NO: 218)


 


asp3F
CCTAGAACGATGTTCGACNNSATTGGAGGCAACGCATAT



(SEQ ID NO: 219)


 


asp4F
AGAACGATGTTCGACGTANNSGGAGGCAACGCATATACT



(SEQ ID NO: 220)


 


asp5F
ACGATGTTCGACGTAATTNNSGGCAACGCATATACTATT



(SEQ ID NO: 221)


 


asp6F
ATGTTCGACGTAATTGGANNSAACGCATATACTATTGGC



(SEQ ID NO: 222)


 


asp7F
TTCGACGTAATTGGAGGCNNSGCATATACTATTGGCGGC



(SEQ ID NO: 223)


 


asp8F
GACGTAATTGGAGGCAACNNSTATACTATTGGCGGCCGG



(SEQ ID NO: 224)


 


asp9F
GTAATTGGAGGCAACGCANNSACTATTGGCGGCCGGTCT



(SEQ ID NO: 225)


 


asp10F
ATTGGAGGCAACGCATATNNSATTGGCGGCCGGTCTAGA



(SEQ ID NO: 226)


 


asp11F
GGAGGCAACGCATATACTNNSGGCGGCCGGTCTAGATGT



(SEQ ID NO: 227)


 


asp12F
GGCAACGCATATACTATTNNSGGCCGGTCTAGATGTTCT



(SEQ ID NO: 228)


 


asp13F
AACGCATATACTATTGGCNNSCGGTCTAGATGTTCTATC



(SEQ ID NO: 229)


 


asp14F
GCATATACTATTGGCGGCNNSTCTAGATGTTCTATCGGA



(SEQ ID NO: 230)


 


asp15F
TATACTATTGGCGGCCGGNNSAGATGTTCTATCGGATTC



(SEQ ID NO: 231)


 


asp16F
ACTATTGGCGGCCGGTCTNNSTGTTCTATCGGATTCGCA



(SEQ ID NO: 232)


 


asp17F
ATTGGCGGCCGGTCTAGANNSTCTATCGGATTCGCAGTA



(SEQ ID NO: 233)


 


asp18F
GGCGGCCGGTCTAGATGTNNSATCGGATTCGCAGTAAAC



(SEQ ID NO: 234)


 


asp19F
GGCCGGTCTAGATGTTCTNNSGGATTCGCAGTAAACGGT



(SEQ ID NO: 235)


 


asp20F
CGGTCTAGATGTTCTATCNNSTTCGCAGTAAACGGTGGC



(SEQ ID NO: 236)


 


asp21F
TCTAGATGTTCTATCGGANNSGCAGTAAACGGTGGCTTC



(SEQ ID NO: 237)


 


asp22F
AGATGTTCTATCGGATTCNNSGTAAACGGTGGCTTCATT



(SEQ ID NO: 238)


 


asp23F
TGTTCTATCGGATTCGCANNSAACGGTGGCTTCATTACT



(SEQ ID NO: 239)


 


asp24F
TCTATCGGATTCGCAGTANNSGGTGGCTTCATTACTGCC



(SEQ ID NO: 240)


 


asp25F
ATCGGATTCGCAGTAAACNNSGGCTTCATTACTGCCGGT



(SEQ ID NO: 241)


 


asp26F
GGATTCGCAGTAAACGGTNNSTTCATTACTGCCGGTCAC



(SEQ ID NO: 242)


 


asp27F
TTCGCAGTAAACGGTGGCNNSATTACTGCCGGTCACTGC



(SEQ ID NO: 243)


 


asp28F
GCAGTAAACGGTGGCTTCNNSACTGCCGGTCACTGCGGA



(SEQ ID NO: 244)


 


asp29F
GTAAACGGTGGCTTCATTNNSGCCGGTCACTGCGGAAGA



(SEQ ID NO: 245)


 


asp30F
AACGGTGGCTTCATTACTNNSGGTCACTGCGGAAGAACA



(SEQ ID NO: 246)


 


asp31F
GGTGGCTTCATTACTGCCNNSCACTGCGGAAGAACAGGA



(SEQ ID NO: 247)


 


asp32F
GGCTTCATTACTGCCGGTNNSTGCGGAAGAACAGGAGCC



(SEQ ID NO: 248)


 


asp33F
TTCATTACTGCCGGTCACNNSGGAAGAACAGGAGCCACT



(SEQ ID NO: 249)


 


asp34F
ATTACTGCCGGTCACTGCNNSAGAACAGGAGCCACTACT



(SEQ ID NO: 250)


 


asp35F
ACTGCCGGTCACTGCGGANNSACAGGAGCCACTACTGCC



(SEQ ID NO: 251)


 


asp36F
GCCGGTCACTGCGGAAGANNSGGAGCCACTACTGCCAAT



(SEQ ID NO: 252)


 


asp37F
GGTCACTGCGGAAGAACANNSGCCACTACTGCCAATCCG



(SEQ ID NO: 253)


 


asp38F
CACTGCGGAAGAACAGGANNSACTACTGCCAATCCGACT



(SEQ ID NO: 254)


 


asp39F
TGCGGAAGAACAGGAGCCNNSACTGCCAATCCGACTGGC



(SEQ ID NO: 255)


 


asp40F
GGAAGAACAGGAGCCACTNNSGCCAATCCGACTGGCACA



(SEQ ID NO: 256)


 


asp41F
AGAACAGGAGCCACTACTNNSAATCCGACTGGCACATTT



(SEQ ID NO: 257)


 


asp42F
ACAGGAGCCACTACTGCCNNSCCGACTGGCACATTTGCA



(SEQ ID NO: 258)


 


asp43F
GGAGCCACTACTGCCAATNNSACTGGCACATTTGCAGGT



(SEQ ID NO: 259)


 


asp44F
GCCACTACTGCCAATCCGNNSGGCACATTTGCAGGTAGC



(SEQ ID NO: 260)


 


asp45F
ACTACTGCCAATCCGACTNNSACATTTGCAGGTAGCTCG



(SEQ ID NO: 261)


 


asp46F
ACTGCCAATCCGACTGGCNNSTTTGCAGGTAGCTCGTTT



(SEQ ID NO: 262)


 


asp47F
GCCAATCCGACTGGCACANNSGCAGGTAGCTCGTTTCCG



(SEQ ID NO: 263)


 


asp48F
AATCCGACTGGCACATTTNNSGGTAGCTCGTTTCCGGGA



(SEQ ID NO: 264)


 


asp49F
CCGACTGGCACATTTGCANNSAGCTCGTTTCCGGGAAAT



(SEQ ID NO: 265)


 


asp50F
ACTGGCACATTTGCAGGTNNSTCGTTTCCGGGAAATGAT



(SEQ ID NO: 266)


 


asp51F
GGCACATTTGCAGGTAGCNNSTTTCCGGGAAATGATTAT



(SEQ ID NO: 267)


 


asp52F
ACATTTGCAGGTAGCTCGNNSCCGGGAAATGATTATGCA



(SEQ ID NO: 268)


 


asp53F
TTTGCAGGTAGCTCGTTTNNSGGAAATGATTATGCATTC



(SEQ ID NO: 269)


 


asp54F
GCAGGTAGCTCGTTTCCGNNSAATGATTATGCATTCGTC



(SEQ ID NO: 270)


 


asp55F
GGTAGCTCGTTTCCGGGANNSGATTATGCATTCGTCCGA



(SEQ ID NO: 271)


 


asp56F
AGCTCGTTTCCGGGAAATNNSTATGCATTCGTCCGAACA



(SEQ ID NO: 272)


 


asp57F
TCGTTTCCGGGAAATGATNNSGCATTCGTCCGAACAGGG



(SEQ ID NO: 273)


 


asp58F
TTTCCGGGAAATGATTATNNSTTCGTCCGAACAGGGGCA



(SEQ ID NO: 274)


 


asp59F
CCGGGAAATGATTATGCANNSGTCCGAACAGGGGCAGGA



(SEQ ID NO: 275)


 


asp60F
GGAAATGATTATGCATTCNNSCGAACAGGGGCAGGAGTA



(SEQ ID NO: 276)


 


asp61F
AATGATTATGCATTCGTCNNSACAGGGGCAGGAGTAAAT



(SEQ ID NO: 277)


 


asp62F
GATTATGCATTCGTCCGANNSGGGGCAGGAGTAAATTTG



(SEQ ID NO: 278)


 


asp63F
TATGCATTCGTCCGAACANNSGCAGGAGTAAATTTGCTT



(SEQ ID NO: 279)


 


asp64F
GCATTCGTCCGAACAGGGNNSGGAGTAAATTTGCTTGCC



(SEQ ID NO: 280)


 


asp65F
TTCGTCCGAACAGGGGCANNSGTAAATTTGCTTGCCCAA



(SEQ ID NO: 281)


 


asp66F
GTCCGAACAGGGGCAGGANNSAATTTGCTTGCCCAAGTC



(SEQ ID NO: 282)


 


asp67F
CGAACAGGGGCAGGAGTANNSTTGCTTGCCCAAGTCAAT



(SEQ ID NO: 283)


 


asp68F
ACAGGGGCAGGAGTAAATNNSCTTGCCCAAGTCAATAAC



(SEQ ID NO: 284)


 


asp69F
GGGGCAGGAGTAAATTTGNNSGCCCAAGTCAATAACTAC



(SEQ ID NO: 285)


 


asp70F
GCAGGAGTAAATTTGCTTNNSCAAGTCAATAACTACTCG



(SEQ ID NO: 286)


 


asp71F
GGAGTAAATTTGCTTGCCNNSGTCAATAACTACTCGGGC



(SEQ ID NO: 287)


 


asp72F
GTAAATTTGCTTGCCCAANNSAATAACTACTCGGGCGGC



(SEQ ID NO: 288)


 


asp73F
AATTTGCTTGCCCAAGTCNNSAACTACTCGGGCGGCAGA



(SEQ ID NO: 289)


 


asp74F
TTGCTTGCCCAAGTCAATNNSTACTCGGGCGGCAGAGTC



(SEQ ID NO: 290)


 


asp75F
CTTGCCCAAGTCAATAACNNSTCGGGCGGCAGAGTCCAA



(SEQ ID NO: 291)


 


asp76F
GCCCAAGTCAATAACTACNNSGGCGGCAGAGTCCAAGTA



(SEQ ID NO: 292)


 


asp77F
CAAGTCAATAACTACTCGNNSGGCAGAGTCCAAGTAGCA



(SEQ ID NO: 293)


 


asp78F
GTCAATAACTACTCGGGCNNSAGAGTCCAAGTAGCAGGA



(SEQ ID NO: 294)


 


asp79F
AATAACTACTCGGGCGGCNNSGTCCAAGTAGCAGGACAT



(SEQ ID NO: 295)


 


asp80F
AACTACTCGGGCGGCAGANNSCAAGTAGCAGGACATACG



(SEQ ID NO: 296)


 


asp81F
TACTCGGGCGGCAGAGTCNNSGTAGCAGGACATACGGCC



(SEQ ID NO: 297)


 


asp82F
TCGGGCGGCAGAGTCCAANNSGCAGGACATACGGCCGCA



(SEQ ID NO: 298)


 


asp83F
GGCGGCAGAGTCCAAGTANNSGGACATACGGCCGCACCA



(SEQ ID NO: 299)


 


asp84F
GGCAGAGTCCAAGTAGCANNSCATACGGCCGCACCAGTT



(SEQ ID NO: 300)


 


asp85F
AGAGTCCAAGTAGCAGGANNSACGGCCGCACCAGTTGGA



(SEQ ID NO: 301)


 


asp86F
GTCCAAGTAGCAGGACATNNSGCCGCACCAGTTGGATCT



(SEQ ID NO: 302)


 


asp87F
CAAGTAGCAGGACATACGNNSGCACCAGTTGGATCTGCT



(SEQ ID NO: 303)


 


asp88F
GTAGCAGGACATACGGCCNNSCCAGTTGGATCTGCTGTA



(SEQ ID NO: 304)


 


asp89F
GCAGGACATACGGCCGCANNSGTTGGATCTGCTGTATGC



(SEQ ID NO: 305)


 


asp90F
GGACATACGGCCGCACCANNSGGATCTGCTGTATGCCGC



(SEQ ID NO: 306)


 


asp91F
CATACGGCCGCACCAGTTNNSTCTGCTGTATGCCGCTCA



(SEQ ID NO: 307)


 


asp92F
ACGGCCGCACCAGTTGGANNSGCTGTATGCCGCTCAGGT



(SEQ ID NO: 308)


 


asp93F
GCCGCACCAGTTGGATCTNNSGTATGCCGCTCAGGTAGC



(SEQ ID NO: 309)


 


asp94F
GCACCAGTTGGATCTGCTNNSTGCCGCTCAGGTAGCACT



(SEQ ID NO: 310)


 


asp95F
CCAGTTGGATCTGCTGTANNSCGCTCAGGTAGCACTACA



(SEQ ID NO: 311)


 


asp96F
GTTGGATCTGCTGTATGCNNSTCAGGTAGCACTACAGGT



(SEQ ID NO: 312)


 


asp97F
GGATCTGCTGTATGCCGCNNSGGTAGCACTACAGGTTGG



(SEQ ID NO: 313)


 


asp98F
TCTGCTGTATGCCGCTCANNSAGCACTACAGGTTGGCAT



(SEQ ID NO: 314)


 


asp99F
GCTGTATGCCGCTCAGGTNNSACTACAGGTTGGCATTGC



(SEQ ID NO: 315)


 


asp100F
GTATGCCGCTCAGGTAGCNNSACAGGTTGGCATTGCGGA



(SEQ ID NO: 316)


 


asp101F
TGCCGCTCAGGTAGCACTNNSGGTTGGCATTGCGGAACT



(SEQ ID NO: 317)


 


asp102F
CGCTCAGGTAGCACTACANNSTGGCATTGCGGAACTATC



(SEQ ID NO: 318)


 


asp103F
TCAGGTAGCACTACAGGTNNSCATTGCGGAACTATCACG



(SEQ ID NO: 319)


 


asp104F
GGTAGCACTACAGGTTGGNNSTGCGGAACTATCACGGCG



(SEQ ID NO: 320)


 


asp105F
AGCACTACAGGTTGGCATNNSGGAACTATCACGGCGCTG



(SEQ ID NO: 321)


 


asp106F
ACTACAGGTTGGCATTGCNNSACTATCACGGCGCTGAAT



(SEQ ID NO: 322)


 


asp107F
ACAGGTTGGCATTGCGGANNSATCACGGCGCTGAATTCG



(SEQ ID NO: 323)


 


asp108F
GGTTGGCATTGCGGAACTNNSACGGCGCTGAATTCGTCT



(SEQ ID NO: 324)


 


asp109F
TGGCATTGCGGAACTATCNNSGCGCTGAATTCGTCTGTC



(SEQ ID NO: 325)


 


asp110F
CATTGCGGAACTATCACGNNSCTGAATTCGTCTGTCACG



(SEQ ID NO: 326)


 


asp111F
TGCGGAACTATCACGGCGNNSAATTCGTCTGTCACGTAT



(SEQ ID NO: 327)


 


asp112F
GGAACTATCACGGCGCTGNNSTCGTCTGTCACGTATCCA



(SEQ ID NO: 328)


 


asp113F
ACTATCACGGCGCTGAATNNSTCTGTCACGTATCCAGAG



(SEQ ID NO: 329)


 


asp114F
ATCACGGCGCTGAATTCGNNSGTCACGTATCCAGAGGGA



(SEQ ID NO: 330)


 


asp115F
ACGGCGCTGAATTCGTCTNNSACGTATCCAGAGGGAACA



(SEQ ID NO: 331)


 


asp116F
GCGCTGAATTCGTCTGTCNNSTATCCAGAGGGAACAGTC



(SEQ ID NO: 332)


 


asp117F
CTGAATTCGTCTGTCACGNNSCCAGAGGGAACAGTCCGA



(SEQ ID NO: 333)


 


asp118F
AATTCGTCTGTCACGTATNNSGAGGGAACAGTCCGAGGA



(SEQ ID NO: 334)


 


asp119F
TCGTCTGTCACGTATCCANNSGGAACAGTCCGAGGACTT



(SEQ ID NO: 335)


 


asp120F
TCTGTCACGTATCCAGAGNNSACAGTCCGAGGACTTATC



(SEQ ID NO: 336)


 


asp121F
GTCACGTATCCAGAGGGANNSGTCCGAGGACTTATCCGC



(SEQ ID NO: 337)


 


asp122F
ACGTATCCAGAGGGAACANNSCGAGGACTTATCCGCACG



(SEQ ID NO: 338)


 


asp123F
TATCCAGAGGGAACAGTCNNSGGACTTATCCGCACGACG



(SEQ ID NO: 339)


 


asp124F
CCAGAGGGAACAGTCCGANNSCTTATCCGCACGACGGTT



(SEQ ID NO: 340)


 


asp125F
GAGGGAACAGTCCGAGGANNSATCCGCACGACGGTTTGT



(SEQ ID NO: 341)


 


asp126F
GGAACAGTCCGAGGACTTNNSCGCACGACGGTTTGTGCC



(SEQ ID NO: 342)


 


asp127F
ACAGTCCGAGGACTTATCNNSACGACGGTTTGTGCCGAA



(SEQ ID NO: 343)


 


asp128F
GTCCGAGGACTTATCCGCNNSACGGTTTGTGCCGAACCA



(SEQ ID NO: 344)


 


asp129F
CGAGGACTTATCCGCACGNNSGTTTGTGCCGAACCAGGT



(SEQ ID NO: 345)


 


asp130F
GGACTTATCCGCACGACGNNSTGTGCCGAACCAGGTGAT



(SEQ ID NO: 346)


 


asp131F
CTTATCCGCACGACGGTTNNSGCCGAACCAGGTGATAGC



(SEQ ID NO: 347)


 


asp132F
ATCCGCACGACGGTTTGTNNSGAACCAGGTGATAGCGGA



(SEQ ID NO: 348)


 


asp133F
CGCACGACGGTTTGTGCCNNSCCAGGTGATAGCGGAGGT



(SEQ ID NO: 349)


 


asp134F
ACGACGGTTTGTGCCGAANNSGGTGATAGCGGAGGTAGC



(SEQ ID NO: 350)


 


asp135F
ACGGTTTGTGCCGAACCANNSGATAGCGGAGGTAGCCTT



(SEQ ID NO: 351)


 


asp136F
GTTTGTGCCGAACCAGGTNNSAGCGGAGGTAGCCTTTTA



(SEQ ID NO: 352)


 


asp137F
TGTGCCGAACCAGGTGATNNSGGAGGTAGCCTTTTAGCG



(SEQ ID NO: 353)


 


asp138F
GCCGAACCAGGTGATAGCNNSGGTAGCCTTTTAGCGGGA



(SEQ ID NO: 354)


 


asp139F
GAACCAGGTGATAGCGGANNSAGCCTTTTAGCGGGAAAT



(SEQ ID NO: 355)


 


asp140F
CCAGGTGATAGCGGAGGTNNSCTTTTAGCGGGAAATCAA



(SEQ ID NO: 356)


 


asp141F
GGTGATAGCGGAGGTAGCNNSTTAGCGGGAAATCAAGCC



(SEQ ID NO: 357)


 


asp142F
GATAGCGGAGGTAGCCTTNNSGCGGGAAATCAAGCCCAA



(SEQ ID NO: 358)


 


asp143F
AGCGGAGGTAGCCTTTTANNSGGAAATCAAGCCCAAGGT



(SEQ ID NO: 359)


 


asp144F
GGAGGTAGCCTTTTAGCGNNSAATCAAGCCCAAGGTGTC



(SEQ ID NO: 360)


 


asp145F
GGTAGCCTTTTAGCGGGANNSCAAGCCCAAGGTGTCACG



(SEQ ID NO: 361)


 


asp146F
AGCCTTTTAGCGGGAAATNNSGCCCAAGGTGTCACGTCA



(SEQ ID NO: 362)


 


asp147F
CTTTTAGCGGGAAATCAANNSCAAGGTGTCACGTCAGGT



(SEQ ID NO: 363)


 


asp148F
TTAGCGGGAAATCAAGCCNNSGGTGTCACGTCAGGTGGT



(SEQ ID NO: 364)


 


asp149F
GCGGGAAATCAAGCCCAANNSGTCACGTCAGGTGGTTCT



(SEQ ID NO: 365)


 


asp150F
GGAAATCAAGCCCAAGGTNNSACGTCAGGTGGTTCTGGA



(SEQ ID NO: 366)


 


asp151F
AATCAAGCCCAAGGTGTCNNSTCAGGTGGTTCTGGAAAT



(SEQ ID NO: 367)


 


asp152F
CAAGCCCAAGGTGTCACGNNSGGTGGTTCTGGAAATTGT



(SEQ ID NO: 368)


 


asp153F
GCCCAAGGTGTCACGTCANNSGGTTCTGGAAATTGTCGG



(SEQ ID NO: 369)


 


asp154F
CAAGGTGTCACGTCAGGTNNSTCTGGAAATTGTCGGACG



(SEQ ID NO: 370)


 


asp155F
GGTGTCACGTCAGGTGGTNNSGGAAATTGTCGGACGGGG



(SEQ ID NO: 371)


 


asp156F
GTCACGTCAGGTGGTTCTNNSAATTGTCGGACGGGGGGA



(SEQ ID NO: 372)


 


asp157F
ACGTCAGGTGGTTCTGGANNSTGTCGGACGGGGGGAACA



(SEQ ID NO: 373)


 


asp158F
TCAGGTGGTTCTGGAAATNNSCGGACGGGGGGAACAACA



(SEQ ID NO: 374)


 


asp159F
GGTGGTTCTGGAAATTGTNNSACGGGGGGAACAACATTC



(SEQ ID NO: 375)


 


asp160F
GGTTCTGGAAATTGTCGGNNSGGGGGAACAACATTCTTT



(SEQ ID NO: 376)


 


asp161F
TCTGGAAATTGTCGGACGNNSGGAACAACATTCTTTCAA



(SEQ ID NO: 377)


 


asp162F
GGAAATTGTCGGACGGGGNNSACAACATTCTTTCAACCA



(SEQ ID NO: 378)


 


asp163F
AATTGTCGGACGGGGGGANNSACATTCTTTCAACCAGTC



(SEQ ID NO: 379)


 


asp164F
TGTCGGACGGGGGGAACANNSTTCTTTCAACCAGTCAAC



(SEQ ID NO: 380)


 


asp165F
CGGACGGGGGGAACAACANNSTTTCAACCAGTCAACCCG



(SEQ ID NO: 381)


 


asp166F
ACGGGGGGAACAACATTCNNSCAACCAGTCAACCCGATT



(SEQ ID NO: 382)


 


asp167F
GGGGGAACAACATTCTTTNNSCCAGTCAACCCGATTTTG



(SEQ ID NO: 383)


 


asp168F
GGAACAACATTCTTTCAANNSGTCAACCCGATTTTGCAG



(SEQ ID NO: 384)


 


asp169F
ACAACATTCTTTCAACCANNSAACCCGATTTTGCAGGCT



(SEQ ID NO: 385)


 


asp170F
ACATTCTTTCAACCAGTCNNSCCGATTTTGCAGGCTTAC



(SEQ ID NO: 386)


 


asp171F
TTCTTTCAACCAGTCAACNNSATTTTGCAGGCTTACGGC



(SEQ ID NO: 387)


 


asp172F
TTTCAACCAGTCAACCCGNNSTTGCAGGCTTACGGCCTG



(SEQ ID NO: 388)


 


asp173F
CAACCAGTCAACCCGATTNNSCAGGCTTACGGCCTGAGA



(SEQ ID NO: 389)


 


asp174F
CCAGTCAACCCGATTTTGNNSGCTTACGGCCTGAGAATG



(SEQ ID NO: 390)


 


asp175F
GTCAACCCGATTTTGCAGNNSTACGGCCTGAGAATGATT



(SEQ ID NO: 391)


 


asp176F
AACCCGATTTTGCAGGCTNNSGGCCTGAGAATGATTACG



(SEQ ID NO: 392)


 


asp177F
CCGATTTTGCAGGCTTACNNSCTGAGAATGATTACGACT



(SEQ ID NO: 393)


 


asp178F
ATTTTGCAGGCTTACGGCNNSAGAATGATTACGACTGAC



(SEQ ID NO: 394)


 


asp179F
TTGCAGGCTTACGGCCTGNNSATGATTACGACTGACTCT



(SEQ ID NO: 395)


 


asp180F
CAGGCTTACGGCCTGAGANNSATTACGACTGACTCTGGA



(SEQ ID NO: 396)


 


asp181F
GCTTACGGCCTGAGAATGNNSACGACTGACTCTGGAAGT



(SEQ ID NO: 397)


 


asp182F
TACGGCCTGAGAATGATTNNSACTGACTCTGGAAGTTCC



(SEQ ID NO: 398)


 


asp183F
GGCCTGAGAATGATTACGNNSGACTCTGGAAGTTCCCCT



(SEQ ID NO: 399)


 


asp184F
CTGAGAATGATTACGACTNNSTCTGGAAGTTCCCCTTAA



(SEQ ID NO: 400)


 


asp185F
AGAATGATTACGACTGACNNSGGAAGTTCCCCTTAACCC



(SEQ ID NO: 401)


 


asp186F
ATGATTACGACTGACTCTNNSAGTTCCCCTTAACCCAAC



(SEQ ID NO: 402)


 


asp187F
ATTACGACTGACTCTGGANNSTCCCCTTAACCCAACAGA



(SEQ ID NO: 403)


 


asp188F
ACGACTGACTCTGGAAGTNNSCCTTAACCCAACAGAGGA



(SEQ ID NO: 404)


 


asp189F
ACTGACTCTGGAAGTTCCNNSTAACCCAACAGAGGACGG



(SEQ ID NO: 405)





Reverse



mutagenesis



primer
DNA sequence, 5′-3′





asp1R
GTTGCCTCCAATTACGTCSNNCATCGTTCTAGGCGTTTC



(SEQ ID NO: 406)


 


asp2R
TGCGTTGCCTCCAATTACSNNGAACATCGTTCTAGGCGT



(SEQ ID NO: 407)


 


asp3R
ATATGCGTTGCCTCCAATSNNGTCGAACATCGTTCTAGG



(SEQ ID NO: 408)


 


asp4R
AGTATATGCGTTGCCTCCSNNTACGTCGAACATCGTTCT



(SEQ ID NO: 409)


 


asp5R
AATAGTATATGCGTTGCCSNNAATTACGTCGAACATCGT



(SEQ ID NO: 410)


 


asp6R
GCCAATAGTATATGCGTTSNNTCCAATTACGTCGAACAT



(SEQ ID NO: 411)


 


asp7R
GCCGCCAATAGTATATGCSNNGCCTCCAATTACGTCGAA



(SEQ ID NO: 412)


 


asp8R
CCGGCCGCCAATAGTATASNNGTTGCCTCCAATTACGTC



(SEQ ID NO: 413)


 


asp9R
AGACCGGCCGCCAATAGTSNNTGCGTTGCCTCCAATTAC



(SEQ ID NO: 414)


 


asp10R
TCTAGACCGGCCGCCAATSNNATATGCGTTGCCTCCAAT



(SEQ ID NO: 415)


 


asp11R
ACATCTAGACCGGCCGCCSNNAGTATATGCGTTGCCTCC



(SEQ ID NO: 416)


 


asp12R
AGAACATCTAGACCGGCCSNNAATAGTATATGCGTTGCC



(SEQ ID NO: 417)


 


asp13R
GATAGAACATCTAGACCGSNNGCCAATAGTATATGCGTT



(SEQ ID NO: 418)


 


asp14R
TCCGATAGAACATCTAGASNNGCCGCCAATAGTATATGC



(SEQ ID NO: 419)


 


asp15R
GAATCCGATAGAACATCTSNNCCGGCCGCCAATAGTATA



(SEQ ID NO: 420)


 


asp16R
TGCGAATCCGATAGAACASNNAGACCGGCCGCCAATAGT



(SEQ ID NO: 421)


 


asp17R
TACTGCGAATCCGATAGASNNTCTAGACCGGCCGCCAAT



(SEQ ID NO: 422)


 


asp18R
GTTTACTGCGAATCCGATSNNACATCTAGACCGGCCGCC



(SEQ ID NO: 423)


 


asp19R
ACCGTTTACTGCGAATCCSNNAGAACATCTAGACCGGCC



(SEQ ID NO: 424)


 


asp20R
GCCACCGTTTACTGCGAASNNGATAGAACATCTAGACCG



(SEQ ID NO: 425)


 


asp21R
GAAGCCACCGTTTACTGCSNNTCCGATAGAACATCTAGA



(SEQ ID NO: 426)


 


asp22R
AATGAAGCCACCGTTTACSNNGAATCCGATAGAACATCT



(SEQ ID NO: 427)


 


asp23R
AGTAATGAAGCCACCGTTSNNTGCGAATCCGATAGAACA



(SEQ ID NO: 428)


 


asp24R
GGCAGTAATGAAGCCACCSNNTACTGCGAATCCGATAGA



(SEQ ID NO: 429)


 


asp25R
ACCGGCAGTAATGAAGCCSNNGTTTACTGCGAATCCGAT



(SEQ ID NO: 430)


 


asp26R
GTGACCGGCAGTAATGAASNNACCGTTTACTGCGAATCC



(SEQ ID NO: 431)


 


asp27R
GCAGTGACCGGCAGTAATSNNGCCACCGTTTACTGCGAA



(SEQ ID NO: 432)


 


asp28R
TCCGCAGTGACCGGCAGTSNNGAAGCCACCGTTTACTGC



(SEQ ID NO: 433)


 


asp29R
TCTTCCGCAGTGACCGGCSNNAATGAAGCCACCGTTTAC



(SEQ ID NO: 434)


 


asp30R
TGTTCTTCCGCAGTGACCSNNAGTAATGAAGCCACCGTT



(SEQ ID NO: 435)


 


asp31R
TCCTGTTCTTCCGCAGTGSNNGGCAGTAATGAAGCCACC



(SEQ ID NO: 436)


 


asp32R
GGCTCCTGTTCTTCCGCASNNACCGGCAGTAATGAAGCC



(SEQ ID NO: 437)


 


asp33R
AGTGGCTCCTGTTCTTCCSNNGTGACCGGCAGTAATGAA



(SEQ ID NO: 438)


 


asp34R
AGTAGTGGCTCCTGTTCTSNNGCAGTGACCGGCAGTAAT



(SEQ ID NO: 439)


 


asp35R
GGCAGTAGTGGCTCCTGTSNNTCCGCAGTGACCGGCAGT



(SEQ ID NO: 440)


 


asp36R
ATTGGCAGTAGTGGCTCCSNNTCTTCCGCAGTGACCGGC



(SEQ ID NO: 441)


 


asp37R
CGGATTGGCAGTAGTGGCSNNTGTTCTTCCGCAGTGACC



(SEQ ID NO: 442)


 


asp38R
AGTCGGATTGGCAGTAGTSNNTCCTGTTCTTCCGCAGTG



(SEQ ID NO: 443)


 


asp39R
GCCAGTCGGATTGGCAGTSNNGGCTCCTGTTCTTCCGCA



(SEQ ID NO: 444)


 


asp40R
TGTGCCAGTCGGATTGGCSNNAGTGGCTCCTGTTCTTCC



(SEQ ID NO: 445)


 


asp41R
AAATGTGCCAGTCGGATTSNNAGTAGTGGCTCCTGTTCT



(SEQ ID NO: 446)


 


asp42R
TGCAAATGTGCCAGTCGGSNNGGCAGTAGTGGCTCCTGT



(SEQ ID NO: 447)


 


asp43R
ACCTGCAAATGTGCCAGTSNNATTGGCAGTAGTGGCTCC



(SEQ ID NO: 448)


 


asp44R
GCTACCTGCAAATGTGCCSNNCGGATTGGCAGTAGTGGC



(SEQ ID NO: 449)


 


asp45R
CGAGCTACCTGCAAATGTSNNAGTCGGATTGGCAGTAGT



(SEQ ID NO: 450)


 


asp46R
AAACGAGCTACCTGCAAASNNGCCAGTCGGATTGGCAGT



(SEQ ID NO: 451)


 


asp47R
CGGAAACGAGCTACCTGCSNNTGTGCCAGTCGGATTGGC



(SEQ ID NO: 452)


 


asp48R
TCCCGGAAACGAGCTACCSNNAAATGTGCCAGTCGGATT



(SEQ ID NO: 453)


 


asp49R
ATTTCCCGGAAACGAGCTSNNTGCAAATGTGCCAGTCGG



(SEQ ID NO: 454)


 


asp50R
ATCATTTCCCGGAAACGASNNACCTGCAAATGTGCCAGT



(SEQ ID NO: 455)


 


asp51R
ATAATCATTTCCCGGAAASNNGCTACCTGCAAATGTGCC



(SEQ ID NO: 456)


 


asp52R
TGCATAATCATTTCCCGGSNNCGAGCTACCTGCAAATGT



(SEQ ID NO: 457)


 


asp53R
GAATGCATAATCATTTCCSNNAAACGAGCTACCTGCAAA



(SEQ ID NO: 458)


 


asp54R
GACGAATGCATAATCATTSNNCGGAAACGAGCTACCTGC



(SEQ ID NO: 459)


 


asp55R
TCGGACGAATGCATAATCSNNTCCCGGAAACGAGCTACC



(SEQ ID NO: 460)


 


asp56R
TGTTCGGACGAATGCATASNNATTTCCCGGAAACGAGCT



(SEQ ID NO: 461)


 


asp57R
CCCTGTTCGGACGAATGCSNNATCATTTCCCGGAAACGA



(SEQ ID NO: 462)


 


asp58R
TGCCCCTGTTCGGACGAASNNATAATCATTTCCCGGAAA



(SEQ ID NO: 463)


 


asp59R
TCCTGCCCCTGTTCGGACSNNTGCATAATCATTTCCCGG



(SEQ ID NO: 464)


 


asp60R
TACTCCTGCCCCTGTTCGSNNGAATGCATAATCATTTCC



(SEQ ID NO: 465)


 


asp61R
ATTTACTCCTGCCCCTGTSNNGACGAATGCATAATCATT



(SEQ ID NO: 466)


 


asp62R
CAAATTTACTCCTGCCCCSNNTCGGACGAATGCATAATC



(SEQ ID NO: 467)


 


asp63R
AAGCAAATTTACTCCTGCSNNTGTTCGGACGAATGCATA



(SEQ ID NO: 468)


 


asp64R
GGCAAGCAAATTTACTCCSNNCCCTGTTCGGACGAATGC



(SEQ ID NO: 469)


 


asp65R
TTGGGCAAGCAAATTTACSNNTGCCCCTGTTCGGACGAA



(SEQ ID NO: 470)


 


asp66R
GACTTGGGCAAGCAAATTSNNTCCTGCCCCTGTTCGGAC



(SEQ ID NO: 471)


 


asp67R
ATTGACTTGGGCAAGCAASNNTACTCCTGCCCCTGTTCG



(SEQ ID NO: 472)


 


asp68R
GTTATTGACTTGGGCAAGSNNATTTACTCCTGCCCCTGT



(SEQ ID NO: 473)


 


asp69R
GTAGTTATTGACTTGGGCSNNCAAATTTACTCCTGCCCC



(SEQ ID NO: 474)


 


asp70R
CGAGTAGTTATTGACTTGSNNAAGCAAATTTACTCCTGC



(SEQ ID NO: 475)


 


asp71R
GCCCGAGTAGTTATTGACSNNGGCAAGCAAATTTACTCC



(SEQ ID NO: 476)


 


asp72R
GCCGCCCGAGTAGTTATTSNNTTGGGCAAGCAAATTTAC



(SEQ ID NO: 477)


 


asp73R
TCTGCCGCCCGAGTAGTTSNNGACTTGGGCAAGCAAATT



(SEQ ID NO: 478)


 


asp74R
GACTCTGCCGCCCGAGTASNNATTGACTTGGGCAAGCAA



(SEQ ID NO: 479)


 


asp75R
TTGGACTCTGCCGCCCGASNNGTTATTGACTTGGGCAAG



(SEQ ID NO: 480)


 


asp76R
TACTTGGACTCTGCCGCCSNNGTAGTTATTGACTTGGGC



(SEQ ID NO: 481)


 


asp77R
TGCTACTTGGACTCTGCCSNNCGAGTAGTTATTGACTTG



(SEQ ID NO: 482)


 


asp78R
TCCTGCTACTTGGACTCTSNNGCCCGAGTAGTTATTGAC



(SEQ ID NO: 483)


 


asp79R
ATGTCCTGCTACTTGGACSNNGCCGCCCGAGTAGTTATT



(SEQ ID NO: 484)


 


asp80R
CGTATGTCCTGCTACTTGSNNTCTGCCGCCCGAGTAGTT



(SEQ ID NO: 485)


 


asp81R
GGCCGTATGTCCTGCTACSNNGACTCTGCCGCCCGAGTA



(SEQ ID NO: 486)


 


asp82R
TGCGGCCGTATGTCCTGCSNNTTGGACTCTGCCGCCCGA



(SEQ ID NO: 487)


 


asp83R
TGGTGCGGCCGTATGTCCSNNTACTTGGACTCTGCCGCC



(SEQ ID NO: 488)


 


asp84R
AACTGGTGCGGCCGTATGSNNTGCTACTTGGACTCTGCC



(SEQ ID NO: 489)


 


asp85R
TCCAACTGGTGCGGCCGTSNNTCCTGCTACTTGGACTCT



(SEQ ID NO: 490)


 


asp86R
AGATCCAACTGGTGCGGCSNNATGTCCTGCTACTTGGAC



(SEQ ID NO: 491)


 


asp87R
AGCAGATCCAACTGGTGCSNNCGTATGTCCTGCTACTTG



(SEQ ID NO: 492)


 


asp88R
TACAGCAGATCCAACTGGSNNGGCCGTATGTCCTGCTAC



(SEQ ID NO: 493)


 


asp89R
GCATACAGCAGATCCAACSNNTGCGGCCGTATGTCCTGC



(SEQ ID NO: 494)


 


asp90R
GCGGCATACAGCAGATCCSNNTGGTGCGGCCGTATGTCC



(SEQ ID NO: 495)


 


asp91R
TGAGCGGCATACAGCAGASNNAACTGGTGCGGCCGTATG



(SEQ ID NO: 496)


 


asp92R
ACCTGAGCGGCATACAGCSNNTCCAACTGGTGCGGCCGT



(SEQ ID NO: 497)


 


asp93R
GCTACCTGAGCGGCATACSNNAGATCCAACTGGTGCGGC



(SEQ ID NO: 498)


 


asp94R
AGTGCTACCTGAGCGGCASNNAGCAGATCCAACTGGTGC



(SEQ ID NO: 499)


 


asp95R
TGTAGTGCTACCTGAGCGSNNTACAGCAGATCCAACTGG



(SEQ ID NO: 500)


 


asp96R
ACCTGTAGTGCTACCTGASNNGCATACAGCAGATCCAAC



(SEQ ID NO: 501)


 


asp97R
CCAACCTGTAGTGCTACCSNNGCGGCATACAGCAGATCC



(SEQ ID NO: 502)


 


asp98R
ATGCCAACCTGTAGTGCTSNNTGAGCGGCATACAGCAGA



(SEQ ID NO: 503)


 


asp99R
GCAATGCCAACCTGTAGTSNNACCTGAGCGGCATACAGC



(SEQ ID NO: 504)


 


asp100R
TCCGCAATGCCAACCTGTSNNGCTACCTGAGCGGCATAC



(SEQ ID NO: 505)


 


asp101R
AGTTCCGCAATGCCAACCSNNAGTGCTACCTGAGCGGCA



(SEQ ID NO: 506)


 


asp102R
GATAGTTCCGCAATGCCASNNTGTAGTGCTACCTGAGCG



(SEQ ID NO: 507)


 


asp103R
CGTGATAGTTCCGCAATGSNNACCTGTAGTGCTACCTGA



(SEQ ID NO: 508)


 


asp104R
CGCCGTGATAGTTCCGCASNNCCAACCTGTAGTGCTACC



(SEQ ID NO: 509)


 


asp105R
CAGCGCCGTGATAGTTCCSNNATGCCAACCTGTAGTGCT



(SEQ ID NO: 510)


 


asp106R
ATTCAGCGCCGTGATAGTSNNGCAATGCCAACCTGTAGT



(SEQ ID NO: 511)


 


asp107R
CGAATTCAGCGCCGTGATSNNTCCGCAATGCCAACCTGT



(SEQ ID NO: 512)


 


asp108R
AGACGAATTCAGCGCCGTSNNAGTTCCGCAATGCCAACC



(SEQ ID NO: 513)


 


asp109R
GACAGACGAATTCAGCGCSNNGATAGTTCCGCAATGCCA



(SEQ ID NO: 514)


 


asp110R
CGTGACAGACGAATTCAGSNNCGTGATAGTTCCGCAATG



(SEQ ID NO: 515)


 


asp111R
ATACGTGACAGACGAATTSNNCGCCGTGATAGTTCCGCA



(SEQ ID NO: 516)


 


asp112R
TGGATACGTGACAGACGASNNCAGCGCCGTGATAGTTCC



(SEQ ID NO: 517)


 


asp113R
CTCTGGATACGTGACAGASNNATTCAGCGCCGTGATAGT



(SEQ ID NO: 518)


 


asp114R
TCCCTCTGGATACGTGACSNNCGAATTCAGCGCCGTGAT



(SEQ ID NO: 519)


 


asp115R
TGTTCCCTCTGGATACGTSNNAGACGAATTCAGCGCCGT



(SEQ ID NO: 520)


 


asp116R
GACTGTTCCCTCTGGATASNNGACAGACGAATTCAGCGC



(SEQ ID NO: 521)


 


asp117R
TCGGACTGTTCCCTCTGGSNNCGTGACAGACGAATTCAG



(SEQ ID NO: 522)


 


asp118R
TCCTCGGACTGTTCCCTCSNNATACGTGACAGACGAATT



(SEQ ID NO: 523)


 


asp119R
AAGTCCTCGGACTGTTCCSNNTGGATACGTGACAGACGA



(SEQ ID NO: 524)


 


asp120R
GATAAGTCCTCGGACTGTSNNCTCTGGATACGTGACAGA



(SEQ ID NO: 525)


 


asp121R
GCGGATAAGTCCTCGGACSNNTCCCTCTGGATACGTGAC



(SEQ ID NO: 526)


 


asp122R
CGTGCGGATAAGTCCTCGSNNTGTTCCCTCTGGATACGT



(SEQ ID NO: 527)


 


asp123R
CGTCGTGCGGATAAGTCCSNNGACTGTTCCCTCTGGATA



(SEQ ID NO: 528)


 


asp124R
AACCGTCGTGCGGATAAGSNNTCGGACTGTTCCCTCTGG



(SEQ ID NO: 529)


 


asp125R
ACAAACCGTCGTGCGGATSNNTCCTCGGACTGTTCCCTC



(SEQ ID NO: 530)


 


asp126R
GGCACAAACCGTCGTGCGSNNAAGTCCTCGGACTGTTCC



(SEQ ID NO: 531)


 


asp127R
TTCGGCACAAACCGTCGTSNNGATAAGTCCTCGGACTGT



(SEQ ID NO: 532)


 


asp128R
TGGTTCGGCACAAACCGTSNNGCGGATAAGTCCTCGGAC



(SEQ ID NO: 533)


 


asp129R
ACCTGGTTCGGCACAAACSNNCGTGCGGATAAGTCCTCG



(SEQ ID NO: 534)


 


asp130R
ATCACCTGGTTCGGCACASNNCGTCGTGCGGATAAGTCC



(SEQ ID NO: 535)


 


asp131R
GCTATCACCTGGTTCGGCSNNAACCGTCGTGCGGATAAG



(SEQ ID NO: 536)


 


asp132R
TCCGCTATCACCTGGTTCSNNACAAACCGTCGTGCGGAT



(SEQ ID NO: 537)


 


asp133R
ACCTCCGCTATCACCTGGSNNGGCACAAACCGTCGTGCG



(SEQ ID NO: 538)


 


asp134R
GCTACCTCCGCTATCACCSNNTTCGGCACAAACCGTCGT



(SEQ ID NO: 539)


 


asp135R
AAGGCTACCTCCGCTATCSNNTGGTTCGGCACAAACCGT



(SEQ ID NO: 540)


 


asp136R
TAAAAGGCTACCTCCGCTSNNACCTGGTTCGGCACAAAC



(SEQ ID NO: 541)


 


asp137R
CGCTAAAAGGCTACCTCCSNNATCACCTGGTTCGGCACA



(SEQ ID NO: 542)


 


asp138R
TCCCGCTAAAAGGCTACCSNNGCTATCACCTGGTTCGGC



(SEQ ID NO: 543)


 


asp139R
ATTTCCCGCTAAAAGGCTSNNTCCGCTATCACCTGGTTC



(SEQ ID NO: 544)


 


asp140R
TTGATTTCCCGCTAAAAGSNNACCTCCGCTATCACCTGG



(SEQ ID NO: 545)


 


asp141R
GGCTTGATTTCCCGCTAASNNGCTACCTCCGCTATCACC



(SEQ ID NO: 546)


 


asp142R
TTGGGCTTGATTTCCCGCSNNAAGGCTACCTCCGCTATC



(SEQ ID NO: 547)


 


asp143R
ACCTTGGGCTTGATTTCCSNNTAAAAGGCTACCTCCGCT



(SEQ ID NO: 548)


 


asp144R
GACACCTTGGGCTTGATTSNNCGCTAAAAGGCTACCTCC



(SEQ ID NO: 549)


 


asp145R
CGTGACACCTTGGGCTTGSNNTCCCGCTAAAAGGCTACC



(SEQ ID NO: 550)


 


asp146R
TGACGTGACACCTTGGGCSNNATTTCCCGCTAAAAGGCT



(SEQ ID NO: 551)


 


asp147R
ACCTGACGTGACACCTTGSNNTTGATTTCCCGCTAAAAG



(SEQ ID NO: 552)


 


asp148R
ACCACCTGACGTGACACCSNNGGCTTGATTTCCCGCTAA



(SEQ ID NO: 553)


 


asp149R
AGAACCACCTGACGTGACSNNTTGGGCTTGATTTCCCGC



(SEQ ID NO: 554)


 


asp150R
TCCAGAACCACCTGACGTSNNACCTTGGGCTTGATTTCC



(SEQ ID NO: 555)


 


asp151R
ATTTCCAGAACCACCTGASNNGACACCTTGGGCTTGATT



(SEQ ID NO: 556)


 


asp152R
ACAATTTCCAGAACCACCSNNCGTGACACCTTGGGCTTG



(SEQ ID NO: 557)


 


asp153R
CCGACAATTTCCAGAACCSNNTGACGTGACACCTTGGGC



(SEQ ID NO: 558)


 


asp154R
CGTCCGACAATTTCCAGASNNACCTGACGTGACACCTTG



(SEQ ID NO: 559)


 


asp155R
CCCCGTCCGACAATTTCCSNNACCACCTGACGTGACACC



(SEQ ID NO: 560)


 


asp156R
TCCCCCCGTCCGACAATTSNNAGAACCACCTGACGTGAC



(SEQ ID NO: 561)


 


asp157R
TGTTCCCCCCGTCCGACASNNTCCAGAACCACCTGACGT



(SEQ ID NO: 562)


 


asp158R
TGTTGTTCCCCCCGTCCGSNNATTTCCAGAACCACCTGA



(SEQ ID NO: 563)


 


asp159R
GAATGTTGTTCCCCCCGTSNNACAATTTCCAGAACCACC



(SEQ ID NO: 564)


 


asp160R
AAAGAATGTTGTTCCCCCSNNCCGACAATTTCCAGAACC



(SEQ ID NO: 565)


 


asp161R
TTGAAAGAATGTTGTTCCSNNCGTCCGACAATTTCCAGA



(SEQ ID NO: 566)


 


asp162R
TGGTTGAAAGAATGTTGTSNNCCCCGTCCGACAATTTCC



(SEQ ID NO: 567)


 


asp163R
GACTGGTTGAAAGAATGTSNNTCCCCCCGTCCGACAATT



(SEQ ID NO: 568)


 


asp164R
GTTGACTGGTTGAAAGAASNNTGTTCCCCCCGTCCGACA



(SEQ ID NO: 569)


 


asp165R
CGGGTTGACTGGTTGAAASNNTGTTGTTCCCCCCGTCCG



(SEQ ID NO: 570)


 


asp166R
AATCGGGTTGACTGGTTGSNNGAATGTTGTTCCCCCCGT



(SEQ ID NO: 571)


 


asp167R
CAAAATCGGGTTGACTGGSNNAAAGAATGTTGTTCCCCC



(SEQ ID NO: 572)


 


asp168R
CTGCAAAATCGGGTTGACSNNTTGAAAGAATGTTGTTCC 



(SEQ ID NO: 573)


 


asp169R
AGCCTGCAAAATCGGGTTSNNTGGTTGAAAGAATGTTGT 



(SEQ ID NO: 574)


 


asp170R
GTAAGCCTGCAAAATCGGSNNGACTGGTTGAAAGAATGT



(SEQ ID NO: 575)


 


asp171R
GCCGTAAGCCTGCAAAATSNNGTTGACTGGTTGAAAGAA



(SEQ ID NO: 576)


 


asp172R
CAGGCCGTAAGCCTGCAASNNCGGGTTGACTGGTTGAAA



(SEQ ID NO: 577)


 


asp173R
TCTCAGGCCGTAAGCCTGSNNAATCGGGTTGACTGGTTG



(SEQ ID NO: 578)


 


asp174R
CATTCTCAGGCCGTAAGCSNNCAAAATCGGGTTGACTGG



(SEQ ID NO: 579)


 


asp175R
AATCATTCTCAGGCCGTASNNCTGCAAAATCGGGTTGAC



(SEQ ID NO: 580)


 


asp176R
CGTAATCATTCTCAGGCCSNNAGCCTGCAAAATCGGGTT



(SEQ ID NO: 581)


 


asp177R
AGTCGTAATCATTCTCAGSNNGTAAGCCTGCAAAATCGG



(SEQ ID NO: 582)


 


asp178R
GTCAGTCGTAATCATTCTSNNGCCGTAAGCCTGCAAAAT



(SEQ ID NO: 583)


 


asp179R
AGAGTCAGTCGTAATCATSNNCAGGCCGTAAGCCTGCAA



(SEQ ID NO: 584)


 


asp180R
TCCAGAGTCAGTCGTAATSNNTCTCAGGCCGTAAGCCTG



(SEQ ID NO: 585)


 


asp181R
ACTTCCAGAGTCAGTCGTSNNCATTCTCAGGCCGTAAGC



(SEQ ID NO: 586)


 


asp182R
GGAACTTCCAGAGTCAGTSNNAATCATTCTCAGGCCGTA



(SEQ ID NO: 587)


 


asp183R
AGGGGAACTTCCAGAGTCSNNCGTAATCATTCTCAGGCC



(SEQ ID NO: 588)


 


asp184R
TTAAGGGGAACTTCCAGASNNAGTCGTAATCATTCTCAG



(SEQ ID NO: 589)


 


asp185R
GGGTTAAGGGGAACTTCCSNNGTCAGTCGTAATCATTCT



(SEQ ID NO: 590)


 


asp186R
GTTGGGTTAAGGGGAACTSNNAGAGTCAGTCGTAATCAT



(SEQ ID NO: 591)


 


asp187R
TCTGTTGGGTTAAGGGGASNNTCCAGAGTCAGTCGTAAT



(SEQ ID NO: 592)


 


asp188R
TCCTCTGTTGGGTTAAGGSNNACTTCCAGAGTCAGTCGT



(SEQ ID NO: 593)


 


asp189R
CCGTCCTCTGTTGGGTTASNNGGAACTTCCAGAGTCAGT



(SEQ ID NO: 594)









Example 16
Construction of Arginine and Cysteine Combinatorial Mutants
In this Example, the construction of multiple arginine and cysteine mutants of ASP is described. These experiments were conducted in order to determine whether the use of surface arginine and cysteine combinatorial libraries would lead to mutants with increased expression at the protein level.
The QuikChange® multi site-directed mutagenesis (QCMS) kit (Stratagene) was used to construct the two libraries. The 5′ phosphorylated primers used to create the two libraries are shown in Table 16-1. It was noted that HPLC, PAGE or any other type of purified primers gave far better results in terms of incorporation of full length primers as well as significant reduction in primer-containing errors. However, in these experiments, purified primers were not used, probably resulting in the production of 12% of clones had undesired mutations.







TABLE 16-1







Primers and Sequences








Primer 



name
Primer sequence





ASPR14L
gcatatactattggcggcctgtctagatgttctatcgga 



(SEQ ID NO: 595)


 


ASPR16Q
actattggcggccggtctcagtgttctatcggattcgc 



(SEQ ID NO: 596)


 


ASPR35F
ctgccggtcactgcggatttacaggagccactactgc 



(SEQ ID NO: 597)


 


ASPR61S
atgattatgcattcgtctcaacaggggcaggagtaaat 



(SEQ ID NO: 598)


 


ASPR79T
ataactactcgggcggcacagtccaagtagcaggacatac 



(SEQ ID NO: 599)


 


ASPR123L
atccagagggaacagtcctgggacttatccgcacgac 



(SEQ ID NO: 600)


 


ASPR127Q
cagtccgaggacttatccagacgacggtttgtgccgaac 



(SEQ ID NO: 601)


 


ASPR159Q
gtggttctggaaattgtcagacggggggaacaacattc 



(SEQ ID NO: 602)


 


ASPR179Q
tgcaggcttacggcctgcagatgattacgactgactc 



(SEQ ID NO: 603)


 


ASPC17S
ttggcggccggtctagatcatctatcggattcgcagta 



(SEQ ID NO: 604)


 


ASPC33S
tcattactgccggtcactcaggaagaacaggagccact 



(SEQ ID NO: 605)


 


ASPC95S
cagttggatctgctgtatctcgctcaggtagcactac 



(SEQ ID NO: 606)


 


ASPC105S
cactacaggttggcattcaggaactatcacggcgctg 



(SEQ ID NO: 607)


 


ASPC131S
cttatccgcacgacggtttcagccgaaccaggtgatag 



(SEQ ID NO: 608)


 


ASPC158S
caggtggttctggaaattcacggacggggggaacaac 



(SEQ ID NO: 609)


 


ASPSEQF1
tgcctcacatttgtgccac 



(SEQ ID NO: 610)


 


ASPSEQF4
caggatgtagctgcaggac 



(SEQ ID NO: 611)


 


ASPSEQR4
ctcggttatgagttagttc 



(SEQ ID NO: 612)









pHPLT-ASP-C1-2 Plasmid Preparation and In Vitro Methylation

To construct the cysteine and arginine libraries using the QCMS kit, the template plasmid pHPLT-ASP-C1-2 was first methylated in vitro since it was derived from a Bacillus strain that does not methylate DNA at GATC sites. This method was used because the more common approach of ensuring methylation in plasmid used in the QCMS protocol involving deriving DNA from dam+E. coli strains was not an option here, because the plasmid pHPLT-ASP-C1-2 does not grown in E. coli. 
Miniprep DNA was prepared from Bacillus cells harboring the pHPLT-ASP-C1-2 plasmid. Specifically, the strain was grown overnight in 5 mL of LB with 10 ppm of neomycin, after which the cells were spun down. The Qiagen spin miniprep DNA kit was used for preparing the plasmid DNA with an additional step wherein 100 uL of 10 mg/mL lysozyme was added after the addition of 250 uL of P1 buffer from the kit. The sample was incubated at 37° C. for 15 min with shaking, after which the remaining steps outlined in the Qiagen miniprep kit manual were carried out. The miniprep DNA was eluted with 30 uL of Qiagen buffer EB provided in the kit.
Next, the pHPLT-ASP-C1-2 plasmid DNA was methylated in vitro using a dam methylase kit from NEB (NEB catalog #MO222S). Briefly, 25 μL of the miniprep DNA (about 1-2 μg) was incubated with 20 μL of the 10×NEB dam methylase buffer, 0.5 μL of S-adenosylmethionine (80 μM), 4 μL of the dam methylase and 150.5 μL of sterile distilled water. The reaction was incubated at 37° C. for 4 hours, after which the DNA was purified using a Qiagen PCR purification kit. The methylated DNA was eluted with 40 μL of buffer EB provided in the kit. To confirm methylation of the DNA, 4 μL of the purified, methylated DNA was digested with MboI (NEB; this enzyme cuts unmethylated GATC sites) or DpnI (Roche; this enzyme cuts methylated GATC sites) in a 20 μL reaction using 2 μL of each enzyme. The reactions were incubated at 37° C. for 2 hours and they were analyzed on a 1.2% E-gel (Invitrogen). A small molecular weight DNA smear/ladder was observed for the DpnI digest, whereas the MboI digest showed intact DNA, which indicated that the pHPLT-ASP-C1-2 plasmid was successfully methylated.
Library Construction
The cysteine (cys) and arginine (arg) combinatorial libraries were constructed as outlined in the Stratagene QCMS kit, with the exception of the primer concentration used in the reactions. Specifically, 4 μL of the methylated, purified pHPLT-ASP-C1-2 plasmid (about 25 to 50 ng) was mixed with 15 μL of sterile distilled water, 1.5 μL of dNTP, 2.5 μL of 10× buffer, 1 μL of the enzyme blend and 1.0 μL arginine or cysteine mutant primer mix (i.e., for a total of 100 ng of primers). The primer mix was prepared using 10 μL of each of the nine arginine primers (100 ng/μL) or each of the six cysteine primers (100 ng/μL); adding 50 ng of each primer for both the arg and cys libraries as recommended in the Stratagene manual resulted in less than 50% of the clones containing mutations in a previous round of mutagenesis. Thus, the protocol was modified in the present round of mutagenesis to include a total of 100 ng of primers in each reaction. The cycling conditions were 95° C. for 1 min, followed by 30 cycles of 95° C. for 1 min, 55° C. for 1 min, and 65° C. for 9 min, in an MJ Research thermocycler using thin-walled 0.2 mL PCR tubes. The reaction product was digested with 1 μL of DpnI from the QCMS kit by incubating at 37° C. overnight. An additional 0.5 μL of DpnI was added, and the reaction was incubated for 1 hour.
To transform the library DNA directly into Bacillus cells with out going through E. coli, the library DNA (single-stranded QCMS product) was amplified using the TempliPhi kit (Amersham cat. #25-6400), because Bacillus requires double-stranded multimeric DNA for transformation. For this purpose, 1 μL of the arginine or cysteine QCMS reaction was mixed with 5 μL of sample buffer from the TempliPhi kit and heated for 3 minutes at 95° C. to denature the DNA. The reaction was placed on ice to cool for 2 minutes and then spun down briefly. Next, 5 μL of reaction buffer and 0.2 μL of phi29 polymerase from the TempliPhi kit were added, and the reactions were incubated at 30° C. in an MJ Research PCR machine for 4 hours. The phi29 enzyme was heat inactivated in the reactions by incubation at 65° C. for 10 min in the PCR machine.
For transformation of the libraries into Bacillus, 0.5 μL of the TempliPhi amplification reaction product was mixed with 100 μL of comK competent cells followed by vigorous shaking at 37° C. for 1 hour. The transformation was serially diluted up to 105 fold, and 50 μL of each dilution was plated on LA plates containing 10 ppm neomycin and 1.6% skim milk. Twenty-four clones from each library were picked for sequencing. Briefly, the colonies were resuspended in 20 μL of sterile distilled water and 2 μL was then used for PCR with ReadyTaq beads (Amersham) in a total volume of 25 μL. Primers ASPF1 and ASPR4 were added at a concentration of 0.5 μM. Cycling conditions were 94° C. for 4 min once, followed by 30 cycles of 94° C. for 1 min, 55° C. for 1 min, and 72° C. for 1 min, followed by one round at 72° C. for 7 min. A 1.5 kb fragment was obtained in each case and the product was purified using a Qiagen PCR purification kit. The purified PCR products were sequenced with ASPF4 and ASPR4 primers.
A total of 48 clones were sequenced (24 from each library). The mutagenesis worked quite well in that only about 15% of the clones were WT. But 20% of the clones had mixed sequences because the plate was crowded with colonies or the TempliPhi amplification resulted in very concentrated DNA for transformation. Also, as indicated above, about 12% of clones had extra mutations. The remaining clones were all mutant, and of these about 60-80% were unique mutants. The sequencing results for the arginine and cysteine libraries are provided below in Tables 16-2, and 16-3.







TABLE 16-2







Arginine Library Sequencing and Skim Milk Plate Results

















Colony
Halo
R14L
R160
R35F
R61S
R79T
R123L
R127Q
R159Q
R1790





R1
medium

X
X




X



R2
yes







X



R3
yes

X



X





R4
yes

X



X





R5
yes

X



X





R6
yes

X



X





R7
yes
X





X
X



R8
yes

X



X





R9
yes











R10
yes
X







X


R11
yes











R12
medium

X
X




X



R13
yes




X






R14
yes











R15
yes











R16
medium











R17
no



X

X
X




R18
medium





X
X

X


R19
medium











R20
yes
X





X
X



R21
medium

X


X

X




R22
small











R23
yes

X


X






R24
yes
















TABLE 16-3







Cysteine Library Sequencing and Skim Milk Plate Results














Colony
Halo?
C17S
C33S
C95S
C105S
C131S
C158S





C1
no
X
X






C2
no








C3
yes








C4
yes








C5
no
X

X





C6
small
X


X




C7
no


X
X
X



C8
yes








C9
no








C10
no








C11
small








C12
no








C13
no
X

X





C14
no
X
X
X


X


C15
no








C16
no





X


C17
no





X


C18
no
X

X
X

X


C19
yes








C20
no








C21
no








C22
no



X




C23
no
X

X





C24
yes









Of the mutants identified in sequencing, the following mutants from the arginine library (See, Table 16-4) were found to be of interest. See the Examples below for additional data regarding the properties of these mutants.







TABLE 16-4







Arginine Mutants of Interest










MUTANT
SEQUENCE







R1
R16Q R35F R159Q



R2
R159Q



R3
R16Q R123L



R7
R14L R127Q R159Q



R10B
R14L R179Q



R18
R123L R127Q R179Q



R21
R16Q R79T R127Q



R23
R16Q R79T



R10
R14L R79T










Importantly, the activity results indicated that mutations in the cysteine residues produced ASP proteases with very low or no activity, suggesting that the disulfide bridges play an important role in the stability of the molecule. However, it is not intended that the present invention be limited to any particular mechanism(s).
Example 17
Expression of Homologous O. turbata Protease in S. lividans 
In this Example, expression of protease produced by O. turbata that is homologous to the protease 69B4 in S. lividans is described. Thus, this Example describes plasmids comprising polynucleotides encoding a polypeptide having proteolytic activity and used such vectors to transform a Streptomyces lividans host cell. The transformation methods used herein are known in the art (See e.g., U.S. Pat. No. 6,287,839; and WO 02/50245, herein incorporated by reference).
The vector (i.e., plasmid) used in these experiments comprised a polynucleotide encoding a protease of the present invention obtained from Oerskovia turbata DSM 20577. This plasmid was used to transform Streptomyces lividans. The final plasmid vector is referred to herein as “pSEA4CT-O. turbata.”
As with previous vectors, the construction of pSEA4CT-O. turbata made use of the pSEGCT plasmid vector (See, above).
An Aspergillus niger (“A4”) regulatory sequence operably linked to the structural gene encoding the Oerskovia turbata protease (Otp) was used to drive the expression of the protease. A fusion between the A4-regulatory sequence and the Oerskovia turbata signal; sequence, N-terminal prosequence and mature protease sequence (i.e., without the C-terminal prosequence) was constructed by fusion-PCR techniques known in the art, as an XbaI-BamHI fragment. The polynucleotide primers for the cloning of Oerskovia turbata protease (Otp) in pSEA4CT were based on SEQ ID NO:67. The primer sequences used were:







A4-turb Fw


(SEQ ID NO: 613)


5′-CAGAGACAGACCCCCGGAGGTAACCATGGCACGATCATTCTGGAGGA


 


CGC-3′


 


A4-turb RV


(SEQ ID NO: 614)


5′-GCGTCCTCCAGAATGATCGTGCCATGGTTACCTCCGGGGGTCTGTCT


 


CTG-3′


 


A4-turb Bam Rv


(SEQ ID NO: 615)


5′-ATCCGCTCGCGGATCCCCATTGTCAGCTCGGGCCCCCACCGTCAGAG


 


GTCACGAG-3′


 


A4-Xba1-FW


(SEQ ID NO: 616)


5′-GCAGCCTGAACTAGTTGCGATCCTCTAGAGATCGAACTTCAT-3′






The fragment was ligated into plasmid pSEA4CT digested with XbaI and BamHI, resulting in plasmid pSEA4CT-O. turbata. 
The host Streptomyces lividans TK23 was transformed with plasmid vector pSEA4CT-O. turbata using the protoplast method described in the previous Example (i.e., using the method of Hopwood et al., supra).
The transformed culture was expanded to provide two fermentation cultures in TS* medium. The composition of TS* medium was (g/L) tryptone (Difco) 16, soytone (Difco) 4, casein hydrolysate (Merck) 20, K2HPO4 10, glucose 15, Basildon antifoam 0.6, pH 7.0. At various time points, samples of the fermentation broths were removed for analysis. For the purposes of this experiment, a skim milk procedure was used to confirm successful cloning. 30 μL of the shake flask supernatant was pipetted in punched out holes in skim milk agar plates and incubated at 37° C.
The incubated plates were visually reviewed after overnight incubation for the presence of clearing zones (halos) indicating the expression of proteolytic enzyme. For purposes of this experiment, the samples were also assayed for protease activity and for molecular weight (SDS-PAGE). At the end of the fermentation, full length protease was observed by SDS-PAGE.
A sample of the fermentation broth was assayed as follows: 10 μL of the diluted supernatant was collected and analyzed using the Dimethylcasein Hydrolysis Assay described in Example 1. The assay results of the fermentation broth of 2 clones clearly show that the polynucleotide from Oerskovia turbata encoding a polypeptide having proteolytic activity was expressed in Streptomyces 
Example 18
Expression of Homologous Cellulomonas and Cellulosimicrobium Proteases in S. lividans 
In this Example, expression of proteases produced by Cellulomonas cellasea DSM 20118 and Cellulosimicrobium cellulans DSM 204244 that are homologous to the protease 69B4 in S. lividans is described. Thus, this Example describes plasmids comprising polynucleotides encoding a polypeptide having proteolytic activity and used such vectors to transform a Streptomyces lividans host cell. The transformation methods used herein are known in the art (See e.g., U.S. Pat. No. 6,287,839; and WO 02/50245, herein incorporated by reference).
The final plasmid vectors are referred to as pSEA4CT-C. cellasea and pSEA4CT-Cm.cellulans. The construction of pSEA4CT-C.cellasea and pSEA4CT-Cm.cellulans made use of the pSEGCT plasmid vector described above.
An Aspergillus niger (“A4”) regulatory sequence operably linked to the structural gene encoding the Cellulomonas cellasea mature protease (Ccp) or alternatively, the structural gene encoding the Cellulosimicrobium cellulans mature protease (Cmcp) was used to drive the expression of the protease. A fusion between the A4-regulatory sequence and the 69B4 protease signal-sequence, N-terminal prosequence of the 69B4 protease gene and mature sequence of the native protease gene obtained from genomic DNA of a strain of Micrococcineae (herein, Cellulomonas cellasea or Cellulosimicrobium cellulans) was constructed by fusion-PCR techniques, as a XbaI-BamHI fragment. The polynucleotide primers for the cloning of Cellulomonas cellasea protease (Ccp) in pSEA4CT were based on SEQ ID NO:63, and are as follows:








Asp-npro fw-cell










5′-AGACCGACGAGACCCCGCGGACCATGGTCGACGTCATCGGCGGCAACGCGTACTAC-3′
(SEQ ID NO: 617)



 


Cell-BH1-rv



5′-TCAGCCGATCCGCTCGCGGATCCCCATTGTCAGCCCAGGACGAGACGCAGACCGTA-3′
(SEQ ID NO: 618)


 


Asp-npro rv-cell



5′-GTAGTACGCGTTGCCGCCGATGACGTCGACCATGGTCCGCGGGGTCTCGTCGGTCT-3′
(SEQ ID NO: 619)


 


Xba-1 fw A4



5′-GCAGCCTGAACTAGTTGCGATCCTCTAGAGATCGAACTTCATGTTCGA-3′
(SEQ ID NO: 620)






The polynucleotide primers for the cloning of Cellulosimicrobium cellulans protease (Cmcp) in pSEA4CT were based on SEQ ID NO: 71, and are as follows,








ASP-npro fw cellu










5′-ACCGACGAGACCCCGCGGACCATGCACGGCGACGTGCGCGGCGGCGACCGCTA-3′
(SEQ ID NO: 621)



 


ASP-npro rv cellu



5′-TAGCGGTCGCCGCCGCGCACGTCGCCGTGCATGGTCCGCGGGGTCTCGTCGGT-3′
(SEQ ID NO: 622)


 


Cellu-BH1-rv



5′-TCAGCCGATCCGCTCGCGGATCCCCATTGTCAGCGAGCCCGACGAGCGCGCTGCCCGAC-3′
(SEQ ID NO: 623)


 


Xba-1 fw A4



5′-GCAGCCTGAACTAGTTGCGATCCTCTAGAGATCGAACTTCATGTTCGA-3′
(SEQ ID NO: 620)






The host Streptomyces lividans TK23 was transformed with plasmid vector pSEA4CT using the protoplast method described above (i.e., Hopwood et al., supra). The transformed culture was expanded to provide two fermentation cultures in TS* medium. The composition of TS* medium was (g/L) tryptone (Difco) 16, soytone (Difco) 4, casein hydrolysate (Merck) 20, K2HPO4 10, glucose 15, Basildon antifoam 0.6, pH 7.0. At various time points, samples of the fermentation broths were removed for analysis. For the purposes of this experiment, a skim milk procedure was used to confirm successful cloning. 30 μL of the shake flask supernatant was pipetted in punched out holes in skim milk agar plates and incubated at 37° C.
The incubated plates were visually reviewed after overnight incubation for the presence of clearing zones (halos) indicating the expression of proteolytic enzyme. For purposes of this experiment, the samples were also assayed for protease activity and for molecular weight (SDS-PAGE). At the end of the fermentation full length protease was observed by SDS-PAGE.
A sample of the fermentation broth was assayed as follows: 10 μL of the diluted supernatant was taken and added to 190 μL AAPF substrate solution (conc. 1 mg/ml, in 0.1 M Tris/0.005% Tween 80, pH 8.6). The rate of increase in absorbance at 410 nm due to release of p-nitroaniline was monitored (25° C.).
As in previous Examples, the results obtained clearly indicated that the polynucleotide from Cellulomonas cellasea or from Cellulosimicrobium cellulans, both encoding polypeptides having proteolytic activity were expressed in Streptomyces lividans. 
Example 19
Determination of the Crystal Structure of ASP Protease
In this Example, methods used to determine the crystal structure of ASP protease are described. Indeed, high quality single crystals were obtained from purified ASP protease. The crystallization conditions were as follows: 25% PEG 8000, 0.2M ammonium sulphate, and 15% glycerol. These crystallization conditions are cryo-protective, so transfer to a cryoprotectant was not required. The crystals were frozen in liquid nitrogen, and kept frozen during data collection using an Xstream (Molecular Structure). Data were collected with a R-axis IV (Molecular Structure), equipped with focusing mirrors. X-ray reflection data were obtained to 1.9 Å resolution. The space group was P212121, with cell dimensions a=35.65 Å, b=51.82 Å and c=76.86 Å. There was one molecule per asymmetric unit.
The crystal structure was solved using the molecular replacement method. The program used was X-MR (Accelrys Inc.). The starting model for the molecular replacement calculations was Streptogrisin. It is clear from the electron density map obtained from X-MR that the molecular replacement solution is correct. Thus, 98% of the model was built correctly, with some minor errors that were fixed manually. The R-factor for data to 1.9 Å was 0.23.
The structure was found to largely consist of β-sheets, with 2 very short α-helices, and a longer helix toward the C-terminal end. There are two sets of β-sheets, with a considerable interface between them. The active-site is found in a cleft formed at this interface. The catalytic triad is formed by His 32, Asp 56, and Ser 137. Table 19-1 provides the atomic coordinates identified for ASP.








TABLE 19-1






Atomic Coordinates of ASP with the Modeled Octapeptide Substrate























CRYST1
35.770
51.730
76.650
90.00
90.00
90.00
P212121



















ATOM
1
N
PHE
A
1
2.421
18.349 
15.176
1.00
16.78
N


ATOM
2
CA 
PHE
A
1
3.695
18.087 
15.905
1.00
18.18
C


ATOM
3
CB 
PHE
A
1
4.875
18.550 
15.048
1.00
16.73
C


ATOM
4
C
PHE
A
1
3.700
18.810 
17.249
1.00
16.36
C


ATOM
5
O
PHE
A
1
3.443
20.011 
17.315
1.00
17.91
O


ATOM
6
CG 
PHE
A
1
6.214
18.292 
15.664
1.00
17.42
C


ATOM
7
CD2
PHE
A
1
6.955
17.180 
15.296
1.00
19.42
C


ATOM
8
CD1
PHE
A
1
6.736
19.160 
16.611
1.00
16.13
C


ATOM
9
CE2
PHE
A
1
8.200
16.933 
15.863
1.00
18.08
C


ATOM
10
CE1
PHE
A
1
7.977
18.922 
17.180 
1.00
18.34
C


ATOM
11
CZ 
PHE
A
1
8.710
17.807 
16.806
1.00
19.32
C


ATOM
12
N
ASP
A
2
3.984
18.076 
18.321
1.00
13.94
N


ATOM
13
CA 
ASP
A
2
4.015
18.670 
19.654
1.00
15.04
C


ATOM
14
CB 
ASP
A
2
3.527
17.677 
20.714
1.00
15.13
C


ATOM
15
C
ASP
A
2
5.403
19.149 
20.063
1.00
14.43
C


ATOM
16
O
ASP
A
2
6.381
18.408 
19.966
1.00
11.44
O


ATOM
17
CG 
ASP
A
2
2.088
17.243 
20.502
1.00
18.25
C


ATOM
18
OD2 
ASP
A
2
1.721
16.150 
20.986
1.00
19.05
O


ATOM
19
OD1 
ASP
A
2
1.320
17.996 
19.874
1.00
15.33
O


ATOM
20
N
VAL
A
3
5.479
20.393 
20.523
1.00
12.30
N


ATOM
21
CA 
VAL
A
3
6.740
20.979 
20.959
1.00
11.83
C


ATOM
22
CB 
VAL
A
3
6.812
22.480 
20.603
1.00
11.52
C


ATOM
23
C
VAL
A
3
6.766
20.795 
22.470
1.00
13.77
C


ATOM
24
O
VAL
A
3
5.912
21.321 
23.183
1.00
11.14
O


ATOM
25
CG1 
VAL
A
3
7.987
23.133 
21.309
1.00
15.13
C


ATOM
26
CG2
VAL
A
3
6.968
22.637 
19.101
1.00
14.21
C


ATOM
27
CB 
ILE 
A
4
7.561
18.267 
24.642
1.00
14.73
C


ATOM
28
CG2
ILE 
A
4
7.799
17.929 
26.099
1.00
14.20
C


ATOM
29
CG1
ILE 
A
4
6.103
17.995 
24.267
1.00
16.79
C


ATOM
30
CD1
ILE 
A
4
5.774
16.518 
24.166
1.00
19.32
C


ATOM
31
C
ILE
A
4
9.334
20.031
24.816
1.00
14.04
C


ATOM
32
O
ILE
A
4
10.289
19.660
24.140
1.00
11.09
O


ATOM
33
N
ILE
A
4
7.745
20.033
22.945
1.00
10.83
N


ATOM
34
CA
ILE
A
4
7.903
19.750
24.365
1.00
13.46
C


ATOM
35
N
GLY
A
5
9.475
20.681
25.965
1.00
11.82
N


ATOM
36
CA
GLY
A
5
10.800
20.995
26.467
1.00
9.81
C


ATOM
37
C
GLY
A
5
11.700
19.785
26.644
1.00
11.77
C


ATOM
38
O
GLY
A
5
11.256
18.737
27.114
1.00
9.20
O


ATOM
39
N
GLY
A
6
12.966
19.927
26.255
1.00
10.03
N


ATOM
40
CA
GLY
A
6
13.917
18.836
26.397
1.00
8.54
C


ATOM
41
C
GLY
A
6
14.070
17.979
25.156
1.00
9.57
C


ATOM
42
O
GLY
A
6
15.020
17.200
25.042
1.00
7.69
O


ATOM
43
N
ASN
A
7
13.131
18.119
24.224
1.00
9.01
N


ATOM
44
CA
ASN
A
7
13.168
17.359
22.985
1.00
10.51
C


ATOM
45
CB
ASN
A
7
11.780
17.293
22.349
1.00
14.65
C


ATOM
46
CG
ASN
A
7
10.897
16.250
22.981
1.00
10.35
C


ATOM
47
OD1
ASN
A
7
9.715
16.144
22.644
1.00
13.61
O


ATOM
48
ND2
ASN
A
7
11.456
15.470
23.896
1.00
6.66
N


ATOM
49
C
ASN
A
7
14.130
17.952
21.976
1.00
12.30
C


ATOM
50
O
ASN
A
7
14.424
19.146
21.991
1.00
15.93
O


ATOM
51
N
ALA
A
8
14.608
17.107
21.079
1.00
11.08
N


ATOM
52
CA
ALA
A
8
15.532
17.564
20.063
1.00
14.32
C


ATOM
53
CB
ALA
A
8
16.336
16.392
19.541
1.00
14.61
C


ATOM
54
C
ALA
A
8
14.766
18.202
18.914
1.00
11.23
C


ATOM
55
O
ALA
A
8
13.567
17.987
18.747
1.00
12.54
O


ATOM
56
N
TYR
A
9
15.468
19.021
18.145
1.00
9.75
N


ATOM
57
CA
TYR
A
9
14.899
19.691
16.988
1.00
12.42
C


ATOM
58
CB
TYR
A
9
14.279
21.059
17.334
1.00
12.79
C


ATOM
59
CG
TYR
A
9
15.216
22.150
17.790
1.00
14.12
C


ATOM
60
CD2
TYR
A
9
15.485
22.333
19.139
1.00
10.17
C


ATOM
61
CE2
TYR
A
9
16.302
23.366
19.572
1.00
12.49
C


ATOM
62
CD1
TYR
A
9
15.791
23.029
16.877
1.00
9.02
C


ATOM
63
CE1
TYR
A
9
16.604
24.066
17.294
1.00
10.92
C


ATOM
64
CZ
TYR
A
9
16.857
24.230
18.644
1.00
13.93
C


ATOM
65
OH
TYR
A
9
17.661
25.261
19.070
1.00
12.50
O


ATOM
66
C
TYR
A
9
16.127
19.792
16.101
1.00
12.21
C


ATOM
67
O
TYR
A
9
17.247
19.589
16.583
1.00
11.38
O


ATOM
68
N
THR
A
10
15.946
20.055
14.816
1.00
11.44
N


ATOM
69
CA
THR
A
10
17.105
20.144
13.946
1.00
13.35
C


ATOM
70
CB
THR
A
10
17.114
18.998
12.916
1.00
14.07
C


ATOM
71
OG1
THR
A
10
15.952
19.098
12.086
1.00
13.63
O


ATOM
72
CG2
THR
A
10
17.121
17.648
13.620
1.00
12.60
C


ATOM
73
C
THR
A
10
17.267
21.452
13.194
1.00
14.66
C


ATOM
74
O
THR
A
10
16.299
22.161
12.907
1.00
12.64
O


ATOM
75
N
ILE
A
11
18.520
21.749
12.881
1.00
14.05
N


ATOM
76
CA
ILE
A
11
18.889
22.954
12.157
1.00
18.00
C


ATOM
77
CB
ILE
A
11
19.649
23.931
13.068
1.00
17.58
C


ATOM
78
CG2
ILE
A
11
19.919
25.230
12.323
1.00
20.00
C


ATOM
79
CG1
ILE
A
11
18.825
24.212
14.327
1.00
21.47
C


ATOM
80
CD1
ILE
A
11
19.560
25.031
15.377
1.00
23.61
C


ATOM
81
C
ILE
A
11
19.802
22.485
11.030
1.00
16.40
C


ATOM
82
O
ILE
A
11
20.913
22.014
11.278
1.00
17.72
O


ATOM
83
N
GLY
A
12
19.330
22.603
9.794
1.00
18.83
N


ATOM
84
CA
GLY
A
12
20.132
22.155
8.673
1.00
17.69
C


ATOM
85
C
GLY
A
12
20.359
20.659
8.791
1.00
18.86
C


ATOM
86
O
GLY
A
12
21.395
20.141
8.376
1.00
19.71
O


ATOM
87
N
GLY
A
13
19.391
19.964
9.380
1.00
17.62
N


ATOM
88
CA
GLY
A
13
19.509
18.525
9.529
1.00
16.37
C


ATOM
89
C
GLY
A
13
20.352
18.060
10.703
1.00
17.10
C


ATOM
90
O
GLY
A
13
20.470
16.861
10.946
1.00
15.94
O


ATOM
91
N
ARG
A
14
20.931
19.002
11.438
1.00
17.27
N


ATOM
92
CA
ARG
A
14
21.772
18.667
12.585
1.00
15.15
C


ATOM
93
CB
ARG
A
14
23.017
19.558
12.586
1.00
19.68
C


ATOM
94
C
ARG
A
14
21.030
18.842
13.908
1.00
16.27
C


ATOM
95
O
ARG
A
14
20.423
19.882
14.159
1.00
12.16
O


ATOM
96
CG
ARG
A
14
24.009
19.273
13.699
1.00
25.94
C


ATOM
97
CD
ARG
A
14
24.879
18.069
13.393
1.00
31.69
C


ATOM
98
NE
ARG
A
14
25.964
17.928
14.360
1.00
40.26
N


ATOM
99
CZ
ARG
A
14
25.802
17.572
15.630
1.00
42.65
C


ATOM
100
NH1
ARG
A
14
26.852
17.483
16.435
1.00
45.09
N


ATOM
101
NH2
ARG
A
14
24.592
17.302
16.091
1.00
41.89
N


ATOM
102
N
SER
A
15
21.075
17.821
14.756
1.00
14.36
N


ATOM
103
CA
SER
A
15
20.407
17.892
16.047
1.00
18.05
C


ATOM
104
CB
SER
A
15
20.033
16.488
16.524
1.00
19.52
C


ATOM
105
C
SER
A
15
21.402
18.533
17.011
1.00
18.51
C


ATOM
106
O
SER
A
15
21.966
17.870
17.882
1.00
16.89
O


ATOM
107
OG
SER
A
15
19.311
16.542
17.742
1.00
24.25
O


ATOM
108
N
ARG
A
16
21.625
19.829
16.842
1.00
15.76
N


ATOM
109
CA 
ARG 
A
16
22.560
20.544
17.695
1.00 
18.30
C


ATOM
110
CB
ARG 
A
16
23.077
21.795
16.976
1.00 
22.82
C


ATOM
111
C
ARG 
A
16
22.006
20.952
19.050
1.00 
17.05
C


ATOM
112
O
ARG 
A
16
22.760
21.064
20.015
1.00 
11.60
O


ATOM
113
CG
ARG 
A
16
23.892
21.498
15.729
1.00 
30.78
C


ATOM
114
CD 
ARG 
A
16
24.503
22.758
15.131
1.00 
36.12
C


ATOM
115
NE
ARG 
A
16
23.494
23.756
14.789
1.00 
41.88
N


ATOM
116
CZ
ARG 
A
16
23.737
24.839
14.058
1.00 
44.68
C


ATOM
117
NH2 
ARG 
A
16
24.954
25.057
13.579
1.00 
46.43
N


ATOM
118
NH1 
ARG 
A
16
22.762
25.698
13.796
1.00 
44.09
N


ATOM
119
N
CYS 
A
17
20.695
21.152
19.130
1.00 
12.26
N


ATOM
120
CA
CYS 
A
17
20.085
21.562
20.388
1.00 
11.02
C


ATOM
121
CB
CYS 
A
17
19.949
23.079
20.394
1.00 
11.05
C


ATOM
122
C
CYS 
A
17
18.744
20.946
20.756
1.00
8.62
C


ATOM
123
O
CYS 
A
17
18.178
20.154
20.008
1.00 
10.24
O


ATOM
124
SG
CYS 
A
17
21.542
23.945
20.503
1.00 
10.83
S


ATOM
125
N
SER 
A
18
18.246
21.338
21.926
1.00
9.44
N


ATOM
126
CA 
SER 
A
18
16.976
20.849
22.441
1.00 
10.14
C


ATOM
127
CB
SER 
A
18
17.226
20.053
23.726
1.00 
11.06
C


ATOM
128
OG
SER 
A
18
18.198
19.042
23.516
1.00 
11.13
O


ATOM
129
C
SER 
A
18
16.019
22.004
22.736
1.00 
10.28
C


ATOM
130
O
SER 
A
18
16.439
23.152
22.882
1.00 
12.80
O


ATOM
131
N
ILE 
A
19
14.731
21.689
22.806
1.00
8.87
N


ATOM
132
CA
ILE 
A
19
13.698
22.676
23.087
1.00
9.04
C


ATOM
133
CB
ILE 
A
19
12.278
22.070
22.951
1.00
9.94
C


ATOM
134
CG2 
ILE 
A
19
11.236
23.126
23.287
1.00 
10.60
C


ATOM
135
CG1 
ILE 
A
19
12.053
21.514
21.543
1.00 
12.49
C


ATOM
136
CD1 
ILE 
A
19
12.083
22.554
20.439
1.00 
10.46
C


ATOM
137
C
ILE 
A
19
13.840
23.154
24.530
1.00
9.36
C


ATOM
138
O
ILE 
A
19
14.039
22.346
25.442
1.00
7.81
O


ATOM
139
N
GLY
A
20
13.748
24.466
24.729
1.00
6.59
N


ATOM
140
CA
GLY
A
20
13.827
25.024
26.067
1.00
7.48
C


ATOM
141
C
GLY
A
20
12.424
25.027
26.649
1.00 
10.12
C


ATOM
142
O
GLY
A
20
12.047
24.128
27.400
1.00
9.28
O


ATOM
143
N
PHE
A
21
11.636
26.037
26.293
1.00 
11.70
N


ATOM
144
CA
PHE
A
21
10.262
26.132
26.770
1.00
9.99
C


ATOM
145
CB
PHE
A
21
10.182
27.019
28.009
1.00 
12.23
C


ATOM
146
CG
PHE
A
21
10.891
26.455
29.197
1.00 
12.14
C


ATOM
147
CD1 
PHE
A
21
10.282
25.493
29.985
1.00 
10.45
C


ATOM
148
CD2 
PHE
A
21
12.174
26.873
29.517
1.00 
11.10
C


ATOM
149
CE1 
PHE
A
21
10.943
24.953
31.078
1.00
9.63
C


ATOM
150
CE2 
PHE
A
21
12.841
26.339
30.606
1.00 
10.44
C


ATOM
151
CZ
PHE
A
21
12.225
25.377
31.390
1.00
5.44
C


ATOM
152
C
PHE
A
21
9.378
26.721
25.692
1.00 
11.93
C


ATOM
153
O
PHE
A
21
9.838
27.500
24.861
1.00 
11.86
O


ATOM
154
N
ALA
A
22
8.105
26.346
25.709
1.00
8.59
N


ATOM
155
CA 
ALA
A
22
7.171
26.861
24.722
1.00 
10.98
C


ATOM
156
CB 
ALA
A
22
5.978
25.920
24.580
1.00
9.33
C


ATOM
157
C
ALA
A
22
6.708
28.233
25.200
1.00
9.72
C


ATOM
158
O
ALA
A
22
6.452
28.431
26.390
1.00 
10.20
O


ATOM
159
N
VAL
A
23
6.621
29.178
24.270
1.00
9.39
N


ATOM
160
CA 
VAL
A
23
6.186
30.542
24.579
1.00 
11.79
C


ATOM
161
CB
VAL
A
23
7.369
31.545
24.567
1.00
8.77
C


ATOM
162
CG1 
VAL
A
23
8.373
31.176
25.644
1.00 
12.30
C


ATOM
163
CG2 
VAL
A
23
8.034
31.557
23.195
1.00
9.56
C


ATOM
164
C
VAL
A
23
5.197
30.943
23.496
1.00 
12.96
C


ATOM
165
O
VAL
A
23
5.047
30.234
22.507
1.00 
15.51
O


ATOM
166
N
ASN
A
24
4.509
32.066
23.668
1.00 
15.64
N


ATOM
167
CA 
ASN
A
24
3.559
32.472
22.642
1.00 
18.48
C


ATOM
168
CB
ASN
A
24
2.848
33.772
23.048
1.00 
23.96
C


ATOM
169
C
ASN
A
24
4.304
32.661
21.319
1.00 
18.42
C


ATOM
170
O
ASN
A
24
5.277
33.410
21.251
1.00 
16.60
O


ATOM
171
CG
ASN
A
24
3.800
34.949
23.182
1.00 
23.94
C


ATOM
172
OD1 
ASN
A
24
4.697
34.951
24.025
1.00 
23.82
O


ATOM
173
ND2 
ASN
A
24
3.602
35.964
22.345
1.00 
25.51
N


ATOM
174
N
GLY
A
25
3.868
31.956
20.278
1.00 
19.39
N


ATOM
175
CA
GLY
A
25
4.509
32.086
18.978
1.00 
18.25
C


ATOM
176
C
GLY
A
25
5.628
31.106
18.649
1.00 
18.73
C


ATOM
177
O
GLY
A
25
6.103
31.065
17.515
1.00 
18.70
O


ATOM
178
N
GLY
A
26
6.064
30.318
19.624
1.00 
14.44
N


ATOM
179
CA
GLY
A
26
7.123
29.362
19.348
1.00 
15.00
C


ATOM
180
C
GLY
A
26
7.779
28.822
20.602
1.00 
11.05
C


ATOM
181
O
GLY
A
26
7.095
28.457
21.554
1.00 
10.68
O


ATOM
182
N
PHE
A
27
9.107
28.759
20.599
1.00 
11.66
N


ATOM
183
CA
PHE
A
27
9.832
28.268
21.761
1.00 
11.72
C


ATOM
184
CB
PHE
A
27
10.056
26.748
21.679
1.00 
10.14
C


ATOM
185
C
PHE
A
27
11.169
28.960
21.934
1.00 
10.62
C


ATOM
186
O
PHE
A
27
11.727
29.509
20.985
1.00 
12.74
O


ATOM
187
CG
PHE
A
27
11.000
26.309
20.580
1.00
9.74
C


ATOM
188
CD1
PHE
A
27
10.524
26.006
19.308
1.00
12.75
C


ATOM
189
CD2
PHE
A
27
12.361
26.158
20.832
1.00
11.98
C


ATOM
190
CE1
PHE
A
27
11.384
25.555
18.312
1.00
8.90
C


ATOM
191
CE2
PHE
A
27
13.228
25.707
19.837
1.00
10.80
C


ATOM
192
CZ
PHE
A
27
12.740
25.406
18.580
1.00
9.83
C


ATOM
193
N
ILE 
A
28
11.675
28.948
23.162
1.00
12.44
N


ATOM
194
CA
ILE 
A
28
12.956
29.573
23.442
1.00
10.82
C


ATOM
195
CB
ILE 
A
28
12.903
30.454
24.707
1.00
10.35
C


ATOM
196
C
ILE 
A
28
13.992
28.469
23.590
1.00
12.26
C


ATOM
197
O
ILE 
A
28
13.667
27.335
23.960
1.00
11.25
O


ATOM
198
CG2
ILE 
A
28
12.081
31.701
24.434
1.00
7.92
C


ATOM
199
CG1
ILE 
A
28
12.278
29.690
25.873
1.00
12.08
C


ATOM
200
CD1
ILE 
A
28
12.175
30.526
27.129
1.00
10.36
C


ATOM
201
N
THR
A
29
15.238
28.804
23.283
1.00
11.02
N


ATOM
202
CA
THR
A
29
16.327
27.845
23.364
1.00
11.15
C


ATOM
203
CB
THR
A
29
16.348
26.988
22.052
1.00
13.72
C


ATOM
204
OG1
THR
A
29
17.364
25.981
22.124
1.00
11.80
O


ATOM
205
CG2
THR
A
29
16.594
27.875
20.841
1.00
9.32
C


ATOM
206
C
THR
A
29
17.630
28.628
23.555
1.00
10.10
C


ATOM
207
O
THR
A
29
17.595
29.818
23.888
1.00
8.90
O


ATOM
208
N
ALA
A
30
18.771
27.974
23.353
1.00
8.93
N


ATOM
209
CA
ALA
A
30
20.069
28.630
23.511
1.00
8.72
C


ATOM
210
CB
ALA
A
30
21.135
27.602
23.862
1.00
9.30
C


ATOM
211
C
ALA
A
30
20.476
29.388
22.252
1.00
8.30
C


ATOM
212
O
ALA
A
30
20.243
28.925
21.133
1.00
11.59
O


ATOM
213
N
GLY
A
31
21.097
30.547
22.448
1.00
10.82
N


ATOM
214
CA
GLY
A
31
21.527
31.366
21.330
1.00
10.68
C


ATOM
215
C
GLY
A
31
22.626
30.770
20.469
1.00
12.90
C


ATOM
216
O
GLY
A
31
22.656
31.014
19.259
1.00
12.57
O


ATOM'
217
N
HIS 
A
32
23.529
29.991
21.065
1.00
9.76
N


ATOM
218
CA
HIS 
A
32
24.615
29.409
20.285
1.00
9.96
C


ATOM
219
CB
HIS 
A
32
25.747
28.891
21.194
1.00
11.85
C


ATOM
220
CG
HIS 
A
32
25.442
27.602
21.896
1.00
9.52
C


ATOM
221
CD2
HIS 
A
32
25.495
26.319
21.464
1.00
11.42
C


ATOM
222
ND1
HIS 
A
32
25.093
27.545
23.226
1.00
12.01
N


ATOM
223
CE1
HIS 
A
32
24.945
26.281
23.588
1.00
12.23
C


ATOM
224
NE2
HIS 
A
32
25.185
25.518
22.538
1.00
12.81
N


ATOM
225
C
HIS 
A
32
24.138
28.301
19.355
1.00
8.20
C


ATOM
226
O
HIS 
A
32
24.917
27.768
18.569
1.00
10.19
O


ATOM
227
N
CYS
A
33
22.850
27.977
19.430
1.00
8.42
N


ATOM
228
CA
CYS
A
33
22.270
26.933
18.589
1.00
9.80
C


ATOM
229
CB
CYS
A
33
20.894
26.536
19.117
1.00
11.66
C


ATOM
230
SG
CYS
A
33
20.964
25.864
20.798
1.00
13.22
S


ATOM
231
C
CYS
A
33
22.131
27.410
17.152
1.00
14.10
C


ATOM
232
O
CYS
A
33
22.338
26.649
16.212
1.00
14.43
O


ATOM
233
N
GLY
A
34
21.775
28.676
16.982
1.00
14.60
N


ATOM
234
CA
GLY
A
34
21.622
29.202
15.643
1.00
13.42
C


ATOM
235
C
GLY
A
34
21.365
30.690
15.632
1.00
13.64
C


ATOM
236
O
GLY
A
34
20.989
31.278
16.652
1.00
12.12
O


ATOM
237
N
ARG
A
35
21.565
31.299
14.467
1.00
12.90
N


ATOM
238
CA
ARG
A
35
21.360
32.728
14.301
1.00
15.08
C


ATOM
239
CB
ARG
A
35
22.458
33.322
13.416
1.00
14.13
C


ATOM
240
C
ARG
A
35
20.003
33.020
13.673
1.00
11.11
C


ATOM
241
O
ARG
A
35
19.367
32.144
13.084
1.00
14.43
O


ATOM
242
CG
ARG
A
35
22.408
32.854
11.971
1.00
19.31
C


ATOM
243
CD
ARG
A
35
23.430
33.597
11.123
1.00
21.41
C


ATOM
244
NE
ARG
A
35
24.800
33.232
11.469
1.00
22.20
N


ATOM
245
CZ
ARG
A
35
25.410
32.135
11.032
1.00
22.78
C


ATOM
246
NH1
ARG
A
35
26.658
31.875
11.400
1.00
21.47
N


ATOM
247
NH2
ARG
A
35
24.779
31.305
10.215
1.00
23.65
N


ATOM
248
N
THR
A
36
19.566
34.265
13.803
1.00
12.06
N


ATOM
249
CA
THR
A
36
18.291
34.688
13.251
1.00
10.87
C


ATOM
250
CB
THR
A
36
18.123
36.212
13.411
1.00
14.79
C


ATOM
251
C
THR
A
36
18.212
34.305
11.774
1.00
11.54
C


ATOM
252
O
THR
A
36
19.195
34.414
11.043
1.00
10.69
O


ATOM
253
OG1
THR
A
36
18.002
36.552
14.802
1.00
19.95
O


ATOM
254
CG2
THR
A
36
16.889
36.705
12.679
1.00
17.55
C


ATOM
255
N
GLY
A
37
17.047
33.839
11.339
1.00
11.25
N


ATOM
256
CA
GLY
A
37
16.896
33.446
9.950
1.00
10.63
C


ATOM
257
C
GLY
A
37
17.140
31.965
9.705
1.00
16.44
C


ATOM
258
O
GLY
A
37
16.711
31.421
8.688
1.00
13.24
O


ATOM
259
N
ALA
A
38
17.837
31.306
10.624
1.00
16.27
N


ATOM
260
CA
ALA
A
38
18.101
29.877
10.481
1.00
17.14
C


ATOM
261
C
ALA
A
38
16.781
29.118
10.602
1.00
15.01
C


ATOM
262
O
ALA
A
38
15.943
29.447
11.442
1.00
14.04
O


ATOM
263
CB
ALA
A
38
19.074
29.416
11.559
1.00
16.54
C


ATOM
264
N
THR
A
39
16.588
28.107
9.764
1.00
15.44
N


ATOM
265 
CA 
THR 
A
39
15.355
27.329
9.811
1.00
16.44
C


ATOM
266 
CB 
THR
A
39
14.867
26.956
8.397
1.00
16.50
C


ATOM
267 
OG1
THR 
A
39
15.848
26.146
7.746
1.00
22.08
O


ATOM
268 
CG2
THR 
A
39
14.615
28.209
7.578
1.00
17.88
C


ATOM
269 
C
THR 
A
39
15.522
26.052
10.622
1.00
14.04
C


ATOM
270 
O
THR 
A
39
16.603
25.467
10.669
1.00
13.48
O


ATOM
271 
N
THR 
A
40
14.437
25.626
11.256
1.00
14.41
N


ATOM
272 
CA 
THR 
A
40
14.445
24.421
12.072
1.00
12.76
C


ATOM
273 
CB 
THR 
A
40
14.081 
24.735 
13.536 
1.00
13.70
C


ATOM
274 
OG1
THR 
A
40
12.745 
25.260 
13.601 
1.00
11.68
O


ATOM
275 
CG2
THR 
A
40
15.043 
25.752 
14.118 
1.00
10.97
C


ATOM
276 
C
THR
A
40
13.437 
23.399 
11.566
1.00
12.70
C


ATOM
277 
O
THR
A
40
12.554 
23.717 
10.773
1.00
15.30
O


ATOM
278 
N
ALA
A
41
13.592 
22.164 
12.033
1.00
12.69
N


ATOM
279
CA
ALA
A
41
12.713 
21.062 
11.667
1.00
13.39
C


ATOM
280
C
ALA
A
41
12.425 
20.346 
12.986
1.00
13.08
C


ATOM
281
O
ALA
A
41
13.234 
20.403 
13.912
1.00
13.32
O


ATOM
282
CB
ALA
A
41
13.403 
20.121 
10.682
1.00
12.91
C


ATOM
283
N
ASN
A
42
11.280 
19.680 
13.075
1.00
13.98
N


ATOM
284
CA
ASN
A
42
10.909 
18.966 
14.296
1.00
15.22
C


ATOM
285
C
ASN
A
42
11.074 
19.886 
15.507
1.00
15.41
C


ATOM
286
O
ASN
A
42
11.835 
19.580 
16.426
1.00
14.69
O


ATOM
287
CB
ASN
A
42
11.792 
17.727 
14.507
1.00
18.61
C


ATOM
288
CG
ASN
A
42
11.862 
16.826 
13.282
1.00
22.16
C


ATOM
289
OD1
ASN
A
42
10.893 
16.685 
12.536
1.00
20.39
O


ATOM
290
ND1
ASN
A
42
13.017 
16.192 
13.085
1.00
21.80
N


ATOM
291
NA
PRO
A
43
10.319 
20.994 
15.558
1.00
12.16
N


ATOM
292
CA
PRO
A
43
9.329
21.449 
14.579
1.00
13.99
C


ATOM
293
CB
PRO
A
43
8.328
22.178 
15.454
1.00
14.60
C


ATOM
294
C
PRO
A
43
9.863
22.387 
13.508
1.00
14.85
C


ATOM
295
O
PRO
A
43
10.949 
22.950 
13.633
1.00
12.84
O


ATOM
296
CD
PRO
A
43
10.287 
21.862 
16.751
1.00
11.35
C


ATOM
297
CG
PRO
A
43
9.259
22.940 
16.356
1.00
12.54
C


ATOM
298
N
THR
A
44
9.074
22.556 
12.454 
1.00
12.78
N


ATOM
299
CA
THR
A
44
9.454
23.436 
11.370
1.00
13.48
C


ATOM
300
CB
THR
A
44
8.441
23.349 
10.217
1.00
15.07
C


ATOM
301
C
THR
A
44
9.387
24.818 
12.010
1.00
13.36
C


ATOM
302
O
THR
A
44
8.430
25.127 
12.721
1.00
12.32
O


ATOM
303
OG1
THR
A
44
8.582
22.082
9.565
1.00
17.67
O


ATOM
304
OG2
THR
A
44
8.660
24.473
9.216
1.00
14.97
C


ATOM
305
N
GLY
A
45
10.412 
25.631 
11.787
1.00
12.10
N


ATOM
306 
CA
GLY
A
45
10.423 
26.958 
12.369
1.00
13.77
C


ATOM
307 
C
GLY
A
45
11.557 
27.824 
11.865
1.00
12.84
C


ATOM
308 
O
GLY
A
45
12.340 
27.412 
11.006
1.00
14.31
O


ATOM
309 
N
THR
A
46
11.648 
29.033 
12.404
1.00
12.18
N


ATOM
310 
CA
THR
A
46
12.686 
29.970 
12.001 
1.00
15.03
C


ATOM
311 
CB
THR
A
46
12.141 
30.953 
10.952 
1.00
15.90
C


ATOM
312 
OG1
THR
A
46
11.528 
30.219
9.884
1.00
20.72
O


ATOM
313 
CG2
THR
A
46
13.257 
31.821 
10.392 
1.00
18.41
C


ATOM
314 
C
THR
A
46
13.167
30.777 
13.203 
1.00
13.19
C


ATOM
315 
O
THR
A
46
12.352 
31.331 
13.944 
1.00
10.72
O


ATOM
316 
N
PHE
A
47
14.480 
30.835 
13.407
1.00
11.27
N


ATOM
317 
CA
PHE
A
47
15.009 
31.596 
14.527
1.00
10.95
C


ATOM
318 
CB
PHE
A
47
16.541 
31.508 
14.596
1.00
11.26
C


ATOM
319 
CG
PHE
A
47
17.054 
30.306 
15.346
1.00
12.89
C


ATOM
320 
CD2
PHE
A
47
17.559 
30.442 
16.633
1.00
8.64
C


ATOM
321 
CD1
PHE
A
47
17.036 
29.046 
14.767
1.00
12.80
C


ATOM
322 
CE2
PHE
A
47
18.040 
29.342 
17.331
1.00
12.73
C


ATOM
323 
CE1
PHE
A
47
17.514 
27.941 
15.457 
1.00
12.73
C


ATOM
324 
CZ
PHE
A
47
18.017 
28.088 
16.740
1.00
14.16
C


ATOM
325 
C
PHE
A
47
14.590 
33.041 
14.291 
1.00
12.22
C


ATOM
326 
O
PHE
A
47
14.737 
33.563 
13.182 
1.00
13.19
O


ATOM
327 
N
ALA
A
48
14.058 
33.673 
15.330 
1.00
11.62
N


ATOM
328 
CA
ALA
A
48
13.613 
35.059 
15.240
1.00
12.91
C


ATOM
329 
CB
ALA
A
48
12.092 
35.126 
15.261
1.00
13.93
C


ATOM
330 
C
ALA
A
48
14.184 
35.856 
16.400 
1.00
15.66
C


ATOM
331 
O
ALA
A
48
13.470 
36.598 
17.072 
1.00
21.12
O


ATOM
332 
N
GLY
A
49
15.482 
35.700 
16.622 
1.00
15.68
N


ATOM
333 
CA
GLY
A
49
16.139 
36.407 
17.701
1.00
16.25
C


ATOM
334 
C
GLY
A
49
17.156 
35.500 
18.352 
1.00
15.88
C


ATOM
335 
O
GLY
A
49
16.820 
34.403 
18.799 
1.00
13.45
O


ATOM'
336 
N
SER 
A
49
18.404 
35.947 
18.405 
1.00
13.85
N


ATOM
337 
CA
SER 
A
50
19.454 
35.144 
19.012
1.00
13.96
C


ATOM
338 
CB
SER 
A
50
20.014 
34.156 
17.984
1.00
17.08
C


ATOM
339 
OG
SER 
A
50
21.045 
33.365 
18.541
1.00
14.72
O


ATOM
340 
C
SER 
A
50
20.574 
36.026 
19.543 
1.00
16.90
C


ATOM
341 
O
SER 
A
50
21.082 
36.894 
18.835
1.00
16.85
O


ATOM
342 
N
SER 
A
51
20.941 
35.802 
20.801
1.00
15.23
N


ATOM
343
CA 
SER 
A
51
22.003
36.561
21.447 
1.00
14.67
C


ATOM
344
CB 
SER 
A
51
21.440
37.431
22.570 
1.00
15.96
C


ATOM
345
OG 
SER 
A
51
22.474
38.172
23.187 
1.00
18.34
O


ATOM
346
C
SER 
A
51
23.062
35.622
22.017 
1.00
11.74
C


ATOM
347
O
SER 
A
51
22.809
34.888
22.969 
1.00
12.45
O


ATOM
348
N
PHE 
A
52
24.247
35.655
21.419 
1.00
8.44
N


ATOM
349
CA
PHE 
A
52
25.367
34.822
21.842 
1.00
11.84
C


ATOM
350
CB
PHE 
A
52
25.090
33.344
21.557 
1.00
9.85
C


ATOM
351
CG
PHE 
A
52
26.264
32.450
21.837 
1.00
14.45
C


ATOM
352
CD1
PHE 
A
52
26.561
32.056
23.133
1.00
14.77
C


ATOM
353
CD2
PHE 
A
52
27.095
32.037
20.808 
1.00
14.93
C


ATOM
354
CE1
PHE 
A
52
27.665
31.267
23.400 
1.00
12.39
C


ATOM
355
CE2
PHE 
A
52
28.203
31.250
21.067
1.00
13.03
C


ATOM
356
CZ
PHE 
A
52
28.489
30.864
22.364 
1.00
15.39
C


ATOM
357
C
PHE 
A
52
26.595
35.245
21.051 
1.00
11.09
C


ATOM
358
O
PHE 
A
52
26.523
35.416
19.830 
1.00
10.06
O


ATOM
359
N
PRO 
A
53
27.737
35.427
21.732 
1.00
13.84
N


ATOM
360
CD
PRO 
A
53
29.034
35.610
21.055 
1.00
13.82
C


ATOM
361
CA
PRO 
A
53
27.919
35.257
23.177 
1.00
11.97
C


ATOM
362
CB
PRO 
A
53
29.433
35.114
23.319 
1.00
15.91
C


ATOM
363
CG
PRO 
A
53
29.953
35.957
22.201 
1.00
16.14
C


ATOM
364
C
PRO 
A
53
27.345
36.429
23.972 
1.00
13.65
C


ATOM
365
O
PRO 
A
53
26.411
37.085
23.516 
1.00
12.98
O


ATOM
366
N
GLY 
A
54
27.909
36.706
25.144 
1.00
13.22
N


ATOM
367
CA
GLY 
A
54
27.385
37.778
25.975
1.00
13.41
C


ATOM
368
C
GLY 
A
54
26.291
37.112
26.781 
1.00
13.11
C


ATOM
369
O
GLY 
A
54
26.403
36.931
27.995 
1.00
12.76
O


ATOM
370
N
ASN 
A
55
25.223
36.740
26.083 
1.00
13.05
N


ATOM
371
CA
ASN 
A
55
24.110
36.013
26.681 
1.00
14.39
C


ATOM
372
CB
ASN 
A
55
22.761
36.681
26.396
1.00
12.65
C


ATOM
373
CG
ASN 
A
55
22.758
38.153
26.682
1.00
11.23
C


ATOM
374
OD1
ASN 
A
55
22.521
38.967
25.784
1.00
16.09
O


ATOM
375
ND2
ASN
A
55
23.001
38.516
27.933
1.00
11.47
N


ATOM
376
C
ASN 
A
55
24.141
34.721
25.888 
1.00
15.51
C


ATOM
377
O
ASN 
A
55
25.076
34.485
25.123 
1.00
11.36
O


ATOM
378
N
ASP 
A
56
23.124
33.890
26.072 
1.00
14.13
N


ATOM
379
CA
ASP 
A
56
23.039
32.631
25.346
1.00
11.90
C


ATOM
380
CB
ASP 
A
56
23.881
31.522
25.993 
1.00
9.70
C


ATOM
381
CG
ASP 
A
56
24.053
30.320
25.070 
1.00
9.97
C


ATOM
382
OD1
ASP 
A
56
24.712
29.330
25.459 
1.00
12.57
O


ATOM
383
OD2
ASP 
A
56
23.526
30.365
23.938
1.00
8.45
O


ATOM
384
C
ASP 
A
56
21.578
32.216
25.279 
1.00
9.86
C


ATOM
385
O
ASP 
A
56
21.158
31.254
25.920 
1.00
11.82
O


ATOM
386
N
TYR
A
57
20.798
32.969
24.509 
1.00
8.71
N


ATOM
387
CA
TYR
A
57
19.379
32.677
24.351
1.00
10.51
C


ATOM
388
CB
TYR
A
57
18.523
33.480
25.348 
1.00
12.30
C


ATOM
389
CG
TYR 
A
57
18.650
34.992
25.271 
1.00
12.51
C


ATOM
390
CD1
TYR 
A
57
19.275
35.708
26.291 
1.00
11.12
C


ATOM
391
CE1
TYR 
A
57
19.366
37.094
26.244 
1.00
11.36
C


ATOM
392
CD2
TYR 
A
57
18.121
35.706
24.197 
1.00
13.29
C


ATOM
393
CE2
TYR 
A
57
18.209
37.096
24.144 
1.00
10.62
C


ATOM
394
CZ
TYR 
A
57
18.832
37.783
25.169 
1.00
13.60
C


ATOM
395
OH
TYR 
A
57
18.921
39.162
25.122 
1.00
12.04
O


ATOM
396
C
TYR 
A
57
18.912
32.963
22.933 
1.00
10.26
C


ATOM
397
O
TYR 
A
57
19.573
33.674
22.172 
1.00
10.59
O


ATOM
398
N
ALA 
A
58
17.767
32.393
22.578 
1.00
9.32
N


ATOM
399
CA
ALA 
A
58
17.200
32.583
21.254
1.00
7.41
C


ATOM
400
CB
ALA 
A
58
17.943
31.732
20.241 
1.00
7.89
C


ATOM
401
C
ALA 
A
58
15.727
32.207
21.271 
1.00
10.96
C


ATOM
402
O
ALA 
A
58
15.260
31.510
22.175 
1.00
11.10
O


ATOM
403
N
PHE 
A
59
15.002
32.702
20.277 
1.00
11.71
N


ATOM
404
CA
PHE 
A
59
13.578
32.435
20.136 
1.00
12.26
C


ATOM
405
CB
PHE 
A
59
12.748
33.707
20.333 
1.00
10.18
C


ATOM
406
CG
PHE 
A
59
11.321
33.576
19.859 
1.00
11.71
C


ATOM
407
CD2
PHE 
A
59
10.871
34.297
18.764
1.00
11.51
C


ATOM
408
CD1
PHE 
A
59
10.441
32.709
20.490
1.00
10.35
C


ATOM
409
CE2
PHE 
A
59
9.566
34.156
18.307
1.00
15.38
C


ATOM
410
CE1
PHE 
A
59
9.140
32.563
20.044
1.00
14.84
C


ATOM
411
CZ
PHE 
A
59
8.700
33.286
18.949 
1.00
13.16
C


ATOM
412
C
PHE 
A
59
13.361
31.931
18.722 
1.00
11.77
C


ATOM
413
O
PHE 
A
59
13.887
32.507
17.771 
1.00
13.80
O


ATOM
414
N
VAL
A
60
12.600
30.852
18.590
1.00
10.53
N


ATOM
415
CA
VAL
A
60
12.310
30.278
17.285 
1.00
11.14
C


ATOM
416
CB
VAL
A
60
12.738
28.796
17.209
1.00
15.19
C


ATOM
417
CG1
VAL
A
60
12.337
28.212
15.856
1.00
10.78
C


ATOM
418
CG2
VAL
A
60
14.248
28.670
17.421
1.00
11.44
C


ATOM
419
C
VAL
A
60
10.801
30.363
17.082
1.00
11.30
C


ATOM
420
O
VAL
A
60
10.034
29.905
17.924
1.00
8.90
O


ATOM
421
N
ARG 
A
61
10.372
30.964
15.979
1.00
12.67
N


ATOM
422
CA
ARG 
A
61
8.944
31.083
15.716
1.00
11.18
C


ATOM
423
CB
ARG 
A
61
8.655
32.314
14.844
1.00
12.63
C


ATOM
424
CG
ARG 
A
61
7.194
32.398
14.379
1.00
17.12
C


ATOM
425
CD
ARG 
A
61
6.967
33.527
13.376
1.00
20.85
C


ATOM
426
NE
ARG 
A
61
5.563
33.614
12.971
1.00
24.18
N


ATOM
427
CZ
ARG 
A
61
4.949
32.744
12.171
1.00
24.05
C


ATOM
428
NH2
ARG 
A
61
3.665
32.904
11.884
1.00
25.34
N


ATOM
429
NH1
ARG 
A
61
5.609
31.708
11.670
1.00
25.91
N


ATOM
430
C
ARG 
A
61
8.424
29.831
15.011
1.00
12.67
C


ATOM
431
O
ARG 
A
61
9.070
29.316
14.096
1.00
11.46
O


ATOM
432
N
THR 
A
62
7.274
29.333
15.461
1.00
13.58
N


ATOM
433
CA
THR 
A
62
6.666
28.147
14.865
1.00
13.24
C


ATOM
434
CB
THR 
A
62
6.495
26.995
15.884
1.00
11.66
C


ATOM
435
OG1
THR 
A
62
5.729
27.450
17.007
1.00
13.55
O


ATOM
436
CG2
THR 
A
62
7.853
26.485
16.349
1.00
13.26
C


ATOM
437
C
THR 
A
62
5.289
28.558
14.335
1.00
13.42
C


ATOM
438
O
THR 
A
62
4.727
29.568
14.770
1.00
16.80
O


ATOM
439
N
GLY 
A
63
4.748
27.778
13.406
1.00
16.51
N


ATOM
440
CA
GLY 
A
63
3.455
28.108
12.834
1.00
15.85
C


ATOM
441
C
GLY 
A
63
2.387
27.033
12.894
1.00
16.64
C


ATOM
442
O
GLY 
A
63
2.137
26.432
13.938
1.00
12.21
O


ATOM
443
N
ALA 
A
64
1.753
26.788
11.753
1.00
15.51
N


ATOM
444
CA
ALA 
A
64
0.678
25.810
11.663
1.00
15.84
C


ATOM
445
C
ALA 
A
64
1.090
24.378
11.977
1.00
15.00
C


ATOM
446
O
ALA 
A
64
2.228
23.977
11.742
1.00
15.60
O


ATOM
447
CB
ALA 
A
64
0.052
25.866
10.279
1.00
16.27
C


ATOM
448
N
GLY 
A
65
0.144
23.614
12.510
1.00
17.17
N


ATOM
449
CA
GLY 
A
65
0.390
22.217
12.828
1.00
19.42
C


ATOM
450
C
GLY 
A
65
1.369
21.946
13.953
1.00
19.21
C


ATOM
451
O
GLY 
A
65
1.691
20.790
14.234
1.00
22.10
O


ATOM
452
N
VAL 
A
66
1.842
23.001
14.603
1.00
15.20
N


ATOM
453
CA
VAL 
A
66
2.788
22.844
15.697
1.00
15.99
C


ATOM
454
CB
VAL 
A
66
4.018
23.746
15.501
1.00
15.02
C


ATOM
455
C
VAL 
A
66
2.116
23.195
17.016
1.00
18.46
C


ATOM
456
O
VAL 
A
66
1.769
24.349
17.257
1.00
16.96
O


ATOM
457
CG1
VAL 
A
66
4.961
23.602
16.688
1.00
13.36
C


ATOM
458
CG2
VAL 
A
66
4.725
23.375
14.195
1.00
11.46
C


ATOM
459
N
ASN 
A
67
1.931
22.193
17.866
1.00
15.34
N


ATOM
460
CA
ASN 
A
67
1.294
22.407
19.158
1.00
16.12
C


ATOM
461
CB
ASN 
A
67
0.474
21.177
19.539
1.00
21.01
C


ATOM
462
C
ASN 
A
67
2.332
22.704
20.228
1.00
17.24
C


ATOM
463
O
ASN 
A
67
3.172
21.862
20.554
1.00
17.97
O


ATOM
464
CG
ASN 
A
67
−0.465
20.748
18.431
1.00
29.21
C


ATOM
465
OD1
ASN 
A
67
−1.308
21.527
17.976
1.00
33.32
O


ATOM
466
ND2
ASN 
A
67
−0.323
19.505
17.982
1.00
33.03
N


ATOM
467
N
LEU 
A
68
2.260
23.915
20.767
1.00
13.94
N


ATOM
468
CA
LEU 
A
68
3.175
24.378
21.807
1.00
14.43
C


ATOM
469
CB
LEU 
A
68
3.317
25.896
21.707
1.00
13.70
C


ATOM
470
C
LEU 
A
68
2.638
23.985
23.178
1.00
15.01
C


ATOM
471
O
LEU 
A
68
1.670
24.568
23.664
1.00
16.08
O


ATOM
472
CG
LEU 
A
68
3.835
26.395
20.358
1.00
8.95
C


ATOM
473
CD1
LEU 
A
68
3.736
27.910
20.284
1.00
8.47
C


ATOM
474
CD2
LEU 
A
68
5.270
25.931
20.179
1.00
12.27
C


ATOM
475
N
LEU 
A
69
3.284
23.005
23.805
1.00
12.99
N


ATOM
476
CA
LEU 
A
69
2.861
22.529
25.119
1.00
12.18
C


ATOM
477
CB
LEU 
A
69
2.88
20.997
25.134
1.00
12.27
C


ATOM
478
CG
LEU 
A
69
2.075
20.310
24.029
1.00
16.54
C


ATOM
479
CD1
LEU 
A
69
2.251
18.802
24.113
1.00
17.85
C


ATOM
480
CD2
LEU 
A
69
0.611
20.679
24.170
1.00
19.65
C


ATOM
481
C
LEU 
A
69
3.665
23.050
26.307
1.00
14.39
C


ATOM
482
O
LEU 
A
69
4.879
23.239
26.228
1.00
14.53
O


ATOM
483
N
ALA 
A
70
2.969
23.271
27.416
1.00
12.89
N


ATOM
484
CA
ALA 
A
70
3.594
23.761
28.635
1.00
14.83
C


ATOM
485
CB
ALA 
A
70
2.585
24.547
29.457
1.00
18.71
C


ATOM
486
C
ALA 
A
70
4.042
22.519
29.391
1.00
12.67
C


ATOM
487
O
ALA 
A
70
3.638
22.293
30.523
1.00
11.15
O


ATOM
488
N
GLN 
A
71
4.876
21.711
28.742
1.00
13.59
N


ATOM
489
CA
GLN 
A
71
5.382
20.483
29.334
1.00
14.04
C


ATOM
490
CB
GLN 
A
71
4.591
19.282
28.809
1.00
14.08
C


ATOM
491
CG
GLN 
A
71
3.114
19.283
29.157
1.00
17.65
C


ATOM
492
CD
GLN 
A
71
2.378
18.099
28.560
1.00
19.50
C


ATOM
493
OE1
GLN 
A
71
1.421
17.592
29.143
1.00
24.87
O


ATOM
494
NE2
GLN 
A
71
2.815
17.658
27.386
1.00
17.48
N


ATOM
495
C
GLN 
A
71
6.849
20.255
29.011
1.00
16.23
C


ATOM
496
O
GLN 
A
71
7.375
20.786
28.035
1.00
15.48
O


ATOM
497
N
VAL 
A
72
7.501
19.451
29.840
1.00
13.56
N


ATOM
498
CA
VAL 
A
72
8.907
19.133
29.648
1.00
12.57
C


ATOM
499 
CB
VAL 
A
72
9.792
19.754 
30.748 
1.00
10.81
C


ATOM
500 
CG1
VAL 
A
72
11.193
19.162 
30.677
1.00
12.46
C


ATOM
501 
CG2
VAL 
A
72
9.862 
21.271 
30.563
1.00
10.56
C


ATOM
502
C
VAL 
A
72
9.007 
17.610 
29.695
1.00
10.65
C


ATOM
503
O
VAL 
A
72
8.415 
16.968 
30.565
1.00
11.44
O


ATOM
504
N
ASN 
A
73
9.736 
17.036 
28.746
1.00
11.79
N


ATOM
505 
CA
ASN 
A
73
9.913 
15.586 
28.673 
1.00
10.87
C


ATOM
506 
CB
ASN 
A
73
10.633
15.229 
27.369
1.00
12.22
C


ATOM
507 
CG
ASN 
A
73
10.598
13.743 
27.065
1.00
12.04
C


ATOM
508 
OD1
ASN 
A
73
10.411
12.916 
27.959
1.00
11.92
O


ATOM
509 
ND2
ASN 
A
73
10.790
13.397 
25.798
1.00
10.15
N


ATOM
510
C
ASN 
A
73
10.751
15.098 
29.863
1.00
11.93
C


ATOM
511
O
ASN 
A
73
11.854
15.597 
30.092
1.00
11.67
O


ATOM
512
N
ASN 
A
74
10.239
14.137 
30.631
1.00
12.17
N


ATOM
513 
CA
ASN 
A
74
11.010
13.640 
31.766
1.00
9.29
C


ATOM
514 
CB
ASN 
A
74
10.109
13.275 
32.958
1.00
13.70
C


ATOM
515 
CG
ASN 
A
74
9.162 
12.126 
32.662
1.00
16.27
C


ATOM
516 
OD1
ASN 
A
74
9.432 
11.274 
31.815
1.00
14.62
O


ATOM
517 
ND2
ASN 
A
74
8.048 
12.088 
33.384
1.00
18.77
N


ATOM
518
C
ASN 
A
74
11.853
12.435 
31.359
1.00
11.15
C


ATOM
519
O
ASN 
A
74
12.528
11.823 
32.189
1.00
10.62
O


ATOM
520
N
TYR 
A
75
11.813
12.115 
30.069
1.00
13.30
N


ATOM
521 
CA
TYR 
A
75
12.556
10.998 
29.495
1.00
13.21
C


ATOM
522 
CB
TYR 
A
75
14.039
11.363 
29.386
1.00
10.04
C


ATOM
523 
CG
TYR 
A
75
14.313
12.223 
28.170
1.00
11.82
C


ATOM
524 
CD1
TYR 
A
75
14.424
11.652 
26.907
1.00
10.82
C


ATOM
525 
CE1
TYR 
A
75
14.591
12.435
25.775
1.00
12.83
C


ATOM
526 
CD2
TYR 
A
75
14.381
13.608 
28.271
1.00
10.15
C


ATOM
527 
CE2
TYR 
A
75
14.545
14.402 
27.142
1.00
10.33
C


ATOM
528 
CZ
TYR 
A
75
14.648
13.805 
25.898
1.00
9.45
C


ATOM
529 
OH
TYR 
A
75
14.793
14.579 
24.770
1.00
10.77
O


ATOM
530
C
TYR 
A
75
12.380
9.652 
30.188
1.00
16.68
C


ATOM
531
O
TYR 
A
75
13.298
8.835 
30.228
1.00
18.39
O


ATOM
532
N
SER 
A
76
11.185
9.433 
30.723
1.00
18.33
N


ATOM
533 
CA
SER 
A
76
10.846 
8.193 
31.411
1.00
20.49
C


ATOM
534 
CB
SER 
A
76
10.811 
8.390 
32.926
1.00
21.53
C


ATOM
535 
OG
SER 
A
76
12.121 
8.424 
33.457
1.00
25.72
O


ATOM
536
C
SER 
A
76
9.470
7.775 
30.919
1.00
21.06
C


ATOM
537
O
SER 
A
76
8.843
6.868 
31.473
1.00
20.62
O


ATOM
538
N
GLY
A
77
9.013
8.452 
29.870
1.00
17.80
N


ATOM
539 
CA
GLY
A
77
7.715 
8.156 
29.295
1.00
18.95
C


ATOM
540
C
GLY
A
77
6.649
9.128 
29.752
1.00
17.33
C


ATOM
541
O
GLY
A
77
5.464
8.942 
29.470
1.00
16.27
O


ATOM
542
N
GLY
A
78
7.059 
10.173 
30.462
1.00
15.79
N


ATOM
543 
CA
GLY 
A
78
6.088 
11.142 
30.939
1.00
16.07
C


ATOM
544
C
GLY 
A
78
6.499 
12.585 
30.734
1.00
17.80
C


ATOM
545
O
GLY 
A
78
7.481 
12.876 
30.041
1.00
15.22
O


ATOM
546 
N
ARG 
A
79
5.742 
13.492 
31.342
1.00
17.07
N


ATOM
547 
CA
ARG 
A
79
6.025 
14.914
31.226
1.00
19.85
C


ATOM
548 
CB
ARG 
A
79
5.199
15.528 
30.090
1.00
23.00
C


ATOM
549 
CG
ARG 
A
79
5.711 
15.176 
28.701
1.00
29.54
C


ATOM
550 
CD
ARG 
A
79
4.683 
14.404 
27.910
1.00
35.50
C


ATOM
551 
NE
ARG 
A
79
5.207 
13.941 
26.626
1.00
39.02
N


ATOM
552 
CZ
ARG 
A
79
6.223 
13.094 
26.493
1.00
41.51
C


ATOM
553 
NH1
ARG 
A
79
6.838 
12.611 
27.566
1.00
38.71
N


ATOM
554 
NH2
ARG 
A
79
6.620 
12.716 
25.285
1.00
43.02
N


ATOM
555
C
ARG 
A
79
5.784 
15.695
32.510
1.00
18.62
C


ATOM
556
O
ARG 
A
79
4.968 
15.313
33.353
1.00
16.21
O


ATOM
557
N
VAL 
A
80
6.517 
16.793 
32.646
1.00
15.48
N


ATOM
558 
CA
VAL 
A
80
6.412 
17.660 
33.810
1.00
14.56
C


ATOM
559 
CB
VAL 
A
80
7.806 
18.040 
34.349
1.00
14.30
C


ATOM
560 
CG1
VAL 
A
80
7.666 
18.967 
35.542
1.00
16.79
C


ATOM
561 
CG2
VAL 
A
80
8.580 
16.787 
34.729
1.00
18.13
C


ATOM
562
C
VAL 
A
80
5.690
18.930 
33.375
1.00
15.88
C


ATOM
563
O
VAL 
A
80
6.106 
19.588
32.421 
1.00
14.01
O


ATOM
564
N
GLN 
A
81
4.602 
19.270
34.057 
1.00
15.03
N


ATOM
565 
CA
GLN 
A
81
3.863 
20.472
33.698 
1.00
18.02
C


ATOM
566 
CB
GLN 
A
81
2.503 
20.512
34.403 
1.00
21.88
C


ATOM
567 
CG
GLN 
A
81
1.422 
19.659 
33.760
1.00
29.23
C


ATOM
568 
CD
GLN 
A
81
1.161 
20.030
32.311
1.00
29.08
C


ATOM
569
OE1
GLN 
A
81
0.928 
21.194
31.984
1.00
31.12
O


ATOM
570 
NE2
GLN 
A
81
1.192 
19.034
31.434
1.00
32.61
N


ATOM
571
C
GLN 
A
81
4.654 
21.722
34.067
1.00
17.67
C


ATOM
572
O
GLN 
A
81
5.278 
21.786
35.128
1.00
18.79
O


ATOM
573 
N
VAL 
A
82
4.636 
22.709
33.179
1.00
15.10
N


ATOM
574 
CA
VAL 
A
82
5.345 
23.960
33.411
1.00
17.88
C


ATOM
575 
CB
VAL 
A
82
5.973 
24.494
32.107
1.00
16.36
C


ATOM
576 
CG1
VAL 
A
82
6.710 
25.792
32.374
1.00
19.17
C


ATOM
577 
CG2
VAL 
A
82
6.927 
23.454 
31.534
1.00
15.85
C


ATOM
578
C
VAL 
A
82
4.309 
24.952 
33.930
1.00
18.78
C


ATOM
579
O
VAL 
A
82
3.512 
25.494 
33.163
1.00
19.15
O


ATOM 
580 
N
ALA 
A
83
4.321 
25.175 
35.240
1.00
20.30
N


ATOM
581 
CA
ALA 
A
83
3.382 
26.091 
35.879
1.00
20.84
C


ATOM
582
CB
ALA 
A
83
3.230 
25.725 
37.348
1.00
20.26
C


ATOM
583
C
ALA 
A
83
3.734 
27.568
35.751 
1.00
20.34
C


ATOM
584
O
ALA 
A
83
2.849 
28.405
35.594 
1.00
21.44
O


ATOM
585
N
GLY 
A
84
5.021 
27.886
35.826 
1.00
18.33
N


ATOM
586 
CA
GLY 
A
84
5.445 
29.267
35.721 
1.00
15.96
C


ATOM
587
C
GLY 
A
84
6.946 
29.381
35.558 
1.00
18.35
C


ATOM
588
O
GLY 
A
84
7.599 
28.433
35.117 
1.00
16.24
O


ATOM
589
N
HIS 
A
85
7.495 
30.534
35.924 
1.00
16.53
N


ATOM
590
CA
HIS 
A
85
8.931 
30.767
35.798 
1.00
14.08
C


ATOM
591 
CB
HIS 
A
85
9.219 
31.528
34.498 
1.00
14.33
C


ATOM
592
C
HIS 
A
85
9.534
31.510 
36.988 
1.00
14.07
C


ATOM
593
O
HIS 
A
85
10.349
32.413 
36.818
1.00
15.60
O


ATOM
594 
CG
HIS 
A
85
8.399
32.770 
34.323
1.00
18.31
C


ATOM
595 
ND1
HIS 
A
85
8.937
34.035 
34.404
1.00
21.61
N


ATOM
596 
CD2
HIS 
A
85
7.079
32.936 
34.064
1.00
19.95
C


ATOM
597 
NE2
HIS 
A
85
6.848
34.289 
33.994
1.00
18.84
N


ATOM
598 
CE1
HIS 
A
85
7.983
34.929 
34.202
1.00
22.08
C


ATOM
599
N
THR
A
86
9.128
31.124 
38.193
1.00
14.33
N


ATOM
600 
CA
THR
A
86
9.640
31.758 
39.405
1.00
13.94
C


ATOM
601 
CB
THR 
A
86
8.754 
31.449 
40.612 
1.00
15.26
C


ATOM
602
C
THR 
A
86
11.044
31.243 
39.690
1.00
14.65
C


ATOM
603
O
THR 
A
86
11.249
30.042 
39.855
1.00
11.10
O


ATOM
604
OG1
THR 
A
86
7.424
31.904 
40.347
1.00
16.89
O


ATOM
605 
CG2
THR 
A
86
9.289
32.147 
41.854
1.00
16.68
C


ATOM
606
N
ALA 
A
87
12.005
32.157 
39.756
1.00
15.86
N


ATOM
607 
CA
ALA 
A
87
13.396
31.801 
40.016
1.00
17.16
C


ATOM
608
C
ALA 
A
87
13.633
31.152 
41.375
1.00
19.39
C


ATOM
609
O
ALA 
A
87
13.113
31.608 
42.395
1.00
18.84
O


ATOM
610 
CB
ALA 
A
87
14.272
33.034 
39.877
1.00
17.44
C


ATOM
611
N
ALA 
A
88
14.431
30.088 
41.373
1.00
16.82
N


ATOM
612
CA
ALA 
A
88
14.766
29.352 
42.584
1.00
14.95
C


ATOM
613
C
ALA 
A
88
16.112
29.832 
43.119
1.00
15.66
C


ATOM
614
O
ALA 
A
88
17.004
30.204 
42.355
1.00
15.62
O


ATOM
615
CB
ALA 
A
88
14.827
27.861 
42.291
1.00
10.05
C


ATOM
616
N
PRO 
A
89
16.275
29.822 
44.447
1.00
15.05
N


ATOM
617
CA
PRO 
A
89
17.510
30.259 
45.100
1.00
16.15
C


ATOM
618 
CB
PRO 
A
89
17.060
30.498 
46.535
1.00
16.59
C


ATOM
619
C
PRO 
A
89
18.661
29.260 
45.022
1.00
17.33
C


ATOM
620
O
PRO 
A
89
18.461
28.076 
44.741
1.00
14.79
O


ATOM 
621 
CD 
PRO 
A
89
15.236
29.522 
45.448 
1.00 
17.98
C


ATOM
622 
CG
PRO 
A
89
16.040
29.425 
46.728
1.00
15.94
C


ATOM
623
N
VAL 
A
90
19.873
29.751 
45.257
1.00
18.24
N


ATOM
624 
CA
VAL 
A
90
21.046
28.894 
45.221
1.00
17.32
C


ATOM
625 
CB
VAL 
A
90
22.312
29.658 
45.672
1.00
16.39
C


ATOM
626 
CG1
VAL 
A
90
23.449
28.678 
45.932
1.00
19.70
C


ATOM
627 
CG2
VAL 
A
90
22.711
30.665 
44.609
1.00
18.18
C


ATOM
628
C
VAL 
A
90
20.764
27.770 
46.211
1.00
17.15
C


ATOM
629
O
VAL 
A
90
20.153
28.005 
47.254
1.00
17.16
O


ATOM
630
N
GLY 
A
91
21.192 
26.556 
45.878
1.00
13.56
N


ATOM
631 
CA
GLY 
A
91
20.971
25.420 
46.755
1.00
13.61
C


ATOM
632
C
GLY 
A
91
19.787
24.583 
46.314
1.00
14.54
C


ATOM
633
O
GLY 
A
91
19.652
23.422 
46.695
1.00
13.48
O


ATOM
634
N
SER 
A
92
18.928
25.175 
45.497
1.00
12.12
N


ATOM
635 
CA
SER 
A
92
17.741
24.486 
45.014
1.00
13.41
C


ATOM
636 
CB
SER 
A
92
16.846
25.457 
44.239
1.00
10.87
C


ATOM
637 
OG
SER 
A
92
16.334
26.463 
45.090
1.00
12.36
O


ATOM
638
C
SER 
A
92
18.040
23.284 
44.134
1.00
13.34
C


ATOM
639
O
SER 
A
92
19.015
23.268 
43.383
1.00
9.90
O


ATOM
640
N
ALA
A
93
17.189
22.274 
44.252
1.00
11.16
N


ATOM
641 
CA
ALA
A
93
17.324
21.057 
43.475
1.00
14.34
C


ATOM
642 
CB
ALA 
A
93
16.554
19.925 
44.136
1.00
14.73
C


ATOM
643
C
ALA 
A
93
16.713
21.389 
42.119
1.00
14.46
C


ATOM
644
O
ALA 
A
93
15.605
21.920 
42.047
1.00
13.83
O


ATOM
645
N 
VAL 
A
94
17.440
21.092 
41.048
1.00
14.27
N


ATOM
646 
CA
VAL 
A
94
16.946
21.370 
39.707
1.00
9.84
O


ATOM
647 
CB
VAL 
A
94
17.617
22.629 
39.113
1.00
11.32
C


ATOM
648 
CG1
VAL 
A
94
17.204
23.859 
39.904
1.00
9.34
C


ATOM
649 
CG2
VAL 
A
94
19.140
22.467 
39.126
1.00
10.97
C


ATOM
650
C
VAL 
A
94
17.216
20.209 
38.763
1.00
9.69
C


ATOM
651
O
VAL 
A
94
18.139
19.421 
38.976
1.00
10.59
O


ATOM
652
N 
CYS 
A
95
16.398
20.094 
37.727
1.00
10.10
N


ATOM
653 
CA
CYS 
A
95
16.573
19.027 
36.752
1.00
9.94
C


ATOM
654 
CB
CYS 
A
95
15.468
17.983 
36.845
1.00
11.63
C


ATOM
655
SG
CYS 
A
95
15.412
17.059 
38.410
1.00
13.27
S


ATOM
656
C
CYS 
A
95
16.566
19.624 
35.359
1.00
10.91
C


ATOM
657
O
CYS 
A
95
15.808
20.551 
35.061
1.00
11.33
O


ATOM
658
N
ARG
A
96
17.424
19.070 
34.515
1.00
9.30
N


ATOM
659
CA
ARG
A
96
17.570
19.496 
33.135
1.00
7.08
C


ATOM
660
CB
ARG 
A
96
19.050
19.767 
32.827
1.00
9.79
C


ATOM
661
CG
ARG 
A
96
19.326
20.069 
31.353
1.00
10.80
C


ATOM
662
CD
ARG 
A
96
20.808
19.966 
31.011
1.00
10.58
C


ATOM
663
NE
ARG 
A
96
21.355
18.643 
31.312
1.00
11.86
N


ATOM
664
CZ
ARG 
A
96
20.957
17.506 
30.747
1.00
10.78
C


ATOM
665
NH1
ARG 
A
96
19.995
17.500 
29.831
1.00
10.18
N


ATOM
666
NH2
ARG 
A
96
21.529
16.365 
31.103
1.00
12.39
N


ATOM
667
C
ARG 
A
96
17.068
18.397 
32.211
1.00
9.14
C


ATOM
668
O
ARG 
A
96
17.237
17.214 
32.499
1.00
10.81
O


ATOM
669
N
SER 
A
97
16.442
18.792 
31.104
1.00
8.35
N


ATOM
670
CA
SER 
A
97
15.925
17.841 
30.134
1.00
8.67
C


ATOM
671
CB
SER 
A
97
14.406
17.976 
29.984
1.00
10.18
C


ATOM
672
OG
SER 
A
97
13.893
16.991 
29.094
1.00
10.36
O


ATOM
673
C
SER 
A
97
16.607
18.169 
28.810
1.00
9.06
C


ATOM
674
O
SER 
A
97
16.564
19.313 
28.353
1.00
10.38
O


ATOM
675
N
GLY
A
98
17.243
17.168 
28.209
1.00
9.45
N


ATOM
676
CA
GLY
A
98
17.939
17.365 
26.947
1.00
8.70
C


ATOM
677
C
GLY
A
98
17.853
16.131 
26.070
1.00
11.12
C


ATOM
678
O
GLY
A
98
17.689
15.021 
26.569
1.00
9.32
O


ATOM
679
N
SER 
A
99
17.993
16.320 
24.762
1.00
13.12
N


ATOM
680
CA
SER 
A
99
17.884
15.222 
23.805
1.00
13.49
C


ATOM
681
CB
SER 
A
99
17.628
15.784 
22.414
1.00
16.87
C


ATOM
682
OG
SER 
A
99
18.805
16.381 
21.906
1.00
16.57
O


ATOM
683
C
SER 
A
99
19.073
14.272 
23.709
1.00
13.85
C


ATOM
684
O
SER 
A
99
18.972
13.230 
23.060
1.00
10.18
O


ATOM
685
N
THR 
A
100
20.195
14.617 
24.331
1.00
9.80
N


ATOM
686
CA
THR
A
100
21.365
13.749 
24.266
1.00
12.00
C


ATOM
687
CB
THR
A
100
22.645
14.572 
24.075
1.00
13.43
C


ATOM
688
OG1
THR
A
100
22.564
15.297 
22.844
1.00
15.69
O


ATOM
689
CG2
THR
A
100
23.860
13.667 
24.044
1.00
13.71
C


ATOM
690
C
THR 
A
100
21.547
12.845 
25.477
1.00
13.37
C


ATOM
691
O
THR 
A
100
21.888
11.667 
25.332
1.00
9.57
O


ATOM
692
N
THR 
A
101
21.319
13.389 
26.668
1.00
11.47
N


ATOM
693
CA
THR 
A
101
21.468
12.613 
27.893
1.00
10.42
C


ATOM
694
CB
THR 
A
101
22.469
13.277 
28.851
1.00
12.08
C


ATOM
695
OG1
THR 
A
101
22.031
14.607 
29.151
1.00
10.91
O


ATOM
696
CG2
THR 
A
101
23.847
13.334 
28.216
1.00
11.99
C


ATOM
697
C
THR 
A
101
20.153
12.410 
28.633
1.00
13.44
C


ATOM
698
O
THR 
A
101
20.078
11.617 
29.566
1.00
12.54
O


ATOM
699
N
GLY 
A
102
19.119
13.128 
28.217
1.00
10.42
N


ATOM
700
CA 
GLY 
A
102
17.829
12.979 
28.860
1.00
9.53
C


ATOM
701
C
GLY 
A
102
17.578
13.835 
30.087
1.00
10.82
C


ATOM
702
O
GLY 
A
102
17.846
15.041 
30.096
1.00
8.61
O


ATOM
703
N
TRP 
A
103
17.067
13.190 
31.132
1.00
9.62
N


ATOM
704
CA
TRP 
A
103
16.716
13.845 
32.383
1.00
11.61
C


ATOM
705
CB
TRP 
A
103
15.370
13.289 
32.865
1.00
11.52
C


ATOM
706
CG
TRP 
A
103
14.837
13.868 
34.145
1.00
13.15
C


ATOM
707
CD2
TRP 
A
103
13.964
14.998 
34.282
1.00
12.58
C


ATOM
708
CE2
TRP 
A
103
13.680
15.147 
35.655
1.00
15.29
C


ATOM
709
CE3
TRP 
A
103
13.387
15.896 
33.375
1.00
11.72
C


ATOM
710
CD1
TRP 
A
103
15.050
13.397 
35.404
1.00
16.94
C


ATOM
711
NE1
TRP 
A
103
14.357
14.156 
36.320
1.00
16.85
N


ATOM
712
CZ2
TRP 
A
103
12.852
16.155 
36.147
1.00
11.23
C


ATOM
713
CZ3
TRP 
A
103
12.561
16.900 
33.865
1.00
12.19
C


ATOM
714
CH2
TRP 
A
103
12.303
17.019 
35.240
1.00
13.20
C


ATOM
715
C
TRP 
A
103
17.790
13.659 
33.448 
1.00
12.90
C


ATOM
716
O
TRP 
A
103
18.082
12.539 
33.872 
1.00
9.69
O


ATOM
717
N
HIS 
A
104
18.386
14.768 
33.872
1.00
10.38
N


ATOM
718
CA
HIS 
A
104
19.434
14.724 
34.890
1.00
12.11
C


ATOM
719
CB
HIS 
A
104
20.806
14.734 
34.226
1.00
12.14
C


ATOM
720
CG
HIS 
A
104
21.106
13.474 
33.477
1.00
12.45
C


ATOM
721
CD2
HIS 
A
104
20.822
13.110 
32.204
1.00
14.29
C


ATOM
722
ND1
HIS 
A
104
21.684
12.375 
34.072
1.00
13.64
N


ATOM
723
CE1
HIS 
A
104
21.740
11.384 
33.197
1.00
14.53
C


ATOM
724
NE2
HIS 
A
104
21.222
11.804 
32.058
1.00
12.11
N


ATOM
725
C
HIS 
A
104
19.283
15.898 
35.839
1.00
12.75
C


ATOM
726
O
HIS 
A
104
18.959
17.014 
35.426
1.00
10.16
O


ATOM
727
N
CYS
A
105
19.545
15.650 
37.114
1.00
10.52
N


1'TOM
728
CA
CYS
A
105
19.408
16.703 
38.102
1.00
13.24
C


ATOM
729
CB
CYS
A
105
18.278
16.318 
39.049
1.00
13.49
C


ATOM
730
SG
CYS
A
105
16.817
15.612 
38.216
1.00
14.12
S


ATOM
731
C
CYS 
A
105
20.657
17.057 
38.896
1.00
13.65
C


ATOM
732
O
CYS 
A
105
21.720
16.465 
38.720
1.00
13.71
O


ATOM
733 
N
GLY 
A
106
20.511
18.042
39.770
1.00
11.96
N


ATOM
734
CA
GLY 
A
106
21.619 
18.499 
40.583
1.00
8.39
C


ATOM
735
C
GLY 
A
106
21.112 
19.662 
41.404
1.00
8.29
C


ATOM
736
O
GLY 
A
106
19.919 
19.720 
41.723
1.00
9.88
O


ATOM
737
N
THR 
A
107
21.997 
20.587 
41.748
1.00
10.00
N


ATOM
738
CA
THR 
A
107
21.593 
21.749 
42.529
1.00
10.90
C


ATOM
739
CB
THR 
A
107
21.979 
21.607 
44.021
1.00
15.03
C


ATOM
740 
OG1
THR 
A
107
23.401 
21.490 
44.138
1.00
19.34
O


ATOM
741 
CG2
THR 
A
107
21.324
20.379 
44.630
1.00
19.07
C


ATOM
742
C
THR 
A
107
22.230 
23.021 
42.003
1.00
11.41
C


ATOM
743
O
THR 
A
107
23.274 
22.986 
41.349
1.00
10.42
O


ATOM
744
N
ILE 
A
108
21.590 
24.150 
42.282
1.00
9.46
N


ATOM
745
CA
ILE 
A
108
22.116 
25.430 
41.835
1.00
8.75
C


ATOM
746
CB
ILE 
A
108
21.050 
26.533 
41.895
1.00
10.61
C


ATOM
747 
CG2
ILE 
A
108
21.696 
27.892 
41.613
1.00
8.96
C


ATOM
748 
CG1
ILE 
A
108
19.926 
26.214 
40.905
1.00
11.64
C


ATOM
749 
CD1
ILE 
A
108
18.797 
27.223 
40.898
1.00
10.98
C


ATOM
750
C
ILE 
A
108
23.240 
25.788 
42.798
1.00
12.89
C


ATOM
751
O
ILE 
A
108
23.030 
25.842 
44.010
1.00
12.63
O


ATOM
752
N
THR
A
109
24.432 
26.026 
42.263
1.00
12.30
N


ATOM
753
CA
THR
A
109
25.570 
26.372 
43.109
1.00
12.21
C


ATOM
754
CB
THR
A
109
26.814 
25.557 
42.714
1.00
16.03
C


ATOM
755 
OG1
THR 
A
109
27.027 
25.662 
41.304
1.00
16.72
O


ATOM
756 
CG2
THR 
A
109
26.623 
24.097 
43.079
1.00
19.29
C


ATOM
757 
C
THR 
A
109
25.916 
27.855 
43.094
1.00
14.26
C


ATOM
758 
O
THR 
A
109
26.590 
28.353 
43.994
1.00
15.02
O


ATOM
759 
N
ALA 
A
110
25.440 
28.563 
42.078
1.00
13.73
N


ATOM
760 
CA
ALA 
A
110
25.708 
29.985 
41.967
1.00
14.67
C


ATOM
761 
CB
ALA 
A
110
27.186 
30.215 
41.668
1.00
15.41
C


ATOM
762 
C
ALA 
A
110
24.853 
30.611 
40.879
1.00
12.70
C


ATOM
763 
O
ALA 
A
110
24.367 
29.924 
39.982
1.00
13.13
O


ATOM
764 
N
LEU 
A
111
24.664 
31.921 
40.982
1.00
13.52
N


ATOM
765 
CA
LEU 
A
111
23.876 
32.680 
40.019
1.00
11.96
C


ATOM
766 
CB
LEU 
A
111
22.639 
33.286 
40.689
1.00
15.77
C


ATOM
767 
CG
LEU 
A
111
21.638 
32.338 
41.357
1.00
19.65
C


ATOM
768 
CD1
LEU 
A
111
20.593 
33.151 
42.113
1.00
17.73
C


ATOM
769 
CD2
LEU 
A
111
20.970 
31.462 
40.313
1.00
14.60
C


ATOM
770 
C
LEU 
A
111
24.775 
33.798 
39.501
1.00
15.77
C


ATOM
771 
O
LEU 
A
111
25.753 
34.169 
40.151
1.00
15.15
O


ATOM
772 
N
ASN 
A
112
24.443 
34.330 
38.332
1.00
12.74
N


ATOM
773 
CA
ASN 
A
112
25.219 
35.409 
37.729
1.00
17.38
C


ATOM
774 
CB
ASN 
A
112
25.168 
36.663 
38.605
1.00
24.14
C


ATOM
775 
CG
ASN 
A
112
23.756 
37.053 
38.980
1.00
26.37
C


ATOM
776 
OD1
ASN 
A
112
23.279 
36.726 
40.067
1.00
33.64
O


ATOM
777 
ND2
ASN 
A
112
23.072 
37.744 
38.076
1.00
34.88
N


ATOM
778 
C
ASN 
A
112
26.672 
35.023 
37.495
1.00
16.99
C


ATOM
779 
O
ASN 
A
112
27.572 
35.850 
37.643
1.00
14.78
O


ATOM
780 
N
SER 
A
113
26.896 
33.766 
37.131
1.00
16.31
N


ATOM
781 
CA
SER 
A
113
28.245 
33.280 
36.872
1.00
19.39
C


ATOM
782 
CB
SER 
A
113
28.315 
31.757 
37.020
1.00
18.03
C


ATOM
783 
OG
SER 
A
113
28.262 
31.349 
38.374
1.00
21.23
O


ATOM
784 
C
SER 
A
113
28.637 
33.650 
35.450
1.00
19.59
C


ATOM
785 
O
SER 
A
113
27.780 
33.946 
34.620
1.00
20.53
O


ATOM
786 
N
SER 
A
114
29.938 
33.634
35.180
1.00
20.43
N


ATOM
787 
CA
SER 
A
114
30.454 
33.957 
33.857
1.00
21.50
C


ATOM
788 
CB
SER 
A
114
31.256 
35.259 
33.878
1.00
23.57
C


ATOM
789 
OG
SER 
A
114
30.407 
36.384 
34.009
1.00
27.72
O


ATOM
790 
C
SER 
A
114
31.356 
32.824 
33.406
1.00
21.25
C


ATOM
791 
O
SER 
A
114
32.019 
32.181 
34.222
1.00
21.50
O


ATOM
792 
N
VAL
A
115
31.364 
32.569 
32.106
1.00
19.94
N


ATOM
793 
CA
VAL
A
115
32.188 
31.515 
31.542
1.00
19.21
C


ATOM
794 
CB
VAL 
A
115
31.394 
30.203 
31.350
1.00
20.02
C


ATOM
795 
CG1
VAL
A
115
30.768 
29.782 
32.663
1.00
23.26
C


ATOM
796 
CG2
VAL 
A
115
30.335 
30.380 
30.276
1.00
19.81
C


ATOM
797 
C
VAL 
A
115
32.675 
31.986 
30.183
1.00
17.61
C


ATOM
798 
O
VAL 
A
115
32.065 
32.857 
29.561
1.00
16.06
O


ATOM
799 
N
THR 
A
116
33.783 
31.419 
29.729
1.00
15.49
N


ATOM
800 
CA
THR 
A
116
34.330 
31.791 
28.441
1.00
15.82
C


ATOM
801 
CB
THR 
A
116
35.750 
32.380 
28.569
1.00
16.00
C


ATOM
802 
OG1
THR 
A
116
35.697 
33.577 
29.355
1.00
19.34
O


ATOM
803 
CG2
THR 
A
116
36.312 
32.721 
27.189
1.00
13.81
C


ATOM
804 
C
THR 
A
116
34.364 
30.572 
27.535
1.00
16.67
C


ATOM
805 
O
THR 
A
116
35.031 
29.576 
27.828
1.00
15.27
O


ATOM
806 
N
TYR 
A
117
33.604 
30.663 
26.451
1.00
14.40
N


ATOM
807 
CA
TYR 
A
117
33.500 
29.609 
25.453
1.00
18.79
C


ATOM
808 
CB
TYR 
A
117
32.077 
29.553 
24.886
1.00
16.32
C


ATOM
809 
CG
TYR 
A
117
30.993 
29.168 
25.871
1.00
19.43
C


ATOM
810 
CD1
TYR 
A
117
29.875 
29.977 
26.057
1.00
17.65
C


ATOM
811
CE1
TYR 
A
117
28.844
29.594
26.911
1.00
19.53
C


ATOM
812
CD2
TYR 
A
117
31.053
27.967
26.569
1.00
19.30
C


ATOM
813
CE2
TYR 
A
117
30.029
27.577
27.421
1.00
23.56
C


ATOM
814
CZ
TYR 
A
117
28.926
28.392
27.588
1.00
21.66
C


ATOM
815
OH
TYR 
A
117
27.898
27.991
28.417
1.00
18.29
O


ATOM
816
C
TYR 
A
117
34.462
29.985
24.330
1.00
16.91
C


ATOM
817
O
TYR 
A
117
34.968
31.107
24.288
1.00
18.67
O


ATOM
818
N
PRO 
A
118
34.727
29.058
23.400
1.00
17.80
N


ATOM
819
CA
PRO 
A
118
35.644
29.417
22.317
1.00
18.47
C


ATOM
820
CB
PRO 
A
118
35.655
28.165
21.434
1.00
19.17
C


ATOM
821
C
PRO 
A
118
35.210
30.679
21.567
1.00
22.88
C


ATOM
822
O
PRO 
A
118
36.052
31.426
21.064
1.00
22.73
O


ATOM
823
CD
PRO 
A
118
34.280
27.663
23.277
1.00
19.00
C


ATOM
824
CG
PRO 
A
118
34.390
27.442
21.799
1.00
22.37
C


ATOM
825
N
GLU 
A
119
33.900
30.923
21.509
1.00
21.24
N


ATOM
826
CA
GLU 
A
119
33.375
32.101
20.819
1.00
22.24
C


ATOM
827
CB
GLU 
A
119
31.888
31.930
20.495
1.00
23.42
C


ATOM
828
C
GLU 
A
119
33.539
33.356
21.665
1.00
24.28
C


ATOM
829
O
GLU 
A
119
33.672
34.464
21.142
1.00
24.95
O


ATOM
830
CG
GLU 
A
119
31.561
30.815
19.522
1.00
25.42
C


ATOM
831
CD
GLU 
A
119
31.812
29.443
20.104
1.00
29.77
C


ATOM
832
OE1
GLU 
A
119
31.546
29.252
21.310
1.00
28.43
O


ATOM
833
OE2
GLU 
A
119
32.260
28.552
19.350
1.00
28.50
O


ATOM
834
N
GLY 
A
120
33.517
33.181
22.979
1.00
21.77
N


ATOM
835
CA
GLY 
A
120
33.658
34.323
23.857
1.00
21.24
C


ATOM
836
C
GLY 
A
120
33.028
34.099
25.215
1.00
18.48
C


ATOM
837
O
GLY 
A
120
32.613
32.991
25.549
1.00
16.77
O


ATOM
838
N
THR 
A
121
32.944
35.169
25.994
1.00
15.75
N


ATOM
839
CA
THR 
A
121
32.388
35.098
27.332
1.00
15.58
C


ATOM
840
CB
THR 
A
121
33.050
36.151
28.242
1.00
20.73
C


ATOM
841
OG1
THR 
A
121
34.472
35.973
28.215
1.00
22.66
O


ATOM
842
CG1
THR 
A
121
32.548
36.020
29.675
1.00
19.40
C


ATOM
843
C
THR 
A
121
30.876
35.292
27.393
1.00
13.13
C


ATOM
844
O
THR 
A
121
30.307
36.105
26.665
1.00
12.67
O


ATOM
845
N
VAL 
A
122
30.235
34.523
28.263
1.00
10.86
N


ATOM
846
CA
VAL 
A
122
28.789
34.591
28.460
1.00
11.99
C


ATOM
847
CB
VAL 
A
122
28.095
33.263
28.093
1.00
10.42
C


ATOM
848
CG1
VAL 
A
122
26.641
33.290
28.534
1.00
11.35
C


ATOM
849
CG2
VAL 
A
122
28.176
33.044
26.587
1.00
7.40
C


ATOM
850
C
VAL 
A
122
28.616
34.875
29.951
1.00
13.37
C


ATOM
851
O
VAL 
A
122
29.219
34.199
30.786
1.00
13.64
O


ATOM
852
N
ARG 
A
123
27.801
35.870
30.288
1.00
14.96
N


ATOM
853
CA
ARG 
A
123
27.581
36.221
31.691
1.00
17.69
C


ATOM
854
CB
ARG 
A
123
27.936
37.693
31.903
1.00
19.72
C


ATOM
855
CG
ARG 
A
123
29.309
38.079
31.374
1.00
28.94
C


ATOM
856
CD
ARG 
A
123
29.620
39.545
31.642
1.00
34.45
C


ATOM
857
NE
ARG 
A
123
30.913
39.934
31.084
1.00
34.81
N


ATOM
858
CZ
ARG 
A
123
31.148
40.109
29.787
1.00
37.65
C


ATOM
859
NH1
ARG 
A
123
30.175
39.936
28.901
1.00
40.89
N


ATOM
860
NH2
ARG 
A
123
32.362
40.450
29.373
1.00
35.27
N


ATOM
861
C
ARG 
A
123
26.160
35.970
32.188
1.00
15.87
C


ATOM
862
O
ARG 
A
123
25.297
35.538
31.430
1.00
14.52
O


ATOM
863
N
GLY 
A
124
25.942
36.231
33.477
1.00
14.67
N


ATOM
864
CA
GLY 
A
124
24.629
36.064
34.084
1.00
12.41
C


ATOM
865
C
GLY 
A
124
24.058
34.659
34.085
1.00
13.04
C


ATOM
866
O
GLY 
A
124
22.841
34.477
34.106
1.00
11.06
O


ATOM
867
N
LEU 
A
125
24.937
33.666
34.092
1.00
10.63
N


ATOM
868
CA
LEU 
A
125
24.520
32.270
34.063
1.00
11.37
C


ATOM
869
CB
LEU 
A
125
25.556
31.455
33.293
1.00
10.04
C


ATOM
870
CG
LEU 
A
125
25.729
31.810
31.820
1.00
8.76
C


ATOM
871
CD1
LEU 
A
125
26.853
30.973
31.236
1.00
12.07
C


ATOM
872
CD2
LEU 
A
125
24.430
31.559
31.072
1.00
10.99
C


ATOM
873
C
LEU 
A
125
24.290
31.595
35.413
1.00
11.50
C


ATOM
874
O
LEU 
A
125
24.887
31.958
36.424
1.00
13.58
O


ATOM
875
N
ILE 
A
126
23.412
30.595
35.398
1.00
9.91
N


ATOM
876
CA
ILE 
A
126
23.078
29.829
36.586
1.00
10.95
C


ATOM
877
CB
ILE 
A
126
21.649
29.253
36.502
1.00
12.17
C


ATOM
878
CG2
ILE 
A
126
21.379
28.348
37.704
1.00
11.45
C


ATOM
879
CG1
ILE 
A
126
20.631
30.394
36.417
1.00
12.38
C


ATOM
880
CD1
ILE 
A
126
19.207
29.931
36.143
1.00
11.96
C


ATOM
881
C
ILE 
A
126
24.066
28.668
36.579
1.00
11.47
C


ATOM
882
O
ILE 
A
126
24.109
27.898
35.620
1.00
11.84
O


ATOM
883
N
ARG 
A
127
24.874
28.557
37.627
1.00
12.21
N


ATOM
884
CA
ARG 
A
127
25.854
27.478
37.716
1.00
14.56
C


ATOM
885
CB
ARG 
A
127
27.106
27.969
38.444
1.00
14.59
C


ATOM
886
CG
ARG 
A
127
28.195
26.921
38.616
1.00
23.58
C


ATOM
887
CD
ARG 
A
127
29.308
27.473
39.493
1.00
26.48
C


ATOM
888
NE
ARG 
A
127
30.349
26.489
39.769
1.00
36.15
N


ATOM
889
CZ
ARG 
A
127
31.209
26.033
38.865
1.00
38.33
C


ATOM
890
NH2
ARG 
A
127
32.127
25.139
39.211
1.00
41.31
N


ATOM
891
NH1
ARG 
A
127
31.156
26.472
37.616
1.00
41.25
N


ATOM
892
C
ARG 
A
127
25.221
26.324
38.485
1.00
12.51
C


ATOM
893
O
ARG 
A
127
24.554
26.548
39.495
1.00
10.73
O


ATOM
894
N
THR 
A
128
25.434
25.098
38.011
1.00
11.75
N


ATOM
895
CA
THR 
A
128
24.867
23.924
38.667
1.00
11.43
C


ATOM
896
CB
THR 
A
128
23.547
23.501
37.998
1.00
12.42
C


ATOM
897
OG1
THR 
A
128
23.835
22.848
36.751
1.00
11.64
O


ATOM
898
CG2
THR 
A
128
22.668
24.719
37.728
1.00
8.69
C


ATOM
899
C
THR 
A
128
25.778
22.698
38.622
1.00
13.02
C


ATOM
900
O
THR 
A
128
26.790
22.680
37.914
1.00
12.78
O


ATOM
901
N
THR 
A
129
25.391
21.674
39.381
1.00
11.69
N


ATOM
902
CA
THR 
A
129
26.132
20.419
39.456
1.00
12.47
C


ATOM
903
CB
THR 
A
129
26.099
19.827
40.878
1.00
12.66
C


ATOM
904
OG1
THR 
A
129
24.737
19.612
41.277
1.00
11.15
O


ATOM
905
CG2
THR 
A
129
26.782
20.766
41.859
1.00
12.84
C


ATOM
906
C
THR 
A
129
25.503
19.399
38.506
1.00
15.23
C


ATOM
907
O
THR 
A
129
25.820
18.211
38.564
1.00
10.87
O


ATOM
908
N
VAL 
A
130
24.601
19.870
37.646
1.00
14.09
N


ATOM
909
CA
VAL 
A
130
23.923
19.006
36.680
1.00
12.55
C


ATOM
910
CB
VAL 
A
130
22.662
19.694
36.103
1.00
13.46
C


ATOM
911
CG1
VAL 
A
130
21.913
18.730
35.195
1.00
15.05
C


ATOM
912
CG2
VAL 
A
130
21.755
20.178
37.234
1.00
10.45
C


ATOM
913
C
VAL 
A
130
24.872
18.692
35.521
1.00
13.62
C


ATOM
914
O
VAL 
A
130
25.655
19.546
35.120
1.00
17.44
O


ATOM
915
N
CYS 
A
131
24.804
17.468
34.997
1.00
10.87
N


ATOM
916
CA
CYS 
A
131
25.658
17.047
33.886
1.00
12.09
C


ATOM
917
CB
CYS 
A
131
25.939
15.541
33.966
1.00
12.10
C


ATOM
918
SG
CYS 
A
131
24.447
14.512
33.745
1.00
14.96
S


ATOM
919
C
CYS 
A
131
24.957
17.343
32.568
1.00
12.93
C


ATOM
920
O
CYS 
A
131
23.739
17.506
32.532
1.00
11.56
O


ATOM
921
N
ALA 
A
132
25.723
17.403
31.486
1.00
13.76
N


ATOM
922
CA
ALA 
A
132
25.141
17.676
30.181
1.00
14.09
C


ATOM
923
CB
ALA 
A
132
24.724
19.141
30.089
1.00
13.62
C


ATOM
924
C
ALA 
A
132
26.086
17.337
29.042
1.00
17.97
C


ATOM
925
O
ALA 
A
132
27.294
17.179
29.237
1.00
15.14
O


ATOM
926
N
GLU 
A
133
25.508
17.215
27.853
1.00
13.21
N


ATOM
927
CA
GLU 
A
133
26.243
16.900
26.639
1.00
18.49
C


ATOM
928
CB
GLU 
A
133
25.732
15.592
26.039
1.00
21.95
C


ATOM
929
CG
GLU 
A
133
26.808
14.614
25.652
1.00
27.91
C


ATOM
930
CD
GLU 
A
133
27.336
13.850
26.840
1.00
31.31
C


ATOM
931
OE1
GLU 
A
133
27.870
14.494
27.767
1.00
28.79
O


ATOM
932
OE2
GLU 
A
133
27.214
12.606
26.846
1.00
28.57
O


ATOM
933
C
GLU 
A
133
25.919
18.051
25.693
1.00
15.23
C


ATOM
934
O
GLU 
A
133
24.915
18.738
25.866
1.00
16.37
O


ATOM
935
N
PRO 
A
134
26.762
18.276
24.680
1.00
16.75
N


ATOM
936
CA
PRO 
A
134
26.527
19.366
23.725
1.00
17.31
C


ATOM
937
CB
PRO 
A
134
27.558
19.082
22.638
1.00
17.01
C


ATOM
938
C
PRO 
A
134
25.093
19.449
23.177
1.00
18.87
C


ATOM
939
O
PRO 
A
134
24.468
20.515
23.204
1.00
21.16
O


ATOM
940
CD
PRO 
A
134
28.022
17.572
24.385
1.00
14.41
C


ATOM
941
CG
PRO 
A
134
28.708
18.528
23.429
1.00
15.96
C


ATOM
942
N
GLY 
A
135
24.577
18.329
22.683
1.00
13.73
N


ATOM
943
CA
GLY 
A
135
23.228
18.315
22.138
1.00
11.51
C


ATOM
944
C
GLY 
A
135
22.114
18.674
23.112
1.00
12.22
C


ATOM
945
O
GLY 
A
135
20.982
18.933
22.696
1.00
10.70
O


ATOM
946
N
ASP 
A
136
22.425
18.676
24.405
1.00
9.59
N


ATOM
947
CA
ASP 
A
136
21.451
19.019
25.441
1.00
10.66
C


ATOM
948
CB
ASP 
A
136
21.957
18.550
26.808
1.00
9.43
C


ATOM
949
C
ASP 
A
136
21.239
20.533
25.485
1.00
9.56
C


ATOM
950
O
ASP 
A
136
20.270
21.018
26.076
1.00
7.80
O


ATOM
951
CG
ASP 
A
136
21.907
17.044
26.969
1.00
12.00
C


ATOM
952
OD2
ASP 
A
136
21.038
16.399
26.348
1.00
14.65
O


ATOM
953
OD1
ASP 
A
136
22.732
16.510
27.737
1.00
11.73
O


ATOM
954
N
SER 
A
137
22.159
21.270
24.867
1.00
11.68
N


ATOM
955
CA
SER 
A
137
22.089
22.728
24.831
1.00
9.45
C


ATOM
956
CB
SER 
A
137
23.167
23.298
23.902
1.00
12.71
C


ATOM
957
C
SER 
A
137
20.723
23.231
24.381
1.00
12.56
C


ATOM
958
O
SER 
A
137
20.110
22.671
23.470
1.00
9.42
O


ATOM
959
OG
SER 
A
137
24.460
23.160
24.466
1.00
11.89
O


ATOM
960
N
GLY
A
138
20.264
24.298
25.027
1.00
12.50
N


ATOM
961
CA
GLY
A
138
18.974
24.873
24.698
1.00
10.84
C


ATOM
962
C
GLY
A
138
17.863
24.228
25.497
1.00
11.17
C


ATOM
963
O
GLY 
A
138
16.759
24.774
25.583
1.00
10.27
O


ATOM
964
N
GLY 
A
139
18.171
23.075
26.090
1.00
9.62
N


ATOM
965
CA
GLY 
A
139
17.202
22.326
26.877
1.00
11.99
C


ATOM
966
C
GLY 
A
139
16.675
22.997
28.135
1.00
9.04
C


ATOM
967
O
GLY 
A
139
17.243
23.698
28.632
1.00
11.52
O


ATOM
968
N
SER 
A
140
15.588
22.444
28.668
1.00
9.88
N


ATOM
969
CA
SER 
A
140
14.942
22.990
29.858
1.00
10.08
C


ATOM
970
CB
SER 
A
140
13.507
22.456
29.977
1.00
9.88
C


ATOM
971
OG
SER 
A
140
12.972
22.057
28.729
1.00
11.61
O


ATOM
972
C
SER 
A
140
15.628
22.710
31.187
1.00
9.54
C


ATOM
973
O
SER 
A
140
16.253
21.670
31.369
1.00
8.61
O


ATOM
974
N
LEU 
A
141
15.492
23.660
32.108
1.00
11.67
N


ATOM
975
CA
LEU 
A
141
16.057
23.559
33.453
1.00
10.92
C


ATOM
976
CB
LEU 
A
141
17.184
24.554
33.718
1.00
10.21
C


ATOM
977
CG
LEU 
A
141
17.665
24.342
35.164
1.00
9.58
C


ATOM
978
CD1
LEU 
A
141
18.252
22.936
35.290
1.00
7.62
C


ATOM
979
CD2
LEU 
A
141
18.682
25.407
35.575
1.00
8.11
C


ATOM
980
C
LEU 
A
141
14.867
23.916
34.330
1.00
10.82
C


ATOM
981
O
LEU 
A
141
14.325
25.021
34.240
1.00
10.91
O


ATOM
982
N
LEU 
A
142
14.455
22.972
35.161
1.00
7.99
N


ATOM
983
CA
LEU 
A
142
13.327
23.175
36.046
1.00
11.41
C


ATOM
984
CB
LEU 
A
142
12.234
22.140
35.741
1.00
12.37
C


ATOM
985
CG
LEU 
A
142
11.432
22.235
34.449
1.00
13.83
C


ATOM
986
CD1
LEU 
A
142
10.710
20.914
34.195
1.00
16.69
C


ATOM
987
CD2
LEU 
A
142
10.443
23.391
34.562
1.00
13.02
C


ATOM
988
C
LEU 
A
142
13.675
23.050
37.518
1.00
10.11
C


ATOM
989
O
LEU 
A
142
14.631
22.377
37.904
1.00
13.30
O


ATOM
990
N
ALA 
A
143
12.875
23.731
38.326
1.00
10.55
N


ATOM
991
CA
ALA 
A
143
12.992
23.746
39.775
1.00
12.59
C


ATOM
992
CB
ALA 
A
143
13.306
25.141
40.284
1.00
14.58
C


ATOM
993
C
ALA 
A
143
11.539
23.388
40.061
1.00
13.64
C


ATOM
994
O
ALA 
A
143
10.677
24.258
40.124
1.00
15.86
O


ATOM
995
N
GLY 
A
144
11.260
22.098
40.178
1.00
14.54
N


ATOM
996
CA
GLY 
A
144
9.890
21.681
40.396
1.00
18.53
C


ATOM
997
C
GLY 
A
144
9.156
21.932
39.092
1.00
18.05
C


ATOM
998
O
GLY 
A
144
9.570
21.445
38.040
1.00
18.26
O


ATOM
999
N
ASN 
A
145
8.071
22.695
39.144
1.00
17.01
N


ATOM
1000
CA
ASN 
A
145
7.316
23.001
37.940
1.00
17.28
C


ATOM
1001
CB
ASN 
A
145
5.821
22.829
38.199
1.00
24.24
C


ATOM
1002
CG
ASN 
A
145
5.380
23.471
39.497
1.00
34.66
C


ATOM
1003
OD1
ASN 
A
145
5.502
24.685
39.682
1.00
36.73
O


ATOM
1004
ND2
ASN 
A
145
4.868
22.655
40.413
1.00
41.73
N


ATOM
1005
C
ASN 
A
145
7.589
24.425
37.477
1.00
16.30
C


ATOM
1006
O
ASN 
A
145
6.844
24.966
36.671
1.00
13.74
O


ATOM
1007
N
GLN 
A
146
8.667
25.022
37.976
1.00
13.23
N


ATOM
1008
CA
GLN 
A
146
9.022
26.388
37.609
1.00
13.97
C


ATOM
1009
CB
GLN 
A
146
9.283
27.205
38.786
1.00
17.49
C


ATOM
1010
CG
GLN 
A
146
8.116
27.191
39.850
1.00
17.44
C


ATOM
1011
CD
GLN 
A
146
6.920
27.948
39.324
1.00
19.58
C


ATOM
1012
OE1
GLN 
A
146
5.781
27.478
39.412
1.00
18.02
O


ATOM
1013
NE2
GLN 
A
146
7.166
29.135
38.781
1.00
13.47
N


ATOM
1014
C
GLN 
A
146
10.238
26.469
36.692
1.00
13.36
C


ATOM
1015
O
GLN 
A
146
11.332
26.026
37.052
1.00
9.49
O


ATOM
1016
N
ALA 
A
147
10.036
27.038
35.508
1.00
10.85
N


ATOM
1017
CA
ALA 
A
147
11.107
27.187
34.527
1.00
11.86
C


ATOM
1018
CB
ALA 
A
147
10.560
27.790
33.231
1.00
9.60
C


ATOM
1019
C
ALA 
A
147
12.212
28.077
35.079
1.00
12.02
C


ATOM
1020
O
ALA 
A
147
11.947
29.181
35.559
1.00
11.92
O


ATOM
1021
N
GLN 
A
148
13.450
27.595
34.990
1.00
9.31
N


ATOM
1022
CA
GLN 
A
148
14.608
28.334
35.478
1.00
8.96
C


ATOM
1023
CB
GLN 
A
148
15.502
27.426
36.317
1.00
9.82
C


ATOM
1024
CG
GLN 
A
148
14.814
26.838
37.532
1.00
9.18
C


ATOM
1025
CD
GLN 
A
148
14.193
27.914
38.392
1.00
8.87
C


ATOM
1026
OE1
GLN 
A
148
12.974
27.953
38.581
1.00
13.35
O


ATOM
1027
NE2
GLN 
A
148
15.024
28.797
38.916
1.00
6.09
N


ATOM
1028
C
GLN 
A
148
15.449
28.925
34.353
1.00
9.54
C


ATOM
1029
O
GLN 
A
148
15.874
30.073
34.424
1.00
10.41
O


ATOM
1030
N
GLY 
A
149
15.707
28.130
33.322
1.00
9.02
N


ATOM
1031
CA
GLY 
A
149
16.522
28.620
32.226
1.00
11.94
C


ATOM
1032
C
GLY 
A
149
16.762
27.559
31.172
1.00
10.03
C


ATOM
1033
O
GLY 
A
149
16.130
26.505
31.198
1.00
11.61
O


ATOM
1034
N
VAL 
A
150
17.670
27.840
30.241
1.00
8.37
N


ATOM
1035
CA
VAL 
A
150
17.977
26.885
29.185
1.00
10.02
C


ATOM
1036
CB
VAL 
A
150
17.557
27.428
27.796
1.00
8.22
C


ATOM
1037
CG1
VAL 
A
150
16.058
27.733
27.799
1.00
9.61
C


ATOM
1038
CG2
VAL 
A
150
18.343
28.682
27.452
1.00
6.21
C


ATOM
1039
C
VAL 
A
150
19.465
26.542
29.211
1.00
11.57
C


ATOM
1040
O
VAL 
A
150
20.309
27.391
29.504
1.00
9.64
O


ATOM
1041
N
THR 
A
151
19.773
25.283
28.925
1.00
11.95
N


ATOM
1042
CA
THR 
A
151
21.153
24.805
28.923
1.00
11.13
C


ATOM
1043
CB
THR 
A
151
21.195
23.325
28.552
1.00
9.19
C


ATOM
1044
OG1
THR 
A
151
20.223
22.622
29.340
1.00
6.16
O


ATOM
1045
CG2 
THR 
A
151
22.578
22.748 
28.828
1.00
8.56
C


ATOM
1046
C
THR 
A
151
22.086
25.577 
27.999
1.00
11.68
C


ATOM
1047
O
THR 
A
151
21.838
25.677 
26.801
1.00
8.62
O


ATOM
1048
N
SER 
A
152
23.172
26.107 
28.560
1.00
11.36
N


ATOM
1049
CA 
SER 
A
152
24.133
26.869 
27.771
1.00
10.56
C


ATOM
1050
CB 
SER 
A
152
24.480
28.183 
28.479
1.00
13.70
C


ATOM
1051
OG 
SER 
A
152
25.434
28.928 
27.735
1.00
11.67
O


ATOM
1052
C
SER 
A
152
25.407
26.080 
27.503
1.00
12.67
C


ATOM
1053
O
SER 
A
152
25.873
26.005 
26.369
1.00
11.22
O


ATOM
1054
N
GLY
A
153
25.967
25.485 
28.547
1.00
11.74
N


ATOM
1055
CA 
GLY
A
153
27.185
24.719 
28.374
1.00
13.59
C


ATOM
1056
C
GLY
A
153
27.807
24.348 
29.699
1.00
13.34
C


ATOM
1057
O
GLY 
A
153
27.226
24.590 
30.758
1.00
11.10
O


ATOM
1058
N
GLY 
A
154
29.001
23.769 
29.644
1.00
12.77
N


ATOM
1059
CA 
GLY 
A
154
29.669
23.370 
30.862
1.00
14.55
C


ATOM
1060
C
GLY 
A
154
30.763
22.360 
30.583
1.00
16.81
C


ATOM
1061
O
GLY 
A
154
31.228
22.235 
29.452
1.00
15.66
O


ATOM
1062
N
SER 
A
155
31.171
21.631 
31.615
1.00
16.15
N


ATOM
1063
CA 
SER 
A
155
32.230
20.641 
31.472
1.00
16.05
C


ATOM
1064
CB 
SER 
A
155
33.475
21.113 
32.214
1.00
16.89
C


ATOM
1065
OG 
SER 
A
155
33.181
21.338 
33.582
1.00
23.41
O


ATOM
1066
C
SER 
A
155
31.799
19.291 
32.026
1.00
16.84
C


ATOM
1067
O
SER 
A
155
30.783
19.187 
32.714
1.00
14.62
O


ATOM
1068
N
GLY
A
156
32.588
18.262 
31.731
1.00
15.26
N


ATOM
1069
CA 
GLY
A
156
32.279
16.928 
32.211
1.00
13.85
C


ATOM
1070
C
GLY
A
156
31.211
16.256 
31.376
1.00
14.91
C


ATOM
1071
O
GLY
A
156
30.935
16.667 
30.251
1.00
17.56
O


ATOM
1072
N
ASN
A
157
30.613 
15.213 
31.931
1.00
14.60
N


ATOM
1073
CA 
ASN
A
157
29.566
14.471 
31.248
1.00
16.61
C


ATOM
1074
CB 
ASN
A
157
30.179
13.445 
30.289
1.00
16.79
C


ATOM
1075
CG 
ASN
A
157
31.168
12.525 
30.974
1.00
16.85
C


ATOM
1076
OD1
ASN 
A
157
30.808
11.768 
31.876
1.00
17.38
O


ATOM
1077
ND2
ASN 
A
157
32.429
12.585 
30.545
1.00
19.44
N


ATOM
1078
C
ASN 
A
157
28.694
13.773 
32.283
1.00
16.81
C


ATOM
1079
O
ASN 
A
157
28.936
13.888 
33.487
1.00
14.11
O


ATOM
1080
N
CYS 
A
158
27.679
13.057 
31.812
1.00
16.33
N


ATOM
1081
CA 
CYS 
A
158
26.773
12.348 
32.704
1.00
17.79
C


ATOM
1082
CB 
CYS 
A
158
25.406
12.202 
32.048
1.00
19.95
C


ATOM
1083
SG 
CYS 
A
158
24.578
13.802 
31.845
1.00
17.50
S


ATOM
1084
C
CYS 
A
158
27.257
10.989 
33.174
1.00
19.67
C


ATOM
1085
O
CYS 
A
158
26.591
10.333 
33.971
1.00
21.67
O


ATOM
1086
N
ARG
A
159
28.403
10.554 
32.672
1.00
18.94
N


ATOM
1087
CA 
ARG
A
159
28.948
9.267
33.070
1.00
19.35
C


ATOM
1088
CB 
ARG
A
159
29.835
8.700
31.953
1.00
19.37
C


ATOM
1089
CG 
ARG 
A
159
29.074
8.300
30.702
1.00
24.85
C


ATOM
1090
CD 
ARG 
A
159
30.003
7.779
29.615
1.00
25.44
C


ATOM
1091
NE 
ARG 
A
159
30.852
8.831
29.068
1.00
26.55
N


ATOM
1092
CZ 
ARG 
A
159
30.414
9.821
28.296 
1.00
28.60
C


ATOM
1093
NH1 
ARG 
A
159
29.130
9.901
27.971
1.00
28.12
N


ATOM
1094
NH2 
ARG 
A
159
31.264
10.734 
27.848
1.00
25.81
N


ATOM
1095
C
ARG 
A
159
29.775
9.461
34.345
1.00
19.70
C


ATOM
1096
O
ARG 
A
159
29.653
8.704
35.309
1.00
20.82
O


ATOM
1097
N
THR 
A
160
30.608
10.494 
34.355
1.00
16.93
N


ATOM
1098
CA 
THR 
A
160
31.445
10.762 
35.517
1.00
18.57
C


ATOM
1099
CB 
THR 
A
160
32.937
10.775 
35.109
1.00
18.72
C


ATOM
1100
OG1 
THR 
A
160
33.136
11.696 
34.028
1.00
19.41
O


ATOM
1101
CG2 
THR 
A
160
33.372
9.387
34.654
1.00
23.02
C


ATOM
1102
C 
THR 
A
160
31.097
12.055 
36.267
1.00
17.76
C


ATOM
1103
O
THR 
A
160
31.730
12.391 
37.269
1.00
14.64
O


ATOM
1104
N
GLY 
A
161
30.079
12.767 
35.792
1.00
15.51
N


ATOM
1105
CA 
GLY 
A
161
29.666
14.000 
36.444
1.00
18.63
C


ATOM
1106
C
GLY 
A
161
30.199
15.264 
35.791
1.00
17.91
C


ATOM
1107
O
GLY 
A
161
31.178
15.231 
35.047
1.00
17.74
O


ATOM
1108
N
GLY 
A
162
29.556
16.392 
36.070
1.00
17.00
N


ATOM
1109
CA 
GLY 
A
162
30.008
17.633 
35.475
1.00
15.39
C


ATOM
1110
C
GLY 
A
162
29.373
18.881 
36.048
1.00
14.86
C


ATOM
1111
O
GLY 
A
162
28.607
18.824 
37.013
1.00
12.41
O


ATOM
1112
N
THR 
A
163
29.716
20.014 
35.445
1.00
11.47
N


ATOM
1113
CA 
THR 
A
163
29.203
21.318 
35.847
1.00
12.96
C


ATOM
1114
CB 
THR 
A
163
30.343
22.255 
36.285
1.00
15.39
C


ATOM
1115
OG1 
THR 
A
163
31.024
21.685 
37.409
1.00
15.28
O


ATOM
1116
CG2 
THR 
A
163
29.793
23.622 
36.664
1.00
11.49
C


ATOM
1117
C
THR 
A
163
28.532
21.921 
34.619
1.00
13.39
C


ATOM
1118
O
THR 
A
163
29.168
22.081 
33.577
1.00
15.12
O


ATOM
1119
N
THR 
A
164
27.252
22.253 
34.741
1.00
10.11
N


ATOM
1120
CA
THR 
A
164
26.518
22.833 
33.624
1.00
10.81
C


ATOM
1121
CB
THR 
A
164
25.362
21.914 
33.192
1.00
8.40
C


ATOM
1122
OG1 
THR 
A
164
25.878
20.612 
32.891
1.00
6.91
O


ATOM
1123
CG2 
THR 
A
164
24.675
22.471
31.955
1.00
6.07
C


ATOM
1124
C
THR 
A
164
25.950
24.203
33.967
1.00
10.82
C


ATOM
1125
O
THR 
A
164
25.401
24.402
35.053
1.00
9.66
O


ATOM
1126
N
PHE 
A
165
26.092
25.139
33.034
1.00
9.84
N


ATOM
1127
CA
PHE 
A
165
25.600
26.502
33.210
1.00
10.47
C


ATOM
1128
CB
PHE 
A
165
26.669
27.513
32.796
1.00
11.30
C


ATOM
1129
CG
PHE 
A
165
27.940
27.419
33.597
1.00
14.89
C


ATOM
1130
CD1 
PHE 
A
165
28.871
26.429
33.335
1.00
15.07
C


ATOM
1131
CD2 
PHE 
A
165
28.188
28.311
34.626
1.00
16.48
C


ATOM
1132
CE1 
PHE 
A
165
30.030
26.330
34.085
1.00
16.20
C


ATOM
1133
CE2 
PHE 
A
165
29.341
28.219
35.379
1.00
16.91
C


ATOM
1134
CZ
PHE 
A
165
30.264
27.223
35.108
1.00
16.17
C


ATOM
1135
C
PHE 
A
165
24.344
26.725
32.373
1.00
11.04
C


ATOM
1136
O
PHE 
A
165
24.224
26.204
31.263
1.00
9.74
O


ATOM
1137
N
PHE 
A
166
23.417
27.517
32.901
1.00
7.30
N


ATOM
1138
CA
PHE 
A
166
22.177
27.796
32.195
1.00
8.16
C


ATOM
1139
CB
PHE 
A
166
20.990
27.127
32.901
1.00
7.36
C


ATOM
1140
CG
PHE 
A
166
21.148
25.650
33.093
1.00
7.82
C


ATOM
1141
CD2 
PHE 
A
166
20.436
24.758
32.302
1.00
10.08
C


ATOM
1142
CD1 
PHE 
A
166
22.018
25.148
34.050
1.00
10.99
C


ATOM
1143
CE2 
PHE 
A
166
20.591
23.383
32.463
1.00
9.63
C


ATOM
1144
CE1 
PHE 
A
166
22.179
23.777
34.218
1.00
7.63
C


ATOM
1145
CZ
PHE 
A
166
21.464
22.894
33.422
1.00
9.75
C


ATOM
1146
C
PHE 
A
166
21.871
29.277
32.077
1.00
9.41
C


ATOM
1147
O
PHE 
A
166
22.183
30.070
32.967
1.00
9.92
O


ATOM
1148
N
GLN 
A
167
21.247
29.634
30.963
1.00
9.95
N


ATOM
1149
CA
GLN 
A
167
20.866
31.010
30.690
1.00
9.14
C


ATOM
1150
CB
GLN 
A
167
20.777
31.231
29.176
1.00
8.25
C


ATOM
1151
CG
GLN 
A
167
19.911
32.403
28.738
1.00
11.98
C


ATOM
1152
CD
GLN 
A
167
20.487
33.751
29.110
1.00
13.09
C


ATOM
1153
OE1 
GLN 
A
167
21.590
34.111
28.690
1.00
12.87
O


ATOM
1154
NE2 
GLN 
A
167
19.746
34.505
29.905
1.00
10.51
N


ATOM
1155
C
GLN 
A
167
19.492
31.178
31.337
1.00
10.77
C


ATOM
1156
O
GLN 
A
167
18.542
30.483
30.977
1.00
7.43
O


ATOM
1157
N
PRO 
A
168
19.375
32.085
32.318
1.00
10.69
N


ATOM
1158
CD
PRO 
A
168
20.431
32.933
32.897
1.00
11.76
C


ATOM
1159
CA
PRO 
A
168
18.092
32.310
32.996
1.00
11.69
C


ATOM
1160
CB
PRO 
A
168
18.392
33.482
33.924
1.00
12.25
C


ATOM
1161
CG
PRO 
A
168
19.837
33.296
34.241
1.00
14.30
C


ATOM
1162
C
PRO 
A
168
16.988
32.628
31.994
1.00
11.80
C


ATOM
1163
O
PRO 
A
168
17.222
33.317
31.006
1.00
9.94
O


ATOM
1164
N
VAL 
A
169
15.784
32.133
32.261
1.00
10.31
N


ATOM
1165
CA
VAL 
A
169
14.650
32.358
31.373
1.00
12.92
C


ATOM
1166
CB
VAL 
A
169
13.528
31.331
31.662
1.00
17.07
C


ATOM
1167
CG1 
VAL 
A
169
13.026
31.491
33.088
1.00
15.81
C


ATOM
1168
CG2 
VAL 
A
169
12.394
31.505
30.678
1.00
19.48
C


ATOM
1169
C
VAL 
A
169
14.028
33.757
31.358
1.00
12.62
C


ATOM
1170
O
VAL 
A
169
13.648
34.253
30.302
1.00
11.62
O


ATOM
1171
N
ASN 
A
170
13.927
34.405
32.510
1.00
12.76
N


ATOM
1172
CA 
ASN 
A
170
13.328
35.736
32.537
1.00
15.21
C


ATOM
1173
CB
ASN 
A
170
13.268
36.249
33.976
1.00
13.89
C


ATOM
1174
CG
ASN 
A
170
12.353
35.396
34.841
1.00
19.50
C


ATOM
1175
OD1 
ASN 
A
170
11.367
34.848
34.347
1.00
19.07
O


ATOM
1176
ND2 
ASN 
A
170
12.667
35.283
36.128
1.00
18.85
N


ATOM
1177
C
ASN 
A
170
13.948
36.764
31.591
1.00
12.70
C


ATOM
1178
O
ASN 
A
170
13.235
37.554
30.977
1.00
14.77
O


ATOM
1179
N
PRO 
A
171
15.278
36.778
31.458
1.00
15.34
N


ATOM
1180
CD
PRO 
A
171
16.339
36.181
32.282
1.00
16.10
C


ATOM
1181
CA
PRO 
A
171
15.826
37.772
30.530
1.00
16.08
C


ATOM
1182
CB
PRO 
A
171
17.336
37.710
30.790
1.00
17.98
C


ATOM
1183
CG
PRO 
A
171
17.539
36.351
31.399
1.00
23.99
C


ATOM
1184
C
PRO 
A
171
15.457
37.465
29.077
1.00
15.20
C


ATOM
1185
O
PRO 
A
171
15.464
38.355
28.228
1.00
10.27
O


ATOM
1186
N
ILE 
A
172
15.139
36.203
28.794
1.00
11.01
N


ATOM
1187
CA
ILE 
A
172
14.769
35.813
27.437
1.00
10.79
C


ATOM
1188
CB
ILE 
A
172
14.784
34.282
27.247
1.00
8.59
C


ATOM
1189
CG2 
ILE 
A
172
14.453
33.943
25.792
1.00
10.32
C


ATOM
1190
CG1 
ILE 
A
172
16.152
33.712
27.617
1.00
7.68
C


ATOM
1191
CD1 
ILE 
A
172
16.184
32.189
27.604
1.00
6.34
C


ATOM
1192
C
ILE 
A
172
13.355
36.310
27.145
1.00
9.04
O


ATOM
1193
O
ILE 
A
172
13.074
36.849
26.070
1.00
9.00
O


ATOM
1194
N
LEU
A
173
12.461
36.112
28.107
1.00
10.13
N


ATOM
1195
CA
LEU
A
173
11.080
36.544
27.951
1.00
12.20
C


ATOM
1196
CB
LEU 
A
173
10.249
36.103
29.157
1.00
9.16
C


ATOM
1197
CG
LEU 
A
173
10.233
34.595
29.436
1.00
10.30
C


ATOM
1198
CD1 
LEU 
A
173
9.469
34.304
30.717
1.00
9.41
C


ATOM
1199
CD2 
LEU 
A
173
9.598
33.873
28.268
1.00
11.50
C


ATOM
1200
C
LEU 
A
173
11.049
38.061
27.824
1.00
13.01
C


ATOM
1201
O
LEU 
A
173
10.295
38.608
27.026
1.00
17.74
O


ATOM
1202
N
GLN 
A
174
11.885
38.733
28.608
1.00
16.26
N


ATOM
1203
CA
GLN 
A
174
11.962
40.190
28.592
1.00
14.31
C


ATOM
1204
CB
GLN 
A
174
12.817
40.681
29.769
1.00
19.36
C


ATOM
1205
CG
GLN 
A
174
12.968
42.198
29.866
1.00
25.15
C


ATOM
1206
CD
GLN 
A
174
11.695
42.891
30.315
1.00
30.84
C


ATOM
1207
OE1
GLN 
A
174
10.628
42.684
29.743
1.00
30.46
O


ATOM
1208
NE2
GLN 
A
174
11.805
43.723
31.348
1.00
34.53
N


ATOM
1209
C
GLN 
A
174
12.556
40.694
27.282
1.00
15.28
C


ATOM
1210
O
GLN 
A
174
12.104
41.694
26.722
1.00
10.15
O


ATOM
1211
N
ALA 
A
175
13.567
39.994
26.786
1.00
13.61
N


ATOM
1212
CA
ALA 
A
175
14.210
40.393
25.544
1.00
16.01
C


ATOM
1213
CB
ALA 
A
175
15.372
39.453
25.234
1.00
14.76
C


ATOM
1214
C
ALA 
A
175
13.245
40.427
24.363
1.00
17.13
C


ATOM
1215
O
ALA 
A
175
13.221
41.387
23.598
1.00
14.51
O


ATOM
1216
N
TYR 
A
176
12.426
39.391
24.229
1.00
16.19
N


ATOM
1217
CA
TYR 
A
176
11.481
39.329
23.124
1.00
17.75
C


ATOM
1218
CB
TYR 
A
176
11.595
37.947
22.476
1.00
15.30
C


ATOM
1219
CG
TYR 
A
176
13.033
37.599
22.138
1.00
15.11
C


ATOM
1220
CD1
TYR 
A
176
13.818
38.482
21.415
1.00
16.28
C


ATOM
1221
CE1
TYR 
A
176
15.134
38.186
21.101
1.00
13.92
C


ATOM
1222
CD2
TYR 
A
176
13.605
36.395
22.548
1.00
12.95
C


ATOM
1223
CE2
TYR 
A
176
14.925
36.086
22.238
1.00
12.38
C


ATOM
1224
CZ
TYR 
A
176
15.682
36.990
21.512
1.00
13.61
C


ATOM
1225
OH
TYR 
A
176
16.983
36.705
21.184
1.00
13.98
O


ATOM
1226
C
TYR 
A
176
10.303
39.653
23.461
1.00
14.14
C


ATOM
1227
O
TYR 
A
176
9.155
39.546
22.604
1.00
16.16
O


ATOM
1228
N
GLY 
A
177
9.780
40.057
24.701
1.00
14.82
N


ATOM
1229
CA
GLY 
A
177
8.424
40.392
25.105
1.00
16.43
C


ATOM
1230
C
GLY 
A
177
7.500
39.207
24.933
1.00
16.44
C


ATOM
1231
O
GLY 
A
177
6.376
39.340
24.439
1.00
17.81
O


ATOM
1232
N
LEU 
A
178
7.987
38.046
25.361
1.00
14.56
N


ATOM
1233
CA
LEU 
A
178
7.261
36.789
25.258
1.00
15.86
C


ATOM
1234
CB
LEU 
A
178
8.209
35.656
24.778
1.00
15.44
C


ATOM
1235
CG
LEU 
A
178
8.886
35.807
23.415
1.00
19.21
C


ATOM
1236
CD1
LEU 
A
178
10.030
34.805
23.331
1.00
18.33
C


ATOM
1237
CD2
LEU 
A
178
7.870
35.553
22.311
1.00
21.44
C


ATOM
1238
C
LEU 
A
178
6.670
36.350
26.586
1.00
16.50
C


ATOM
1239
O
LEU 
A
178
7.086
36.808
27.650
1.00
16.26
O


ATOM
1240
N
ARG
A
179
5.700
35.447
26.504
1.00
17.69
N


ATOM
1241
CA
ARG
A
179
5.040
34.911
27.684
1.00
15.79
C


ATOM
1242
CB
ARG
A
179
3.565
35.312
27.729
1.00
21.75
C


ATOM
1243
CG
ARG
A
179
3.321
36.700
28.298
1.00
30.60
C


ATOM
1244
CD
ARG 
A
179
1.837
36.960
28.493
1.00
37.51
C


ATOM
1245
NE
ARG 
A
179
1.586
38.213
29.199
1.00
47.17
N


ATOM
1246
CZ
ARG 
A
179
2.011
39.405
28.790
1.00
49.75
C


ATOM
1247
NH1
ARG 
A
179
2.715
39.516
27.672
1.00
52.12
N


ATOM
1248
NH2
ARG 
A
179
1.731
40.488
29.500
1.00
50.23
N


ATOM
1249
C
ARG 
A
179
5.153
33.398
27.640
1.00
15.02
C


ATOM
1250
O
ARG 
A
179
5.039
32.787
26.574
1.00
14.80
O


ATOM
1251
N
MET 
A
180
5.401
32.800
28.799
1.00
13.59
N


ATOM
1252
CA
MET 
A
180
5.529
31.356
28.909
1.00
16.64
C


ATOM
1253
CB
MET 
A
180
5.991
30.969
30.316
1.00
17.26
C


ATOM
1254
CG
MET 
A
180
7.358
31.449
30.714
1.00
22.61
C


ATOM
1255
SD
MET 
A
180
8.603
30.324
30.120
1.00
24.38
S


ATOM
1256
CE
MET 
A
180
8.143
28.828
30.998
1.00
21.48
C


ATOM
1257
C
MET 
A
180
4.156
30.739
28.706
1.00
16.31
C


ATOM
1258
O
MET 
A
180
3.167
31.255
29.225
1.00
17.83
O


ATOM
1259
N
ILE 
A
181
4.076
29.656
27.942
1.00
14.71
N


ATOM
1260
CA
ILE 
A
181
2.778
29.019
27.740
1.00
13.74
C


ATOM
1261
CB
ILE 
A
181
2.794
28.044
26.559
1.00
16.62
C


ATOM
1262
CG2
ILE 
A
181
1.570
27.130
26.622
1.00
15.86
C


ATOM
1263
CG1
ILE 
A
181
2.829
28.835
25.247
1.00
17.95
C


ATOM
1264
CD1
ILE 
A
181
2.732
27.982
24.009
1.00
26.87
C


ATOM
1265
C
ILE 
A
181
2.589
28.256
29.049
1.00
15.69
C


ATOM
1266
O
ILE 
A
181
3.452
27.469
29.438
1.00
13.45
O


ATOM
1267
N
THR
A
182
1.468
28.483
29.727
1.00
17.74
N


ATOM
1268
CA
THR 
A
182
1.210
27.812
30.998
1.00
23.56
C


ATOM
1269
C
THR 
A
182
0.141
26.728
31.019
1.00
26.44
C


ATOM
1270
O
THR 
A
182
−0.071
26.088
32.052
1.00
29.65
O


ATOM
1271
CB
THR 
A
182
0.841
28.841
32.073
1.00
24.55
C


ATOM
1272
OG1
THR 
A
182
−0.378
29.497
31.701
1.00
27.19
O


ATOM
1273
CG2
THR 
A
182
1.940
29.877
32.211
1.00
28.36
C


ATOM
1274
N
THR 
A
183
−0.540
26.517
29.901
1.00
27.09
N


ATOM
1275
CA
THR 
A
183
−1.573
25.494
29.866
1.00
33.19
C


ATOM
1276
C
THR 
A
183
−1.835
25.008
28.447
1.00
33.29
C


ATOM
1277
O
THR 
A
183
−1.707
25.765
27.484
1.00
34.57
O


ATOM
1278
CB
THR 
A
183
−2.888
26.020
30.477
1.00
33.38
C


ATOM
1279
OG1 
THR 
A
183
−3.822
24.942
30.602
1.00
39.37
O


ATOM
1280
CG2
THR 
A
183
−3.486
27.105
29.600
1.00
36.39
C


ATOM
1281
N
ASP 
A
184
−2.210
23.739
28.330
1.00
33.90
N


ATOM
1282
CA
ASP 
A
184
−2.489
23.133
27.035
1.00
36.90
C


ATOM
1283
C
ASP 
A
184
−3.988
23.017
26.769
1.00
36.93
C


ATOM
1284
O
ASP 
A
184
−4.744
23.965
26.985
1.00
38.19
O


ATOM
1285
CB
ASP 
A
184
−1.841
21.749
26.980
1.00
36.49
C


ATOM
1286
CG
ASP 
A
184
−0.497
21.713
27.682
1.00
39.84
C


ATOM
1287
OD1
ASP 
A
184
0.341
22.597
27.400
1.00
40.10
O


ATOM
1288
OD2
ASP 
A
184
−0.279
20.804
28.515
1.00
34.43
O


TER
1289
O
ASP 
A
184

























ATOM
1290
O
*1

1
13.322
21.904
47.897
1.00
25.15
LIGA
O


ATOM
1291
H
*1

1
12.748
22.438
47.362
1.00
20.00
LIGA
H


ATOM
1292
S
*1

1
14.827
22.185
47.500
1.00
22.18
LIGA
S


ATOM
1293
O
*1

1
15.755
21.317
48.284
1.00
26.48
LIGA
O


ATOM
1294
O
*1

1
15.030
21.926
46.041
1.00
26.21
LIGA
O


ATOM
1295
O
*1

1
15.058
23.692
47.860
1.00
25.81
LIGA
O


ATOM
1296
H
*1

1
15.899
23.969
47.521
1.00
20.00
LIGA
H


TER
1297

*1

1









ATOM
1298
O
*1

1
8.257
10.233
23.934
1.00
51.93
LIGA
O


ATOM
1299
H
*1

1
8.965
10.771
24.260
1.00
20.00
LIGA
H


ATOM
1300
S
*1

1
7.968
9.064
24.968
1.00
52.38
LIGA
S


ATOM
1301
O
*1

1
6.699
8.342
24.628
1.00
53.41
LIGA
O


ATOM
1302
O
*1

1
9.106
8.094
25.015
1.00
51.29
LIGA
O


ATOM
1303
O
*1

1
7.802
9.828
26.339
1.00
52.66
LIGA
O


ATOM
1304
H
*1

1
7.532
9.218
27.014
1.00
20.00
LIGA
H


TER
1305

*1

1









ATOM
1306
O
*1

1
31.870
41.807
26.377
1.00
77.97
LIGA
O


ATOM
1307
H
*1

1
32.101
42.067
27.259
1.00
20.00
LIGA
H


ATOM
1308
S
*1

1
33.167
41.279
25.641
1.00
81.24
LIGA
S


ATOM
1309
O
*1

1
33.774
40.123
26.382
1.00
80.04
LIGA
O


ATOM
1310
O
*1

1
32.867
40.862
24.230
1.00
80.50
LIGA
O


ATOM
1311
O
*1

1
34.119
42.548
25.670
1.00
79.65
LIGA
O


ATOM
1312
H
*1

1
34.951
42.330
25.269
1.00
20.00
LIGA
H


TER
1313

*1

1









ATOM
1314
O
HOH 
W
1
19.154
20.019
28.345
1.00
14.14
S
O


ATOM
1315
O
HON 
W
2
23.228
15.643
36.576
1.00
16.94
S
O


ATOM
1316
O
HOH 
W
3
9.851
19.721
10.708
1.00
13.00
S
O


ATOM
1317
O
HOH 
W
4
8.807
18.269
21.008
1.00
14.72
S
O


ATOM
1318
O
HOH 
W
5
4.955
20.914
9.889
1.00
26.47
S
O


ATOM
1319
O
HOH 
W
6
17.303
10.248
31.329
1.00
20.21
S
O


ATOM
1320
O
HOH 
W
7
21.419
36.535
33.815
1.00
20.37
S
O


ATOM
1321
O
HOH 
W
8
17.558
29.940
39.867
1.00
20.33
S
O


ATOM
1322
O
HOH 
W
9
6.195
26.062
12.062
1.00
15.73
S
O


ATOM
1323
O
HOH 
W
10
27.195
16.076
37.425
1.00
23.27
S
O


ATOM
1324
O
HOH 
W
11
7.569
24.195
27.699
1.00
15.49
S
O


ATOM
1325
O
HOH 
W
12
9.918
10.244
27.897
1.00
14.73
S
O


ATOM
1326
O
HOH 
W
13
18.578
40.541
22.823
1.00
17.35
S
O


ATOM
1327
O
HOH 
W
14
12.929
31.417
36.841
1.00
14.91
S
O


ATOM
1328
O
HOH 
W
15
18.919
21.848
17.030
1.00
16.90
S
O


ATOM
1329
O
HOH 
W
16
16.648
20.485
10.072
1.00
19.27
S
O


ATOM
1330
O
HOH 
W
17
22.460
33.500
36.980
1.00
16.01
S
O


ATOM
1331
O
HOH 
W
18
3.488
17.715
36.292
1.00
27.12
S
O


ATOM
1332
O
HOH 
W
19
19.370
14.862
9.712
1.00
13.10
S
O


ATOM
1333
O
HOH 
W
20
19.355
40.188
27.351
1.00
20.79
S
O


ATOM
1334
O
HOH 
W
21
16.874
12.423
21.691
1.00
24.23
S
O


ATOM
1335
O
HOH 
W
22
18.521
38.452
20.251
1.00
22.43
S
O


ATOM
1336
O
HOH 
W
23
10.797
19.540
36.865
1.00
27.07
S
O


ATOM
1337
O
HOH 
W
24
11.234
19.209
19.064
1.00
21.16
S
O


ATOM
1338
O
HOH 
W
25
11.110
10.795
24.566
1.00
21.70
S
O


ATOM
1339
O
HOH 
W
26
10.089
25.686
42.195
1.00
27.30
S
O


ATOM
1340
O
HOH 
W
27
5.885
26.924
28.544
1.00
17.14
S
O


ATOM
1341
O
HOH 
W
28
22.189
13.924
20.647
1.00
19.65
S
O


ATOM
1342
O
HOH 
W
29
2.839
15.407
25.779
1.00
24.76
S
O


ATOM
1343
O
HOH 
W
30
20.416
36.872
30.702
1.00
22.38
S
O


ATOM
1344
O
HOH 
W
31
14.010
25.569
46.267
1.00
20.18
S
O


ATOM
1345
O
HOH 
W
32
19.103
14.781
19.716
1.00
25.71
S
O


ATOM
1346
O
HOH 
W
33
14.999
33.688
35.037
1.00
17.93
S
O


ATOM
1347
O
HOH 
W
35
23.578
36.561
29.922
1.00
21.76
S
O


ATOM
1348
O
HOH 
W
36
20.341
32.322
45.950
1.00
21.47
S
O


ATOM
1349
O
HOH 
W
37
0.497
25.775
19.401
1.00
26.47
S
O


ATOM
1350
O
HOH 
W
38
11.741
34.995
39.424
1.00
22.41
S
O


ATOM
1351
O
HOH 
W
39
22.467
9.409
26.630
1.00
15.84
S
O


ATOM
1352
O
HOH 
W
40
22.662
11.866
36.367
1.00
45.41
S
O


ATOM
1353
O
HOH 
W
41
3.122
26.816
16.542
1.00
23.85
S
O


ATOM
1354
O
HOH 
W
42
6.805
20.983
12.758
1.00
24.18
S
O


ATOM
1355
O
HOH 
W
43
29.143
24.285
40.975
1.00
26.61
S
O


ATOM
1356
O
HOH 
W
44
24.253
18.985
43.742
1.00
21.24
S
O


ATOM
1357
O
HOH 
W
45
16.923 
33.119 
42.439
1.00
24.24
S
O


ATOM
1358
O
HOH 
W
46
−0.710
24.821 
24.871
1.00
22.41
S
O


ATOM
1359
O
HOH 
W
47
28.123 
37.039 
34.996
1.00
23.29
S
O


ATOM
1360
O
HOH 
W
48
22.509 
29.264 
12.288
1.00
22.20
S
O


ATOM
1361
O
HOH 
W
49
18.268 
20.963 
47.186
1.00
27.23
S
O


ATOM
1362
O
HOH 
W
50
25.603 
33.211 
43.598
1.00
26.97
S
O


ATOM
1363
O
HOH 
W
51
20.065 
33.475
8.796
1.00
27.50
S
O


ATOM
1364
O
HOH 
W
52
27.258 
11.820 
29.311
1.00
24.08
S
O


ATOM
1365
O
HOH 
W
53
10.875 
28.986 
41.865
1.00
23.77
S
O


ATOM
1366
O
HOH 
W
54
5.763 
34.393 
31.210
1.00
25.54
S
O


ATOM
1367
O
HOH 
W
55
13.975 
14.195 
21.784
1.00
27.82
S
O


ATOM
1368
O
HOH 
W
56
12.541 
23.538
8.045
1.00
22.43
S
O


ATOM
1369
O
HOH 
W
57
24.567 
16.480 
39.993
1.00
26.58
S
O


ATOM
1370
O
HOH 
W
58
24.532 
38.285 
35.829
1.00
57.74
S
O


ATOM
1371
O
HOH 
W
59
25.710 
22.863 
22.059
1.00
31.50
S
O


ATOM
1372
O
HOH 
W
60
12.323 
34.306 
43.203
1.00
31.10
S
O


ATOM
1373
O
HOH 
W
61
4.395 
14.949 
17.739
1.00
29.65
S
O


ATOM
1374
O
HOH 
W
62
6.745 
20.043
6.966
1.00
84.14
S
O


ATOM
1375
O
HOH 
W
63
5.532 
20.170 
37.794
1.00
41.49
S
O


ATOM
1376
O
HOH 
W
64
26.003 
16.001 
22.248
1.00
29.03
S
O


ATOM
1377
O
HOH 
W
65
5.525 
35.401 
19.570
1.00
33.21
S
O


ATOM
1378
O
HOH 
W
66
31.845 
33.895 
37.644
1.00
34.28
S
O


ATOM
1379
O
HOH 
W
67
20.183 
13.414 
38.159
1.00
27.70
S
O


ATOM
1380
O
HOH 
W
68
20.038 
18.219 
20.060
1.00
50.13
S
O


ATOM
1381
O
HOH 
W
70
0.763 
17.179 
17.010
1.00
37.46
S
O


ATOM
1382
O
HOH 
W
71
24.671 
21.255 
26.579
1.00
25.15
S
O


ATOM
1383
O
HOH 
W
72
8.061 
13.765 
23.048
1.00
31.32
S
O


ATOM
1384
O
HOH 
W
73
21.384 
36.182 
15.238
1.00
25.91
S
O


ATOM
1385
O
HOH 
W
74
32.543 
19.236 
37.104 
1.00
32.62
S
O


ATOM
1386
O
HOH 
W
75
3.201 
29.276 
38.786
1.00
44.04
S
O


ATOM
1387
O
HOH 
W
76
2.482 
32.835 
31.391
1.00
47.33
S
O


ATOM
1388
O
HOH 
W
77
22.558
9.563 
30.212
1.00
60.39
S
O


ATOM
1389
O
HOH 
W
78
24.502 
25.394 
46.538
1.00
35.15
S
O


ATOM
1390
O
HOH 
W
79
7.028 
39.862 
28.058
1.00
31.57
S
O


ATOM
1391
O
HOH 
W
80
33.571
14.425
35.307
1.00
26.04
S
O


ATOM
1392
O
HOH 
W
81
2.732 
10.198 
34.775 
1.00
30.67
S
O


ATOM
1393
O
HOH 
W
82
34.746 
11.362 
31.655
1.00
34.37
S
O


ATOM
1394
O
HOH 
W
83
27.003 
10.835 
24.556
1.00
34.40
S
O


ATOM
1395
O
HOH 
W
84
11.607 
15.263 
18.090
1.00
53.18
S
O


ATOM
1396
O
HOH 
W
85
18.961 
26.409
8.948
1.00
30.64
S
O


ATOM
1397
O
HOH 
W
86
8.329 
30.456 
11.682
1.00
24.79
S
O


ATOM
1398
O
HOH 
W
87
28.267 
25.545
24.816
1.00
34.18
S
O


ATOM
1399
O
HOH 
W
88
27.826 
26.788 
46.520
1.00
45.31
S
O


ATOM
1400
O
HOH 
W
89
13.822 
23.152
43.665
1.00
23.81
S
O


ATOM
1401
O
HOH 
W
90
15.013 
32.301 
6.825
1.00
36.86
S
O


ATOM
1402
O
HOH 
W
91
7.321 
15.444 
19.576
1.00
38.03
S
O


ATOM
1403
O
HOH 
W
92
9.274 
4.160
30.626 
1.00
34.25
S
O


ATOM
1404
O
HOH 
W
93
1.045 
23.765 
33.021
1.00
29.72
S
O


ATOM
1405
O
HOH 
W
94
0.274 
28.435 
36.491
1.00
39.40
S
O


ATOM
1406
O
HOH 
W
95
26.351 
23.628 
18.493
1.00
37.43
S
O


ATOM
1407
O
HOH 
W
96
34.940 
15.280 
30.014
1.00
38.02
S
O


ATOM
1408
O
HOH 
W
97
20.426 
30.014 
49.201
1.00
33.87
S
O


ATOM
1409
O
HOH 
W
98
13.509 
20.866 
41.132 
1.00
41.63
S
O


ATOM
1410
O
HOH 
W
99
28.366 
18.133 
31.889
1.00
32.04
S
O


ATOM
1411
O
HOH 
W
100
0.422 
36.030 
31.986
1.00
42.04
S
O


ATOM
1412
O
HOH 
W
103
13.872 
24.846 
4.468
1.00
45.22
S
O


ATOM
1413
O
HOH 
W
104
25.742 
19.925 
19.691
1.00
41.00
S
O


ATOM
1414
O
HOH 
W
105
5.894 
32.368 
37.453
1.00
28.07
S
O


ATOM
1415
O
HOH 
W
108
27.692 
30.529 
45.176
1.00
36.94
S
O


ATOM
1416
O
HOH 
W
109
30.999 
38.392
25.165
1.00
26.39
S
O


ATOM
1417
O
HOH 
W
111
13.400
10.503 
34.273
1.00
29.92
S
O


ATOM
1418
O
HOH 
W
112
20.748 
36.914 
39.970
1.00
40.16
S
O


ATOM
1419
O
HOH 
W
113
24.634 
31.190 
17.336
1.00
36.87
S
O


ATOM
1420
O
HOH 
W
114
5.642 
30.898 
42.120
1.00
38.57
S
O


ATOM
1421
O
HOH 
W
115
8.972 
40.592 
30.979
1.00
32.13
S
O


ATOM
1422
O
HOH 
W
116
2.047 
31.605 
35.777
1.00
62.75
S
O


ATOM
1423
O
HOH 
W
117
27.060 
7.939
28.519
1.00
31.51
S
O


ATOM
1424
O
HOH 
W
118
4.134 
24.143 
10.395
1.00
19.77
S
O


ATOM
1425
O
HOH 
W
119
17.406 
32.729 
38.273
1.00
19.77
S
O


ATOM
1426
O
HOH 
W
120
21.370 
42.268 
22.477
1.00
19.75
S
O


ATOM
1427
O
HOH 
W
121
23.854 
15.724 
43.136
1.00
19.76
S
O


ATOM
1428
O
HOH 
W
122
19.654 
34.836 
37.602
1.00
19.76
S
O


ATOM
1429
O
HOH 
W
123
21.170 
42.930 
27.470
1.00
19.75
S
O


ATOM
1430
O
HOH 
W
124
25.304 
8.005
25.551
1.00
19.75
S
O


ATOM
1431
O
HOH 
W
125
20.739 
40.152 
30.476 
1.00
19.73
S
O


ATOM
1432
O
HOH 
W
126
19.238 
15.779
6.587
1.00
19.76
S
O


ATOM
1433
O
HOH 
W
127
7.151
28.097 
9.617
1.00
19.75
S
O


ATOM
1434
O
HOH 
W
128
7.122 
17.869
11.543
1.00
19.75
S
O


ATOM
1435
O
HOH 
W
129
9.467
35.418 
37.012
1.00
19.76
S
O


ATOM
1436
O
HOH 
W
130
5.720
23.417
6.558
1.00
19.76
S
O


ATOM
1437
O
HOH 
W
131
3.123
12.568 
32.283
1.00
19.76
S
O


ATOM
1438
O
HOH 
W
132
12.909
18.142 
39.232
1.00
19.75
S
O


ATOM
1439
O
HOH 
W
133
18.190
34.668 
45.077
1.00
19.77
S
O


ATOM
1440
O
HOH 
W
134
16.371
23.490
8.743
1.00
19.77
S
O


ATOM
1441
O
HOH 
W
135
25.889
26.341 
15.721
1.00
19.77
S
O


ATOM
1442
O
HOH 
W
138
18.831
37.368 
35.694
1.00
19.75
S
O


ATOM
1443
O
HOH 
W
139
−1.837
27.004 
34.243
1.00
19.78
S
O


ATOM
1444
O
HOH 
W
140
29.965
21.328 
39.814
1.00
19.75
S
O


ATOM
1445
O
HOH 
W
141
29.084
22.512 
22.380
1.00
19.74
S
O


ATOM
1446
O
HOH 
W
144
26.825
34.183 
16.982
1.00
19.75
S
O


ATOM
1447
O
HOH 
W
146
28.060
21.125 
26.874
1.00
19.76
S
O


ATOM
1448
O
HOH 
W
147
7.953
28.465 
43.320
1.00
19.76
S
O


ATOM
1449
O
HOH 
W
148
25.139
13.555 
38.510
1.00
19.76
S
O


ATOM
1450
O
HOH 
W
154
27.898
15.263 
40.931
1.00
19.75
S
O


ATOM
1451
O
HOH 
W
157
29.305
18.029 
39.665
1.00
19.76
S
O


ATOM
1452
O
HOH 
W
158
22.038
30.753
9.108
1.00
19.76
S
O


ATOM
1453
O
HOH 
W
159
18.399
11.163 
36.207
1.00
19.76
S
O


ATOM
1454
O
HOH 
W
164
26.335
11.937 
35.945
1.00
19.75
S
O


ATOM
1455
O
HOH 
W
165
1.758
29.855 
17.357
1.00
19.75
S
O


ATOM
1456
O
HOH 
W
166
24.163
39.471 
32.170
1.00
19.76
S
O


ATOM
1457
O
HOH 
W
170
16.077
17.918
7.749
1.00
19.75
S
O


ATOM
1458
O
HOH 
W
172
32.921
14.044 
27.295
1.00
19.76
S
O


ATOM
1459
O
HOH 
W
177
32.795
38.969 
32.954
1.00
19.77
S
O


ATOM
1460
O
HOH 
W
179
4.059
6.708 
28.892
1.00
19.75
S
O


ATOM
1461
O
HOH 
W
180
25.397
29.865 
14.090
1.00
19.76
S
O


ATOM
1462
O
HOH 
W
182
11.078
20.731 
43.859
1.00
19.77
S
O


ATOM
1463
O
HOH 
W
184
30.825
30.779 
39.402
1.00
19.77
S
O


ATOM
1464
O
HOH 
W
187
10.289
21.108
7.474
1.00
19.75
S
O


ATOM
1465
O
HOH 
W
189
27.314
38.906 
38.135
1.00
19.76
S
O


ATOM
1466
O
HOH 
W
197
25.884
26.959 
11.320
1.00
19.70
S
O


ATOM
1467
O
HOH 
W
209
9.364
16.866 
38.731
1.00
19.73
S
O


ATOM
1468
O
HOH 
W
219
32.352
16.134 
38.786
1.00
19.73
S
O


ATOM
1469
O
HOH 
W
221
15.972
35.898 
37.609
1.00
19.69
S
O


ATOM
1470
O
HOH 
W
223
3.319
35.758 
13.483
1.00
19.71
S
O


TER
1471

HOH 
W
223









END





















The surface accessible residues of ASP were determined from the crystallographic coordinates provided above, using the program DS Modeling (Accelrys), using the default settings. The total surface accessibility (SA) for ASP was found to be 8044.777 Angstroms. Table 19-2 provides the total SA, side chain SA, and percent SAS is the percentage of an amino acid's total surface that is accessible to solvent.







TABLE 19-2







Total Surface Accessibility of ASP










Residue
Total SA ang2
SideChain SA ang2
Percent SAS













asp 1: Phe
89.992
66.420
36.954


asp 2: Asp
85.970
68.625
48.199


asp 4 : Ile
17.921
12.076
9.714


asp 7: Asn
40.541
40.541
21.246


asp 8: Ala
41.497
24.153
35.259


asp 10: Thr
35.846
35.846
21.190


asp 11: Ile
29.424
18.114
17.028


asp 12: Gly
81.658
30.191
73.513


asp 13: Gly
75.236
18.114
67.615


asp 14: Arg
124.289
124.289
55.664


asp 15: Ser
29.424
29.424
19.554


asp 16: Arg
105.411
88.447
38.127


asp 22: Ala
11.690
0.000
9.932


asp 24: Asn
71.105
65.067
47.079


asp 25: Gly
53.190
30.191
43.325


asp 32: His
34.693
17.728
19.568


asp 34: Gly
18.114
12.076
20.656


asp 35: Arg
177.087
171.242
69.918


asp 36: Thr
87.506
64.886
45.401


asp 37: Gly
58.465
24.153
55.659


asp 38: Ala
18.114
12.076
16.195


asp 39: Thr
99.579
87.889
55.002


asp 40: Thr
11.310
0.000
6.469


asp 41: Ala
36.229
36.229
38.182


asp 42: Asn
86.537
74.844
43.919


asp 43: Pro
6.038
0.000
4.599


asp 44: Thr
111.082
99.582
59.375


asp 45: Gly
6.038
6.038
5.436


asp 46: Thr
52.427
52.427
28.958


asp 47: Phe
5.655
0.000
2.715


asp 48: Ala
58.848
30.191
52.705


asp 49: Gly
12.076
12.076
12.937


asp 50: Ser
51.274
0.000
37.049


asp 51: Ser
17.348
17.348
11.573


asp 52: Phe
52.040
12.076
25.034


asp 53: Pro
53.193
36.229
40.511


asp 54: Gly
30.191
30.191
27.274


asp 55: Asn
34.499
34.499
18.613


asp 57: Tyr
28.658
28.658
11.861


asp 59: Phe
18.114
18.114
9.808


asp 61: Arg
146.706
141.051
59.429


asp 62: Thr
22.619
5.655
12.939


asp 63: Gly
17.538
6.038
17.646


asp 64: Ala
112.229
60.381
90.564


asp 65: Gly
70.535
30.191
60.226


asp 66: Val
16.965
0.000
10.967


asp 67: Asn
69.002
62.964
39.692


asp 68: Leu
34.503
6.038.
16.536


asp 69: Leu
42.267
42.267
20.295


asp 71: Gln
39.774
39.774
18.552


asp 73: Asn
17.345
17.345
8.760


asp 74: Asn
41.301
41.301
25.351


asp 75: Tyr
93.544
47.922
37.830


asp 76: Ser
97.666
52.044
76.965


asp 77: Gly
81.275
24.153
73.294


asp 78: Gly
17.921
12.076
18.067


asp 79: Arg
139.911
94.292
56.632


asp 80: Val
36.229
30.191
22.621


asp 81: Gln
82.421
70.921
37.295


asp 83: Ala
41.117
24.153
33.386


asp 84: Gly
12.076
12.076
12.151


asp 85: His
71.298
65.454
36.451


asp 86: Thr
111.082
93.544
65.517


asp 87: Ala
64.886
42.267
52.523


asp 88: Ala
12.076
6.038
10.760


asp 89: Pro
90.572
78.496
58.405


asp 90: Val
94.694
66.420
53.062


asp 91: Gly
58.082
18.114
49.593


asp 92: Ser
34.886
23.003
27.450


asp 93: Ala
83.381
60.381
70.846


asp 95: Cys
26.565
26.565
15.773


asp 99: Ser
39.584
0.000
29.907


asp 100: Thr
87.123
47.155
48.121


asp 101: Thr
34.696
6.038
22.060


asp 102: Gly
12.076
12.076
13.771


asp 103: Trp
70.728
47.919
27.630


asp 104: His
47.726
41.687
23.152


asp 105: Cys
54.609
31.799
33.796


asp 106: Gly
23.386
12.076
23.531


asp 107: Thr
47.155
47.155
29.873


asp 108: Ile
5.655
0.000
2.888


asp 109: Thr
64.503
30.191
35.741


asp 110: Ala
24.153
24.153
21.668


asp 111: Leu
71.115
48.305
36.142


asp 112: Asn
138.770
104.841
66.301


asp 113: Ser
17.731
11.693
12.794


asp 114: Ser
92.391
52.427
63.967


asp 115: Val
30.191
24.153
18.166


asp 116: Thr
128.237
82.618
66.534


asp 117: Tyr
35.846
24.153
15.603


asp 118: Pro
159.964
102.648
93.188


asp 119: Glu
132.745
87.123
63.766


asp 120: Gly
18.114
18.114
20.611


asp 121: Thr
93.924
76.579
48.828


asp 123: Arg
129.748
129.748
59.619


asp 124: Gly
29.231
12.076
26.315


asp 126: Ile
6.038
6.038
3.084


asp 127: Arg
99.943
99.943
36.957


asp 128: Thr
5.655
0.000
3.450


asp 129: Thr
76.579
59.615
45.219


asp 130: Val
0.000
0.000
0.000


asp 131: Cys
25.568
19.723
18.583


asp 132: Ala
11.693
6.038
9.495


asp 133: Glu
40.734
29.041
20.057


asp 134: Pro
114.531
102.648
68.994


asp 135: Gly
11.883
6.038
11.979


asp 137: Ser
5.655
5.655
3.915


asp 143: Ala 
17.731
6.038
18.763


asp 144: Gly
59.612
36.229
63.599


asp 145: Asn
81.832
70.142
44.061


asp 146: Gln
52.810
52.810
27.510


asp 147: Ala
5.655
0.000
4.797


asp 148: Gln
11.500
5.845
5.335


asp 152: Ser
5.655
0.000
4.092


asp 153: Gly
24.153
18.114
25.819


asp 154: Gly
63.927
12.076
64.322


asp 155: Ser
88.656
70.541
69.864


asp 156: Gly
52.807
18.114
50.090


asp 157: Asn
35.263
35.263
20.195


asp 158: Cys
34.312
6.038
21.893


asp 159: Arg
199.716
154.094
79.090


asp 160: Thr
135.044
89.422
85.862


asp 161: Gly
35.462
24.153
33.699


asp 162: Gly
23.576
6.038
21.225


asp 163: Thr
46.005
46.005
25.438


asp 164: Thr
5.655
5.655
3.127


asp 165: Phe
24.153
24.153
10.669


asp 167: Gln
5.845
5.845
3.042


asp 168: Pro
48.305
48.305
31.227


asp 170: Asn
59.032
53.377
31.882


asp 171: Pro
59.615
42.267
42.027


asp 173: Leu
17.731
12.076
8.274


asp 174: Gln
145.572
122.569
80.497


asp 175: Ala
52.044
6.038
44.291


asp 176: Tyr
64.886
36.229
29.811


asp 177: Gly
69.775
24.153
70.340


asp 178: Leu
11.693
6.038
5.788


asp 179: Arg
182.932
182.932
72.390


asp 180: Met
34.886
12.076
17.253


asp 181: Ile
36.229
30.191
19.053


asp 182: Thr
99.389
76.579
60.785


asp 183: Thr 
104.854
93.544
68.979


asp 184: Asp 
122.008
23.386
52.822









The ASP co-ordinates, and those of homologous structures were loaded into MOE (Chemical Computing Group). Co-ordinates for waters and ligands were removed. Using MOE align, the structures were aligned using actual secondary structure, with structural alignment enabled and superpose chains enabled. This resulted in the following structural alignment. The numbers indicated refer to the mature ASP protease amino-acid sequence.











PDB ID









1        10         20             30        40 


ASP
FDVIGGNAYTIG-GRSRCSIGFAVN-----GGFITAGHCGRTGATTAN------PTGTFA


1HPG
--VLGGGAIYGG-GSR-CSAAFNVTK-GGARYFVTAGHCTNISANWSASS-GGSVVGVRE


1SGP
--ISGGDAIYSS-TGR-CSLGFNVRS-GSTYYFLTAGHCTDGATTWWANSARTTVLGTTS


1TAL
ANIVGGIEYSINNASL-CSVGFSVTR-GATKGFVTAGHCGTVNATARIG---GAVVGTFA


2SFA
--IAGGEAIYAAGGGR-CSLGFNVRSSSGATYALTAGHCTEIASTWYTNSGQTSLLGTRA


2SGA
--IAGGEAITT-GGSR-CSLGFNVSV-NGVAHALTAGHCTNISASWS--------IGTRT


 



 50        60         70        80        90        100


ASP
GSSFPGNDYAFVRTGAG-VNLLAQVNNYSGGRVQVAGHTAAPVGSAVCRSGSTTGWHCGT


1HPG
GTSFPTNDYGIVRYTDG-SSPAGTVDLYNGSTQDISSAANAVVGQAIKKSGSTTKVTSGT


1SGP
GSSFPNNDYGIVRYTNTTIPKDGTVG-----GQDITSAANATVGMAVTRRGSTTGTHSGS


1TAL
ARVFPGNDRAWVSLTSA-QTLLPRVANG-SSFVTVRGSTEAAVGAAVCRSGRTTGYQCGT


2SFA
GTSFPGNDYGLIRHSNA-SAADGRVYLYNGSYRDITGAGNAYVGQTVQRSGSTTGLHSGR


2SGA
GTSFPNNDYGIIRHSNP-AAADGRVYLYNGSYQDITTAGNAFVGQAVQRSGSTTGLRSGS


 



  110        120        130      140        150            160


ASP
ITALNSSVTYPE-GTVRGLIRTTVCAEPGDSGGSLLA-GNQAQGVTSGGSG-----NCRT


1HPG
VTAVNVTVNYGD-GPVYNMVRTTACSAGGDSGGAHFA-GSVALGIHSGSSG------CSG


1SGP
VTALNATVNYGGGDVVYGMIRTNVCAEPGDSGGPLYS-GTRAIGLTSGGSG-----NCSS


1TAL
ITAKNVTANYAE-GAVRGLTQGNACMGRGDSGGSWITSAGQAQGVMSGGNVQSNGNNCGI


2SFA
VTGLNATVNYGGGDIVSGLIQTNVCAEPGDSGGALFA-GSTALGLTSGGSG-----NCRT


2SGA
VTGLNATVNYGSSGIVYGMIQTNVCAQPGDSGGSLFA-GSTALGLTSGGSG-----NCRT


 



            170       180


ASP
G---GTTFFQPVNPILQAYGLRMITTD (SEQ ID NO: 624)


1HPG
TA--GSAIHQPVTEALSAYGVTVY--- (SEQ ID NO: 625)


1SGP
G---GTTFFQPVTEALVAYGVSVY--- (SEQ ID NO: 626)


1TAL
PASQRSSLFERLQPILSQYGLSLVTG- (SEQ ID NO: 627)


2SFA
G---GTTFFQPVTEALSAYGVSIL--- (SEQ ID NO: 628)


2SGA
G---GTTFYQPVTEALSAYGATVL--- (SEQ ID NO: 629)





In the above alignment, the codes are as follows:


1HPG = Streptomyces griseus glutamic acid specific protease.


1SGP = Streptomyces griseus proteinase B


1SGT = Streptomyces griseus strain K1 trypsin


1TAL = Lysobacter enzymogenes alpha-lytic protease


2SFA = Streptomyces fradiae serine proteinase


2SGA = Streptomyces griseus protease A






Example 20
Enzyme Substrate Modeling and Mapping of the ASP Active-Site
In this Example, enzyme-substrate modeling and mapping of the ASP active site methods are described. Preliminary inspection of the active-site revealed a large P1 binding pocket that is large enough to accommodate large hydrophobic groups such as the side-chains of Trp, Tyr, and Phe.
The crystal structure of Streptogrisin A with the turkey third domain of the ovomucoid inhibitor (pdb code 2SGB) was been determined. 2SGB was structurally aligned to ASP, using MOE (Chemical Computing Corp), which places the inhibitor in the active-site of ASP. All of the 2SGB co-ordinates were removed, except for those which define a hexa-peptide bound in the ASP active-site, corresponding to binding at the S4 to S2′ binding sites. The Pro-ASP protein self-cleaves the pro domain-mature domain junction, to release the mature protease enzyme. The last four residues of the pro domain are expected to occupy the S1-S4 sites, and the first two residues of the mature protease occupy the S1′ and S2′ sites. Therefore the hexapeptide in the active-site was in-silico mutated to sequence PRTMFD (SEQ ID NO:630).
From inspection of the structure of the initial substrate bound model, the backbone amide of Gly135 and Asp136 would be expected to form the oxy-anion hole. However, the amide nitrogen of Gly135 appears to point in the wrong direction. Comparison with streptogrisin A confirms this. Thus, it is presumed that a conformational change in ASP is required to form the oxy-anion hole. However, it is not intended that the present invention be limited by any particular mechanism nor hypothesis. The peptide backbone between residues 134 and 135 was altered to that of a similar orientation to that of structurally equivalent atoms in the streptogrisin A structure. The enzyme substrate model was then energy minimized.
Residues within 6 Å of the modeled substrate were determined using the proximity tools within the program QUANTA. These residues were identified as: Arg14, Ser15, Arg16, Cys17, His32, Cys33, Phe52, Asp56, Thr100, Val115, Thr116, Tyr117, Pro118, Glu119, Ala132, Glu133, Pro134, Gly135, Asp136, Ser137, Thr151, Ser152, Gly153, Gly154, Ser155, Gly156, Asn157, Thr164, Phe165. Of these, His 32, Asp56, and Ser137 form the catalytic triad.
The P1 pocket is formed by Cys131, Ala132, Glu133, Pro134, Gly135, Thr151, Ser152, Gly153, Gly154, Ser155, Gly156, Asn157 and Gly 162, Thr 163, Thr164. The P2 pocket is defined by Phe52, Tyr117, Pro118 and Glu119. The P3 pocket has main-chain to main chain hydrogen bonding from Gly 154 to the substrate main-chain. The P1′ pocket is defined by Arg16, and His32. The P2′ pocket is defined by Thr100, and Pro134. The atomic coordinates of ASP with the modeled octapeptide substrate are provided in Table 20-1 below.







Table 20-1





Atomic Coordinates of ASP with the Modeled Octapeptide Substrate

























ATOM
1
N
PHE
A
1
2.452
18.495
15.165
0.00
N1+


ATOM
2
CA
PHE
A
1
3.712
18.208
15.901
0.00
C


ATOM
3
CB
PHE
A
1
4.906
18.646
15.055
0.00
C


ATOM
4
C
PHE
A
1
3.743
18.914
17.254
0.00
C


ATOM
5
O
PHE
A
1
3.539
20.133
17.340
0.00
O


ATOM
6
CG
PHE
A
1
6.232
18.405
15.707
0.00
C


ATOM
7
CD2
PHE
A
1
6.963
17.268
15.411
0.00
C


ATOM
8
CD1
PHE
A
1
6.750
19.312
16.618
0.00
C


ATOM
9
CE2
PHE
A
1
8.192
17.035
16.010
0.00
C


ATOM
10
CE1
PHE
A
1
7.981
19.086
17.222
0.00
C


ATOM
11
CZ
PHE
A
1
8.702
17.946
16.917
0.00
C


ATOM
12
N
ASP
A
2
4 .000
18.148
18.311
0.00
N


ATOM
13
CA 
ASP
A
2
4.052
18.708
19.659
0.00
C


ATOM
14
CB
ASP
A
2
3.584
17.678
20.688
0.00
C


ATOM
15
C
ASP
A
2
5.422
19.210
20.066
0.00
C


ATOM
16
O
ASP
A
2
6.415
18.508
19.925
0.00
O


ATOM
17
CG
ASP
A
2
2.109
17.354
20.560
0.00
C


ATOM
18
OD2
ASP
A
2
1.597
16.558
21.379
0.00
O1−


ATOM
19
OD1
ASP
A
2
1.459
17.889
19.638
0.00
O


ATOM
20
N
VAL
A
3
5.464
20.440
20.562
0.00
N


ATOM
21
CA
VAL
A
3
6.707
21.057
21.009
0.00
C


ATOM
22
CB
VAL
A
3
6.736
22.574
20.718
0.00
C


ATOM
23
C
VAL
A
3
6.737
20.837
22.513
0.00
C


ATOM
24
O
VAL
A
3
5.806
21.233
23.216
0.00
O


ATOM
25
CG1
VAL
A
3
7.921
23.222
21.425
0.00
C


ATOM
26
CG2
VAL
A
3
6.840
22.810
19.220
0.00
C


ATOM
27
CB
ILE
A
4
7.602
18.448
24.730
0.00
C


ATOM
28
CG2
ILE
A
4
7.684
18.189
26.227
0.00
C


ATOM
29
CG1
ILE
A
4
6.196
18.137
24.220
0.00
C


ATOM
30
CD1
ILE
A
4
5.768
16.711
24.456
0.00
C


ATOM
31
C 
ILE
A
4
9.379
20.168
24.911
0.00
C


ATOM
32
O 
ILE
A
4
10.346
19.836
24.229
0.00
C


ATOM
33
N
ILE
A
4
7.801
20.200
22.997
0.00
N


ATOM
34
CA
ILE
A
4
7.955
19.916
24.423
0.00
C


ATOM
35
N
GLY
A
5
9.499
20.743
26.103
0.00
N


ATOM
36
CA
GLY
A
5
10.807
21.030
26.653
0.00
C


ATOM
37
C
GLY
A
5
11.655
19.787
26.819
0.00
C


ATOM
36
O
GLY
A
5
11.171
18.750
27.277
0.00
O


ATOM
39
N
GLY
A
6
12.927
19.885
26.443
0.00
N


ATOM
40
CA
GLY
A
6
13.817
18.747
26.572
0.00
C


ATOM
41
C
GLY
A
6
14.007
17.948
25.294
0.00
C


ATOM
42
O
GLY
A
6
14.990
17.217
25.157
0.00
O


ATOM
43
N
ASN
A
7
13.069
18.082
24.359
0.00
N


ATOM
44
CA
ASN
A
7
13.155
17.351
23.100
0.00
C


ATOM
45
CB
ASN
A
7
11.784
17.247
22.450
0.00
C


ATOM
46
CG
ASN
A
7
10.918
16.210
23.102
0.00
C


ATOM
47
OD1
ASN
A
7
9.741
16.069
22.760
0.00
O


ATOM
40
ND2
ASN
A
7
11.492
15.464
24.049
0.00
N


ATOM
49
C
ASN
A
7
14.124
17.933
22.086
0.00
C


ATOM
50
O
ASN
A
7
14.466
19.114
22.119
0.00
C


ATOM
51
N
ALA
A
8
14.561
17.077
21.176
0.00
N


ATOM
52
CA
ALA
A
8
15.486
17.487
20.138
0.00
C


ATOM
53
CB 
ALA
A
8
16.212
16.271
19.577
0.00
C


ATOM
54
C
ALA
A
8
14.716
18.174
19.023
0.00
C


ATOM
55
O
ALA
A
8
13.509
17.988
18.874
0.00
O


ATOM
56
N
TYR
A
9
15.423
18.993
18.262
0.00
N


ATOM
57
CA
TYR
A
9
14.847
19.714
17.143
0.00
C


ATOM
58
CB
TYR
A
9
14.253
21.064
17.580
0.00
C


ATOM
59
CG
TYR
A
9
15.221
22.148
17.963
0.00
C


ATOM
60
CD2
TYR
A
9
15.517
22.398
19.301
0.00
C


ATOM
61
CE2
TYR
A
9
16.341
23.443
19.663
0.00
C


ATOM
62
CD1
TYR
A
9
15.785
22.972
16.993
0.00
C


ATOM
63
CE1
TYR
A
9
16.609
24.021
17.343
0.00
C


ATOM
64
CZ
TYR
A
9
16.883
24.255
18.678
0.00
C


ATOM
65
OH
TYR
A
9
17.688
25.309
19.029
0.00
O


ATOM
66
C
TYR
A
9
16.072
19.837
16.262
0.00
C


ATOM
67
O 
TYR
A
9
17.188
19.678
16.753
0.00
O


ATOM
68
N
THR
A
10
15.886
20.077
14.970
0.00
N


ATOM
69
CA
THR
A
10
17.034
20.183
14.082
0.00
C


ATOM
70
CB
THR
A
10
17.031
19.031
13.041
o.oo
C


ATOM
71
OG1
THR
A
10
15.822
19.082
12.269
0.00
O


ATOM
72
CG2
THR
A
10
17.129
17.676
13.741
0.00
C


ATOM
73
C
THR
A
10
17.205
21.486
13.329
0.00
C


ATOM
74
O
THR
A
10
16.249
22.243
13.104
0.00
O


ATOM
75
N
ILE
A
11
18.453
21.734
12.936
0.00
N


ATOM
76
CA
ILE
A
11
18.828
22.930
12.197
0.00
C


ATOM
77
CB
ILE
A
11
19.609
23.914
13.093
0.00
C


ATOM
78
CG
ILE
A
11
19.855
25.221
12.343
0.00
C


ATOM
79
CG1
ILE
A
11
18.811
24.187
14.369
0.00
C


ATOM
80
CD1
ILE
A
11
19.546
25.036
15.385
0.00
C


ATOM
81
C 
ILE
A
11
19.712
22.442
11.054
0.00
C


ATOM
82
O
ILE
A
11
20.772
21.856
11.284
0.00
O


ATOM
83
N 
GLY
A
12
19.274
22.668
9.821
0.00
N


ATOM
84
CA
GLY
A
12
20.048
22.193
8.689
0.00
C


ATOM
85
C
GLY
A
12
20.344
20.705
8.845
0.00
C


ATOM
86
O
GLY
A
12
21.439
20.239
8.523
0.00
O


ATOM
87
N
GLY
A
13
19.373
19.957
9.361
0.00
N


ATOM
88
CA
GLY
A
13
19.564
18.531
9.545
0.00
C


ATOM
89
C
GLY
A
13
20.373
18.127
10.769
0.00
C


ATOM
90
O
GLY
A
13
20.438
16.945
11.103
0.00
O


ATOM
91
N
ARG
A
14
20.984
19.091
11.449
0.00
N


ATOM
92
CA
ARG
A
14
21.787
18.782
12.627
0.00
C


ATOM
93
CB
ARG
A
14
23.036
19.670
12.669
0.00
C


ATOM
94
C
ARG
A
14
21.018
18.938
13.935
0.00
C


ATOM
95
O
ARG
A
14
20.441
19.982
14.212
0.00
O


ATOM
96
CG
ARG
A
14
24.251
19.072
11.964
0.00
C


ATOM
97
CD
ARG
A
14
24.065
19.084
10.450
0.00
C


ATOM
98
NE
ARG
A
14
24.173
17.752
9.858
0.00
N1+


ATOM
99
CZ
ARG
A
14
25.316
17.100
9.660
0.00
C


ATOM
100
NH1
ARG
A
14
26.474
17.655
10.004
0.00
N


ATOM
101
NH2
ARG
A
14
25.302
15.886
9.120
0.00
N


ATOM
102
N
SER
A
15
21.016
17.878
14.733
0.00
N


ATOM
103
CA
SER
A
15
20.335
17.870
16.017
0.00
C


ATOM
104
CB
SER
A
15
20.062
16.429
16.454
0.00
C


ATOM
105
C
SER
A
15
21.312
18.525
16.983
0.00
C


ATOM
106
O
SER
A
15
21.933
17.849
17.803
0.00
O


ATOM
107
OG
SER
A
15
19.396
16.382
17.701
0.00
O


ATOM
108
N
ARG
A
16
21.454
19.841
16.867
0.00
N


ATOM
109
CA
ARG
A
16
22.362
20.594
17.724
0.00
C


ATOM
110
CB
ARG
A
16
22.741
21.927
17.073
0.00
C


ATOM
111
C
ARG
A
16
21.815
20.907
19.104
0.00
C


ATOM
112
O
ARG
A
16
22.550
20.867
20.088
0.00
O


ATOM
113
CG
ARG
A
16
23.719
21.851
15.915
0.00
C


ATOM
114
CD
ARG
A
16
24.200
23.253
15.549
0.00
C


ATOM
115
NE
ARG
A
16
24.625
23.984
16.745
0.00
N1+


ATOM
116
CZ
ARG
A
16
25.242
25.166
16.739
0.00
C


ATOM
117
NH2
ARG
A
16
25.581
25.735
17.888
0.00
N


ATOM
118
NH1
ARG
A
16
25.528
25.781
15.597
0.00
N


ATOM
119
N
CYS
A
17
20.526
21.215
19.178
0.00
N


ATOM
120
CA
CYS
A
17
19.928
21.546
20.455
0.00
C


ATOM
121
CB
CYS
A
17
19.800
23.068
20.553
0.00
C


ATOM
122
C
CYS
A
17
10.539
20.911
20.003
0.00
C


ATOM
123
O
CYS
A
17
18.071
20.077
20.071
0.00
O


ATOM
124
SG
CYS
A
17
21.393
23.932
20.696
0.00
S


ATOM
125
N
SER
A
18
18.066
21.348
21.942
0.00
N


ATOM
126
CA
SER
A
18
16.799
20.865
22.455
0.00
C


ATOM
127
CB
SER
A
18
17.042
20.053
23.723
0.00
C


ATOM
128
CG
SER
A
18
18.081
19.111
23.521
0.00
O


ATOM
129
C
SER
A
18
15.871
22.030
22.769
0.00
C


ATOM
130
O
SER
A
18
16.312
23.175
22.890
0.00
O


ATOM
131
N
ILE
A
19
14.584
21.728
22.892
0.00
N


ATOM
132
CA
ILE
A
19
13.582
22.737
23.195
0.00
C


ATOM
133
CB
ILE
A
19
12.150
22.152
23.125
0.00
C


ATOM
134
CG2
ILE
A
19
11.133
23.215
23.532
0.00
C


ATOM
135
CG1
ILE
A
19
11.852
21.634
21.715
0.00
C


ATOM
136
CD1
ILE
A
19
11.832
22.709
20.655
0.00
C


ATOM
137
C
ILE
A
19
13.794
23.273
24.614
0.00
C


ATOM
138
O
ILE
A
19
14.070
22.505
25.545
0.00
O


ATOM
139
N
GLY
A
20
13.670
24.589
24.774
0.00
N


ATOM
140
CA
GLY
A
20
13.818
25.185
26.088
0.00
C


ATOM
141
C
GLY
A
20
12.443
25.203
26.722
0.00
C


ATOM
142
O
GLY
A
20
12.122
24.389
27.585
0.00
O


ATOM
143
N
PHE
A
21
11.616
26.137
26.274
0.00
N


ATOM
144
CA
PHE
A
21
10.253
26.258
26.763
0.00
C


ATOM
145
CB
PHE
A
21
10.196
27.160
27.992
0.00
C


ATOM
146
CG
PHE
A
21
10.855
26.559
29.195
0.00
C


ATOM
147
CD1
PHE
A
21
10.269
25.491
29.857
0.00
C


ATOM
148
CD2
PHE
A
21
12.086
27.025
29.638
0.00
C


ATOM
149
CE1
PHE
A
21
10.898
24.898
30.936
0.00
C


ATOM
150
CE2
PHE
A
21
12.713
26.435
30.715
0.00
C


ATOM
151
CZ
PHE
A
21
12.122
25.370
31.366
0.00
C


ATOM
152
C
PHE
A
21
9.391
26.825
25.664
0.00
C


ATOM
153
O
PHE
A
21
9.865
27.597
24.830
0.00
O


ATOM
154
N
ALA
A
22
8.131
26.413
25.646
0.00
N


ATOM
155
CA
ALA
A
22
7.194
26.882
24.647
0.00
C


ATOM
156
CB
ALA
A
22
6.014
25.915
24.533
0.00
C


AT CM
157
C
ALA
A
22
6.719
28.230
25.138
0.00
C


ATOM
158
O
ALA
A
22
6.416
28.388
26.320
0.00
O


ATOM
159
N
VAL
A
23
6.677
29.202
24.239
0.00
N


ATOM
160
CA
VAL
A
23
6.233
30.546
24.582
0.00
C


ATOM
161
CB
VAL
A
23
7.402
31.570
24.551
0.00
C


ATOM
162
CG1
VAL
A
23
8.328
31.338
25.728
0.00
C


ATOM
163
CG2
VAL
A
23
8.182
31.442
23.248
0.00
C


ATOM
164
C
VAL
A
23
5.206
30.945
23.545
0.00
C


ATOM
165
O
VAL
A
23
5.053
30.267
22.526
0.00
O


ATOM
166
N
ASN
A
24
4.495
32.036
23.791
0.00
N


ATOM
167
CA
ASN
A
24
3.492
32.476
22.832
0.00
C


ATOM
168
CB
ASN
A
24
2.807
33.759
23.328
0.00
C


ATOM
169
C
ASN
A
24
4.177
32.715
21.484
0.00
C


ATOM
170
O
ASN
A
24
5.050
33.576
21.365
0.00
O


ATOM
171
CG
ASN
A
24
3.737
34.963
23.334
0.00
C


ATOM
172
OD1
ASN
A
24
4.697
35.029
24.107
0.00
O


ATOM
173
ND2
ASN
A
24
3.451
35.927
22.462
0.00
N


ATOM
174
N
GLY
A
25
3.801
31.929
20.477
0.00
N


ATOM
175
CA
GLY
A
25
4.396
32.084
19.158
0.00
C


ATOM
176
C
GLY
A
25
5.503
31.104
18.788
0.00
C


ATOM
177
O
GLY
A
25
5.925
31.054
17.635
0.00
O


ATOM
178
N
GLY
A
26
5.989
30.327
19.748
0.00
N


ATOM
179
CA
GLY
A
26
7.043
29.377
19.433
0.00
C


ATOM
180
C
GLY
A
26
7.702
28.795
20.666
0.00
C


ATOM
181
O
GLY
A
26
7.028
28.328
21.582
0.00
O


ATOM
182
N
PHE
A
27
9.028
28.813
20.688
0.00
N


ATOM
183
CA
PHE
A
27
9.757
28.294
21.832
0.00
C


ATOM
184
CB
PHE
A
27
9.973
26.783
21.710
0.00
C


ATOM
185
C
PHE
A
27
11.103
28.975
21.954
0.00
C


ATOM
186
O
PHE
A
27
11.660
29.459
20.963
0.00
O


ATOM
187
CG
PHE
A
27
10.949
26.376
20.624
0.00
C


ATOM
188
CD1
PHE
A
27
10.504
26.078
19.336
0.00
C


ATOM
189
CD2
PHE
A
27
12.306
26.246
20.905
0.00
C


ATOM
190
CE1
PHE
A
27
11.391
25.650
18.352
0.00
C


ATOM
191
CE2
PHE
A
27
13.202
25.819
19.926
0.00
C


ATOM
192
CZ
PHE
A
27
12.742
25.518
18.648
0.00
C


ATOM
193
H
ILE
A
28
11.615
29.020
23.180
0.00
N


ATOM
194
CA
ILE
A
28
12.904
29.640
23.445
0.00
C


ATOM
195
CB
ILE
A
28
12.843
30.524
24.704
0.00
C


ATOM
196
C
ILE
A
28
13.953
28.542
23.603
0.00
C


ATOM
197
O
ILE
A
2R
13.640
27.426
24.011
0.00
O


ATOM
198
CG2
ILE
A
28
11.915
31.688
24.450
0.00
C


ATOM
199
CG1
ILE
A
28
12.350
29.718
25.904
0.00
C


ATOM
200
CD1
ILE
A
28
12.270
30.524
27.176
0.00
C


ATOM
201
N
THR
A
29
15.195
28.866
23.265
0.00
N


ATOM
202
CA
THR
A
29
16.293
27.916
23.353
0.00
C


ATOM
203
CB
THR
A
29
16.329
27.054
22.052
0.00
C


ATOM
204
OG1
THR
A
29
17.423
26.126
22.095
0.00
O


ATOM
205
CG2
THR
A
29
16.459
27.950
20.831
0.00
C


ATOM
206
C
THR
A
29
17.601
28.695
23.538
0.00
C


ATOM
207
O
THR
A
29
17.565
29.881
23.842
0.00
O


ATOM
208
N
ALA
A
30
18.743
28.029
23.362
0.00
N


ATOM
209
CA
ALA
A
30
20.059
28.662
23.510
0.00
C


ATOM
210
CB
ALA
A
30
21.121
27.601
23.765
0.00
C


ATOM
211
C
ALA
A
30
20.447
29.486
22.282
0.00
C


ATOM
212
O
ALA
A
30
20.232
29.061
21.141
0.00
O


ATOM
213
N
GLY
A
31
21.028
30.659
22.520
0.00
N


ATOM
214
CA
GLY
A
31
21.427
31.522
21.423
0.00
C


ATOM
215
C
GLY
A
31
22.508
30.942
20.528
0.00
C


ATOM
216
O
GLY
A
31
22.527
31.212
19.322
0.00
O


ATOM
217
N
HIS
A
32
23.410
30.143
21.099
0.00
N


ATOM
218
CA
HIS
A
32
24.490
29.558
20.310
0.00
C


ATOM
219
CB
HIS
A
32
25.648
29.091
21.215
0.00
C


ATOM
220
CG
HIS
A
32
25.412
27.772
21.885
0.00
C


ATOM
221
CD2
HIS
A
32
24.715
27.451
23.001
0.00
C


ATOM
222
ND1
HIS
A
32
25.946
26.589
21.419
0.00
N


ATOM
223
CE1
HIS
A
32
25.590
25.601
22.216
0.00
C


ATOM
224
NE2
HIS
A
32
24.842
26.098
23.188
0.00
N


ATOM
225
C
HIS
A
32
24.029
28.401
19.413
0.00
C


ATOM
226
O
HIS
A
32
24.805
27.870
18.630
0.00
O


ATOM
227
N
CYS
A
33
22.762
28.025
19.525
0.00
N


ATOM
228
CA
CYS
A
33
22.210
26.940
18.723
0.00
C


ATOM
229
CB
CYS
A
33
20.836
26.522
19.251
0.00
C


ATOM
230
SG
CYS
A
33
20.853
25.876
20.942
0.00
S


ATOM
231
C
CYS
A
33
22.062
27.395
17.283
0.00
C


ATOM
232
O
CYS
A
33
22.149
26.603
16.356
0.00
O


ATOM
233
N
GLY
A
34
21.822
28.680
17.095
0.00
N


ATOM
234
CA
GLY
A
34
21.664
29.181
15.749
0.00
C


ATOM
235
C
GLY
A
34
21.360
30.656
15.763
0.00
C


ATOM
236
O
GLY
A
34
20.984
31.213
16.794
0.00
O


ATOM
237
N
ARG
A
35
21.523
31.288
14.608
0.00
N


ATOM
238
CA
ARG
A
35
21.284
32.716
14.478
0.00
C


ATOM
239
CB
ARG
A
35
22.417
33.355
13.680
0.00
C


ATOM
240
C
ARG
A
35
19.951
33.012
13.798
0.00
C


ATOM
241
O
ARG
A
35
19.348
32.138
13.173
0.00
O


ATOM
242
CG
ARG
A
35
22.437
32.937
12.219
0.00
C


ATOM
243
CD
ARG
A
35
23.488
33.715
11.458
0.00
C


ATOM
244
NE
ARG
A
35
24.832
33.237
11.755
0.00
N1+


ATOM
245
CZ
ARG
A
35
25.406
32.207
11.139
0.00
C


ATOM
246
NH1
ARG
A
35
26.634
31.832
11.471
0.00
N


ATOM
247
NH2
ARG
A
35
24.759
31.559
10.178
0.00
N


ATOM
248
N
THR
A
36
19.513
34.258
13.918
0.00
N


ATOM
249
CA
THR
A
36
18.259
34.714
13.335
0.00
C


ATOM
250
CB
THR
A
36
18.124
36.242
13.522
0.00
C


ATOM
251
C
THR
A
36
18.161
34.353
11.856
0.00
C


ATOM
252
O
THR
A
36
19.123
34.512
11.099
0.00
O


ATOM
253
OG1
THR
A
36
18.120
36.536
14.923
0.00
O


ATOM
254
CG2
THR
A
36
16.844
36.773
12.880
0.00
C


ATOM
255
N
GLY
A
37
16.999
33.855
11.449
0.00
N


ATOM
256
CA
GLY
A
37
16.813
33.479
10.059
0.00
C


ATOM
257
C
GLY
A
37
17.046
32.001
9.799
0.00
C


ATOM
258
O
GLY
A
37
16.521
31.451
8.839
0.00
O


ATOM
259
N
ALA
A
38
17.842
31.349
10.640
0.00
N


ATOM
260
CA
ALA
A
38
18.095
29.924
10.470
0.00
C


ATOM
261
C
ALA
A
38
16.745
29.222
10.565
0.00
C


ATOM
262
O
ALA
A
38
15.881
29.657
11.324
0.00
O


ATOM
263
CB
ALA
A
38
19.026
29.426
11.566
0.00
C


ATOM
264
N
THR
A
39
16.553
28.151
9.800
0.00
N


ATOM
265
CA
THR
A
39
15.281
27.432
9.842
0.00
C


ATOM
266
CB
THR
A
39
14.779
27.066
8.425
0.00
C


ATOM
267
OG1
THR
A
39
15.582
26.012
7.887
0.00
O


ATOM
268
CG2
THR
A
39
14.857
28.277
7.504
0.00
C


ATOM
269
C
THR
A
39
15.433
26.157
10.664
0.00
C


ATOM
270
O
THR
A
39
16.533
25.637
10.821
0.00
O


ATOM
271
N
THR
A
40
14.328
25.649
11.186
0.00
N


ATOM
272
CA
THR
A
40
14.382
24.437
11.990
0.00
C


ATOM
273
CB
THR
A
40
14.143
24.753
13.473
0.00
C


ATOM
274
OG1
THR
A
40
12.807
25.242
13.636
0.00
O


ATOM
275
CG2
THR
A
40
15.124
25.799
13.962
0.00
C


ATOM
276
C
THR
A
40
13.332
23.421
11.581
0.00
C


ATOM
277
O
THR
A
40
12.345
23.760
10.927
0.00
O


ATOM
278
N
ALA
A
41
13.546
22.178
11.994
0.00
N


ATOM
279
CA
ALA
A
41
12.629
21.084
11.698
0.00
C


ATOM
280
C
ALA
A
41
12.368
20.368
13.030
0.00
C


ATOM
281
O
ALA
A
41
13.211
20.394
13.936
0.00
O


ATOM
282
CB
ALA
A
41
13.247
20.133
10.684
0.00
C


ATOM
283
N
ASN
A
42
11.206
19.734
13.149
0.00
N


ATOM
284
CA
ASN
A
42
10.839
19.022
14.370
0.00
C


ATOM
285
C
ASN
A
42
11.037
19.959
15.555
0.00
C


ATOM
286
O
ASN
A
42
11.861
19.693
16.424
0.00
O


ATOM
287
CB
ASN
A
42
11.720
17.780
14.584
0.00
C


ATOM
288
CG
ASN
A
42
11.686
16.812
13.408
0.00
C


ATOM
289
OD1
ASN
A
42
10.687
16.713
12.695
0.00
O


ATOM
290
ND2
ASN
A
42
12.779
16.076
13.217
0.00
N


ATOM
291
N
PRO
A
43
10.258
21.046
15.635
0.00
N


ATOM
292
CA
PRO
A
43
9.206
21.493
14.718
0.00
C


ATOM
293
CB
PRO
A
43
8.274
22.244
15.649
0.00
C


ATOM
294
C
PRO
A
43
9.697
22.416
13.612
0.00
C


ATOM
295
O
PRO
A
43
10.816
22.920
13.660
0.00
O


ATOM
296
CD
PRO
A
43
10.319
21.934
16.809
0.00
C


ATOM
297
CG
PRO
A
43
9.278
23.008
16.480
0.00
C


ATOM
298
N
THR
A
44
8.841
22.652
12.621
0.00
N


ATOM
299
CA
THR
A
44
9.208
23.533
11.522
0.00
C


ATOM
300
CB
THR
A
44
8.225
23.421
10.345
0.00
C


ATOM
301
C
THR
A
44
9.142
24.934
12.110
0.00
C


ATOM
302
O
THR
A
44
8.162
25.293
12.772
0.00
O


ATOM
303
OG1
THR
A
44
8.437
22.176
9.671
0.00
O


ATOM
304
CG2
THR
A
44
8.423
24.566
9.366
0.00
C


ATOM
305
N
GLY
A
45
10.196
25.710
11.893
0.00
N


ATOM
306
CA
GLY
A
45
10.233
27.057
12.425
0.00
C


ATOM
307
C
GLY
A
45
11.421
27.851
11.920
0.00
C


ATOM
308
O
GLY
A
45
12.226
27.355
11.120
0.00
O


ATOM
309
N
THR
A
46
11.537
29.084
12.401
0.00
N


ATOM
310
CA
THR
A
46
12.615
29.979
11.998
0.00
C


ATOM
311
CB
THR
A
46
12.134
30.919
10.867
0.00
C


ATOM
312
OG1
THR
A
46
11.720
30.132
9.741
0.00
O


ATOM
313
CG2
THR
A
46
13.246
31.872
10.438
0.00
C


ATOM
314
C
THR
A
46
13.097
30.831
13.171
0.00
C


ATOM
315
O
THR
A
46
12.287
31.407
13.909
0.00
O


ATOM
316
N
PHE
A
47
14.412
30.903
13.358
0.00
N


ATOM
317
CA
PHE
A
47
14.954
31.702
14.451
0.00
C


ATOM
318
CB
PHE
A
47
16.478
31.585
14.530
0.00
C


ATOM
319
CG
PHE
A
47
16.959
30.410
15.339
0.00
C


ATOM
320
CD2
PHE
A
47
17.538
30.606
16.590
0.00
C


ATOM
321
CD1
PHE
A
47
16.843
29.115
14.857
0.00
C


ATOM
322
CE2
PHE
A
47
17.996
29.532
17.345
0.00
C


ATOM
323
CE1
PHE
A
47
17.300
28.030
15.608
0.00
C


ATOM
324
CZ
PHE
A
47
17.878
26.241
16.855
0.00
C


ATOM
325
C
PHE
A
47
14.567
33.160
14.226
0.00
C


ATOM
326
O
PHE
A
47
14.665
33.686
13.111
0.00
O


ATOM
327
N
ALA
A
46
14.102
33.795
15.291
0.00
N


ATOM
328
CA
ALA
A
48
13.690
35.184
15.245
0.00
C


ATOM
329
CB
ALA
A
48
12.161
35.280
15.133
0.00
C


ATOM
330
C
ALA
A
48
14.174
35.828
16.532
0.00
C


ATOM
331
O
ALA
A
48
13.389
36.116
17.433
0.00
O


ATOM
332
N
GLY
A
49
15.481
36.038
16.609
0.00
N


ATOM
333
CA
GLY
A
49
16.072
36.635
17.791
0.00
C


ATOM
334
C
GLY
A
49
17.068
35.674
18.415
0.00
C


ATOM
335
O
GLY
A
49
16.698
34.589
18.867
0.00
O


ATOM
336
N
SER
A
50
18.333
36.073
18.438
0.00
N


ATOM
337
CA
SER
A
50
19.387
35.248
18.999
0.00
C


ATOM
338
CB
SER
A
50
19.976
34.360
17.899
0.00
C


ATOM
339
OG
SER
A
50
21.019
33.552
18.406
0.00
O


ATOM
340
C
SER
A
50
20.484
36.112
19.633
0.00
C


ATOM
341
O
SER
A
50
20.999
37.045
19.012
0.00
O


ATOM
342
N
SER
A
51
20.832
35.794
20.877
0.00
N


ATOM
343
CA
SER
A
51
21.860
36.529
21.603
0.00
C


ATOM
344
CB
SER
A
51
21.228
37.337
22.741
0.00
C


ATOM
345
OG
SER
A
51
22.179
38.189
23.359
0.00
O


ATOM
346
C
SER
A
53
22.938
35.596
22.162
0.00
C


ATOM
347
O
SER
A
51
22.700
34.819
22.089
0.00
O


ATOM
348
N
PHE
A
52
24.127
35.692
21.579
0.00
N


ATOM
349
CA
PHE
A
52
25.277
34.869
21.970
0.00
C


ATOM
350
CB
PHE
A
52
25.031
33.414
21.643
0.00
C


ATOM
351
CG
PHE
A
52
26.204
32.518
21.941
0.00
C


ATOM
352
CD1
PHE
A
52
26.485
32.124
23.238
0.00
C


ATOM
353
CD2
PHE
A
52
27.034
32.081
20.922
0.00
C


ATOM
354
CE1
PHE
A
52
27.575
31.312
23.516
0.00
C


ATOM
355
CE2
PHE
A
52
28.131
31.266
21.193
0.00
C


ATOM
356
CZ
PHE
A
52
28.400
30.883
27.492
0.00
C


ATOM
357
C
PHE
A
52
26.468
35.390
21.167
0.00
C


ATOM
358
O
PHE
A
52
26.370
35.589
19.960
0.00
C


ATOM
359
N
PRO
A
53
27.612
35.603
21.827
0.00
N


ATOM
360
CD
PRO
A
53
28.893
35.756
21.110
0.00
C


ATOM
361
CA
PRO
A
53
27.831
35.405
23.266
0.00
C


ATOM
362
CB
PRO
A
53
29.351
35.249
23.361
0.00
C


ATOM
363
CG
PRO
A
53
29.851
36.088
22.223
0.00
C


ATOM
364
C
PRO
A
53
27.268
36.543
24.132
0.00
C


ATOM
365
O
PRO
A
53
26.346
37.235
23.713
0.00
O


ATOM
366
N
GLY
A
54
27.814
36.744
25.328
0.00
N


ATOM
367
CA
GLY
A
54
27.288
37.777
26.211
0.00
C


ATOM
368
C
GLY
A
54
26.143
37.138
26.980
0.00
C


ATOM
369
O
GLY
A
54
26.210
36.964
28.197
0.00
O


ATOM
370
N
ASN
A
55
25.079
36.806
26.254
0.00
N


ATOM
371
CA
ASN
A
55
23.922
36.103
26.810
0.00
C


ATOM
372
CB
ASN
A
55
22.579
36.740
26.404
0.00
C


ATOM
373
CG
ASN
A
55
22.516
38.240
26.641
0.00
C


ATOM
374
OD1
ASN
A
55
22.161
39.005
25.734
0.00
O


ATOM
375
ND2
ASN
A
55
22.833
38.667
27.857
0.00
N


ATOM
376
C
ASN
A
55
24.011
34.788
26.037
0.00
C


ATOM
377
O
ASN
A
55
24.998
34.538
25.333
0.00
O


ATOM
378
N
ASP
A
56
22.980
33.958
26.171
0.00
N


ATOM
379
CA
ASP
A
56
22.917
32.682
25.473
0.00
C


ATOM
380
CB
ASP
A
56
23.774
31.595
26.119
0.00
C


ATOM
381
CG
ASP
A
56
23.987
30.395
25.179
0.00
C


ATOM
382
OD1
ASP
A
56
24.631
29.408
25.585
0.00
O


ATOM
383
OD2
ASP
A
56
23.504
30.443
24.024
0.00
O1−


ATOM
384
C
ASP
A
56
21.470
32.221
25.379
0.00
C


ATOM
385
O
ASP
A
56
21.078
31.195
25.930
0.00
O


ATOM
386
N
TYR
A
57
20.672
33.008
24.671
0.00
N


ATOM
387
CA
TYR
A
57
19.266
32.693
24.485
0.00
C


ATOM
388
CB
TYR
A
57
18.396
33.484
25.463
0.00
C


ATOM
389
CG
TYR
A
57
18.527
34.993
25.374
0.00
C


ATOM
390
CD1
TYR
A
57
19.153
35.711
26.390
0.00
C


ATOM
391
CE1
TYR
A
57
19.231
37.092
26.352
0.00
C


ATOM
392
CD2
TYR
A
57
17.986
35.706
24.303
0.00
C


ATOM
393
CE2
TYR
A
57
18.060
37.093
24.255
0.00
C


ATOM
394
CZ
TYR
A
57
18.682
37.781
25.289
0.00
C


ATOM
395
OH
TYR
A
57
18.732
39.165
25.286
0.00
O


ATOM
396
C
TYR
A
57
18.820
32.998
23.062
0.00
C


ATOM
397
O
TYR
A
57
19.438
33.800
22.355
0.00
O


ATOM
398
N
ALA
A
58
17.742
32.344
22.652
0.00
N


ATOM
399
CA
ALA
A
58
17.187
32.532
21.323
0.00
C


ATOM
400
CB
ALA
A
58
17.899
31.645
20.312
0.00
C


ATOM
401
C
ALA
A
58
15.706
32.191
21.360
0.00
C


ATOM
402
O
ALA
A
58
15.228
31.521
22.284
0.00
O


ATOM
403
N
PHE
A
59
14.989
32.683
20.359
0.00
N


ATOM
404
CA
PHE
A
59
13.564
32.453
20.225
0.00
C


ATOM
405
CB
PHE
A
59
12.762
33.735
20.438
0.00
C


ATOM
406
CO
PHE
A
59
11.333
33.629
19.970
0.00
C


ATOM
407
CD2
PHE
A
59
10.859
34.437
18.947
0.00
C


ATOM
408
CD1
PHE
A
59
10.475
32.698
20.531
0.00
C


ATOM
409
CE2
PHE
A
59
9.553
34.316
18.491
0.00
C


ATOM
410
CE1
PHE
A
59
9.175
32.573
20.084
0.00
C


ATOM
411
CZ
PHE
A
59
8.712
33.382
19.063
0.00
C


ATOM
412
C
PHE
A
59
13.294
31.942
18.816
0.00
C


ATOM
413
O
PHE
A
59
13.693
32.562
17.820
0.00
O


ATOM
414
N
VAL
A
60
12.616
30.809
18.731
0.00
N


ATOM
415
CA
VAL
A
60
12.308
30.253
17.434
0.00
C


ATOM
416
CB
VAL
A
60
12.702
28.776
17.340
0.00
C


ATOM
417
CG1
VAL
A
60
12.503
28.279
15.908
0.00
C


ATOM
418
CG2
VAL
A
60
14.147
28.593
17.796
0.00
C


ATOM
419
C
VAL
A
60
10.816
30.361
17.236
0.00
C


ATOM
420
O
VAL
A
60
10.043
29.927
18.087
0.00
O


ATOM
421
N
ARG
A
61
10.406
30.960
16.126
0.00
N


ATOM
422
CA
APR
A
61
8.987
31.098
15.851
0.00
C


ATOM
423
CB
ARG
A
61
8.704
32.313
14.962
0.00
C


ATOM
424
CG
ARG
A
61
7.255
32.374
14.430
0.00
C


ATOM
425
CD
ARG
A
61
7.019
33.543
13.521
0.00
C


ATOM
426
NE
ARG
A
61
5.615
33.660
13.118
0.00
N1+


ATOM
427
CZ
ARG
A
61
4.989
32.815
12.303
0.00
C


ATOM
428
NH2
ARG
A
61
3.711
33.007
12.004
0.00
N


ATOM
429
NH1
ARG
A
61
5.636
31.777
11.787
0.00
N


ATOM
430
C
ARG
A
61
8.509
29.847
15.128
0.00
C


ATOM
431
O
ARG
A
61
9.193
29.338
14.238
0.00
O


ATOM
432
N
THR
A
62
7.338
29.357
15.527
0.00
N


ATOM
433
CA
THR
A
62
6.740
28.170
14.923
0.00
C


ATOM
434
CB
THR
A
62
6.514
27.046
15.956
0.00
C


ATOM
435
OG1
THR
A
62
5.808
27.570
17.089
0.00
O


ATOM
436
CG2
THR
A
62
7.845
26.460
16.396
0.00
C


ATOM
437
C
THR
A
62
5.391
28.597
14.352
0.00
C


ATOM
438
O
THR
A
62
4.857
29.645
14.724
0.00
O


ATOM
439
N
GLY
A
63
4.837
27.791
13.455
0.00
N


ATOM
440
CA
GLY
A
63
3.562
28.146
12.859
0.00
C


ATOM
441
C
GLY
A
63
2.522
27.046
12.880
0.00
C


ATOM
442
O
GLY
A
63
2.375
26.326
13.873
0.00
O


ATOM
443
N
ALA
A
64
1.806
26.909
11.767
0.00
N


ATOM
444
CA
ALA
A
64
0.744
25.916
11.643
0.00
C


ATOM
445
C
ALA
A
64
1.213
24.496
11.895
0.00
C


ATOM
446
O
ALA
A
64
2.370
24.154
11.651
0.00
O


ATOM
447
CB
ALA
A
64
0.111
26.009
10.268
0.00
C


ATOM
448
N
GLY
A
65
0.291
23.672
12.381
0.00
N


ATOM
449
CA
GLY
A
65
0.596
22.281
12.657
0.00
C


ATOM
450
C
GLY
A
65
1.469
22.050
13.877
0.00
C


ATOM
451
O
GLY
A
65
1.797
20.908
14.199
0.00
O


ATOM
452
N
VAL
A
66
1.837
23.119
14.572
0.00
N


ATOM
453
CA
VAL
A
66
2.699
22.976
15.736
0.00
C


ATOM
454
CB
VAL
A
66
3.946
23.854
15.595
0.00
C


ATOM
455
C
VAL
A
66
2.031
23.307
17.063
0.00
C


ATOM
456
O
VAL
A
66
1.737
24.467
17.337
0.00
O


ATOM
457
CGI
VAL
A
66
4.832
23.683
16.818
0.00
C


ATOM
458
CG2
VAL
A
66
4.698
23.482
14.324
0.00
C


ATOM
459
N
ASN
A
67
1.806
22.283
17.882
0.00
N


ATOM
460
CA
ASN
A
67
1.176
22.454
19.185
0.00
C


ATOM
461
CB
ASN
A
67
0.403
21.188
19.564
0.00
C


ATOM
462
C
ASN
A
67
2.240
22.745
20.237
0.00
C


ATOM
463
O
ASN
A
67
3.120
21.920
20.491
0.00
O


ATOM
464
CG
ASN
A
67
−0.405
20.634
18.404
0.00
C


ATOM
465
OD1
ASN
A
67
−1.160
21.361
17.750
0.00
O


ATOM
466
ND2
ASN
A
67
−0.253
19.340
18.140
0.00
N


ATOM
467
N
LEU
A
68
2.148
23.923
20.845
0.00
N


ATOM
468
CA
LEU
A
68
3.087
24.366
21.876
0.00
C


ATOM
469
CB
LEU
A
68
3.279
25.883
21.759
0.00
C


ATOM
470
C
LEU
A
68
2.571
23.996
23.273
0.00
C


ATOM
471
O
LEU
A
68
1.620
24.597
23.770
0.00
O


ATOM
472
CG
LEU
A
68
3.688
26.430
20.380
0.00
C


ATOM
473
CD1
LEU
A
68
3.724
27.950
20.406
0.00
C


ATOM
474
CD2
LEU
A
68
5.051
25.888
19.987
0.00
C


ATOM
475
N
LEU
A
69
3.218
23.027
23.917
0.00
N


ATOM
476
CA
LEU
A
69
2.803
22.584
25.250
0.00
C


ATOM
477
CB
LEU
A
69
2.769
21.052
25.286
0.00
C


ATOM
478
CG
LEU
A
69
2.045
20.369
24.116
0.00
C


ATOM
479
CD1
LEU
A
69
2.109
18.857
24.274
0.00
C


ATOM
460
CD2
LEU
A
69
0.604
20.841
24.050
0.00
C


ATOM
481
C
LEU
A
69
3.612
23.078
26.449
0.00
C


ATOM
482
O
LEU
A
69
4.635
23.216
26.394
0.00
O


ATOM
483
N
ALA
A
70
2.907
23.332
27.544
0.00
N


ATOM
484
CA
ALA
A
70
3.534
23.796
28.773
0.00
C


ATOM
485
CB
ALA
A
70
2.507
24.496
29.646
0.00
C


ATOM
486
C
ALA
A
70
4.048
22.539
29.473
0.00
C


ATOM
487
O
ALA
A
70
3.712
22.273
30.618
0.00
O


ATOM
488
N
GLN
A
71
4.862
21.763
28.770
0.00
N


ATOM
489
CA
GLN
A
71
5.408
20.536
29.325
0.00
C


ATOM
490
CB
GLN
A
71
4.618
19.333
28.808
0.00
C


ATOM
491
CG
GLN
A
71
3.169
19.299
29.255
0.00
C


ATOM
492
CD
GLN
A
71
2.407
18.116
28.692
0.00
C


ATOM
493
OE1
GLN
A
71
1.460
17.634
29.308
0.00
O


ATOM
494
NE2
GLN
A
71
2.809
17.646
27.515
0.00
N


ATOM
495
C
GLN
A
71
6.869
20.310
28.998
0.00
C


ATOM
496
O
GLN
A
71
7.395
20.825
28.009
0.00
O


ATOM
497
N
VAL
A
72
7.520
19.529
29.850
0.00
N


ATOM
498
CA
VAL
A
12
8.924
19.199
29.676
0.00
C


ATOM
499
CB
VAL
A
12
9.809
19.799
30.777
0.00
C


ATOM
500
CG1
VAL
A
72
11.240
19.342
30.580
0.00
C


ATOM
501
CG2
VAL
A
72
9.726
21.309
30.758
0.00
C


ATOM
502
C
VAL
A
72
8.997
17.685
29.772
0.00
C


ATOM
503
O
VAL
A
72
8.419
17.086
30.680
0.00
O


ATOM
504
N
ASN
A
73
9.699
17.075
28.824
0.00
N


ATOM
505
CA
ASN
A
73
9.867
15.629
28.771
0.00
C


ATOM
506
CB
ASN
A
73
10.543
15.250
27.452
0.00
C


ATOM
507
CG
ASN
A
73
10.513
13.756
27.182
0.00
C


ATOM
508
OD1
ASN
A
73
10.470
12.947
28.106
0.00
O


ATOM
509
ND2
ASN
A
73
10.551
13.387
25.906
0.00
N


ATOM
510
C
ASN
A
73
10.735
15.146
29.931
0.00
C


ATOM
511
O
ASN
A
73
11.843
15.651
30.123
0.00
O


ATOM
512
N
ASN
A
74
10.244
14.175
30.703
0.00
N


ATOM
513
CA
ASN
A
74
11.028
13.663
31.823
0.00
C


ATOM
514
CB
ASN
A
74
10.151
13.368
33.049
0.00
C


ATOM
515
CG
ASN
A
74
9.191
12.217
32.830
0.00
C


ATOM
516
OD1
ASN
A
74
9.486
11.265
32.108
0.00
O


ATOM
517
ND2
ASN
A
74
8.032
12.291
33.477
0.00
N


ATOM
518
C
ASN
A
74
11.791
12.408
31.417
0.00
C


ATOM
519
O
ASN
A
74
12.332
11.695
32.266
0.00
O


ATOM
520
N
TYR
A
75
11.830
12.156
30.112
0.00
N


ATOM
521
CA
TYR
A
75
12.514
11.005
29.528
0.00
C


ATOM
522
CB
TYR
A
75
14.008
11.321
29.354
0.00
C


ATOM
523
CG
TYR
A
75
14.268
12.239
28.181
0.00
C


ATOM
524
CD1
TYR
A
75
14.228
11.756
26.873
0.00
C


ATOM
525
CE1
TYR
A
75
14.371
12.597
25.792
0.00
C


ATOM
526
CD2
TYR
A
75
14.466
13.599
28.370
0.00
C


ATOM
527
CE2
TYR
A
75
14.608
14.451
27.290
0.00
C


ATOM
528
CZ
TYR
A
75
14.557
13.945
26.005
0.00
C


ATOM
529
OH
TYR
A
75
14.679
14.796
24.931
0.00
O


ATOM
530
C
TYR
A
75
12.326
9.680
30.260
0.00
C


ATOM
531
O
TYR
A
75
13.253
8.875
30.378
0.00
O


ATOM
532
N
SER
A
76
11.112
9.464
30.747
0.00
N


ATOM
533
CA
SER
A
76
10.773
8.244
31.458
0.00
C


ATOM
534
CB
SER
A
76
10.737
8.471
32.968
0.00
C


ATOM
535
OG
SER
A
76
12.053
8.566
33.47S
0.00
O


ATOM
536
C
SER
A
76
9.407
7.816
30.963
0.00
C


ATOM
537
O
SER
A
76
8.784
6.905
31.512
0.00
O


ATOM
538
N
GLY
A
77
8.960
8.483
29.905
0.00
N


ATOM
539
CA
GLY
A
77
7.671
8.180
29.320
0.00
C


ATOM
540
C
GLY
A
77
6.610
9.168
29.755
0.00
C


ATOM
541
O
GLY
A
77
5.464
9.090
29.311
0.00
O


ATOM
542
N
GLY
A
78
6.992
10.102
30.622
0.00
N


ATOM
543
CA
GLY
A
78
6.043
11.087
31.101
0.00
C


ATOM
544
C
GLY
A
78
6.492
12.522
30.887
0.00
C


ATOM
545
O
GLY
A
78
7.545
12.775
30.293
0.00
O


ATOM
546
N
ARG
A
79
5.681
13.462
31.315
0.00
N


ATOM
547
CA
ARG
A
79
5.986
14.879
31.240
0.00
C


ATOM
548
CB
ARG
A
79
5.099
15.537
30.166
0.00
C


ATOM
549
CG
ARG
A
75
5.356
15.117
20.732
0.00
C


ATOM
550
CD
ARG
A
79
4.770
13.772
28.432
0.00
C


ATOM
551
NE
ARG
A
79
4.975
13.348
27.048
0.00
N1+


ATOM
552
CZ
ARG
A
79
6.081
12.764
26.593
0.00
C


ATOM
553
NH1
ARG
A
79
7.095
12.529
27.412
0.00
N


ATOM
554
NH2
ARG
A
79
6.165
12.399
25.319
0.00
N


ATOM
555
C
ARG
A
79
5.797
15.627
32.550
0.00
C


ATOM
556
O
ARG
A
79
5.129
15.149
33.466
0.00
O


ATOM
557
N
VAL
A
80
6.398
16.809
32.620
0.00
N


ATOM
558
CA
VAL
A
80
6.317
17.656
33.794
0.00
C


ATOM
559
CB
VAL
A
80
7.714
17.891
34.389
0.00
C


ATOM
560
CG1
VAL
A
80
7.630
18.858
35.546
0.00
C


ATOM
561
CG2
VAL
A
80
8.300
16.566
34.853
0.00
C


ATOM
562
C
VAL
A
80
5.682
18.989
33.398
0.00
C


ATOM
563
O
VAL
A
80
6.182
19.701
32.517
0.00
O


ATOM
564
N
GLN
A
81
4.561
19.307
34.036
0.00
N


ATOM
565
CA
GLN
A
81
3.846
20.546
33.760
0.00
C


ATOM
566
CB
GLN
A
81
2.516
20.579
34.527
0.00
C


ATOM
567
CG
GLN
A
81
1.415
19.722
33.930
0.00
C


ATOM
568
CD
GLN
A
81
1.138
20.067
32.473
0.00
C


ATOM
569
OE1
GLN
A
81
1.086
21.239
32.101
0.00
O


ATOM
570
NE2
GLN
A
81
0.950
19.044
31.644
0.00
N


ATOM
571
C
GLN
A
81
4.650
21.785
34.126
0.00
C


ATOM
572
O
GLN
A
81
5.333
21.817
35.154
0.00
O


ATOM
573
N
VAL
A
82
4.578
22.798
33.272
0.00
N


ATOM
574
CA
VAL
A
82
5.288
24.047
33.510
0.00
C


ATOM
575
CB
VAL
A
82
5.925
24.610
32.219
0.00
C


ATOM
576
CG1
VAL
A
82
6.695
25.885
32.535
0.00
C


ATOM
577
CG2
VAL
A
82
6.841
23.580
31.605
0.00
C


ATOM
578
C
VAL
A
82
4.222
25.022
34.003
0.00
C


ATOM
579
O
VAL
A
82
3.399
25.500
33.221
0.00
O


ATOM
580
N
ALA
A
83
4.242
25.305
35.302
0.00
N


ATOM
581
CA
ALA 
A
83
3.277
26.213
35.919
0.00
C


ATOM
582
CB
ALA
A
83
3.009
25.768
37.342
0.00
C


ATOM
583
C
ALA 
A
83
3.677
27.688
35.909
0.00
C


ATOM
584
O
ALA
A
83
2.820
28.571
35.886
0.00
O


ATOM
585
N
GLY
A
84
4.975
27.960
35.934
0.00
N


ATOM
586
CA
GLY
A
84
5.413
29.339
35.930
0.00
C


ATOM
587
C
GLY
A
84
6.913
29.458
35.790
0.00
C


ATOM
588
O
GLY
A
84
7.601
28.463
35.536
0.00
O


ATOM
589
N
HIS
A
85
7.426
30.673
35.960
0.00
N


ATOM
590
CA
HIS
A
85
8.856
30.903
35.841
0.00
C


ATOM
591
CB
HIS
A
85
9.154
31.684
34.557
0.00
C


ATOM
592
C
HIS
A
85
9.476
31.610
37.037
0.00
C


ATOM
593
O
HIS
A
85
10.275
32.530
36.866
0.00
O


ATOM
594
CG
HIS
A
85
8.328
32.920
34.391
0.00
C


ATOM
595
ND1
HIS
A
85
6.864
34.188
34.455
0.00
N


ATOM
596
CD2
HIS
A
85
7.006
33.083
34.147
0.00
C


ATOM
597
NE2
HIS
A
85
6.772
34.434
34.067
0.00
N


ATOM
598
CE1
HIS
A
85
7.909
35.078
34.256
0.00
C


ATOM
599
N
THR
A
86
9.115
31.163
38.243
0.00
N


ATOM
600
CA
THR
A
86
9.631
31.738
39.491
0.00
C


ATOM
601
CB
THR
A
86
8.743
31.356
40.683
0.00
C


ATOM
602
C
THR
A
86
11.055
31.244
39.779
0.00
C


ATOM
603
O
THR
A
86
11.289
30.047
39.908
0.00
O


ATOM
604
OG1
THR
A
86
7.417
31.837
40.456
0.00
O


ATOM
605
CG2
THR 
A
86
9.283
31.957
41.971
0.00
C


ATOM
606
N
ALA
A
87
11.996
32.175
39.894
0.00
N


ATOM
607
CA
ALA
A
87
13.391
31.830
40.160
0.00
C


ATOM
608
C
ALA
A
87
13.621
31.173
41.519
0.00
C


ATOM
609
O
ALA
A
87
13.145
31.649
42.546
0.00
O


ATOM
610
CB
ALA
A
87
14.265
33.077
40.032
0.00
C


ATOM
611
N
ALA
A
88
14.360
30.074
41.517
0.00
N


ATOM
612
CA
ALA
A
88
14.653
29.360
42.747
0.00
C


ATOM
613
C
ALA
A
88
16.009
29.831
43.263
0.00
C


ATOM
614
O
ALA
A
88
16.904
27.855
42.490
0.00
C


ATOM
615
CB
ALA
A
88
14.662
30.156
42.482
0.00
O


ATOM
616
N
PRO
A
89
16.173
29.877
44.592
0.00
N


ATOM
617
CA
PRO
A
89
17.404
30.310
45.260
0.00
C


ATOM
618
CB
PRO
A
89
16.939
30.560
46.690
0.00
C


ATOM
619
C
PRO
A
89
18.545
29.304
45.204
0.00
C


ATOM
620
O
PRO
A
89
18.323
28.106
45.007
0.00
O


ATOM
621
CD
PRO
A
89
15.136
29.511
45.576
0.00
C


ATOM
622
CG
PRO
A
89
15.917
29.473
46.866
0.00
C


ATOM
623
N
VAL
A
90
19.767
29.809
45.361
0.00
N


ATOM
624
CA
VAL
A
90
20.960
28.973
45.343
0.00
C


ATOM
625
CB
VAL 
A
90
22.197
29.747
45.873
0.00
C


ATOM
626
CG1
VAL
A
90
23.347
28.779
46.162
0.00
C


ATOM
627
CG2
VAL
A
90
22.623
30.803
44.858
0.00
C


ATOM
628
C
VAL
A
90
20.680
21.810
46.279
0.00
C


ATOM
629
O
VAL
A
90
19.988
27.976
47.287
0.00
O


ATOM
630
N
GLY
A
91
21.205
26.635
45.954
0.00
N


ATOM
631
CA
GLY
A
91
20.976
25.482
46.806
0.00
C


ATOM
632
C
GLY
A
91
19.734
24.699
46.425
0.00
C


ATOM
633
O
GLY
A
91
19.576
23.550
46.836
0.00
O


ATOM
634
N
SER 
A
92
18.853
25.314
45.642
0.00
N


ATOM
635
CA
SER 
A
92
17.620
24.659
45.210
0.00
C


ATOM
636
CB
SER 
A
92
16.720
25.634
44.438
0.00
C


ATOM
637
OG
SER 
A
92
16.414
26.792
45.196
0.00
O


ATOM
638
C
SER 
A
92
17.929
23.479
44.306
0.00
C


ATOM
639
O
SER 
A
92
18.881
23.512
43.521
0.00
O


ATOM
640
N
ALA
A
93
17.117
22.435
44.417
0.00
N


ATOM
641
CA
ALA
A
93
17.301
21.247
43.600
0.00
C


ATOM
642
CB
ALA
A
93
16.576
20.054
44.231
0.00
C


ATOM
643
C
ALA
A
93
16.715
21.559
42.226
0.00
C


ATOM
644
O
ALA
A
93
15.669
22.202
42.126
0.00
O


ATOM
645
N
VAL
A
94
17.396
21.125
41.170
0.00
N


ATOM
646
CA
VAL
A
94
16.916
21.371
39.814
0.00
C


ATOM
647
CB
VAL
A
94
17.544
22.639
39.212
0.00
C


ATOM
648
CG1
VAL
A
91
17.072
23.872
39.971
0.00
C


ATOM
649
CG2
VAL
A
94
19.050
22.534
39.241
0.00
C


ATOM
650
C
VAL
A
94
17.204
20.211
38.867
0.00
C


ATOM
651
O
VAL
A
94
18.143
19.432
39.071
0.00
O


ATOM
652
N
CYS
A
95
16.378
20.090
37.834
0.00
N


ATOM
653
CA
CYS
A
95
16.548
19.030
36.851
0.00
C


ATOM
654
CB
CYS
A
95
15.428
17.983
36.942
0.00
C


ATOM
655
SG
CYS
A
95
15.344
17.009
38.470
0.00
S


ATOM
656
C
CYS
A
95
16.552
19.647
35.464
0.00
C


ATOM
657
O
CYS
A
95
15.820
20.598
35.194
0.00
O


ATOM
658
N
ARG
A
96
17.391
19.092
34.598
0.00
N


ATOM
659
CA
ARG
A
96
17.531
19.551
33.228
0.00
C


ATOM
660
CB
ARG
A
96
19.003
19.840
32.935
0.00
C


ATOM
661
CG
ARG
A
96
19.300
20.110
31.465
0.00
C


ATOM
662
CD
ARG
A
96
20.778
19.955
31.151
0.00
C


ATOM
663
NE
ARG
A
96
21.272
18.625
31.499
0.00
N1+


ATOM
664
CZ
ARG
A
96
20.875
17.493
30.927
0.00
C


ATOM
665
NH1
ARG
A
96
19.967
17.507
29.960
0.00
N


ATOM
666
NH2
ARG
A
96
21.377
16.341
31.341
0.00
N


ATOM
667
C
ARG
A
96
17.027
18.487
32.258
0.00
C


ATOM
668
O
ARG
A
96
17.160
17.288
32.509
0.00
O


ATOM
669
N
SER 
A
97
16.458
18.924
31.142
0.00
N


ATOM
670
CA
SER 
A
97
15.950
17.983
30.157
0.00
C


ATOM
671
CB
SER 
A
97
14.418
18.054
30.082
0.00
C


ATOM
672
OG
SER 
A
97
13.913
17.133
29.134
0.00
O


ATOM
673
C
SER 
A
97
16.564
18.272
28.797
0.00
C


ATOM
674
O
SER 
A
97
16.429
19.378
26.267
0.00
O


ATOM
675
N
GLY
A
98
17.246
17.265
28.251
0.00
N


ATOM
676
CA
GLY
A
98
17.900
17.390
26.960
0.00
C


ATOM
677
C
GLY
A
98
17.767
16.133
26.115
0.00
C


ATOM
678
O
GLY
A
98
17.481
15.042
26.624
0.00
O


ATOM
679
N
SER 
A
99
17.997
16.290
24.814
0.00
N


ATOM
680
CA
SER 
A
99
17.865
15.196
23.864
0.00
C


ATOM
681
CB
SER 
A
99
17.547
15.749
22.478
0.00
C


ATOM
682
OG
SER 
A
99
18.663
16.457
21.968
0.00
O


ATOM
683
C
SER 
A
99
19.067
14.279
23.740
0.00
C


ATOM
684
O
SER 
A
99
18.992
13.275
23.039
0.00
O


ATOM
685
N
THR
A
100
20.174
14.612
24.396
0.00
N


ATOM
686
CA
THR
A
100
21.358
13.766
24.307
0.00
C


ATOM
687
CB
THR
A
100
22.641
14.602
24.125
0.00
C


ATOM
688
OG1
THR
A
100
22.516
15.417
22.953
0.00
O


ATOM
689
CG2
THR
A
100
23.860
13.690
23.967
0.00
C


ATOM
690
C
THR
A
100
21.531
12.872
25.524
0.00
C


ATOM
691
O
THR
A
100
21.890
11.697
25.368
0.00
O


ATOM
692
N
THR
A
101
21.269
13.422
26.707
0.00
N


ATOM
693
CA
THR
A
101
21.401
12.662
27.950
0.00
C


ATOM
694
CB
THR
A
101
22.375
13.340
28.936
0.00
C


ATOM
695
OG1
THR
A
101
21.791
14.549
29.441
0.00
O


ATOM
696
CG2
THR
A
101
23.692
13.642
28.244
0.00
C


ATOM
697
C
THR
A
101
20.083
12.442
28.677
0.00
C


ATOM
698
O
THR
A
101
19.990
11.581
29.548
0.00
O


ATOM
699
N
GLY
A
102
19.068
13.227
28.342
0.00
N


ATOM
700
CA
GLY
A
102
17.784
13.044
28.985
0.00
C


ATOM
701
C
GLY
A
102
17.522
13.868
30.231
0.00
C


ATOM
702
O
GLY
A
102
17.759
15.075
30.245
0.00
O


ATOM
703
N
TRP 
A
103
17.023
13.207
31.274
0.00
N


ATOM
704
CA
TRP 
A
103
16.681
13.847
32.540
0.00
C


ATOM
705
CB
TRP 
A
103
15.339
13.286
33.027
0.00
C


ATOM
706
CG
TRP 
A
103
14.790
13.876
34.311
0.00
C


ATOM
707
CD2
TRP 
A
103
13.889
14.989
34.434
0.00
C


ATOM
708
CE2
TRP 
A
103
13.601
15.141
35.807
0.00
C


ATOM
709
CE3
TRP 
A
103
13.293
15.858
33.517
0.00
C


ATOM
710
CD1
TRP
A
103
15.010
13.424
35.581
0.00
C


ATOM
711
NE1
TRP
A
103
14.295
14.179
36.483
0.00
N


ATOM
712
CZ2
TRP
A
103
12.749
16.138
36.284
0.00
C


ATOM
713
CZ3
TRP
A
103
12.444
16.848
33.991
0.00
C


ATOM
714
CH2
TRP
A
103
12.178
16.977
35.363
0.00
C


ATOM
715
C
TRP
A
103
17.776
13.652
33.593
0.00
C


ATOM
716
O
TRP
A
103
18.112
12.526
33.955
0.00
C


ATOM
717
N
HIS
A
104
18.333
14.762
34.074
0.00
N


ATOM
718
CA
HIS
A
104
19.391
14.731
35.081
0.00
C


ATOM
719
CB
HIS
A
104
20.755
14.780
34.405
o.oo
C


ATOM
720
CG
HIS
A
104
21.064
13.554
33.619
0.00
C


ATOM
721
CD2
HIS
A
104
20.741
13.202
32.352
0.00
C


ATOM
722
ND1
HIS
A
104
21.704
12.465
34.167
0.00
N


ATOM
723
CE1
HIS
A
104
21.759
11.496
33.273
0.00
C


ATOM
724
NE2
HIS
A
104
21.181
11.917
32.162
0.00
N


ATOM
725
C
HIS
A
104
19.229
15.894
36.038
0.00
C


ATOM
726
O
HIS
A
104
18.859
16.995
35.636
0.00
O


ATOM
111
N
CYS
A
105
19.512
15.647
37.309
0.00
N


ATOM
728
CA
CYS
A
105
19.373
16.692
38.303
0.00
C


ATOM
729
CB
CYS
A
105
18.223
16.317
39.234
0.00
C


ATOM
730
SG
CYS
A
105
16.804
15.602
38.342
0.00
S


ATOM
731
C
CYS
A
105
20.635
17.014
39.099
0.00
C


ATOM
732
O
CYS
A
105
21.665
16.361
38.964
0.00
O


ATOM
733
N
GLY
A
106
20.533
18.046
39.923
0.00
N


ATOM
734
CA
GLY
A
106
21.642
18.487
40.739
0.00
C


ATOM
735
C
GLY
A
106
21.125
19.693
41.490
0.00
C


ATOM
736
O
GLY
A
106
19.916
19.805
41.707
0.00
O


ATOM
737
N
THR
A
107
22.011
20.598
41.888
0.00
N


ATOM
738
CA
THR
A
107
21.572
21.784
42.603
0.00
C


ATOM
739
CB
THR
A
107
21.865
21.681
44.117
0.00
C


ATOM
740
OG1
THR
A
107
23.250
21.386
44.328
0.00
O


ATOM
741
CG2
THR
A
107
21.021
20.583
44.746
0.00
C


ATOM
742
C
THR
A
107
22.215
23.046
42.058
0.00
C


ATOM
743
O
THR
A
107
23.238
23.001
41.372
0.00
O


ATOM
744
N
ILE
A
108
21.588
24.178
42.344
0.00
N


ATOM
745
CA
ILE
A
108
22.103
25.452
41.886
0.00
C


ATOM
746
CB
ILE
A
108
21.039
26.550
41.982
0.00
C


ATOM
747
CG2
ILE
A
108
21.651
27 .904
41.614
0.00
C


ATOM
748
CG1
ILE
A
108
19.850
26.200
41.091
0.00
C


ATOM
749
CD1
ILE
A
108
18.691
27.166
41.228
0.00
C


ATOM
750
C
ILE
A
108
23.216
25.803
42.852
0.00
C


ATOM
751
O
ILE
A
108
23.001
25.774
44.065
0.00
O


ATOM
752
N
THR
A
109
24.399
26.118
42.331
0.00
N


ATOM
753
CA
THR
A
109
25.516
26.472
43.197
0.00
C


ATOM
754
CB
THR
A
109
26.787
25.662
42.849
0.00
C


ATOM
755
OG1
THR
A
109
27.055
25.748
41.443
0.00
O


ATOM
756
CG2
THR
A
109
26.599
24.201
43.244
0.00
C


ATOM
757
C
THR
A
109
25.827
27.965
43.128
0.00
C


ATOM
758
O
THR
A
109
26.599
28.481
43.928
0.00
O


ATOM
759
N
ALA
A
110
25.214
28.664
42.181
0.00
N


ATOM
760
CA
ALA
A
110
25.457
30.090
42.053
0.00
C


ATOM
761
CB
ALA
A
110
26.949
30.348
41.846
0.00
C


ATOM
762
C
ALA
A
110
24.650
30.767
40.946
0.00
C


ATOM
763
O
ALA
A
110
24.191
30.132
40.001
0.00
O


ATOM
764
N
LEU
A
111
24.491
32.078
41.092
0.00
N


ATOM
765
CA
LEU
A
111
23.755
32.893
40.145
0.00
C


ATOM
766
CB
LEU
A
111
22.550
33.530
40.844
0.00
C


ATOM
767
CG
LEU
A
111
21.547
32.545
41.465
0.00
C


ATOM
768
CD1
LEU
A
111
20.462
33.315
42.227
0.00
C


ATOM
769
CD2
LEU
A
111
20.925
31.681
40.371
0.00
C


ATOM
770
C
LEU
A
111
24.688
33.970
39.593
0.00
C


ATOM
771
O
LEU
A
111
25.661
34.363
40.243
0.00
O


ATOM
772
N
ASN
A
112
24.380
34.439
38.390
0.00
N


ATOM
773
CA
ASN
A
112
25.171
35.468
37.719
0.00
C


ATOM
774
CB
ASN
A
112
25.123
36.788
38.490
0.00
C


ATOM
775
CG
ASN
A
112
23.725
37.149
38.930
0.00
C


ATOM
776
OD1
ASN
A
112
23.244
36.663
39.950
0.00
O


ATOM
777
ND2
ASN
A
112
23.057
37.994
38.155
0.00
N


ATOM
778
C
ASN
A
112
26.629
35.074
37.507
0.00
C


ATOM
779
O
ASN
A
112
27.526
35.891
37.680
0.00
O


ATOM
780
N
SER
A
113
26.870
33.820
37.144
0.00
N


ATOM
781
CA
SER
A
113
28.237
33.383
36.916
0.00
C


ATOM
782
CB
SER
A
113
28.375
31.875
37.126
0.00
C


ATOM
783
OG
SER
A
113
28.149
31.522
38.479
0.00
O


ATOM
784
C
SER
A
113
28.604
33.726
35.480
0.00
C


ATOM
785
O
SER
A
113
27.733
34.045
34.663
0.00
O


ATOM
786
N
SER
A
114
29.899
33.691
35.192
0.00
N 


ATOM
787
CA
SER
A
114
30.403
33.985
33.859
0.00
C


ATOM
788
CB
SER
A
114
31.288
35.234
33.847
0.00
C


ATOM
789
OG
SER
A
114
30.524
36.417
33.962
0.00
O


ATOM
790
C
SER
A
114
31.237
32.795
33.440
0.00
C


ATOM
791
O
SER
A
114
31.708
32.027
34.277
0.00
O


ATOM
792
N
VAL
A
115
31.400
32.631
32.138
0.00
N


ATOM
793
CA
VAL
A
115
32.185
31.535
31.611
0.00
C


ATOM
794
CB
VAL
A
115
31.342
30.241
31.427
0.00
C


ATOM
795
CG1
VAL
A
115
30.850
29.751
32.772
0.00
C


ATOM
796
CG2
VAL
A
115
30.174
30.492
30.485
0.00
C


ATOM
797
C
VAL
A
115
32.678
32.010
30.266
0.00
C


ATOM
798
O
VAL
A
115
32.121
32.945
29.691
0.00
O


ATOM
799
N
THR
A
116
33.735
31.387
29.768
0.00
N


ATOM
800
CA
THR
A
116
34.268
31.779
28.482
0.00
C


ATOM
801
CB
THR
A
116
35.671
32.359
28.634
0.00
C


ATOM
802
OG1
THR
A
116
35.602
33.538
29.446
0.00
O


ATOM
803
CG2
THR
A
116
36.247
32.726
27.277
0.00
C


ATOM
804
C
THR
A
116
34.278
30.589
27.535
0.00
C


ATOM
805
O
THR
A
116
34.931
29.573
27.787
0.00
O


ATOM
806
N
TYR
A
117
33.512
30.721
26.459
0.00
N


ATOM
807
CA
TYR
A
117
33.397
29.691
25.443
0.00
C


ATOM
808
CB
TYR
A
117
31.972
29.664
24.885
0.00
C


ATOM
809
CG
TYR
A
117
30.892
29.244
25.874
0.00
C


ATOM
810
CD1
TYR
A
117
29.803
30.072
26.134
0.00
C


ATOM
811
CE1
TYR
A
117
28.780
29.675
26.981
0.00
C


ATOM
812
CD2
TYR
A
117
30.930
27.998
26.499
0.00
C


ATOM
813
CE2
TYR
A
117
29.908
27.589
27.353
0.00
C


ATOM
814
CZ
TYR
A
117
28.834
28.432
27.590
0.00
C


ATOM
815
OH
TYR
A
117
27.814
28.036
28.431
0.00
O


ATOM
816
C
TYR
A
117
34.368
30.064
24.333
0.00
C


ATOM
817
O
TYR
A
117
34.922
31.161
24.329
0.00
O


ATOM
818
N
PRO
A
118
34.609
29.151
23.384
0.00
N


ATOM
819
CA
PRO
A
118
35.541
29.528
22.318
0.00
C


ATOM
820
CB
PRO
A
118
35.655
28.249
21.472
0.00
C


ATOM
821
C
PRO
A
118
35.080
30.751
21.520
0.00
C


ATOM
822
O
PRO
A
118
35.875
31.369
20.805
0.00
O


ATOM
823
CD
PRO
A
118
34.313
27.711
23.347
0.00
C


ATOM
824
CG
PRO
A
118
34.472
27.405
21.890
0.00
C


ATOM
825
N
GLU
A
119
33.804
31.109
21.651
0.00
N


ATOM
826
CA
GLU
A
119
33.266
32.265
20.935
0.00
C


ATOM
827
CB
GLU
A
119
31.785
32.079
20.612
0.00
C


ATOM
828
C
GLU
A
119
33.416
33.514
21.789
0.00
C


ATOM
829
O
GLU
A
119
33.498
34.634
21.275
0.00
O


ATOM
830
CG
GLU
A
119
31.470
30.965
19.611
0.00
C


ATOM
831
CD
GLU
A
119
31.686
29.606
20.179
0.00
C


ATOM
832
OE1
GLU
A
119
31.593
29.460
21.415
0.00
O1−


ATOM
833
OE2
GLU
A
119
31.932
28.667
19.393
0.00
O


ATOM
834
N
GLY
A
120
33.437
33.315
23.102
0.00
N


ATOM
835
CA
GLY
A
120
33.575
34.438
24.002
0.00
C


ATOM
836
C
GLY
A
120
32.984
34.167
25.368
0.00
C


ATOM
837
O
GLY
A
120
32.714
33.020
25.727
0.00
O


ATOM
838
N
THR
A
121
32.782
35.233
26.133
0.00
N


ATOM
839
CA
THR
A
121
32.233
35.110
27.471
0.00
C


ATOM
840
CB
THR
A
121
32.932
36.084
28.435
0.00
C


ATOM
841
OG1
THR
A
121
34.319
35.733
28.527
0.00
O


ATOM
842
CG2
THR
A
121
32.293
36.026
29.831
0.00
C


ATOM
843
C
THR
A
121
30.739
35.358
27.526
0.00
C


ATOM
844
O
THR
A
121
30.198
36.160
26.771
0.00
O


ATOM
845
N
VAL
A
122
30.075
34.632
28.417
0.00
N


ATOM
846
CA
VAL
A
122
28.635
34.754
28.607
0.00
C


ATOM
847
CB
VAL
A
122
27.899
33.451
28.196
0.00
C


ATOM
848
CG1
VAL
A
122
26.412
33.553
28.519
0.00
C


ATOM
849
CG2
VAL
A
122
28.091
33.207
26.695
0.00
C


ATOM
850
C
VAL
A
122
28.515
35.016
30.103
0.00
C


ATOM
851
O
VAL
A
122
29.182
34.363
30.902
0.00
O


ATOM
852
N
ARG
A
123
27.689
35.978
30.493
0.00
N


ATOM
853
CA
ARG
A
123
27.546
36.282
31.915
0.00
C


ATOM
854
CB
ARG
A
123
27.917
37.748
32.153
0.00
C


ATOM
855
CG
ARG
A
123
26.856
38.724
31.655
0.00
C


ATOM
856
CD
ARG
A
123
25.712
38.852
32.671
0.00
C


ATOM
857
NE
ARG
A
123
24.459
39.336
32.089
0.00
N1+


ATOM
858
CZ
ARG
A
123
24.345
40.426
31.336
0.00
C


ATOM
859
NH1
ARG
A
123
25.414
41.162
31.054
0.00
N


ATOM
860
NH2
ARG
A
123
23.156
40.796
30.877
0.00
N


ATOM
861
C
ARG
A
123
26.151
36.018
32.468
0.00
C


ATOM
862
O
ARG
A
123
25.252
35.605
31.739
0.00
O


ATOM
863
N
GLY
A
124
25.991
36.272
33.767
0.00
N


ATOM
864
CA
GLY
A
124
24.714
36.080
34.438
0.00
C


ATOM
865
C
GLY
A
124
24.094
34.712
34.238
0.00
C


ATOM
866
O
GLY
A
124
22.910
34.603
33.936
0.00
O


ATOM
867
N
LEU
A
125
24.891
33.666
34.415
0.00
N


ATOM
868
CA
LEU
A
125
24.412
32.304
34.238
0.00
C


ATOM
869
CB
LEU
A
125
25.411
31.495
33.406
0.00
C


ATOM
870
CG
LEU
A
125
25.597
31.779
31.913
0.00
C


ATOM
871
CD1
LEU
A
125
26.780
30.977
31.383
0.00
C


ATOM
872
CD2
LEU
A
125
24.333
31.411
31.152
0.00
C


ATOM
873
C
LEU
A
125
24.189
31.574
35.554
0.00
C


ATOM
874
O
LEU
A
125
24.828
31.869
36.573
0.00
O


ATOM
875
N
ILE
A
126
23.270
30.615
35.516
0.00
N


ATOM
876
CA
ILE
A
126
22.949
29.813
36.665
0.00
C


ATOM
877
CB
ILE
A
126
21.506
29.276
36.600
0.00
C


ATOM
878
CG2
ILE
A
126
21.268
28.230
37.672
0.00
C


ATOM
879
CG1
ILE
A
126
20.517
30.441
36.754
0.00
C


ATOM
880
CD1
ILE
A
126
19.074
30.045
36.578
0.00
C


ATOM
881
C
ILE
A
126
23.947
28.646
36.668
0.00
C


ATOM
882
O
ILE
A
126
24.009
27.881
35.701
0.00
O


ATOM
883
N
ARG
A
127
24.746
28.536
37.723
0.00
N


ATOM
884
CA
ARG
A
127
25.738
27.473
37.831
0.00
C


ATOM
885
CB
ARG
A
127
26.989
28.007
38.528
0.00
C


ATOM
886
CG
ARG
A
127
28.129
27.015
38.679
0.00
C


ATOM
887
CD
ARG
A
127
29.261
27.678
39.441
0.00
C


ATOM
888
NE
ARG
A
127
30.312
26.748
39.830
0.00
N1+


ATOM
889
CZ
ARG
A
127
31.098
26.112
36.971
0.00
C


ATOM
890
NH2
ARG
A
127
32.033
25.279
39.417
0.00
N


ATOM
891
NH1
ARG
A
127
30.949
26.310
37.669
0.00
N


ATOM
892
C
ARG
A
127
25.132
26.328
36.633
0.00
C


ATOM
893
O
ARG
A
127
24.507
26.553
39.676
0.00
O


ATOM
894
N
THR
A
128
25.325
25.103
38.151
0.00
N


ATOM
895
CA
THR
A
128
24.784
23.929
38.628
0.00
C


ATOM
896
CB
THR
A
128
23.447
23.475
38.189
0.00
C


ATOM
897
OG1
THR
A
128
23.718
22.755
36.977
0.00
O


ATOM
896
CG2
THR
A
128
22.568
24.674
37.863
0.00
C


ATOM
899
C
THR
A
128
25.720
22.729
38.782
0.00
C


ATOM
900
O
THR
A
128
26.763
22.759
38.135
0.00
O


ATOM
901
N
THR
A
129
25.317
21.667
39.472
0.00
N


ATOM
902
CA
THR
A
129
26.084
20.429
39.533
0.00
C


ATOM
903
CB
THR
A
129
26.055
19.838
40.946
0.00
C


ATOM
904
OG1
THR
A
129
24.691
19.639
41.355
0.00
O


ATOM
905
CG2
THR
A
129
26.758
20.779
41.924
0.00
C


ATOM
906
C
THR
A
129
25.474
19.411
38.565
0.00
C


ATOM
907
O
THR
A
129
25.792
18.227
38.607
0.00
O


ATOM
908
N
VAL
A
130
24.589
19.886
37.696
0.00
N


ATOM
909
CA
VAL
A
130
23.930
19.027
36.722
0.00
C


ATOM
910
CB
VAL
A
130
22.663
19.707
36.164
0.00
C


ATOM
911
CG1
VAL
A
130
21.972
18.790
35.162
0.00
C


ATOM
912
CG2
VAL
A
130
21.715
20.054
37.308
0.00
C


ATOM
913
C
VAL
A
130
24.857
18.691
35.561
0.00
C


ATOM
914
O
VAL
A
130
25.623
19.536
35.109
0.00
O


ATOM
915
N
CYS
A
131
24.790
17.449
35.086
0.00
N


ATOM
916
CA
CYS
A
131
25.626
17.016
33.975
0.00
C


ATOM
917
CB
CYS
A
131
25.889
15.507
34.034
0.00
C


ATOM
918
SG
CYS
A
131
24.399
14.468
33.874
0.00
S


ATOM
919
C
CYS
A
131
24.893
17.340
32.690
0.00
C


ATOM
920
O
CYS
A
131
23.670
17.436
32.678
0.00
O


ATOM
921
N
ALA
A
132
25.636
17.514
31.607
0.00
N


ATOM
922
CA
ALA
A
132
25.020
17.821
30.329
0.00
C


ATOM
923
CB
ALA
A
132
24.707
19.313
30.237
0.00
C


ATOM
924
C
ALA
A
132
25.920
17.404
29.176
0.00
C


ATOM
925
O
ALA
A
132
27.113
17.139
29.356
0.00
O


ATOM
926
N
GLU
A
133
25.323
17.353
27.992
0.00
N


ATOM
927
CA
GLU
A
133
26.017
16.981
26.774
0.00
C


ATOM
928
CB
GLU
A
133
25.434
15.686
26.219
0.00
C


ATOM
929
CG
GLU
A
133
26.457
14.695
25.730
0.00
C


ATOM
930
CD
GLU
A
133
27.077
13.909
26.862
o.oo
C


ATOM
931
OE1
GLU
A
133
27.702
14.533
27.741
0.00
O1−


ATOM
932
OE2
GLU
A
133
26.937
12.667
26.871
0.00
O


ATOM
933
C
GLU
A
133
25.750
18.114
25.792
0.00
C


ATOM
934
O
GLU
A
133
24.778
18.851
25.946
0.00
O


ATOM
935
N
PRO
A
134
26.602
18.268
24.769
0.00
N


ATOM
936
CA
PRO
A
134
26.395
19.343
23.789
0.00
C


ATOM
937
CB
PRO
A
134
27.471
19.059
22.742
0.00
C


ATOM
938
C
PRO
A
134
24.975
19.390
23.185
0.00
C


ATOM
939
O
PRO
A
134
24.331
20.446
23.159
0.00
O


ATOM
940
CD
PRO
A
134
27.856
17.539
24.501
0.00
C


ATOM
941
CG
PRO
A
134
28.586
16.478
23.572
0.00
C


ATOM
942
N
GLY
A
135
24.490
16.250
22.708
0.00
N


ATOM
943
CA
GLY
A
135
23.167
18.218
22.117
0.00
C


ATOM
944
C
GLY
A
135
22.074
18.675
23.063
0.00
C


ATOM
945
O
GLY
A
135
20.979
19.053
22.631
0.00
O


ATOM
946
N
ASP
A
136
22.369
18.638
24.359
0.00
N


ATOM
947
CA
ASP
A
136
21.414
19.046
25.387
0.00
C


ATOM
946
CB
ASP
A
136
21.914
18.588
26.770
0.00
C


ATOM
949
C
ASP
A
136
21.162
20.564
25.400
0.00
C


ATOM
950
O
ASP
A
136
20.124
21.024
25.886
0.00
O


ATOM
951
CG
ASP
A
136
21.783
17.075
26.982
0.00
C


ATOM
952
OD2
ASP
A
136
20.834
16.471
26.436
0.00
O1−


ATOM
953
OD1
ASP
A
136
22.618
16.492
27.714
0.00
O


ATOM
954
N
SER
A
137
22.109
21.332
24.868
0.00
N


ATOM
955
CA
SER
A
137
21.989
22.791
24.823
0.00
C


ATOM
956
CB
SER
A
137
23.048
23.390
23.896
0.00
C


ATOM
957
C
SER
A
137
20.610
23.287
24.388
0.00
C


ATOM
956
O
SER
A
137
19.993
22.752
23.456
0.00
O


ATOM
959
OG
SER
A
137
24.352
23.234
24.427
0.00
O


ATOM
960
N
GLY
A
138
20.148
24.332
25.070
0.00
N


ATOM
961
CA
GLY
A
138
18.854
24.904
24.782
0.00
C


ATOM
962
C
GLY
A
138
17.803
24.224
25.629
0.00
C


ATOM
963
O
GLY
A
138
16.706
24.748
25.609
0.00
O


ATOM
964
N
GLY
A
139
18.150
23.057
26.160
0.00
N


ATOM
965
CA
GLY
A
139
17.222
22.297
26.982
0.00
C


ATOM
966
C
GLY
A
139
16.617
23.021
28.176
0.00
C


ATOM
967
O
GLY
A
139
17.104
24.070
28.604
0.00
O


ATOM
968
N
SER
A
140
15.555
22.438
28.729
0.00
N


ATOM
969
CA
SER
A
140
14.858
23.024
29.870
0.00
C


ATOM
970
CB
SER
A
140
13.423
22.500
29.948
0.00
C


ATOM
971
OG
SER
A
140
12.971
22.037
28.691
0.00
O


ATOM
972
C
SER
A
140
15.532
22.736
31.198
0.00
C


ATOM
973
O
SER
A
140
16.162
21.691
31.389
0.00
O


ATOM
974
N
LEU
A
141
15.393
23.683
32.115
0.00
N


ATOM
975
CA
LEU
A
141
15.967
23.558
33.448
0.00
C


ATOM
976
CB
LEU
A
141
17.175
24.482
33.639
0.00
C


ATOM
977
CG
LEU
A
141
17.722
24.420
35.073
0.00
C


ATOM
978
CD1
LEU
A
141
18.323
23.047
35.334
0.00
C


ATOM
979
CD2
LEU
A
141
18.749
25.518
35.297
0.00
C


ATOM
980
C
LEU
A
141
14.851
23.945
34.405
0.00
C


ATOM
981
O
LEU
A
141
14.398
25.081
34.422
0.00
O


ATOM
982
N
LEU
A
142
14.409
22.987
35.199
0.00
N


ATOM
983
CA
LEU
A
142
13.341
23.220
36.150
0.00
C


ATOM
984
CB
LEU
A
142
12.230
22.198
35.913
0.00
C


ATOM
985
CG
LEU
A
142
11.289
22.306
34.719
0.00
C


ATOM
986
CD1
LEU
A
142
10.674
20.933
34.463
0.00
C


ATOM
987
CD2
LEU
A
142
10.219
23.350
34.996
0.00
C


ATOM
988
C
LEU
A
142
13.702
23.168
37.629
0.00
C


ATOM
989
O
LEU
A
142
14.745
22.671
38.029
0.00
O


ATOM
990
N
ALA
A
143
12.788
23.701
38.424
0.00
N


ATOM
991
CA
ALA
A
143
12.880
23.759
39.875
0.00
C


ATOM
992
CB
ALA
A
143
13.159
25.178
40.345
0.00
C


ATOM
993
C
ALA
A
143
11.434
23.368
40.165
0.00
C


ATOM
994
O
ALA
A
143
10.557
24.221
40.225
0.00
O


ATOM
995
N
GLY
A
144
11.175
22.072
40.287
0.00
N


ATOM
996
CA
GLY
A
144
9.610
21.642
40.513
0.00
C


ATOM
997
C
GLY
A
144
9.058
21.945
39.232
0.00
C


ATOM
998
O
GLY
A
144
9.457
21.487
38.154
0.00
O


ATOM
999
N
ASN
A
145
7.984
22.723
39.322
0.00
N


ATOM
1000
CA
ASN
A
145
7.241
23.066
38.122
0.00
C


ATOM
1001
CB
ASN
A
145
5.736
22.846
38.321
0.00
C


ATOM
1002
CG
ASN
A
145
5.144
23.751
39.384
0.00
C


ATOM
1003
OD1
ASN
A
145
5.382
24.962
39.396
0.00
O


ATOM
1004
ND2
ASN
A
145
4.351
23.166
40.281
0.00
N


ATOM
1005
C
ASN
A
145
7.503
24.496
37.650
0.00
C


ATOM
1006
O
ASN
A
145
6.716
25.049
36.886
0.00
O


ATOM
1007
N
GLN
A
146
8.613
25.086
38.093
0.00
N


ATOM
1006
CA
GLN
A
146
8.968
26.455
37.702
0.00
C


ATOM
1009
CB
GLN
A
146
9.234
27.314
38.942
0.00
C


ATOM
1010
CG
GLN
A
146
8.080
27.367
39.916
0.00
C


ATOM
1011
CD
GLN
A
146
6.875
28.097
39.363
0.00
C


ATOM
1012
OE1
GLN
A
146
5.735
27.705
39.615
0.00
O


ATOM
1013
NE2
GLN
A
146
7.117
29.172
38.617
0.00
N


ATOM
1014
C
GLN
A
146
10.205
26.492
36.798
0.00
C


ATOM
1015
O
GLN
A
146
11.277
25.999
37.169
0.00
O


ATOM
1016
N
ALA
A
147
10.055
27.084
35.618
0.00
N


ATOM
1017
CA
ALA
A
147
11.160
27.188
34.660
0.00
C


ATOM
1018
CB
ALA
A
147
10.642
27.698
33.309
0.00
C


ATOM
1019
C
ALA
A
147
12.253
28.124
35.163
0.00
C


ATOM
1020
O
ALA
A
147
11.958
29.233
35.625
0.00
O


ATOM
1021
N
GLN
A
148
13.506
27.679
35.124
0.00
N


ATOM
1022
CA
GLN
A
148
14.637
28.481
35.593
0.00
C


ATOM
1023
CB
GLN
A
148
15.576
27.627
36.435
0.00
C


ATOM
1024
CG
GLN
A
148
14.939
27.023
37.667
0.00
C


ATOM
1025
CD
GLN
A
148
14.283
28.062
38.553
0.00
C


ATOM
1026
OE1
GLN
A
148
13.074
28.016
38.797
0.00
O


ATOM
1027
NE2
GLN
A
148
15.075
29.005
39.040
0.00
N


ATOM
1028
C
GLN
A
148
15.440
29.128
34.466
0.00
C


ATOM
1029
O
GLN
A
148
15.723
30.325
34.501
0.00
O


ATOM
1030
N
GLY
A
149
15.818
28.330
33.472
0.00
N


ATOM
1031
CA
GLY
A
149
16.578
28.852
32.349
0.00
C


ATOM
1032
C
GLY
A
149
16.762
27.783
31.290
0.00
C


ATOM
1033
O
GLY
A
149
16.104
26.742
31.348
0.00
O


ATOM
1034
N
VAL
A
150
17.628
28.041
30.311
0.00
N


ATOM
1035
CA
VAL
A
150
17.896
27.061
29.261
0.00
C


ATOM
1036
CB
VAL
A
150
17.568
27.604
27.848
0.00
C


ATOM
1037
CG1
VAL
A
150
16.053
27.737
27.694
0.00
C


ATOM
1038
CG2
VAL
A
150
18.256
28.936
27.611
0.00
C


ATOM
1039
C
VAL
A
150
19.351
26.630
29.329
0.00
C


ATOM
1040
O
VAL
A
150
20.210
27.368
29.809
0.00
O


ATOM
1041
N
THR
A
151
19.630
25.431
28.844
0.00
N


ATOM
1042
CA
THR
A
151
20.985
24.914
28.873
0.00
C


ATOM
1043
CB
THR
A
151
20.980
23.429
28.536
0.00
C


ATOM
1044
OG1
THR
A
151
20.057
22.771
29.412
0.00
O


ATOM
1045
CG2
THR
A
151
22.361
22.828
28.748
0.00
C


ATOM
1046
C
THR
A
151
21.954
25.675
27.984
0.00
C


ATOM
1047
O
THR
A
151
21.756
25.796
26.779
0.00
O


ATOM
1048
N
SER
A
152
23.007
26.192
28.607
0.00
N


ATOM
1049
CA
SER
A
152
24.020
26.956
27.898
0.00
C


ATOM
1050
CB
SER
A
152
24.382
28.193
28.723
0.00
C


ATOM
1051
OG
SER
A
152
25.262
29.047
28.021
0.00
O


ATOM
1052
C
SER
A
152
25.264
26.127
27.603
0.00
C


ATOM
1053
O
SER
A
152
25.673
26.005
26.450
0.00
O


ATOM
1054
N
GLY
A
153
25.859
25.561
28.648
0.00
N


ATOM
1055
CA
GLY
A
153
27.047
24.740
28.483
0.00
C


ATOM
1056
C
GLY
A
153
27.702
24.415
29.817
0.00
C


ATOM
1057
O
GLY
A
153
27.151
24.724
30.872
0.00
O


ATOM
1058
N
GLY
A
154
28.876
23.794
29.771
0.00
N


ATOM
1059
CA
GLY
A
154
29.589
23.443
30.984
0.00
C


ATOM
1060
C
GLY
A
154
30.688
22.439
30.708
0.00
C


ATOM
1061
O
GLY
A
154
31.161
22.322
29.579
0.00
O


ATOM
1062
N
SER
A
155
31.101
21.703
31.736
0.00
N


ATOM
1063
CA
SER
A
155
32.149
20.701
31.572
0.00
C


ATOM
1064
CB
SER
A
155
33.416
21.148
32.296
0.00
C


ATOM
1065
OG
SER
A
155
33.180
21.231
33.688
0.00
O


ATOM
1066
C
SER
A
155
31.700
19.354
32.128
0.00
C


ATOM
1067
O
SER
A
155
30.690
19.268
32.836
0.00
O


ATOM
1068
N
GLY
A
156
32.460
18.307
31.805
0.00
N


ATOM
1069
CA
GLY
A
156
32.142
16.970
32.283
0.00
C


ATOM
1070
C
GLY
A
156
31.101
16.231
31.458
0.00
C


ATOM
1071
O
GLY
A
156
30.856
16.564
30.302
0.00
O


ATOM
1072
N
ASN
A
157
30.495
15.211
32.051
0.00
N


ATOM
1073
CA
ASN
A
157
29.470
14.434
31.369
0.00
C


ATOM
1074
CB
ASN
A
157
30.115
13.390
30.450
0.00
C


ATOM
1075
CG
ASN
A
157
31.077
12.472
31.188
0.00
C


ATOM
1076
OD1
ASN
A
157
30.669
11.681
32.038
0.00
O


ATOM
1077
ND2
ASN
A
157
32.365
12.575
30.863
0.00
N


ATOM
1078
C
ASN
A
157
28.578
13.766
32.410
0.00
C


ATOM
1079
O
ASN
A
157
28.788
13.932
33.610
0.00
O


ATOM
1080
N
CYS
A
158
27.580
13.021
31.948
0.00
N


ATOM
1081
CA
CYS
A
158
26.661
12.337
32.840
0.00
C


ATOM
1082
CB
CYS
A
158
25.304
12.169
32.155
0.00
C


ATOM
1083
SG
CYS
A
158
24.426
13.754
31.973
0.00
S


ATOM
1084
C
CYS
A
158
27.181
11.000
33.350
0.00
C


ATOM
1085
O
CYS
A
158
26.592
10.393
34.244
0.00
O


ATOM
1086
N
ARG
A
159
28.288
10.538
32.784
0.00
N


ATOM
1087
CA
ARG
A
159
28.862
9.271
33.215
0.00
C


ATOM
1088
CB
ARG
A
159
29.714
6.662
32.099
0.00
C


ATOM
1089
CG
ARG
A
159
28.922
8.198
30.890
0.00
C


ATOM
1090
CD
ARG
A
159
29.852
7.660
29.817
0.00
C


ATOM
1091
NE
ARG
A
159
30.711
8.703
29.260
0.00
N1+


ATOM
1092
CZ
ARG
A
159
30.273
9.714
28.517
0.00
C


ATOM
1093
NH1
ARG
A
159
28.980
9.826
28.237
0.00
N


ATOM
1094
NH2
ARG
A
159
31.128
10.610
28.049
0.00
N


ATOM
1095
C
ARG
A
159
29.716
9.462
34.467
0.00
C


ATOM
1096
O
ARG
A
159
29.634
8.675
35.405
0.00
O


ATOM
1097
N
THR
A
160
30.533
10.510
34.481
0.00
N


ATOM
1098
CA
THR
A
160
31.400
10.785
35.628
0.00
C


ATOM
1099
CB
THR
A
160
32.874
10.925
35.185
0.00
C


ATOM
1100
OG1
THR
A
160
32.966
11.897
34.138
0.00
O


ATOM
1101
CG2
THR
A
160
33.404
9.605
34.676
0.00
C


ATOM
1102
C
THR
A
160
31.000
12.048
36.396
0.00
C


ATOM
1103
O
THR
A
160
31.525
12.320
37.475
0.00
O


ATOM
1104
N
GLY
A
161
30.068
12.812
35.836
0.00
N


ATOM
1105
CA
GLY
A
161
29.622
14.028
36.490
0.00
C


ATOM
1106
C
GLY
A
161
30.200
15.295
35.878
0.00
C


ATOM
1107
O
GLY
A
161
31.194
15.262
35.150
0.00
O


ATOM
1108
N
GLY
A
162
29.577
16.426
36.180
0.00
N


ATOM
1109
CA
GLY
A
162
30.061
17.681
35.646
0.00
C


ATOM
1110
C
GLY
A
162
29.414
18.909
36.250
0.00
C


ATOM
1111
O
GLY
A
162
28.785
18.856
37.318
0.00
O


ATOM
1112
N
THR
A
163
29.592
20.023
35.547
0.00
N


ATOM
1113
CA
THR
A
163
29.057
21.322
35.934
0.00
C


ATOM
1114
CB
THR
A
163
30.179
22.248
36.440
0.00
C


ATOM
1115
OG1
THR
A
163
30.861
21.621
37.535
0.00
O


ATOM
1116
CG2
THR
A
163
29.604
23.576
36.896
0.00
C


ATOM
1117
C
THR
A
163
28.414
21.948
34.695
0.00
C


ATOM
1118
O
THR
A
163
29.052
22.077
33.650
0.00
O


ATOM
1119
N
THR
A
164
27.152
22.342
34.810
0.00
N


ATOM
1120
CA
THR
A
164
26.452
22.945
33.680
0.00
C


ATOM
1121
CB
THR
A
164
25.290
22.053
33.208
0.00
C


ATOM
1122
OG1
THR
A
164
25.744
20.700
33.075
0.00
C


ATOM
1123
CG2
THR
A
164
24.768
22.536
31.869
0.00
C


ATOM
1124
C
THR
A
164
25.878
24.298
34.060
0.00
C


ATOM
1125
O
THR
A
164
25.354
24.466
35.167
0.00
O


ATOM
1126
N
PHE
A
165
25.981
25.256
33.139
0.00
N


ATOM
1127
CA
PHE
A
165
25.470
26.607
33.369
0.00
C


ATOM
1128
CB
PHE
A
165
26.521
27.662
33.021
0.00
C


ATOM
1129
CG
PHE
A
165
27.818
27.507
33.769
0.00
C


ATOM
1130
CD1
PHE
A
165
28.758
26.554
33.380
0.00
C


ATOM
1131
CD2
PHE
A
165
28.107
28.319
34.858
0.00
C


ATOM
1132
CE1
PHE
A
165
29.957
26.427
34.071
0.00
C


ATOM
1133
CE2
PHE
A
165
29.302
28.187
35.541
0.00
C


ATOM
1134
CZ
PHE
A
165
30.224
27.242
35.146
0.00
C


ATOM
1135
C
PHE
A
165
24.242
26.834
32.505
0.00
C


ATOM
1136
O
PHE
A
165
24.170
26.334
31.386
0.00
O


ATOM
1137
N
PHE
A
166
23.276
27.586
33.020
0.00
N


ATOM
1138
CA
PHE
A
166
22.067
27.854
32.262
0.00
C


ATOM
1139
CB
PHE
A
166
20.860
27.130
32.880
0.00
C


ATOM
1140
CG
PHE
A
166
21.062
25.643
33.076
0.00
C


ATOM
1141
CD2
PHE
A
166
20.371
24.721
32.295
0.00
C


ATOM
1142
CD1
PHE
A
166
21.936
25.165
34.050
0.00
C


ATOM
1143
CE2
PHE
A
166
20.549
23.339
32.484
0.00
C


ATOM
1144
CE1
PHE
A
166
22.117
23.788
34.243
0.00
C


ATOM
1145
CZ
PHE
A
166
21.423
22.879
33.460
0.00
C


ATOM
1146
C
PHE
A
166
21.765
29.344
32.173
0.00
C


ATOM
1147
O
PHE
A
166
22.066
30.122
33.086
0.00
O


ATOM
1148
N
GLN
A
167
21.187
29.736
31.046
0.00
N


ATOM
1149
CA
GLN
A
167
20.822
31.122
30.802
0.00
C


ATOM
1150
CB
GLN
A
167
20.737
31.366
29.291
0.00
C


ATOM
1151
CG
GLN
A
167
19.786
32.464
28.875
0.00
C


ATOM
1152
CD
GLN
A
167
20.300
33.840
29.234
0.00
C


ATOM
1153
OE1
GLN
A
167
21.329
34.282
28.722
0.00
O


ATOM
1154
NE2
GLN
A
167
19.589
34.525
30.126
0.00
N


ATOM
1155
C
GLN
A
167
19.449
31.262
31.442
0.00
C


ATOM
1156
O
GLN
A
167
18.500
30.600
31.020
0.00
O


ATOM
1157
N
PRO
A
168
19.322
32.107
32.481
0.00
N


ATOM
1158
CD
PRO
A
168
20.334
32.973
33.113
0.00
C


ATOM
1159
CA
PRO
A
168
18.013
32.270
33.132
0.00
C


ATOM
1160
CB
PRO
A
168
18.261
33.395
34.138
0.00
C


ATOM
1161
CG
PRO
A
168
19.716
33.237
34.470
0.00
C


ATOM
1162
C
PRO
A
168
16.926
32.611
32.108
0.00
C


ATOM
1163
O
PRO
A
168
17.180
33.288
31.116
0.00
O


ATOM
1164
N
VAL
A
169
15.711
32.149
32.373
0.00
N


ATOM
1165
CA
VAL
A
169
14.578
32.379
31.487
0.00
C


ATOM
1166
CB
VAL
A
169
13.465
31.328
31.802
0.00
C


ATOM
1167
CG1
VAL
A
169
12.914
31.542
33.204
0.00
C


ATOM
1168
CG2
VAL
A
169
12.370
31.401
30.786
0.00
C


ATOM
1169
C
VAL
A
169
13.962
33.788
31.427
0.00
C


ATOM
1170
O
VAL
A
169
13.638
34.273
30.358
0.00
O


ATOM
1171
N
ASN
A
170
13.802
34.455
32.560
0.00
N


ATOM
1172
CA
ASN
A
170
13.213
35.793
32.554
0.00
C


ATOM
1173
CB
ASN
A
170
13.128
36.329
33.979
0.00
C


ATOM
1174
CG
ASN
A
170
12.215
35.486
34.843
0.00
C


ATOM
1175
OD1
ASN
A
170
11.177
35.008
34.373
0.00
O


ATOM
1176
ND2
ASN
A
170
12.587
35.298
36.107
0.00
N


ATOM
1177
C
ASN
A
170
13.859
36.822
31.635
0.00
C


ATOM
1178
O
ASN
A
170
13.166
37.586
30.975
0.00
O


ATOM
1179
N
PRO
A
171
15.192
36.873
31.590
0.00
N


ATOM
1180
CD
PRO
A
171
16.217
36.299
32.476
0.00
C


ATOM
1181
CA
PRO
A
171
15.768
37.872
30.684
0.00
C


ATOM
1182
CB
PRO
A
171
17.258
37.857
31.043
0.00
C


ATOM
1183
CG
PRO
A
171
17.466
36.517
31.673
0.00
C


ATOM
1184
C
PRO
A
171
15.484
37.551
29.209
0.00
C


ATOM
1185
O
PRO
A
171
15.601
38.418
28.338
0.00
O


ATOM
1186
N
ILE
A
172
15.101
36.307
28.935
0.00
N


ATOM
1187
CA
ILE
A
172
14.798
35.896
27.570
0.00
C


ATOM
1188
CB
ILE
A
172
14.811
34.374
27.403
0.00
C


ATOM
1189
CG2
ILE
A
172
14.516
34.018
25.947
0.00
C


ATOM
1190
CG1
ILE
A
172
16.164
33.801
27.813
0.00
C


ATOM
1191
CD1
ILE
A
172
16.196
32.269
27.764
0.00
C


ATOM
1192
C
ILE
A
172
13.386
36.369
27.251
0.00
C


ATOM
1193
O
ILE
A
172
13.113
36.885
26.166
0.00
O


ATOM
1194
N
LEU
A
173
12.488
36.179
28.210
0.00
N


ATOM
1195
CA
LEU
A
173
11.102
36.587
28.038
0.00
C


ATOM
1196
CB
LEU
A
173
10.279
36.158
29.252
0.00
C


ATOM
1197
CG
LEU
A
173
10.263
34.645
29.486
0.00
C


ATOM
1198
CD1
LEU
A
173
9.465
34.308
30.759
0.00
C


ATOM
1199
CD2
LEU
A
173
9.671
33.965
28.251
0.00
C


ATOM
1200
C
LEU
A
173
11.031
38.105
27.869
0.00
C


ATOM
1201
O
LEU
A
173
10.287
38.617
27.037
0.00
O


ATOM
1202
N
GLN
A
174
11.831
38.807
28.662
0.00
N


ATOM
1203
CA
GLN
A
174
11.896
40.259
28.641
0.00
C


ATOM
1204
CB
GLN
A
174
12.665
40.752
29.870
0.00
C


ATOM
1205
CG
GLN
A
174
12.868
42.259
29.923
0.00
C


ATOM
1206
CD
GLN
A
174
11.664
43.006
30.461
0.00
C


ATOM
1207
OE1
GLN
A
174
10.532
42.811
30.003
0.00
O


ATOM
1208
NE2
GLN
A
174
11.904
43.876
31.438
0.00
N


ATOM
1209
C
GLN
A
174
12.555
40.817
27.381
0.00
C


ATOM
1210
O
GLN
A
174
12.219
41.909
26.933
0.00
O


ATOM
1211
N
ALA
A
175
13.493
40.078
26.808
0.00
N


ATOM
1212
CA
ALA
A
175
14.164
40.552
25.604
0.00
C


ATOM
1213
CB
ALA
A
175
15.378
39.681
25.306
0.00
C


ATOM
1214
C
ALA
A
175
13.238
40.580
24.394
0.00
C


ATOM
1215
O
ALA
A
175
13.276
41.512
23.595
0.00
O


ATOM
1216
N
TYR
A
176
12.396
39.561
24.276
0.00
N


ATOM
1217
CA
TYP
A
176
11.462
39.458
23.161
0.00
C


ATOM
1218
CB
TYP
A
176
11.571
38.063
22.535
0.00
C


ATOM
1219
CG
TYR
A
176
12.990
37.700
22.173
0.00
C


ATOM
1220
CD1
TYR
A
176
13.761
38.551
21.381
0.00
C


ATOM
1221
CE1
TYP
A
176
15.075
38.249
21.073
0.00
C


ATOM
1222
CD2
TYR
A
176
13.574
36.528
22.643
0.00
C


ATOM
1223
CE2
TYR
A
176
14.890
36.213
22.335
0.00
C


ATOM
1224
CZ
TYP
A
176
15.636
37.083
21.553
0.00
C


ATOM
1225
OH
TYR
A
176
16.959
36.817
21.285
0.00
O


ATOM
1226
C
TYP
A
176
10.004
39.742
23.500
0.00
C


ATOM
1227
O
TYP
A
176
9.135
39.574
22.646
0.00
O


ATOM
1228
N
GLY
A
177
9.736
40.165
24.733
0.00
N


ATOM
1229
CA
GLY
A
177
8.366
40.457
25.131
0.00
C


ATOM
1230
C
GLY
A
177
7.469
39.232
25.065
0.00
C


ATOM
1231
O
GLY
A
177
6.295
39.326
24.711
0.00
O


ATOM
1232
N
LEU
A
178
8.033
38.080
25.421
0.00
N


ATOM
1233
CA
LEU
A
178
7.323
36.807
25.390
0.00
C


ATOM
1234
CB
LEU
A
178
8.275
35.694
24.937
0.00
C


ATOM
1235
CG
LEU
A
178
8.981
35.724
23.591
0.00
C


ATOM
1236
CD1
LEU
A
178
10.077
34.688
23.584
0.00
C


ATOM
1237
CD2
LEU
A
178
8.006
35.441
22.454
0.00
C


ATOM
1238
C
LEU
A
178
6.737
36.403
26.741
0.00
C


ATOM
1239
O
LEU
A
178
7.221
36.821
27.794
0.00
O


ATOM
1240
N
ARG
A
179
5.698
35.573
26.668
0.00
N


ATOM
1241
CA
ARG
A
179
5.008
35.060
27.875
0.00
C


ATOM
1242
CB
ARG
A
179
3.519
35.439
27.872
0.00
C


ATOM
1243
CG
ARG
A
179
3.193
36.849
28.356
0.00
C


ATOM
1244
CD
ARG
A
179
1.760
37.239
27.989
0.00
C


ATOM
1245
NE
ARG
A
179
1.401
38.565
28.490
0.00
N1+


ATOM
1246
CZ
ARG
A
179
1.070
38.825
29.751
0.00
C


ATOM
1247
NH1
ARG
A
179
1.044
37.844
30.646
0.00
N


ATOM
1248
NH2
ARG
A
179
0.773
40.066
30.117
0.00
N


ATOM
1249
C
ARG
A
179
5.118
33.541
27.794
0.00
C


ATOM
1250
O
ARG
A
179
5.043
32.978
26.707
0.00
O


ATOM
1251
N
MET
A
180
5.313
32.882
28.931
0.00
N


ATOM
1252
CA
MET
A
180
5.422
31.428
28.955
0.00
C


ATOM
1253
CB
MET
A
180
5.866
30.936
30.329
0.00
C


ATOM
1254
CG
MET
A
180
7.257
31.311
30.768
0.00
C


ATOM
1255
SD
MET
A
180
8.400
30.052
30.227
0.00
S


ATOM
1256
CE
MET
A
180
7.622
28.610
30.892
0.00
C


ATOM
1257
C
MET
A
180
4.034
30.856
28.712
0.00
C


ATOM
12S8
O
MET
A
180
3.034
31.444
29.118
0.00
O


ATOM
1259
N
ILE
A
181
3.967
29.715
28.042
0.00
N


ATOM
1260
CA
ILE
A
181
2.690
29.085
27.781
0.00
C


ATOM
1261
CB
ILE
A
181
2.726
28.266
26.464
0.00
C


ATOM
1262
CG2
ILE
A
181
1.534
27.294
26.399
0.00
C


ATOM
1263
CG1
ILE
A
181
2.711
29.237
25.270
0.00
C


ATOM
1264
CD1
ILE
A
181
2.740
28.556
23.910
0.00
C


ATOM
1265
C
ILE
A
181
2.613
28.211
29.023
0.00
C


ATOM
1266
O
ILE
A
181
3.458
27.357
29.238
0.00
O


ATOM
1267
N
THR
A
182
1.598
28.457
29.845
0.00
N


ATOM
1268
CA
THR
A
182
1.398
27.724
31.090
0.00
C


ATOM
1269
C
THR
A
182
0.212
26.770
31.163
0.00
C


ATOM
1270
O
THR
A
182
−0.098
26.231
32.241
0.00
O


ATOM
1271
CB
THR
A
182
1.315
28.733
32.273
0.00
C


ATOM
1272
OG1
THR
A
182
0.199
28.407
33.111
0.00
O


ATOM
1273
CG2
THR
A
182
1.137
30.133
31.739
0.00
C


ATOM
1274
N
THR
A
183
−0.448
26.534
30.036
0.00
N


ATOM
1275
CA
THR
A
183
−1.593
25.623
30.045
0.00
C


ATOM
1276
C
THR
A
183
−1.754
25.043
28.647
0.00
C


ATOM
1277
O
THR
A
183
−1.274
25.608
27.675
0.00
O


ATOM
1278
CB
THR
A
183
−2.909
26.342
30.433
0.00
C


ATOM
1279
OG1
THR
A
183
−3.716
25.460
31.228
0.00
O


ATOM
1280
CG2
THR
A
183
−3.690
26.738
29.184
0.00
C


ATOM
1281
N
ASP
A
184
−2.402
23.896
28.532
0.00
N


ATOM
1282
CA
ASP
A
184
−2.573
23.318
27.213
0.00
C


ATOM
1283
C
ASP
A
184
−4.035
23.091
26.918
0.00
C


ATOM
1284
O
ASP
A
184
−4.380
22.208
26.174
0.00
O


ATOM
1285
CB
ASP
A
184
−1.810
22.005
27.113
0.00
C


ATOM
1286
CG
ASP
A
184
−0.464
22.056
27.794
0.00
C


ATOM
1287
OD1
ASP
A
184
0.296
23.029
27.577
0.00
O


ATOM
1288
OD2
ASP
A
184
−0.152
21.080
28.527
0.00
O1−


TER
1289

ASP
A
184







ATOM
1290
N
ALA
B
14
37.553
22.457
29.194
0.00
N1+


ATOM
1291
H
ALA
B
14
36.582
22.364
28.935
0.00
H


ATOM
1292
H
ALA
B
14
37.991
23.157
28.614
0.00
H


ATOM
1293
H
ALA
B
14
38.021
21.572
29.065
0.00
H


ATOM
1294
CA
ALA
B
14
37.649
22.863
30.616
0.00
C


ATOM
1295
C
ALA
B
14
36.345
22.665
31.400
0.00
C


ATOM
1296
O
ALA
B
14
36.364
21.816
32.304
0.00
O


ATOM
1297
CB
ALA
B
14
38.235
24.270
30.658
0.00
C


ATOM
1298
N
ALA
B
15
35.261
23.393
31.094
0.00
N


ATOM
1299
CA
ALA
B
15
35.165
24.394
30.026
0.00
C


ATOM
1300
C
ALA
B
15
34.368
23.941
28.790
0.00
C


ATOM
1301
O
ALA
B
15
34.957
23.330
27.892
0.00
O


ATOM
1302
CB
ALA
B
15
34.779
25.773
30.573
0.00
C


ATOM
1303
N
ALA
B
16
33.028
24.069
28.763
0.00
N


ATOM
1304
CA
ALA
B
15
52.304
23.388
27.683
0.00
C


ATOM
1305
C
ALA
B
16
31.144
24.054
26.918
0.00
C


ATOM
1306
O
ALA
B
16
30.114
24.490
27.453
0.00
O


ATOM
1307
CB
ALA
B
16
32.420
21.850
27.713
0.00
C


ATOM
1308
H
ALA
B
16
32.544
24.608
29.452
0.00
H


ATOM
1309
N
HIS
B
17
31.370
24.111
25.600
0.00
N


ATOM
1310
CA
HIS
B
17
30.508
24.676
24.521
0.00
C


ATOM
1311
C
HIS
B
17
29.820
23.558
23.756
0.00
C


ATOM
1312
O
HIS
B
17
30.487
22.621
23.291
0.00
O


ATOM
1313
CB
HIS
B
17
31.473
25.545
23.683
0.00
C


ATOM
1314
CG
HIS
B
17
30.806
26.351
22.601
0.00
C


ATOM
1315
ND1
HIS
B
17
30.728
26.028
21.264
0.00
N


ATOM
1316
CD2
HIS
B
17
30.170
27.551
22.772
0.00
C


ATOM
1317
CE1
HIS
B
17
30.054
27.014
20.648
0.00
C


ATOM
1318
NE2
HIS
B
17
29.694
27.965
21.525
0.00
N


ATOM
1319
H
HIS
B
17
32.233
23.710
25.292
0.00
H


ATOM
1320
N
TYR
B
18
28.491
23.661
23.613
0.00
N


ATOM
1321
CA
TYR
B
18
27.651
22.538
23.244
0.00
C


ATOM
1322
C
TYR
B
18
26.791
22.741
21.978
0.00
C


ATOM
1323
O
TYR
B
18
25.936
21.904
21.762
0.00
O


ATOM
1324
CB
TYR
B
18
26.869
22.044
24.476
0.00
C


ATOM
1325
CG
TYR
B
18
27.638
21.257
25.527
0.00
C


ATOM
1326
CD1
TYR
B
18
27.073
20.996
26.793
0.00
C


ATOM
1327
CD2
TYR
B
18
28.818
20.596
25.160
0.00
C


ATOM
1328
CE1
TYR
B
18
27.702
20.099
27.685
0.00
C


ATOM
1329
CE2
TYR
B
18
29.420
19.668
26.020
0.00
C


ATOM
1330
CZ
TYR
B
18
28.855
19.410
27.276
0.00
C


ATOM
1331
OH
TYR
B
18
29.519
18.595
28.139
0.00
O


ATOM
1332
H
TYR
B
18
28.022
24.521
23.872
0.00
H


ATOM
1333
N
ASP
B
19
27.328
23.446
20.986
0.00
N


ATOM
1334
CA
ASP
B
19
27.252
23.065
19.573
0.00
C


ATOM
1335
C
ASP
B
19
25.957
22.335
19.178
0.00
C


ATOM
1336
O
ASP
B
19
24.855
22.851
19.367
0.00
O


ATOM
1337
CB
ASP
B
19
27.381
24.271
18.655
0.00
C


ATOM
1338
CG
ASP
B
19
28.399
25.369
18.926
0.00
C


ATOM
1339
OD1
ASP
B
19
28.777
25.568
20.105
0.00
O


ATOM
1340
OD2
ASP
B
19
28.588
26.117
17.941
0.00
O1−


ATOM
1341
H
ASP
B
19
28.092
24.050
21.252
0.00
H


ATOM
1342
N
GLU
B
20
26.024
21.140
18.622
0.00
N


ATOM
1343
CA
GLU
B
20
27.219
20.341
18.451
0.00
C


ATOM
1344
C
GLU
B
20
27.848
20.634
17.079
0.00
C


ATOM
1345
O
GLU
B
20
27.311
20.147
16.091
0.00
O


ATOM
1346
CB
GLU
B
20
26.641
18.934
18.532
0.00
C


ATOM
1347
CG
GLU
B
20
26.790
18.174
19.836
0.00
C


ATOM
1348
CD
GLU
B
20
26.391
16.720
19.643
0.00
C


ATOM
1349
OE1
GLU
B
20
26.614
16.043
20.673
0.00
O1−


ATOM
1350
OE2
GLU
B
20
26.569
16.221
18.501
0.00
O


ATOM
1351
H
GLU
B
20
25.129
20.696
16.442
0.00
H


ATOM
1352
N
ALA
B
21
29.122
21.069
17.024
0.00
N


ATOM
1353
CA
ALA
3
21
29.859
21.221
15.768
0.00
C


ATOM
1354
C
ALA
3
21
30.422
19.894
15.208
0.00
C


ATOM
1355
O
ALA
B
21
31.618
19.821
14.879
0.00
O


ATOM
1356
CB
ALA
B
21
30.954
22.295
15.900
0.00
C


ATOM
1357
OXT
ALA
B
21
29.677
18.897
15.088
0.00
O1−


ATOM
1358
H
ALA
B
21
29.585
21.298
17.880
0.00
H


TER
1359

ALA
B
21









Example 21
Oxidative Stability of ASP
This Example describes experiments conducted to determine the oxidative stability of the ASP protease and mutant proteases. The resistance to oxidation of Cellulomonas 69B4 protease was compared to that of: a BPN′-variant protease (BPN′-variant 1; Genencor; See, U.S. Pat. No. RE 34,606 [incorporated herein by reference], for a description of this enzyme); a GG36 variant protease (GG36-variant 1; Genencor; See e.g., U.S. Pat. Nos. 5,955,340 and 5,700,676, herein incorporated by reference); and PURAFECT protease (Genencor).
The assay was conducted by incubating a sample of the protease with 0.1 M H2O2. A 2.0 ml volume of 0.1 M Borate buffer (45.4 gm NaB4O710H2O), pH 9.45 containing 0.1 M H2O2 and 100 ppm protease was incubated at 25° C. for 20 minutes and assayed for enzyme activity.
The enzyme activity was determined as follows: 50 μl of the incubation mixture was combined with 950 μl 0.1 M Tris buffer, pH 8.6 and a sample from 10 μl was taken and added to 990 μl AAPF substrate solution, conc. 1 mg/ml, in 0.1 M Tris/0.005% TWEEN®, pH 8.6. The rate of increase in absorbance at 410 nm due to release of p-nitroaniline was monitored. The results obtained for these proteases are provided in FIG. 31. As indicated in this graph, protease 69B4 showed greatly enhanced stability under oxidative conditions relative to the subtilisin proteases.
Example 22
Chelate Stability of ASP
In this Example, experiments to determine the chelate stability of ASP are described. The resistance to the presence of a chelator of 69B4 protease was assayed by incubating an aliquot of the enzyme with 10 mM EDTA in 50 mM Tris, pH 8.2. The same enzyme preparations as used in Example 21 were used in these experiments.
In specific, a volume of 2.0 ml 50 mM Tris buffer, pH 8.2, containing 10 mM EDTA and 100 ppm protease was incubated at 45° C. for 100 minutes and assayed for enzyme activity as follows: 50 μl of the incubation mixture was combined with 950 μl 0.1 M Tris buffer, pH 8.6 and a sample from 10 μl was taken and added to 990 μl AAPF substrate solution, conc. 1 mg/ml, in 0.1 M Tris/0.005% TWEEN®, pH 8.6
The rate of increase in absorbance at 410 nm due to release of p-nitroaniline was monitored. The results obtained for these four proteases are shown in FIG. 32. As indicated by these results, protease 69B4 showed greatly enhanced stability in the presence of a chelator than BPN′ variant-1, PURAFECT®, or GG36 variant-1.
Example 23
Thermal Stability of ASP
In this Example, experiments conducted to determine the thermostability of ASP protease are described. In one set of experiments, 69B4 protease was tested for resistance to thermal inactivation in solution. As in Examples 21 and 22, a BPN′ variant (BPN′-variant-1), PURAFECT®, and a GG36 variant (GG36-variant-1) were also tested and compared with ASP.
The thermal inactivation was performed by incubating a volume of 2.0 ml 50 mM Tris buffer, pH 8.0, containing 100 ppm protease at 45° C. for 300 minutes and assayed for enzyme activity as follows: 50 μl of the incubation mixture was combined with 950 μl 0.1 M Tris buffer, pH 8.6 and a sample from 10 μl was taken and added to 990 μl AAPF substrate solution, conc. 1 mg/ml, in 0.1 M Tris/0.005% TWEEN®, pH 8.6. The rate of increase in absorbance at 410 nm due to release of p-nitroaniline was monitored. The results of these four proteases are shown in FIG. 33. As shown by these results, protease 6984 showed enhanced or comparative thermal stability at 45 degrees centigrade than the BPN′ variant, PURAFECT®, or the GG36 variant.
In addition to the above experiments, an alternative method for determining the thermostability of ASP was also tested. In these experiments, a temperature gradient between 57°-62° C. was used. The thermal inactivation (using a Thermocycler-MTP plate DNA Engine Tetad; MJ Research) was performed by incubating a volume of 180 μl 100 mM Tris buffer, pH 8.6, containing 1 mM CaCl2 and 5 ppm protease for 60 minutes and assayed for enzyme activity as follows: 10 μl was taken and added to 190 μl AAPF substrate solution, conc. 1 mg/ml, in 0.1 M Tris/0.005% TWEEN®, pH 8.6. The rate of increase in absorbance at 410 nm due to release of p-nitroaniline was monitored (at 25° C.). The results of 4 proteases are shown in FIG. 34.
Example 24
pH Profile of ASP Protease on DMC Substrate
In this Example, experiments conducted to determine the pH profile of the ASP protease are described. The Cellulomonas 69B4 protease of the present invention, isolated and purified by methods described herein and three currently used subtilisin proteases (PURAFECT®, BPN′-varianl 1, GG36-variant-1) described in Examples 21-23, were analyzed for their ability to hydrolyze a commercial synthetic substrate, di-methyl casein (“DMC”/Sigma C-9801) in the pH range from 4 to 12.
The DMC method described at the beginning of the Experimental section was used, with modifications, as indicated below. Briefly, a 5 mg/ml DMC substrate solution was prepared in the appropriate buffer (5 mg/ml DMC, 0.005% (w/w) TWEEN-80® (polyoxyethylene sorbitan mono-oleate, Sigma P-1754)). The appropriate DMC buffers were composed as follows: 40 mM MES for pH 4 and 5; 40 mM HEPES for pH 6 and 7, 40 mM TRIS for pH 8 and 9; and 40 mM Carbonate for pH 10, 11 and 12.
For the determination, 180 μl of each pH-substrate solution was transferred into 96 well microtiter plate and were pre-incubated at 37° C. for twenty minutes prior to enzyme addition. The respective enzyme solutions (BPN′-variant-1; GG36-variant-1; PURAFECT®; and 69B4 protease) were prepared, containing about 25 ppm and 20 μl of these enzyme solutions. These enzyme solutions were pipetted into the substrate containing wells in order to achieve a 2.5 ppm final enzyme concentration in each well. The 96 well plate containing enzyme-substrate mixtures was incubated at 37° C. and 300 rpm for one hour in an IKS-Multitron incubator/shaker.
A 2,4,6-trinitrobenzene sulfonate (“TNBS”) color reaction method was used to determine the amount of peptides and amino acids release from DMC substrate. The free amino groups (of the peptides and amino acids) react with 2,4,6-trinitro-benzene sulfonic acid to form a yellow colored complex. The absorbance was measured at 405 nm in a SpectraMax 250 MTP Reader.
The TNBS assay was conducted as follows. A 1 mg/ml solution of TNBS (5% 2,4,6 trinitrobenzene sulfonic acid/Sigma-P2297) was prepared in reagent buffer A (2.4 g NaOH, 45.4 g Na2B4O7.10H2O dissolved by heating in 1000 ml). Then, 60 μl per well were aliquoted into a 96-well plate and 10 μl of the incubation mixture described above were added to each well and mixed for 20 minutes at room temperature. Then, 200 μl of reagent B (70.4 g NaH2PO4.H2O and 1.2 g Na2SO3 in 2000 ml) were added to each well and mixed to stop the reaction. The absorbance at 405 nm was measured in a SpectraMax 250 MTP Reader. The absorbance value was corrected for a blank (without enzyme).
The data in Table 24-1 show the comparative ability of the 69B4 protease to hydrolyze such substrate versus proteases from a known mutant variants (BPN′ variant-1 and GG36 variant-1).
Also, as shown in FIG. 35, the serine protease of the present invention showed comparative or increased hydrolysis of DMC substrate with an optimal DMC-hydrolysis activity over a broad pH range from 7 to 12.







TABLE 24-1







TNBS Response









TNBS response (OD405 nm)
















Enzyme
pH4
pH5
pH6
pH7
pH8
pH9
pH10
pH11
pH12



















BPN′
0.095
0.174
0.482
0.749
0.813
0.847
0.730
0.683
0.590


variant-1











GG36
0.228
0.172
0.499
0.740
0.958
1.062
1.068
1.175
1.136


variant-1











Purafect ®
0.042
0.202
0.545
0.783
0.956
1.130
1.102
1.188
1.174


69B4
0.252
0.218
0.575
0.742
0.803
0.965
0.762
0.741
0.729









Example 25
pH Stability of ASP Protease
In this Example, experiments conducted to determine the pH stability of the ASP protease are described. As in Examples 21-24, two currently used subtilisin proteases (PURAFECT® and BPN′-variant-1) were also tested.
The respective enzyme solutions (i.e., BPN′-variant-1, PURAFECT®, and 69B4 protease) were prepared containing 90 ppm protease in 0.1 M Citrate buffer, pH 3, 4, 5 and 6. Then, 10 ml tubes containing 1 ml of buffered enzyme solutions were placed in a GFL 1083 water bath set at 25° C., 35° C. and 45° C. respectively, for 60 minutes. AAPF activity was determined for each enzyme sample at time 0 and 60 minutes as described above. The remaining enzyme activity was calculated and the results are provided in Table 25-1 below, and are shown in FIGS. 25-28).
As indicated by the data in Table 25-1, the ASP protease is exceptional stable at pH 3, 4, 5, and 6, at temperatures between 25° C. and 45° C., as compared to the BPN′ variant-1 and PURAFECT®.







TABLE 25-1







pH Stability Data











BPN′ Variant-1
PURAFECT ®
ASP
















pH
25°
35°
45°
25°
35°
45°
25°
35°
45°



















pH 3
39
1
0
42
2
0
97
109
95


pH 4
92
35
1
55
7
0
106
105
102


pH 5
112
82
12
95
68
8
114
115
106


pH 6
113
99
59
104
96
63
95
104
104









Example 26
Stability and Specificity of ASP
In this Example, experiments conducted to determine the stability and specificity differences between ASP, ASP mutants, and FNA are described. These experiments were performed by formulating liquid TIDE® detergent (Procter & Gamble) with calcium formate (an anionic surfactant titrant), borate (a P1 binder/inhibitor), and glycerol (water ordering), either independently of or in combination with each other. The enzyme was tested under these conditions and the residual enzyme activity was determined over time at a fixed temperature.
The experiments are described in greater detail below. Unformulated liquid TIDE® detergent (i.e., without added enzyme stabilizing chemicals) was divided into eleven aliquots. Then, glycerol, borax, or calcium formate were added to the detergent aliquots in the proportions shown in Table 26-1.







TABLE 26-1







Detergent Additives (%)















% Calcium



Aliquot #
% Glycerol
% Borax
Formate
















1
5
0
.1



2
2.5 
1.5
.05



3
5
3
0



4
0
3
0



5
2.5 
1.5
.05



6
0
0
.1



7
0
3
.1



8 
0
0
0



9
5
0
0



10
2.5
1.5
.05



11
5
3
.1










Each aliquot was pre-warmed to 90° F., and either FNA, ASP (wild-type) or an ASP R18 variant was added to approximately one gram per liter protease. After thorough mixing, a portion was removed and assayed for activity with synthetic AAPF-pNA substrate, as described above. After the assay, each aliquot was placed back into a 90° F. oven. The assay process was repeated over time, and the decline in activity at T0 was plotted as a % T0 activity remaining.
Surprisingly, it was found that ASP did not have the same calcium formate or glycerol dependency as FNA. Furthermore, it was determined that borate (alone) had the most dramatic effect on stabilizing ASP. It was also found that the addition of stabilizing chemicals provided significant benefits to the wild-type ASP, as well as the ASP R18 variant, indicating that the variant site is independent of the borate-activated site.
Example 27
LAS Stability of ASP
In this Example, experiments conducted to determine the stability of ASP to anionic surfactants are described. LAS (linear alkyl sulfonate), an anionic surfactant, is a component of HDL detergents known to inactivate enzymes. The methods used are described above.
It was determined that wild-type ASP incubated in LAS dissolved in Tris HCl pH 8.6 is inactivated (See, Table 27-1, below). Further study revealed that inactivation is rapid (See, Table 27-2). As LAS is a negatively charged molecule, the hypothesis that electrostatic attraction of LAS with positively charged amino-acid side chains of ASP was the cause of the LAS sensitivity, was developed. To test this hypothesis, arginine residues (wild-type ASP contains no lysine residues), were mutated to other amino-acids.
Incubation of these mutants in 0.05% (w/v) LAS in Tris HCl pH8.6, for one hour revealed that all arginine replacement mutants were more stable than wild-type ASP. In contrast, non-arginine replacement mutations that were also tested for LAS stability were generally not improved compared to wild-type (See, Table 27-3). Subsequent multiple arginine replacement mutations revealed that the enzyme is substantially more stable than the wild-type enzyme, and more stable that single arginine replacement mutations (See, Table 27-4).
Another anionic surfactant that is used in HDL detergents is AES. Wild-type ASP was found to be unstable in high concentrations of AES (See, Table 27-5). The mutant ASP R18 was found to be more stable than wild-type in AES (See, Table 27-5). Also, the rate of inactivation of activity by 5% AES was found to be higher for the wild-type than the ASP R18 mutant (See, Table 27-6). These results confirm that replacement of arginine residues of ASP improves the stability of ASP in anionic detergents in general. It is not intended that the present invention be limited to any specific anionic detergents or mutations. Indeed, it is contemplated that various anionic detergents (as well as other detergents) will find use in the present invention, as will various ASP mutants.







TABLE 27-1







Inactivation of ASP by LAS in Tris HCl pH 8.6










% LAS (w/v)
% Activity of Control







Control (0 LAS)
100 



0.01
87



0.03
77



0.06
59



0.10
47



0.30
31



0.60
20



1.00
12

















TABLE 27-2







Time-course of ASP Inactivation by 0.1% LAS










Time (secs)
% Remaining Activity







 0
100 



 60
45



120
26



240
20



600
11

















TABLE 27-3







Stability of ASP and Single Mutants


(Incubated 0.05% LAS in Tris HCl, pH 8.6, for 60 mins.)










Mutant
% Remaining Activity of 0 LAS Control














Wild-type
18



R14L
47



R16I
49



R16L
56



R16Q
51



R35F
43



R127A
59



R127K
31



R127Q
52



R159K
25



T36S
11



G65Q
22



Y75G
7



N76L
17



S76V
17

















TABLE 27-4







Stability of ASP and Multiple Arginine Replacements


(Incubated 0.05% LAS in Tris HCl, pH 8.6. for 60 mins)










Mutant
% Remaining Activity of 0 LAS Control














Wild-type
27.5



ASP R-1
98.8



ASP R-2
69.6



ASP R-3
100.2



ASP R-7
103.9



ASP R-10B
98.9



ASP R-18
100.9



ASP R23
79.4







In this Table,



R-1 = R16Q/R35F/R159Q



R-2 = R159Q



R-3 = R16Q/R123L



R-7 = R14L/R127Q/R159Q



R-10B = R14L/R179Q



R-18 = R123L/R127Q/R179Q.



R-21 = R16Q/R79T/R127Q



R-23 = R16Q/R79T













TABLE 27-5







Inactivation of ASP and ASP Mutant R-18 by AES in Tris HCl pH 8.6









% Remaining activity of 0% AES control









% AES (v/w)
Wild-type ASP
ASP R-18












0
100
100


1
70
94


5
32
57
















TABLE 27-6







Time-course of ASP and Mutant R-18 Inactivation


by 5% AES in Tris HCl, pH 8.6









% Remaining Activity of 0% AES Control









Time (Mins)
Wild-type ASP
ASP R-128












0
100
100


90
99
105


4020
15
83









Example 28
Determination of ASP Autolysis Sites in the Presence and Absence of LAS Detergent
In this Example, experiments conducted to determine the ASP autolysis sits in the presence and absence of LAS are described. ASP autolysis was evaluated in a buffer with and without LAS (dodecylbenzene-sulfonic acid). Autolysis peptide assignments were made based on molecular weight and sequence of each peptide (from MS and MS/MS data, respectively).
ASP (at concentration of 0.35 ug/uL) was incubated (at 4° C.) in a 100 mM Tris pH 8.6 with and without 0.1% LAS (dodecylbenzene-sulfonic acid). Aliquots were taken at time periods from 0 to 30 min of incubation and autolysis was terminated by an addition of TFA (final concentration 1%). Aliquots (10 μL) were analyzed by liquid chromatography coupled with electrospray tandem mass spectrometry (LC-ESI-MS/MS). Peptides were resolved using an HPLC system (model 1100, Agilent Technologies) using a reversed-phase column (Vydac C4, 0.3 mmID×150 mm), and a gradient from 0 to 100% solvent B (0.1% formic acid in acetonitrile) in 60 min at a flow rate of 5 μL/min (generated using a static split from a pump flow rate of 250uL/min). Solvent A consisted of 0.1% formic acid in water; and solvent B was 0.1% formic acid in acetonitrile.
Mass spectra were acquired using ion trap mass spectrometer (model LCQ Classic, Thermo). The mass spectrometer was tuned for optimum detection of m/z of 785 and operated with spray voltage of 2.5 kV, and a heated capillary at 250° C. Mass spectra were acquired with injection time of 500 msec and 5 microscans. Tandem MS spectra were acquired in data-dependent mode, with the most intense peak selected and fragmented with a normalized collision energy of 35%. For relative peptide quantitation, peak areas were determined using vendor software. The identity of the autolysis peptides was determined using a database search program (TurboSequest, Thermo) run on a database containing ASP sequence. Database searches were performed with no enzyme selected, threshold of 10000, dta file parameters (peptide m/z error of 1.7, group 11, minimum ion count 15), and database parameters (peptide error of 2.2, MS/MS ions error of 0.0, both B,Y ions).
Without LAS in the sample buffer, ASP cleavages were primarily observed at the termini and in the middle of the molecule (positions Y9, F47, Y59, F165, Q174, Y176; See Table 28-1, below). Relative quantitative data for observed peptides and intact ASP was plotted over the course of the experiment (See, FIG. 25, Panel A). The majority of the ASP remained intact and only 1% was in the form of cleaved peptides (protein:peptide ratio of 99:1) These data indicated that the majority of ASP remains intact, folded, and resistant to further autolytic cleavage.
With 0.1% LAS in the sample buffer, ASP cleavages were observed throughout the protein (positions Y9, T40, F47, Y57, F59, R61, L69, F165, Q174, Y176). The majority of the ASP was in the peptide form after 10 min (See, FIG. 25, Panel B). After 60 min, the protein:peptide ratio was <1:99. These data indicate that ASP is totally unfolded in the presence of LAS detergent, thus extensive cleavage throughout the sequence was observed. The observed autolysis cleavage sites under the two conditions are summarized in the following Table. In this Table, the amino acids preceding and following the periods are the amino acids that immediately precede and follow the autolysis peptide. The sequence between the periods indicates the sequence of the autolysis peptides observed.







TABLE 28-1







ASP Autolysis Peptides Observed With and Without 0.1% LAS

















Observed




Calculated
Measured
Observed in
in


Peptide Sequence
Start-End
Mass (Da)
Mass (Da)
0.1% LAS
0% LAS















-.FDVIGGNAY.T (SEQ ID NO: 631)
[1-9]
954.5
954.4
Y
Y


 


T.ANPTGTF.A (SEQ ID NO: 632)
[41-47]
706.3
706.3
Y
N


 


F.AGSSFPGNDY.A (SEQ ID NO: 633)
[48-57]
1013.4
1013.3
Y
N


 


F.AGSSFPGNDYAF.V (SEQ ID NO: 634)
[48-59]
1231.5
1231.4
Y
Y


 


R.TGAGVNLL.A (SEQ ID NO: 635)
[62-69]
743.3
743.4
Y
N


 


F.FQPVNPI.L (SEQ ID NO: 636)
[166-172]
813.4

N
N


 


F.FQPVNPILQ.A (SEQ ID NO: 637)
[166-174]
1054.6
1054.5
Y
Y


 


F.FQPVNPILQAY.G (SEQ ID NO: 638)
[166-176]
1288.7
1288.5
Y
Y









Example 29
Use of Reversible Inhibitors to Reduce LAS-Induced Degradation of ASP
In this Example, experiments conducted to assess the use of reversible inhibitors to reduce LAS-induced degradation of ASP are described. Benzamidine (BZA) is a known reversible inhibitor of serine proteases. Using the standard succ-AAPF-pNA assay as described above, BZA was shown to inhibit the activity of approximately 2 μg/ml ASP, with complete inhibition occurring at 1000 mM (1M), as indicated in Table 29-1, below:







TABLE 29-1







Inhibition of ASP










BZA Conc. mM
Assay Rate














0
0.83



1
0.85



10
0.82



100
0.42



1000
0.02










Approximately 200 μg/ml ASP was then incubated with 0.1% LAS and with, and without 1M BZA for up to 4 days. Enzyme activity was measured at different time points by addition of 10 μl incubated sample to 990 μl of assay solution. This reduces the BZA concentration to 10 mM, which by reference to the table above is not inhibitory. Therefore, any loss of activity will be due to enzyme degradation. As indicated in the results below, enzyme incubated with 0.1% LAS and without BZA lost all activity (i.e., it was degraded), while enzyme incubated with 0.1% LAS and 1M BZA, retained activity over the 4 day time-course of the study, demonstrating that inhibition of ASP activity prevents degradation by LAS.







TABLE 29-2







Assay Rate Results for Enzyme Incubated with 0.1% LAS,


With/Without BZA









Time
ASP + 0.1% LAS
ASP + 0.1% LAS + 1M BZA













30
secs
0.755
0.761


30
mins
0.685
0.781


18
hrs
0.067
0.761


4
days
0.004
0.853









Example 30
Testing of Mutant ASPs
In addition to the tests described above, tests were conducted on various mutants of ASP. The methods described above in Example 1 were used. In the following Tables, “Variant Code” provides the wild-type amino acid, the position in the amino acid sequence, and the replacement amino acid (i.e., “F001A” indicates that the phenylalanine at position 1 in the amino acid sequence has been replaced by alanine in this particular variant).
Keratin Hydrolysis
The table (Table 30-1) below provides the keratin hydrolysis data obtained for the ASP variants which show activity on this substrate in the keratin assay as described above (“Protease Assay with Keratin in Microtiter Plates”). The values are relative to wild type (WT) and calculated as described in the assay procedure. Values greater than 1 are indicative of better activity than WT ASP.







TABLE 30-1







Keratin Hydrolysis Results











Keratin



Variant
hydrolysis



code
relative







F001T
1.24



F001D
1.13



F001H
1.04



F001M
1.01



F001E
1.01



V003L
1.08



I004E
1.00



N007L
1.18



A008E
1.18



A008G
1.13



A008D
1.04



T010N
1.27



T010E
1.20



T010D
1.13



T010G
1.04



I011A
1.01



G012D
1.17



G013S
1.16



G013M
1.03



G013A
1.01



R014L
1.52



R014Q
1.49



R014I
1.40



R014D
1.36



R014N
1.29



R014G
1.28



R014T
1.21



R014M
1.21



R014K
1.18



R014A
1.12



R014S
1.12



R014W
1.07



R014P
1.04



R014H
1.03



S015W
1.20



S015T
1.05



R016A
1.04



R016S
1.03



R016Q
1.03



I019V
1.11



N024E
2.44



N024A
1.72



N024T
1.55



N024Q
1.40



N024V
1.28



N024L
1.26



N024H
1.26



N024M
1.14



N024F
1.05



N024S
1.03



R035E
1.60



R035L
1.47



R035Q
1.42



R035F
1.41



R035A
1.37



R035K
1.26



R035T
1.22



R035H
1.18



R035M
1.17



R035Y
1.16



R035W
1.13



R035S
1.12



R035D
1.07



R035N
1.03



R035V
1.02



T036I
6.82



T036S
1.34



T036G
1.34



T036N
1.22



T036D
1.16



T036H
1.13



T036P
1.03



T036L
1.01



A038R
1.77



A038D
1.51



A038H
1.30



A038N
1.28



A038F
1.22



A038L
1.19



A038S
1.18



A038Y
1.17



A038T
1.10



A038V
1.07



A038G
1.03



A038I
1.01



T040V
1.11



A041N
1.17



A041D
1.17



A041I
1.07



A041L
1.03



T044E
1.03



A048E
1.09



G049A
1.36



G049S
1.26



G049H
1.16



G049F
1.13



G049L
1.04



G049T
1.00



S051D
1.33



S051Q
1.18



S051H
1.12



S051V
1.11



S051T
1.09



S051M
1.01



G054D
1.71



G054E
1.23



G054N
1.06



G054L
1.02



G054I
1.00



N055E
1.30



N055F
1.25



N055Q
1.05



R061M
1.20



R061T
1.16



R061E
1.15



R061H
1.10



R061S
1.09



R061N
1.08



R061K
1.07



R061V
1.01



T062I
1.00



G063D
1.18



G063V
1.07



A064I
1.40



A064N
1.21



A064Y
1.19



A064L
1.17



A064V
1.17



A064H
1.16



A064F
1.15



A064P
1.15



A064T
1.13



A064Q
1.13



A064M
1.13



A064S
1.11



A064W
1.09



A064G
1.01



G065P
1.42



G065D
1.29



G065Q
1.29



G065S
1.25



G065T
1.25



G065V
1.23



G065L
1.21



G065Y
1.16



G065A
1.05



G065R
1.02



N067D
1.36



N067G
1.20



N067T
1.12



N067E
1.12



N067S
1.10



N067H
1.09



N067A
1.08



N067Q
1.07



N067L
1.05



L068H
1.07



L069S
1.35



L069H
1.23



L069V
1.03



A070D
1.20



A070H
1.16



A070G
1.13



A070S
1.04



Q071G
1.20



Q071H
1.14



Q071D
1.13



Q071S
1.10



Q071A
1.07



Q071N
1.06



Q071I
1.06



V072I
1.11



N073T
1.95



N073S
1.07



N074G
1.75



Y075G
1.42



Y075F
1.24



S076D
1.69



S076V
1.48



S076E
1.47



S076Y
1.45



S076T
1.25



S076L
1.25



S076N
1.24



S076I
1.22



S076W
1.17



S076Q
1.13



S076A
1.08



G077T
2.13



G077S
1.21



G077N
1.06



G078D
1.35



G078A
1.27



G078S
1.07



G078N
1.07



G078V
1.03



G078T
1.00



R079G
1.48



R079D
1.44



R079P
1.43



R079A
1.31



R079E
1.31



R079L
1.25



R079V
1.25



R079T
1.23



R079M
1.23



R079S
1.23



R079C
1.02



V080L
1.03



Q081E
1.22



Q081D
1.12



Q081V
1.10



Q081H
1.10



Q081P
1.01



A083E
1.27



A083L
1.05



A083I
1.03



H085Q
1.26



H085T
1.22



H085L
1.14



H085M
1.10



H085A
1.06



H085S
1.02



T086D
1.33



T086E
1.24



T086I
1.08



T086L
1.07



T086Q
1.07



T086G
1.06



T086A
1.05



T086N
1.01



A088E
1.01



A088F
1.00



P089E
1.04



V090P
1.51



V090S
1.42



V090I
1.34



V090T
1.22



V090N
1.10



V090A
1.08



V090L
1.06



S092G
1.20



S092A
1.12



S092C
1.06



A093D
1.20



A093S
1.12



A093E
1.09



S099N
1.27



S099V
1.23



S099D
1.21



S099T
1.21



S099I
1.08



T101S
1.14



W103M
1.17



T107E
1.32



T107S
1.30



T107V
1.23



T107H
1.23



T107M
1.21



T107I
1.17



T107N
1.12



T107A
1.10



T107Q
1.03



T107K
1.01



T109E
1.36



T109I
1.11



T109G
1.10



T109A
1.10



T109L
1.08



T109P
1.05



T109H
1.03



T109N
1.00



A110S
1.10



A110T
1.03



A110H
1.01



L111E
1.08



N112E
1.61



N112D
1.42



N112Q
1.36



N112L
1.27



N112V
1.23



N112Y
1.20



N112I
1.13



N112S
1.06



N112R
1.04



S113T
1.21



S114A
1.12



V115A
1.15



T116E
1.34



T116Q
1.28



T116F
1.09



T116S
1.02



T121E
1.35



T121D
1.15



T121S
1.05



R123E
1.63



R123D
1.57



R123I
1.48



R123F
1.40



R123A
1.30



R123L
1.30



R123Q
1.29



R123N
1.24



R123H
1.22



R123T
1.16



R123Y
1.15



R123S
1.12



R123G
1.11



R123V
1.09



R123W
1.07



R123K
1.07



G124A
1.06



I126L
1.06



R127A
1.38



R127Q
1.23



R127H
1.19



R127S
1.19



R127K
1.17



R127Y
1.15



R127E
1.14



R127F
1.11



R127T
1.04



R127C
1.01



T129S
1.31



A132S
1.03



P134A
1.04



S140A 
1.02



L142V
1.31



A143N
1.07



N145E
1.33



N145D
1.14



N145T
1.10



N145S
1.07



N145Q
1.07



V150L
1.01



N157D
1.01



R159E
1.61



R159F
1.37



R159N
1.30



R159Q
1.28



R159D
1.23



R159K
1.20



R159C
1.19



R159S
1.10



R159A
1.10



R159L
1.09



R159Y
1.08



R159H 
1.08



R159V
1.08



R159G
1.06



R159M
1.06



T160E
1.19



T160D
1.02



G161K
1.04



T163D
1.11



T163I
1.08



T163C
1.03



Q167T
1.02



N170Y
2.23



N170D
1.38



N170L
1.12



N170A
1.06



N170C
1.04



N170G
1.04



I172T
6.27



A175E
1.04



G177M
1.01



R179V
1.60



R179T
1.53



R179D
1.48



R179N
1.42



R179E
1.42



R179M
1.41



R179A
1.39



R179I
1.38



R179K
1.32



R179Y
1.27



R179L
1.11



R179W
1.06



I181L
1.96



I181S
1.07



T182V
1.14



T182L
1.02



T183E
1.19



T183I
1.17



T183Q
1.07



T183D
1.05



D184E
1.02



S185N
1.11



S185D
1.03



S185M
1.03



S185G
1.01



G186N
2.05



S187H
1.05



S187E
1.01



S188E
1.08










DMC Assay

The following table (Table 30-2) provides the variants with improved specific activity on casein. The activity on casein as substrate for all variants was determined as described above (“Protease Assay with Dimethylcasein (96 wells), With or Without Preheating of the Protease for Activity and Thermostability Assays”). The values in the table provide relative values for each variant compared to the activity of the WT enzyme (i.e., each value is the quotient of (variant activity)/(wild type activity)). Every variant with a value higher than 1 is better than WT.







TABLE 30-2







DMC Assay Results











Casein



Variant
specific activity



code
relative to wild type














F001T
1.19



F001A
1.11



F001G
1.00



D002G
1.24



D002Q
1.24



D002A
1.12



D002H
1.10



D002N
1.10



V003L
1.33



V003I
1.28



V003T
1.17



I004V
1.07



I004Q
1.02



N007L
1.56



N007S
1.25



N007A
1.22



N007H
1.11



N007I
1.11



N007V
1.06



A008G
1.12



A008K
1.09



Y009V
1.06



T010G
1.18



T010K
1.12



T010Q
1.01



I011Q
1.28



I011A
1.26



I011T
1.16



I011S
1.11



I011L
1.06



G012W
1.11



G012R
1.02



G013M
1.09



G013S
1.08



R014E
1.27



S015F
1.09



S015A
1.03



I019V
1.04



N024A
2.48



N024E
2.37



N024T
1.70



N024Q
1.70



N024V
1.62



N024M
1.48



N024H
1.45



N024L
1.34



N024F
1.21



N024S
1.10



I028L
1.16



A030S
1.11



R035F
1.20



R035D
1.01



T036I
14.08



T036G
2.46



T036N
2.13



T036S
2.08



T036W
1.84



T036P
1.69



T036H
1.67



T036D
1.61



T036Y
1.48



T036V
1.48



T036R
1.38



T036F
1.36



T036L
1.33



T036C
1.12



A038R
3.72



A038F
1.45



A038D
1.39



A038S
1.38



A038H
1.36



A038L
1.30



A038N
1.24



A038K
1.17



A038V
1.17



A038Y
1.14



A038I
1.11



A038I
1.11



A038G
1.09



A038T
1.00



T039A
1.01



T040V
1.21



T040S
1.09



A041N
1.13



A041I
1.02



N042H
1.18



N042K
1.01



T046K
1.01



F047I
1.17



F047M
1.13



F047V
1.01



G049F
1.32



G049K
1.16



G049A
1.16



G049L
1.12



G049W
1.08



G049H
1.07



G049T
1.06



G049S
1.01



S051A
1.47



S051Q
1.14



S051F
1.13



S051H
1.09



G054D
1.66



G054R
1.33



G054L
1.32



G054H
1.32



G054K
1.24



G054M
1.24



G054A
1.23



G054I
1.22



G054Q
1.21



G054N
1.05



G054E
1.03



N055F
1.54



N055Q
1.17



N055K
1.11



N055H
1.09



N055E
1.00



Y057M
1.00



R061M
1.20



R061S
1.08



R061T
1.02



T062I
1.22



G063V
1.21



G063W
1.12



G063Q
1.09



G063D
1.08



G063H
1.07



G063R
1.05



A064W
1.34



A064H
1.28



A064N
1.26



A064Y
1.26



A064R
1.22



A064F
1.21



A064K
1.19



A064M
1.19



A064S
1.18



A064L
1.18



A064I
1.16



A064Q
1.11



A064T
1.11



A064V
1.10



A064P
1.01



A064G
1.00



G065P
1.57



G065R
1.56



G065V
1.48



G065Y
1.46



G065S
1.40



G065T
1.38



G065Q
1.37



G065L
1.26



G065A
1.16



G065H
1.12



G065I
1.07



G065D
1.05



V066H
1.46



V066D
1.45



V066I
1.29



V066L
1.25



V066E
1.24



V066A
1.23



V066M
1.10



V066N
1.10



V066G
1.08



V066T
1.03



N067G
1.38



N067L
1.30



N067K
1.29



N067A
1.25



N067H
1.22



N067T
1.19



N067D
1.18



N067S
1.16



N067Q
1.14



N067R
1.13



N067Y
1.12



N067V
1.12



N067F
1.11



N067M
1.06



N067E
1.05



L068W
1.10



L068H
1.05



L068P
1.04



L069S
2.13



L069H
1.60



L069V
1.27



L069W
1.14



L069K
1.05



L069R
1.02



L069N
1.01



A070H
1.53



A070S
1.33



A070D
1.24



A070G
1.09



A070P
1.07



A070W
1.04



Q071I
1.46



Q071K
1.41



Q071G
1.40



Q071M
1.33



Q071H
1.28



Q071A
1.26



Q071N
1.26



Q071S
1.19



Q071D
1.16



Q071F
1.14



Q071L
1.11



Q071R
1.10



Q071T
1.06



V072I
1.17



N073T
2.73



N073S
1.28



N073H
1.12



N074G
1.87



Y075I
1.37



Y075G
1.36



Y075F
1.34



S076W
1.77



S076Y
1.69



S076V
1.51



S076L
1.44



S076N
1.20



S076T
1.18



S076I
1.18



S076E
1.17



S076R
1.14



S076A
1.13



S076Q
1.11



S076K
1.09



S076K
1.09



S076H
1.05



G077T
2.50



G077S
1.34



G077Y
1.21



G077N
1.18



G077Q
1.02



G077R
1.02



G078A
1.64



G078S
1.35



G078H
1.31



G078T
1.29



G078D
1.25



G078N
1.23



G078I
1.19



G078V
1.19



G078R
1.18



G078M
1.01



R079P
1.24



R079G
1.20



V080H
1.24



V080L
1.22



V080F
1.15



Q081V
1.33



Q081K
1.30



Q081H
1.24



Q081I
1.13



Q081D
1.11



Q081P
1.07



Q081E
1.03



Q081R
1.01



A083N
1.13



A083M
1.09



A083G
1.08



A083L
1.08



A083H
1.07



A083I
1.03



A083E
1.02



A083V
1.02



H085Q
1.41



H085T
1.26



H085R
1.22



H085L
1.22



H085K
1.15



H085M
1.01



T086A
1.21



T086G
1.08



T086N
1.08



T086I
1.08



T086L
1.08



T086E
1.03



T086K
1.03



T086H
1.02



A088K
1.05



P089N
1.19



P089V
1.05



P089Y
1.02



P089T
1.00



V090P
1.62



V090I
1.30



V090S
1.26



V090A
1.12



V090T
1.11



V090L
1.10



V090F
1.02



S092G
1.25



S092C
1.07



A093Q
1.08



A093T
1.07



A093H
1.01



S099T
1.02



G102Q
1.09



W103M
1.54



W103I
1.33



W103Y
1.01



H104K
1.22



H104R
1.04



T107S
1.17



T107V
1.14



T107M
1.12



T107H
1.12



T107R
1.07



T107K
1.03



T107N
1.01



T107Q
1.01



T109I
1.30



T109H
1.23



T109A
1.22



T109P
1.20



T109R
1.19



T109L
1.19



T109G
1.16



T109N
1.09



T109V
1.07



T109E
1.06



A110T
1.11



A110S
1.11



N112I
1.11



N112R
1.08



N112G
1.06



N112L
1.04



N112Q
1.03



N112H
1.00



S114G
1.37



T116F
1.45



T116R
1.06



T116H
1.04



T116G
1.01



P118A
1.45



P118F
1.39



P118R
1.37



P118H
1.24



P118I
1.19



P118Q
1.17



P118K
1.16



P118E
1.13



P118G
1.00



E119R
1.94



E119K
1.28



E119Q
1.04



E119G
1.02



E119L
1.00



R123E
1.20



R123I
1.11



R123K
1.05



R123D
1.03



I126L
1.20



R127F
1.20



T129S
1.20



E133Q
1.10



P134R
1.06



S140G
1.03



L142V
1.12



L142M
1.08



A143N
1.12



A143S
1.11



N145I
1.26



N145Q
1.25



N145E
1.24



N145G
1.16



N145T
1.14



N145L
1.11



N145S
1.07



N145F
1.04



N145R
1.04



N145P
1.00



Q146D
1.06



V150L
1.26



V150M
1.14



T151L
1.13



S155H
1.01



R159F
1.49



R159E
1.10



R159Y
1.07



R159K
1.04



R159N
1.01



G161K
1.08



T163I
1.13



F166Y
1.07



Q167N
1.16



Q167E
1.09



N170Y
2.76



N170D
1.15



N170L
1.12



N170A
1.11



N170C
1.05



N170R
1.03



N170P
1.01



P171T
1.02



Q174I
1.08



Q174L
1.02



A175V
1.04



A175T
1.02



A175H
1.02



G177M
1.42



G177S
1.09



G177R
1.04



R179V
1.63



R179M
1.36



R179D
1.33



R179I
1.31



R179N
1.29



R179Y
1.29



R179T
1.27



R179L
1.23



R179K
1.23



R179A
1.22



R179E
1.22



R179W
1.06



R179F
1.06



T182V
1.20



T182W
1.02



T182Q
1.01



T183I
1.35



T183K
1.19



T183M
1.14



T183R
1.09



T183L
1.07



T183Q
1.07



T183E
1.05



T183H
1.02



D184F
1.18



D184R
1.18



D184H
1.14



D184Q
1.10



D184T
1.03



D184I
1.03



D184V
1.01



S185I
1.15



S185V
1.11



S185W
1.09



S185N
1.07



S185K
1.06



S185P
1.03



S185L
1.02



P189Y
1.06



P189W
1.02



P189R
1.01



I181H
1.37



I181G
1.12



I181N
1.15



G186V
1.49



G186E
1.54



G186I
1.41



G186L
1.05



G186N
1.01



S187P
1.63



S187E
1.12



S187T
1.29



S187L
1.12



S188M
1.25



S188L
1.04










Thermostability Assays

The data in the following table (Table 30-3) represent the relative thermostability data of variants of ASP relative to the stability of the WT ASP stability under these conditions. The stability was measured by determining casein activity before and after incubation at elevated temperature (See, “Thermostability Assays” above). The table contains the relative thermostability values compared to WT under these conditions. It is the quotient of (Variant residual activity/WT residual activity). A value greater than one indicates higher thermostability.







TABLE 30-3







Thermostability Assay Results










Variant
Thermostability



code
relative







V003R
1.53



I004D
1.89



I004P
1.89



I004G
1.66



A008G
1.16



Y009E
2.04



Y009P
2.04



T010Y
1.64



T010F
1.53



T010W
1.49



T010L
1.26



T010C
1.21



T010E
1.10



T010D
1.09



T010M
1.06



T010V
1.06



T010S
1.03



G012D
1.86



G012A
1.15



G012H
1.14



G012V
1.06



G012I
1.06



G012S
1.00



R014H
1.08



R014I
1.08



R014K
1.08



R014N
1.08



R014Q
1.08



R014S
1.08



R014T
1.08



S015Q
1.23



S015R
1.23



S015C
1.22



S015T
1.16



S015N
1.16



S015H
1.13



S015F
1.07



S015A
1.04



S015M
1.04



S015I
1.03



R016K
1.07



R016I
1.06



S018E
2.18



A022C
2.27



A022S
1.94



A022T
1.55



N024T
1.49



N024S
1.25



N024E
1.12



N024G
1.12



N024Q
1.04



N024K
1.04



N024A
1.01



N024V
1.01



G025S
1.25



G026I
2.50



G026K
2.50



G026L
2.50



G026Q
2.50



G026V
2.50



G026W
2.50



G026E
2.11



F027V
2.50



F027W
2.50



F027I
1.36



I028P
2.50



I028W
1.99



I028T
1.78



T029E
2.50



A030M
2.13



A030N
2.13



A030P
1.75



A030Y
1.57



G031M
2.13



G031H
1.65



G031V
1.63



G031N
1.55



G031A
1.15



H032A
1.37



H032C
1.01



H032R
1.01



C033M
2.13



C033L
2.04



C033N
1.85



C033E
1.85



C033D
1.36



C033T
1.01



C033K
1.01



R035H
1.08



R035Q
1.08



R035V
1.08



R035W
1.08



R035H
1.08



R035T
1.08



R035Y
1.05



T036V
1.13



T036I
1.09



T036K
1.08



T036P
1.08



A038D
1.60



A038C
1.43



A038Y
1.07



T039R
1.72



T039V
1.19



T039Q
1.11



T039K
1.07



T039W
1.07



T039L
1.03



T039P
1.03



T040D
2.33



T040Q
2.33



T040H
2.24



T040P
1.73



T040N
1.55



T040G
1.07



A041S
1.31



A041D
1.07



P043D
2.33



P043H
2.33



P043K
2.33



P043L
2.33



P043N
2.12



P043G
1.53



T044V
1.03



G045V
2.06



G045A
1.82



T046Y
1.68



T046V
1.66



T046W
1.43



T046F
1.32



T046Q
1.01



A048P
1.96



A048V
1.05



A048E
1.04



G049A
1.22



S051V
1.32



S051C
0.99



P053N
1.00



G054E
1.00



Y057N
1.65



Y057M
1.55



F059K
2.17



F059W
1.33



F059C
1.07



T062R
1.92



T062G
1.44



A070P
1.89



A070G
1.43



Q071Y
1.35



Q071A
1.21



Q071F
1.06



N073P
2.08



N074F
1.36



S076A
1.00



R079T
1.58



R079V
1.31



R079M
1.01



Q081A
1.92



Q081S
1.65



Q081P
1.57



Q081G
1.54



Q081H
1.52



Q081D
1.51



Q081F
1.43



Q081E
1.39



Q081C
1.13



Q081T
1.08



A083H
1.62



A083M
1.35



A083E
1.23



A083F
1.20



A083R
1.14



A083S
1.00



G084C
2.08



G084P
2.08



G084V
1.17



G084M
1.17



T086S
1.39



T086I
1.20



T086M
1.12



T086A
1.11



T086H
1.08



T086D
1.06



T086N
1.05



T086V
1.04



A087S
1.20



A087E
1.12



P089W
2.22



P089A
1.27



V090A
1.35



V090M
1.18



V090I
1.11



V090T
1.03



G091L
2.22



G091K
1.06



S092T
1.14



A093S
1.66



A093D
1.19



A093Q
1.06



A093Q
1.06



A093N
1.06



A093G
1.02



R096C
1.92



R096F
1.75



R096E
1.57



S099A
1.80



S099G
1.17



T100A
1.70



T100D
1.18



T100Q
1.16



T100E
1.08



T101S
1.14



W103N
1.20



C105E
1.89



C105G
1.89



C105K
1.89



C105M
1.89



C105N
1.89



C105S
1.89



C105P
1.72



C105W
1.69



C105T
1.28



C105Y
1.22



C105A
1.21



C105L
1.18



T107S
1.30



T107L
1.24



T107Q
1.24



T107A
1.17



T107F
1.14



T107R
1.11



T107K
1.10



T107H
1.02



T107M
1.00



A110G
1.15



L111K
1.17



L111R
1.10



N112D
1.08



N112E
1.08



N112G
1.08



N112H
1.08



N112Q
1.08



N112R
1.07



N112L
1.03



N112P
1.03



N112F
1.01



S113M
1.08



S113N
1.08



S113R
1.08



S113T
1.08



S113C
1.04



S113H
1.01



S113F
1.00



S113I
0.99



V115I
1.18



V115L
1.14



V115T
1.05



T116Q
1.13



T116E
1.09



T116L
1.03



Y117K
1.41



Y117Q
1.41



Y117R
1.41



Y117V
1.41



P118T
1.12



P118R
1.08



P118Q
1.03



P118S
1.02



E119L
1.24



E119V
1.03



T121E
1.54



T121D
1.23



T121A
1.15



T121S
1.05



T121H
1.03



V122C
1.02



R123W
1.73



R123F
1.67



R123Y
1.58



R123N
1.53



R123L
1.39



R123I
1.39



R123T
1.35



R123Q
1.20



R123K
1.18



R123V
1.11



L125A
1.45



L125M
1.38



R127K
1.41



R127Q
1.41



R127F
1.21



R127Y
1.09



R127D
1.03



R127E
1.03



T128A
1.89



T128V
1.89



T128G
1.88



T128S
1.48



T128C
1.47



T129W
2.50



T129Y
1.30



V130T
1.13



V130C
1.07



A132C
1.19



P134W
1.18



S137R
1.92



S140P
1.88



L141C
1.33



L142M
1.10



A143H
1.19



G144A
1.14



G144V
1.10



G144D
1.02



G144I
1.00



G144E
0.99



Q146P
1.53



Q146Y
1.02



A147E
2.00



A147C
1.08



V150N
1.12



T151C
1.30



T151A
1.07



G153K
1.23



G153V
1.23



G154L
1.17



G154R
1.14



G154E
1.13



S155P
1.92



S155R
1.92



S155W
1.78



S155K
1.69



S155Y
1.66



S155F
1.48



S155T
1.18



S155V
0.99



G156I
1.92



G156L
1.92



G156P
1.81



G156V
1.08



G156E
1.03



C158H
2.00



C158G
1.57



C158M
1.49



R159K
1.56



R159T
1.26



R159V
1.15



R159Q
1.14



T160I
1.48



T160E
1.27



T160Q
1.14



T160L
1.09



T160D
1.04



T160R
1.04



G161L
2.13



G161V
2.13



G161I
1.50



G161K
1.24



G162P
1.32



G162L
1.11



T163I
1.19



T163V
1.02



T164G
1.83



T164L
1.54



F165T
1.01



F165D
0.99



F166S
1.44



F166C
1.29



F166A
1.20



F166G
1.01



Q167L
1.79



Q167N
1.08



P168Y
1.45



P168I
1.17



N170E
1.32



N170D
1.17



N170L
1.06



N170V
0.99



Q174H
1.11



Q174L
1.06



Q174R
1.06



Q174V
1.03



Y176P
1.48



Y176K
1.06



Y176D
1.03



G177N
1.18



G177K
1.03



R179K
1.21



M180L
1.30



T182L
1.14



T182V
1.01



T183P
1.26



T183I
1.17



T183A
1.13



T183S
1.11



T183V
1.06



D184E
1.04



S185R
1.32



S185Q
1.08



G186S
1.65



G186P
1.23



S187R
1.02



S187G
1.00



S188A
1.44



S188E
1.42



S188V
1.42



S188T
1.36



S188M
1.26



S188G
1.23



S188C
1.16



S188H
1.01



P189S
1.16



P189S
1.16



P189D
1.04



P189K
1.04



P189Y
1.03



P189F
0.99










BMI-LVJ 1 Performance Assays

The following table (Table 30-4) provides the data obtained for selected variants in the BMI-LVJ 1 performance assay (See, “Microswatch Assay for Testing Protease Performance”). The table shows performance indices, which where calculated as described above for the variants, which show improved performance compare to the WT enzyme. Those variants, which have a performance index greater than 1, have an improved performance.







TABLE 30-4







BMI-LVJ 1 Performance Assay Results











BMI US LVJ-1



Variant
liquid detergent



code
[perf. Index]







F001T 
1.06 



D002Q 
1.14



D002E 
1.05



D002P 
1.01



V003L 
1.24 



V003I
1.12



N007L 
1.14 



A008G 
1.09



A008D
1.07



A008E
1.04



A008M
1.03



A008K
1.01



T010E
1.10



T010Q
1.08



T010L
1.08



T010D
1.02



T010G
1.01



I011Q
1.18



I011A
1.13



I011T
1.12



I011S
1.11



I011L
1.06



G012D
1.08



G012Y
1.07



G012N
1.05



G012L
1.03



G012Q
1.00



R014I
1.25



R014M
1.20



R014L
1.11



R014E
1.09



R014N
1.08



R014P
1.08



R014G
1.03



R014Q
1.03



S015E
1.09



S015G
1.04



R016Q
1.14



R016L
1.14



R016N
1.11



R016G
1.11



R016I
1.09



R016A
1.09



R016M
1.03



I019V
1.02



N024E
1.36



N024A
1.31



N024T
1.21



N024Q
1.19



N024V
1.17



N024H
1.14



N024M
1.13



N024L
1.12



N024S
1.07



N024W
1.00



R035F
1.23



R035L
1.14



R035A
1.06



R035D
1.03



R035H
1.03



T036I
9.16



T036N
1.77



T036G
1.64



T036S
1.61



T036P
1.49



T036D
1.41



T036H
1.25



T036Y
1.25



T036L
1.18



T036W
1.15



T036F
1.05



A038R
1.55



A038L
1.18



A038S
1.16



A038Y
1.12



A038N
1.10



A038D
1.09



A038F
1.08



A038V
1.06



T040V
1.10



T040S
1.02



A041N
1.09



A041I
1.04



N042H
1.11



T046Q
1.05



F047V
1.02



A048Q
1.26



G049A
1.15



G049F
1.11



G049H
1.09



G049S
1.07



G049T
1.02



G049V
1.01



S050F
1.04



S051Q
1.10



S051T
1.07



S051D
1.05



S051A
1.05



S051V
1.03



S051M
1.01



S051H
1.01



G054D
1.48



G054Q
1.17



G054E
1.16



G054N
1.14



G054I
1.14



G054L
1.11



G054M
1.09



G054A
1.08



G054H
1.00



N055F
1.07



N055E
1.01



Y057M
1.00



R061V
1.13



R061K
1.12



R061M
1.11



R061H
1.08



R061S
1.06



R061T
1.06



T062I
1.02



A064H
1.15



A064F
1.14



A064Y
1.13



A064W
1.10



A064N
1.10



A064T
1.09



A064S
1.08



A064V
1.06



A064Q
1.04



A064I
1.04



A064L
1.02



A064G
1.02



G065P
1.28



G065Q
1.27



G065T
1.19



G065Y
1.19



G065S
1.17



G065L
1.13



G065V
1.11



G065R
1.10



V066H
1.15



V066D
1.07



V066E
1.02



N067L
1.25



N067S
1.23



N067A
1.19



N067Y
1.16



N067G
1.14



N067V
1.14



N067Q
1.13



N067T
1.10



N067F
1.08



N067M
1.07



N067K
1.06



N067D
1.02



N067H
1.02



N067C
1.01



L068H
1.02



L069S
1.29



L069H
1.14



L069W
1.06



L069V
1.02



A070G
1.12



A070P
1.09



A070D
1.01



Q071I
1.14



Q071H
1.13



Q071F
1.12



Q071D
1.11



Q071L
1.09



Q071T
1.06



Q071Y
1.04



Q071S
1.04



Q071A
1.03



V072I
1.04



N073T
1.67



N074G
1.28



Y075G
1.37



Y075F
1.30



Y075I
1.18



S076W
1.49



S076L
1.39



S076Y
1.37



S076T
1.30



S076V
1.30



S076I
1.25



S076D
1.22



S076N
1.20



S076A
1.16



S076E
1.14



G077T
1.48



G077S
1.11



G077N
1.07



G078D
1.24



G078A
1.12



G078N
1.10



G078H
1.04



G078S
1.02



R079P
1.20



R079G
1.13



R079D
1.12



R079C
1.02



V080L
1.11



V080H
1.09



V080Q
1.04



Q081P
1.22



Q081V
1.02



Q081K
1.01



Q081H
1.01



A083N
1.03



A083E
1.03



H085Q
1.42



H085L
1.30



H085R
1.23



H085K
1.19



H085F
1.13



H085Y
1.11



H085T
1.10



H085M
1.05



H085V
1.02



T086N
1.07



T086D
1.05



T086R
1.01



T086Q
1.00



T086I
1.00



T086V
1.00



A088F
1.12



A088H
1.04



P089D
2.85



P089N
1.11



P089V
1.07



P089T
1.00



V090I
1.12



V090P
1.10



V090T
1.06



V090L
1.01



S092A
1.21



S092G
1.11



A093T
2.26



A093S
2.15



A093D
1.10



A093E
1.06



A093Q
1.05



A093H
1.00



R096K
1.02



S099W
1.50



S099N
1.38



S099A
1.22



S099D
1.15



S099T
1.14



S099G
1.09



S099E
1.03



S099V
1.01



G102Q
1.01



W103M
1.01



H104S
1.05



T107H
1.02



T107N
1.01



T107S
1.01



T109E
1.15



T109N
1.03



T109I
1.02



L111E
1.10



L111D
1.07



L111T
1.01



N112E
1.14



N112L
1.11



N112Q
1.09



N112D
1.08



N112G
1.07



N112A
1.01



N112H
1.01



S113A
1.13



S113G
1.12



S113M
1.04



S114G
1.25



S114A
1.05



V115T
1.03



T116F
1.15



T116E
1.02



P118E
1.02



T121D
1.13



T121E
1.11



T121S
1.04



R123I
1.24



R123F
1.22



R123L
1.15



R123Q
1.12



R123E
1.10



R123K
1.07



R123P
1.03



R123W
1.02



R123H
1.01



G124A
1.01



L125V
1.06



R127A
1.44



R127S
1.36



R127Q
1.36



R127K
1.28



R127L
1.25



R127H
1.25



R127Y
1.23



R127F
1.22



R127T
1.19



R127G
1.16



R127V
1.01



T129S
1.03



A132V
1.22



A132C
1.03



P134E
1.14



P134G
1.06



P134A
1.01



S140A
1.07



L142V
1.09



L142M
1.02



A143N
1.21



A143S
1.05



A143H
1.01



N145D
1.06



N145S
1.02



V150L
1.07



N157D
1.17



R159F
1.63



R159E
1.43



R159K
1.29



R159H
1.28



R159N
1.22



R159Y
1.17



R159D
1.17



R159V
1.12



R159C
1.11



R159L
1.10



R159A
1.06



R159W
1.02



T160E
1.12



T160D
1.02



G161K
1.15



G161E
1.10



T163D
1.13



T163I
1.06



N170Y
1.34



N170D
1.09



N170L
1.08



N170G
1.03



N170A
1.00



P171S
1.03



P171V
1.01



A175V
1.05



G177M
1.03



R179V
1.19



R179T
1.11



R179K
1.10



R179N
1.09



R179D
1.07



R179E
1.06



R179A
1.03



R179I
1.01



R179M
1.01



R179F
1.00



I181Q
1.24



I181H
1.07



I181T
1.00



T182V
1.05



T182L
1.00



T183I
1.10



T183V
1.03



T183S
1.01



D184F
1.34



D184H
1.15



D184R
1.11



D184T
1.08



D184I
1.07



D184Q
1.06



D184L
1.05



S185I
1.09



S185W
1.08



S185L
1.05



S185L
1.05



S185M
1.04



S185G
1.01



G186V
1.27



G186E
1.20



G186I
1.17



G186L
1.06



G186T
1.03



G186Q
1.01



G186R
1.00



S187P
1.24



S187Q
1.13



S187E
1.08



S187T
1.04



S188Q
1.06



S188M
1.03



S188L
1.01



P189T
1.05



P189N
1.02



P189I
1.01










BMI-Low pH Performance Assays

The table below (Table 30-5) provides the data obtained for the ASP variants which show activity on this substrate in the microswatch assays under low pH conditions (See, Microswatch Assay for Testing Protease Performance) using TIDE®. The table provides performance indices, which were calculated as described above for the variants which show improved performance compared to WT. Variants that have a performance index greater than 1 have improved performance.







TABLE 30-5







BMI-Low pH Performance Assays











BMI US LVJ-1



Variant
liquid detergent



code
[perf. Index]







F001T
1.06



V003L
1.11



V003I
1.03



I004M
1.11



N007L
1.08



A008R
1.53



A008V
1.46



A008T
1.44



A008S
1.25



A008E
1.20



A008L
1.20



A008N
1.19



A008H
1.15



A008P
1.13



A008D
1.08



A008Q
1.07



T010Q
1.04



T010L
1.04



T010D
1.01



I011T
1.14



I011S
1.05



G012D
1.00



R014L
1.32



R014M
1.25



R014E
1.21



R014I
1.16



R014Q
1.16



R014N
1.07



R014K
1.05



R014D
1.01



S015E
1.05



R016Q
1.22



R016L
1.08



R016I
1.07



R016W
1.05



R016N
1.02



I019V
1.04



N024E
1.61



N024A
1.52



N024T
1.35



N024Q
1.25



N024L
1.21



N024M
1.15



N024V
1.15



N024H
1.14



N024F
1.06



N024S
1.03



R035F
1.36



R035L
1.21



R035A
1.14



R035E
1.13



R035D
1.08



R035H
1.07



T036I
9.02



T036N
1.69



T036G
1.63



T036S
1.59



T036P
1.41



T036D
1.28



T036V
1.19



T036W
1.07



T036H
1.06



T036L
1.02



T036F
1.02



A038R
1.89



A038F
1.41



A038S
1.32



A038L
1.26



A038D
1.25



A038H
1.20



A038N
1.13



A038I
1.10



A038Y
1.08



A038V
1.02



A038T
1.00



T040V
1.14



T040S
1.01



A041N
1.10



A041I
1.04



F047I
1.01



A048E
1.04



G049L
1.16



G049A
1.10



G049F
1.06



G049N
1.06



G049T
1.04



G049S
1.04



S051A
1.35



S051D
1.25



S051Q
1.12



S051F
1.09



S051T
1.08



S051H
1.06



G054D
1.67



G054I
1.22



G054L
1.21



G054E
1.20



G054Q
1.16



G054A
1.16



G054M
1.10



G054N
1.06



G054H
1.01



G054K
1.01



N055F
1.69



N055E
1.35



N055S
1.25



N055Q
1.15



N055V
1.09



N055T
1.02



F059W
1.01



R061M
1.35



R061T
1.22



R061V
1.15



R061S
1.07



R061N
1.02



R061K
1.02



R061Q
1.02



T062I
1.14



G063V
1.25



G063D
1.18



G063P
1.13



G063Q
1.12



A064N
1.28



A064H
1.24



A064S
1.23



A064Q
1.21



A064R
1.19



A064M
1.15



A064T
1.15



A064I
1.14



A064W
1.14



A064F
1.11



A064L
1.11



A064V
1.09



A064K
1.06



A064Y
1.05



G065P
1.66



G065Q
1.49



G065S
1.35



G065Y
1.32



G065T
1.26



G065R
1.22



G065D
1.16



G065A
1.12



G065L
1.05



G065V
1.04



V066D
1.21



V066E
1.08



N067G
1.41



N067V
1.39



N067L
1.32



N067T
1.31



N067D
1.25



N067M
1.25



N067F
1.24



N067S
1.24



N067Y
1.23



N067C
1.20



N067A
1.18



N067Q
1.13



N067R
1.11



N067K
1.07



N067E
1.07



N067H
1.06



L068T
1.03



L068H
1.01



L069S
1.79



L069H
1.64



L069W
1.26



L069V
1.21



L069Q
1.12



A070S
1.18



A070P
1.12



A070G
1.10



Q071M
1.15



Q071D
1.10



Q071S
1.03



N073T
1.77



N074G
1.61



Y075G
1.58



Y075F
1.40



S076V
1.71



S076Y
1.71



S076I
1.55



S076D
1.55



S076L
1.46



S076W
1.42



S076N
1.40



S076E
1.25



S076C
1.22



S076T
1.18



S076Q
1.17



S076A
1.11



S076K
1.07



S076H
1.00



G077T
1.86



G077Q
1.13



G077N
1.10



G077S
1.03



G078D
1.23



R079P
1.89



R079C
1.34



R079G
1.32



R079E
1.29



R079D
1.28



R079L
1.12



R079A
1.02



Q081V
1.31



Q081I
1.11



Q081E
1.10



Q081H
1.10



Q081L
1.07



Q081K
1.06



Q081D
1.06



Q081A
1.01



A083N
1.27



A083I
1.16



A083D
1.12



A083M
1.07



A083L
1.04



A083E
1.02



A083G
1.00



H085Q
1.24



H085L
1.19



H085R
1.12



H085N
1.08



H085T
1.08



H085F
1.05



H085K
1.04



T086A
1.27



T086I
1.24



T086L
1.22



T086F
1.21



T086E
1.15



T086M
1.11



T086D
1.07



1086C
1.04



T086Q
1.04



T086G
1.03



A088F
1.15



P089V
1.07



P089T
1.07



V090P
1.50



V090S
1.38



V090I
1.32



V090T
1.23



V090N
1.22



V090L
1.16



V090A
1.07



S092C
1.20



S092G
1.12



S092A
1.02



A093D
1.31



A093E
1.30



A093Q
1.09



R096K
1.15



T101S
1.16



W103M
1.18



W103Y
1.18



H104M
1.01



H104K
1.00



T107N
1.42



T107S
1.30



T107M
1.24



T107A
1.20



T107E
1.20



T107Q
1.15



T107H
1.11



T107V
1.10



T109E
1.46



T109I
1.31



T109A
1.28



T109G
1.21



T109H
1.13



T109N
1.13



T109L
1.10



T109F
1.01



A110S
1.19



A110T
1.13



A110N
1.05



N112E
1.24



N112D
1.20



N112Q
1.07



N112A
1.05



N112L
1.04



S113A
1.07 



S113G
1.04



S113M
1.03



S113E
1.00



S114G
1.20



S114T
1.05



S114A
1.03



T116F
1.12



T116G
1.06



T116E
1.06



T116Q
1.00



P118E
1.00



T121E
1.46



T121D
1.31



T121L
1.12



T121G
1.06



R123E
1.42



R123D
1.35



R123I
1.34



R123F
1.29



R123L
1.20



R123P
1.18



R123Q
1.14



R123A
1.12



R123H
1.12



R123K
1.11



R123N
1.01



G124N
1.04



G124T
1.00



L125V
1.17



I126L
1.26



R127A
1.38



R127S
1.31



R127Q
1.26



R127L
1.26



R127K
1.26



R127H
1.25



R127Y
1.21



R127T
1.19



R127F
1.18



R127G
1.06



R127V
1.04



T129S
1.20



T129G
1.14



A132V
1.19



A132S
1.08



E133D
1.06



P134A
1.25



P134E
1.23



P134D
1.15



P134G
1.09



S140A
1.15



L142V
1.28



L142M
1.02



A143N
1.25



A143M
1.03



A143S
1.03



N145S
1.36



N145E
1.32



N145Q
1.15



N145G
1.13



N145P
1.12



N145T
1.09



N145L
1.06



N145F
1.01



Q146D
1.12



Q146F
1.02



T151V
1.18



N157D
1.04



R159F
1.67



R159E
1.60



R159C
1.50



R159Y
1.31



R159D
1.30



R159K
1.25



R159Q
1.22



R159N
1.20



R159H
1.17



R159A
1.16



R159L
1.09



R159V
1.08



R159W
1.06



R159P
1.06



R159M
1.03



T160E
1.08



G161E
1.33



G161K
1.11



G161Q
1.05



T163D
1.25



T163I
1.00



T163C
1.00



F166Y
1.12



P168S
1.06



N170Y
2.54



N170D
1.20



N170C
1.19



N170L
1.06



N170L
1.06



N170P
1.02



N170H
1.00



P171M
1.18



P171V
1.03



I172V
1.28



A175T
1.13



A175V
1.12



A175F
1.02



Y176L
1.08



G177M
1.62



G177S
1.08



G177Q
1.08



R179V
1.70



R179M
1.44



R179I
1.39



R179Y
1.37



R179N
1.35



R179T
1.30



R179L
1.30



R179K
1.30



R179A
1.30



R179D
1.27



R179E
1.22



R179W
1.20



R179G
1.08



R179F
1.06



M180D
1.31



I181Q
1.07



I181C
1.01



I181L
1.00



I181T
1.00



T182V
1.23



T182W
1.11



T182L
1.07



T182Q
1.06



T182P
1.05



T183I
1.25



T183E
1.16



T183Q
1.14



T183K
1.10



T183L
1.10



T183A
1.05



T183D
1.05



T183V
1.05



T183R
1.04



T183M
1.03



D184F
1.00



G186E
1.42



G186V
1.34



G186I
1.21



G186L
1.11



G186P
1.09



G186T
1.09



G186A
1.03



S187P
1.39



S187T
1.18



S187E
1.11



S187L
1.07



S187Q
1.04



S187V
1.02



S188E
1.09



S188P
1.04










Scrambled Egg Assay (ADW) Performance

The following table (Table 30-6) provides the data obtained for selected variants in the scrambled egg performance assay (See, “Scrambled Egg Assay”) using Detergent Composition I. The table shows performance indices, which where calculated as described above for the variants, which show improved performance compare to the WT enzyme. Those variants, which have a performance index greater than 1, have an improved performance.







TABLE 30-6







Scrambled Egg Assay Performance Results










Variant
ADW



code
[perf. Index]







F001T
1.00



D002A
1.06



D002N
1.05



T010G
1.36



T010A
1.25



T010L
1.14



T010F
1.03



T010M
1.03



T010V
1.03



T010Q
1.02



T010S
1.01



I011A
1.20



I011S
1.20



I011T
1.18



I011L
1.02



G012I
1.12



G012Y
1.08



G012R
1.05



G012Q
1.04



R014M
1.26



R014G
1.14



R014A
1.10



S015G
1.14



S015F
1.14



S015E
1.13



S015H
1.08



R016K
1.15



R016N
1.12



R016A
1.10



R016H
1.03



I019V
1.02



A022V
1.23



N024E
1.67



N024T
1.46



N024Q
1.31



N024A
1.28



N024L
1.15



N024V
1.12



N024H
1.03



G034A
1.28



T036I
6.72



T036S
1.32



T036G
1.30



T036N
1.18



T036V
1.11



T036W
1.06



T036Y
1.05



T036D
1.02



T036P
1.02



T036F
1.01



A038T
1.27



A038F
1.24



A038M
1.00



T046K
1.10



F047I
1.05



F047V
1.02



G049F
1.17



G049A
1.13



G049L
1.10



G049H
1.05



G049S
1.02



G049V
1.01



S051A
1.13



Y057M
1.05



G063V
1.08



G063W
1.01



G063D
1.01



A064H
1.11



A064R
1.08



A064Y
1.07



A064W
1.07



A064V
1.06



A064T
1.05



A064N
1.05



A064K
1.04



A064Q
1.04



A064L
1.04



A064I
1.02



G065V
1.17



G065T
1.11



G065S
1.10



G065L
1.10



G065A
1.07



G065P
1.04



G065D
1.03



L069S
1.39



L069H
1.19



L069V
1.06



A070S
1.09



Q071I
1.15



Q071F
1.09



Q071M
1.05



Q071H
1.03



Q071D
1.02



Q071L
1.01



N073T
1.89



N074G
1.12



Y075F
1.10



Y075G
1.07



S076W
1.26



S076V
1.22



S076Y
1.21



S076D
1.13



S076L
1.12



S076E
1.09



S076R
1.09



S076N
1.08



S076A
1.07



S076Q
1.05



S076H
1.05



S076T
1.05



S076I
1.04



S076K
1.04



G077T
1.87



G078T
1.08



G078A
1.06



G078S
1.05



G078R
1.03



G078D
1.00



R079L
1.07



R079G
1.07



R079S
1.05



R079T
1.04



R079V
1.03



R079D
1.01



R079A
1.01



V080A
1.13



V080L
1.11



H085T
1.05



T086Q
1.03



T086A
1.02



A088F
1.04



P089A
1.03



V090I
1.17



V090P
1.13



A093S
1.04



A093Q
1.02



S099N
1.14



S099V
1.12



S099Q
1.05



S099I
1.01



T107R
1.13



T107K
1.12



T107S
1.10



T107H
1.09



T107F
1.09



T107I
1.07



T107M
1.07



T107V
1.06



T107A
1.06



T107L
1.04



T107W
1.02



T109R
1.07



T109I
1.06



T109V
1.02



A110S
1.01



N112S
1.31



S114A
1.11



S114T
1.09



V115A
1.04



T116A
1.10



T116S
1.03



P118F
1.06



P118R
1.05



E119R
1.27



T121L
1.05



T121S
1.03



T121Q
1.02



G124T
1.03



L125Q
1.02



R127F
1.15



T128S
1.10



T129S
1.11



P134R
1.89



P134E
1.49



P134L
1.48



P134H
1.29



P134V
1.23



P134D
1.13



P134T
1.11



P134S
1.08



S140A
1.33



L142V
1.24



A143S
1.06



N145D
1.01



V150L
1.12



T151L
1.07



G154S
1.01



R159F
1.39



R159K
1.15



R159Y
1.06



R159Q
1.00



G161K
1.11



T163I
1.15



T164G
1.11



F166Y
1.13



F166V
1.07



Q167N
1.13



N170Y
1.24



N170D
1.03



N170G
1.02



L178V
1.08



R179V
1.16



R179K
1.10



R179T
1.05



T182V
1.22



T182L
1.04



T183I
1.08



T183S
1.02



D184T
1.05



D184Q
1.03



G186S
1.34



G186E
1.26



G186V
1.19



G186I
1.11



G186A
1.05



G186L
1.02



S187E
1.02



S187T
1.02



S188A
1.19



S188M
1.07



S188G
1.02










Las Stability

The following table (Table 30-7) shows all variants, which have an improved stability compared to the WT-ASP. All variants were tested and the calculations determined according to the protocol shown above (See, “LAS Stability Assay”). The table provides the residual activity after incubation for the variants. Under these conditions the average of the WT value was found to be 10.59% residual activity. All variants with a higher activity are improved with respect to the WT molecule.







TABLE 30-7







LAS Stability Assay Results











LAS




stability




[residual



Variant
Activity



code
(%)]







F001P
21.73



F001N
16.59



F001R
11.13



D002P
22.43



D002I
20.86



D002V
20.15



D002T
19.97



D002M
15.20



D002N
13.27



D002F
12.71



D002A
12.13



D002C
11.50



A008G
33.00



A008T
20.39



A008R
18.33



A008P
14.19



T010L
24.24



T010C
24.00



T010Y
20.40



T010Q
19.48



T010D
18.06



T010E
17.48



T010F
17.10



T010M
14.94



T010W
12.63



I011W
50.85



I011E
26.05



I011T
23.20



I011Q
22.59



G012D
41.99



G012Q
28.25



G012N
27.52



G012V
27.44



G012S
24.06



G012I
23.30



G012H
19.43



G012Y
16.33



G012P
15.10



G012R
13.43



G012A
12.15



G012L
11.15



G012W
10.66



G013E
18.82



G013D
16.72



G013K
10.79



G013K
10.79



R014E
71.80



R014D
64.85



R014T
45.51



R014G
31.47



R014S
30.62



R014I
26.03



R014A
25.60



R014Q
25.38



R014C
23.91



R014N
23.61



R014M
18.47



R014H
15.72



R014L
15.35



R014P
12.43



S015R
57.77



S015H
53.39



S015C
50.38



S015E
25.99



S015Y
23.97



S015M
19.73



S015F
17.11



S015N
16.21



S015G
14.44



S015L
12.00



S015A
11.84



S015T
11.83



S015I
10.89



R016E
34.61



R016T
27.36



R016C
25.97



R016V
25.79



R016D
22.22



R016Q
19.87



R016I
19.83



R016S
10.71



A022C
27.48



A022S
25.99



N024E
23.54



N024T
18.16



N024G
15.54



N024S
14.04



N024F
13.05



N024V
11.86



I028V
14.49



R035E
88.92



R035D
76.48



R035Q
49.08



R035V
49.02



R035S
47.13



R035T
44.84



R035N
42.49



R035A
42.38



R035C
41.31



R035P
32.50



R035H
27.88



R035M
25.29



R035K
15.26



T036C
25.91



T036V
20.77



A038D
47.40



A038C
34.28



A038T
12.27



A041D
24.80



A041C
23.37



A041T
18.58



A041S
15.58



N042D
15.04



N042C
13.16



T044E
33.74



T044C
17.24



T046V
40.22



T046F
34.46



T046E
34.01



T046Y
27.10



T046C
23.20



F047R
46.98



F047V
20.38



F047I
12.72



A048E
29.23



G049C
64.06



G049Q
49.53



G049E
48.76



G049H
47.79



G049A
43.93



G049V
43.28



G049N
29.58



G049L
24.93



G049S
19.86



G049F
16.65



G049K
15.46



G049T
11.73



S051L
19.79



S051A
15.12



S051C
14.59



S051G
14.33



P053C
11.51



P053N
10.68



G054C
26.41



G054E
19.88



G054Q
12.71



G054K
11.71



N055G
33.29



N055A
15.31



D056L
42.96



D056F
17.11



Y057G
27.33



F059W
31.25



R061E
30.95



R061V
26.22



R061M
26.01



R061T
23.33



R061K
20.21



R061Q
18.05



G063D
13.79



A064C
15.65



G065D
14.73



V066N 
16.37



A070M
21.09



A070G
15.83



A070P
14.86



Q071L
11.17



Y075W
10.97



G078H
12.06



R079T
16.18



R079V
15.24



R079L
12.03



V080E
10.65



Q081P
18.28



Q081G
15.49



Q081A
14.60



Q081E
14.36



Q081H
14.02



Q081S
13.51



Q081D
13.17



Q081Y
13.15



Q081F
12.61



Q081I
11.93



Q081W
11.89



Q081C
11.40



A083H
17.04



A083D
15.14



A083E
14.66



A083Y
12.54



A083V
11.93



A083N
11.52



A083M
11.35



A083F
11.21



A083I
10.80



H085P
10.62



T086E
16.60



T086I
13.95



T086C
13.70



T086W
13.45



T086V
12.92



T086Y
10.97



T086F
10.78



T086D
10.70



A087E
20.99



A087C
17.19



A087P
11.78



A088F
18.06



A088E
14.11



A088V
13.47



A088H
10.95



P089D
10.88



V090C
12.71



G091Q
23.98



S092T
17.35



S092I
11.15



S092C
10.93



S092L
10.60



A093H
14.05



S099A
28.58



S099G
22.20



S099K
17.98



S099Q
17.50



S099H
15.09



T100A
27.16



T100R
22.31



T100K
22.07



T100Q
15.53



T100C
11.47



W103L
20.25



H104M
10.65



T107R
26.61



T107H
12.35



T109E
24.23



T109K
17.25



N112P
25.16



N112E
17.68



N112D
15.90



S113C
35.77



S113A
16.28



S113D
14.68



S113H
13.27



S114C
22.24



S114E
16.60



S114D
11.86



T116C
16.41



T116N
14.90



T116G
14.42



T116A
11.29



P118R
28.25



P118K
23.28



P118C
16.70



P118A
15.98



P118W
15.50



P118G
14.55



P118H
13.73



P118F
12.80



P118Y
11.29



E119G
32.98



E119Y
29.43



E119R
26.97



E119T
26.28



E119V
24.47



E119N
20.71



E119A
19.95



E119L
15.83



E119S
15.80



E119Q
14.68



T121E
36.49



T121L
34.33



T121F
23.82



T121A
17.78



T121D
16.73



T121V
14.25



T121Q
12.39



T121G
12.17



T121S
11.93



T121N
11.51



R123D
48.24



R123Y
47.97



R123C
46.46



R123E
44.33



R123N
40.60



R123H
39.41



R123T
34.97



R123W
33.83



R123F
30.58



R123S
30.56



R123Q
25.60



R123V
24.71



R123M
18.54



R123A
17.24



R123K
16.38



R123G
16.12



R123I
16.04



G124D
25.10



G124N
12.84



L125Q
25.77



L125M
14.90



R127E
36.18



R127S
31.24



R127D
29.46



R127Q
27.92



R127K
25.25



R127A
21.74



R127C
16.40



R127T
14.31



R127Y
13.61



R127H
12.89



R127F
10.69



T128A
21.49



T128V
12.94



V130C
12.97



A132S
19.09



A132P
11.71



P134R
22.20



S140P
21.06



L141M
18.59



L141C
12.46



A143H
10.95



G144E
12.63



N145E
12.29



Q146D
12.05



T151L
46.42



T151C
26.57



T151V
17.57



S155C
38.40



S155W
30.61



S155Y
23.95



S155I
22.60



S155V
21.53



S155E
19.78



S155T
17.58



S155F
17.11



S155Q
12.59



N157D
18.83



R159T
28.61



R159E
27.00



R159Q
25.25



R159D
23.12



R159V
22.92



R159S
22.29



R159K
20.78



R159N
19.95



R159C
19.24



R159A
19.09



R159M
15.74



R159L
14.00 



R159H
12.56



R159Y
11.23



T160D
15.18



T160E
11.72



T163D
23.84



T163C
19.09



T163Q
14.20



T163R
11.15



F165W
28.00



F165E
23.57



F165H
21.46



F165S
14.33



Q167E
64.13



Q167S
12.59



V169A
12.75



N170D
29.08



N170C
23.07



N170L
14.63



N170G
13.30



N170A
12.77



N170P
12.72



I172A
20.40



Q174C
16.62



Q174S
14.76



Q174T
14.54



Q174V
13.40



Q174H
11.18



A175T
16.19



G177D
24.74



G177E
21.37



G177C
14.01



G177N
11.53



R179E
25.06



R179D 
24.16



R179C 
20.71



R179V 
20.09



R179I 
19.51



R179T 
19.20



R179Y 
17.89



R179M 
16.74



R179S 
16.12



R179N 
16.11



R179F 
15.67



R179W 
15.56



R179L 
15.12



R179A 
14.35



R179K 
12.30



M180L 
25.64



M180I 
12.31



I181C 
11.51



T182L 
12.63



T183D 
13.51



T183E 
13.32



S185D 
14.31



S185C
13.10



S185Y
10.74



S185N 
10.73



G186E 
14.36



G186P 
13.48



G186C 
11.96



S187E 
15.92



S187F 
13.28



S187L 
12.26



S187C 
11.34



S187W 
11.21



S187G 
10.83



S187A 
10.72



S187V 
10.71



S187H 
10.66



S188E 
15.00



S188C 
12.56



S188T 
11.89



S188G 
11.15



S188V 
10.68










Example 31
Determination of ASP Cleaning Activity
In this Example, experiments conducted to determine the cleaning activity of ASP under various conditions, as well as the properties of the various wash conditions are described.
There is a wide variety of wash conditions including varying detergent formulations, wash water volume, wash water temperature, and length of wash time. Thus, detergent components such as proteases must be able to tolerate and function under adverse environmental conditions. For example, detergent formulations used in different areas have different concentrations of their relevant components present in the wash water. For example, a European detergent typically has about 3000-8000 ppm of detergent components in the wash water, while a Japanese detergent typically has less than 800 (e.g., 667 ppm) of detergent components in the wash water. In North America, particularly the United States, detergent typically have about 800 to 2000 (e.g., 975 ppm) of detergent components present in the wash water.
Latin American detergents are generally high suds phosphate builder detergents and the range of detergents used in Latin America can fall in both the medium and high detergent concentrations, as they range from 1500 ppm to 6000 ppm of detergent components in the wash water. Brazilian detergents typically has approximately 1500 ppm of detergent components present in the wash water. However, other high suds phosphate builder detergent geographies, not limited to other Latin American countries, may have high detergent concentration systems up to about 6000 ppm of detergent components present in the wash water.
In light of the foregoing, it is evident that concentrations of detergent compositions in typical wash solutions throughout the world varies from less than about 800 ppm of detergent composition (“low detergent concentration geographies”), for example about 667 ppm in Japan, to between about 800 ppm to about 2000 ppm (“medium detergent concentration geographies”), for example about 975 ppm in U.S. and about 1500 ppm in Brazil, to greater than about 2000 ppm (“high detergent concentration geographies”), for example about 3000 ppm to about 8000 ppm in Europe and about 6000 ppm in high suds phosphate builder geographies.
The concentrations of the typical wash solutions are determined empirically. For example, in the U.S., a typical washing machine holds a volume of about 64.4 L of wash solution. Accordingly, in order to obtain a concentration of about 975 ppm of detergent within the wash solution, about 62.79 g of detergent composition must be added to the 64.4 L of wash solution. This amount is the typical amount measured into the wash water by the consumer using the measuring cup provided with the detergent.
As a further example, different geographies use different wash temperatures. The temperature of the wash water in Japan is typically less than that used in Europe. For example, the temperature of the wash water in North America and Japan can be between 10 and 30° C. (e.g., about 20° C.), whereas the temperature of wash water in Europe is a typically between 30 and 50° C. (e.g., about 40° C.).
As a further example, different geographies may have different water hardness. Water hardness is typically described as grains per gallon mixed Ca2+/Mg2+. Hardness is a measure of the amount of calcium (Ca2+) and magnesium (Mg2+) in the water. Most water in the United States is hard, but the degree of hardness varies from area to area. Moderately hard (60-120 ppm) to hard (121-181 ppm) water has 60 to 181 parts per million (i.e., parts per million converted to grains per U.S. gallon is ppm # divided by 17.1 equals grains per gallon) of hardness minerals. Table 31-1 provides ranges of water hardness.







TABLE 31-1







Water Hardness Ranges









Water
Grains per Gallon
Parts per Million





Soft
less than 1.0
less than 17


Slightly hard
1.0 to 3.5
 17 to 60 


Moderately hard
3.5 to 7.0
 60 to 120


Hard
 7.0 to 10.5
120 to 180


Very hard
greater than 10.5
greater than 180









European water hardness is typically greater than 10.5 (e.g., 10.5-20.0) grains per gallon mixed Ca2+/Mg2+ (e.g., about 15 grains per gallon mixed Ca2+/Mg2+). North American water hardness is typically greater than Japanese water hardness, but less than European water hardness. For example, North American water hardness can be between 3 to 10 grains, 3-8 grains or about 6 grains. Japanese water hardness is typically lower than North American water hardness, typically less than 4, for example 3 grains per gallon mixed Ca2+/Mg2+.
The present invention provides protease variants that provide improved wash performance in at least one set of wash conditions and typically in multiple wash conditions.
As described herein, the protease variants are tested for performance in different types of detergent and wash conditions using a microswatch assay (See above, and U.S. patent application Ser. No. 09/554,992; and WO 99/34011, both of which are incorporated by reference herein). Protease variants are tested for other soil substrates also in a similar fashion.
In the experiments conducted to determine cleaning activity of ASP, the following methods were used. Incubators (Innova 4330 Model Incubator, New Brunswick) was pre-warmed for 60 minutes to 40° C. for “European” conditions and for 20° C. for “Japanese” conditions. Blood-Milk-Ink swatches (EMPA 116) were obtained from the Swiss Federal Laboratories for Material Testing and from CFT Research, and were modified by exposure to 0.03% hydrogen peroxide for 30 minutes at 60° C., then dried. Circles of 1⁄4′′ diameter were cut from the dried swatches and placed vertically, one per well, in a 96 well microplate.
Protease samples of ASP were diluted in 10 mM NaCl, 0.005% TWEEN®-80 to provide the desired concentration of 10 ppm (protein). To provide “North American wash conditions,” 1 gram per liter TIDE® laundry detergent (Procter & Gamble) without bleach was prepared in deionized water, and a concentrated stock of calcium and magnesium was added to result in a final water hardness value of 6 grains per gallon. To provide “European wash conditions,” 7.6 gram per liter ARIEL® REGULAR laundry detergent (Procter & Gamble) without bleach was prepared in deionized water, and a concentrated stock of calcium and magnesium was added to result in a final water hardness value of 15 grains per gallon. To provide “Japanese wash conditions,” 0.67 gram per liter PURE CLEAN laundry detergent (Procter & Gamble) without bleach was prepared in deionized water, and a concentrated stock of calcium and magnesium was added to result in a final water hardness value of 3 grains per gallon.
In yet another detergent composition to provide “Japanese wash conditions with North American detergent formulation,” 0.66 gram per liter Detergent Composition III without bleach was prepared in deionized water, and a concentrated stock of calcium and magnesium was added to result in a final water hardness value of 3 grains per gallon.
The detergent solutions were allowed to mix for 15 minutes and were then filtered through a 0.2 micron cellulose acetate filter. A 190 ul of the respective detergent solution was then added to the appropriate wells of a microplate. Then, 10 ul of the enzyme preparation were added to the filtered detergent in order to obtain a final concentration 0.25-3.0 ppm (micrograms per milliliter) of enzyme, for a total volume of 200 μl. The microplate was then sealed to prevent leakage, placed in a holder on an incubator/shaker set to 20° C. and 350/400 RPM and allowed to shake for one hour.
The plate was then removed from the incubator/shaker and an aliquot of 100 μl of solution was removed from each well, and placed on a fresh Costar microtiter plate (Corning). The absorbance at 405 nm wavelength was read for each aliquot on a Microtiter plate reader (SpectraMax 340, Molecular Devices), and reported. The detergent composition and incubation conditions in the microswatch assay are set forth in Table 31-2.







TABLE 31-2







Detergent Composition and Incubation Conditions














Water
Enzyme
Temper-



Geography
Detergent
Hardness
dosage
ature
Swatch















Powder







detergent







European
Ariel Regular
15 gpg
0.25-3.0
40°
Superfix



7.6 g/l
Ca/Mg = 4/1
ppm




North 
Detergent
6 gpg
0.25-3.0
20°
3K


American
Comp. III
Ca/Mg = 3/1
ppm





1.0 g/l






Japanese
Pure Clean
3 gpg
0.25-3.0
20°
3K



0.66 g/l
Ca/Mg = 3/1
ppm




Japanese
Detergent
3 gpg
0.25-3.0
20°
3K



Comp. III

ppm




(pseudo)
0.66 g/l
Ca/Mg = 3/1





Liquid 
Liquid-Tide ® 
6 gpg
0.25-3.0
20°
3K


detergent 


ppm




(1.5 ml/L) 














The dose response curves depicting absorbance at 405 nm as a function of concentration (ppm in well), for PURAFECT® (Genencor), OPTIMASE® (Genencor), RELASETM (Genencor; GG36-variant described above), and ASP are provided in FIGS. 23-27).
As indicated in FIG. 26, under North American conditions, in liquid TIDE® detergent, the ASP protease showed enhanced cleaning performance as compared to PURAFECT®, RELASETM and OPTIMASETM proteases under the same conditions. Under Japanese conditions, in Detergent Comp. III powder (0.66 g/l), ASP showed enhanced or the same cleaning performance as compared to PURAFECT®, RELASETM and OPTIMASETM proteases under the same conditions (See, FIG. 27). Under European conditions, in ARIEL® REGULAR powder detergent, the ASP protease showed enhanced cleaning performance as compared to PURAFECT®, RELASETM and OPTIMASETM proteases under the same conditions (See, FIG. 28). In both tests, ASP and OPTIMASETM provided results that were 2 to 10 times the absorbance at 405 nm as compared to PURAFECT® and RELASETM. Under Japanese conditions, in PURE CLEAN powder detergent (See, FIG. 29), the ASP protease showed enhanced and comparative cleaning performance as compared to PURAFECT®, RELASETM and OPTIMASETM proteases under the same conditions. Under North American conditions, in Detergent Composition III powder detergent (See, FIG. 30), the ASP protease showed enhanced or comparative cleaning performance as compared to PURAFECT®, RELASETM and OPTIMASETM proteases under the same conditions.
Example 32
Liquid Fabric Cleaning Compositions
This Example provides liquid fabric cleaning compositions that find use in conjunction with the present invention. These compositions are contemplated to find particular utility under Japanese machine wash conditions, as well as for applications involving cleaning of fine and/or delicate fabrics. Table 32-1 provides a suitable composition. However, it is not intended that the present invention be limited to this specific formulation, as many other formulations find use with the present invention.







TABLE 32-1







Liquid Fabric Cleaning Composition










Component
Amount (%)














AE2.5S
2.16



AS
3.30



N-Cocoyl N-methyl glucamine
1.10



Nonionic surfactant
10.00



Citric acid
0.40



Fatty acid
0.70



Base
0.85



Monoethanolamine
1.01



1,2-Propanediol
1.92



EtOH
0.24



HXS
2.09



Protease.sup.1
0.01



Amylase
0.06



Minors/inerts to 100%











Example 33
Liquid Dishwashing Compositions
This Example provides liquid dishwashing compositions that find use in conjunction with the present invention. These compositions are contemplated to find particular utility under Japanese dish washing conditions. Table 33-1 provide suitable compositions. However, it is not intended that the present invention be limited to this specific formulation, as many other formulations find use with the present invention.







TABLE 33-1







Liquid Dishwashing Compositions









Component
A
B












AE1.4S
24.69
24.69


N-cocoyl N-methyl glucamine
3.09
3.09


Amine oxide
2.06
2.06


Betaine
2.06
2.06


Nonionic surfactant
4.11
4.11


Hydrotrope
4.47
4.47


Magnesium
0.49
0.49


Ethanol
7.2
7.2


LemonEase
0.45
0.45


Geraniol/BHT
—
0.60/0.02


Amylase
0.03
0.005


Protease
0.01
0.43


Balance to 100%











Example 34
Liquid Fabric Cleaning Compositions
The proteases of the present invention find particular use in cleaning compositions. For example, it is contemplated that liquid fabric cleaning composition of particular utility under Japanese machine wash conditions be prepared in accordance with the invention. In some preferred embodiments, these compositions comprise the following components shown in Table 34-1.







TABLE 34-1







Liquid Fabric Cleaning Composition










Component
Amount (%)














AE2.5S
15.00



AS
5.50



N-Cocoyl N-methyl glucamine
5.50



Nonionic surfactant
4.50



Citric acid
3.00



Fatty acid
5.00



Base
0.97



Monoethanolamine
5.10



1,2-Propanediol
7.44



EtOH
5.50



HXS
1.90



Boric Acid
3.50



Ethoxylated tetraethylenepentaimine
3.00



SRP
0.30



Protease
0.069



Amylase
0.06



Cellulase
0.08



Lipase
0.18



Brightener
0.10



Minors/inerts to 100%










Example 35
Granular Fabric Cleaning Compositions
In this Example, various granular fabric cleaning compositions that find use with the present invention are provided. The following Tables provide suitable compositions. However, it is not intended that the present invention be limited to these specific formulations, as many other formulations find use with the present invention.







TABLE 35-1







Granular Fabric Cleaning Compositions









Formulations











Component
A
B
C
D














Protease1
0.10
0.20
0.03
0.05


Protease2


0.2
0.15


C13 linear alkyl benzene sulfonate
22.00
22.00
22.00
22.00


Phosphate (as sodium 
23.00
23.00
23.00
23.00


tripolyphosphate)






Sodium carbonate
23.00
23.00
23.00
23.00


Sodium silicate
14.00
14.00
14.00
14.00


Zeolite
8.20
8.20
8.20
8.20


Chelant (diethylaenetriamine- 
0.40
0.40
0.40
0.40


petaacetic acid)






Sodium sulfate
5.50
5.50
5.50
5.50








Water
Balance to 100%
















TABLE 35-2







Granular Fabric Cleaning Compositions









Formulations











Component
A
B
C
D














Protease1
0.10
0.20
0.30
0.05


Protease2


0.2
0.1


C12 alkyl benzene sulfonate
12.00
12.00
12.00
12.00


Zeolite A (1-10 micrometer)
26.00
26.00
26.00
26.00


C12-C14 secondary (2,3)
5.00
5.00
5.00
5.00


alkyl sulfate, Na salt






Sodium citrate
5.00
5.00
5.00
5.00


Optical brightenere
0.10
0.10
0.10
0.10


Sodium sulfate
17.00 
17.00 
17.00 
17.00








Fillers, water, minors
Balance to 100%









The following laundry detergent compositions are contemplated to provide particular utility under European machine wash conditions.







TABLE 35-3







Granular Fabric Cleaning Compositions











Formulations












Component
A
B
C
















LAS
7.0
5.61
4.76



TAS


1.57



C45AS
6.0
2.24
3.89



C25E25
1.0
0.76
1.18



C45E7


2.0



C25E3
4.0
5.5




QAS
0.8
2.0
2.0



STPP






Zeolite
25.0
19.5
19.5



Citric acid
2.0
2.0
2.0



NaSKS-6
8.0
10.6
10.6



Carbonate I
8.0
10.0
8.6



MA/AA
1.0
2.6
1.6



CMC
0.5
0.4
0.4



PB4

12.7




Percarbonate


19.7



TAED

3.1
5.0



Citrate
7.0





DTPMP
0.25
0.2
0.3



HEDP
0.3
0.3
0.3



QEA 1
0.9
1.2
1.0



Protease1
0.02
0.05
0.035



Lipase
0.15
0.25
0.15



Cellulase
0.28
0.28
0.28



Amylase
0.4
0.7
0.3



PVPI/PVNO
0.4

0.1



Photoactivated
15 ppm
27 ppm
27 ppm



bleach (ppm)






Brightener 1
0.08
0.19
0.19



Brightener 2

0.04
0.04



Perfume
0.3
0.3
0.3



Effervescent
15
15
5



granules (malic






acid 40%,






sodium






bicarbonate






40%, sodium






carbonate






20%)






Silicon
0.5
2.4
2.4



antifoam













Minors/inerts to
Balance to 100%












100%













Example 36
Detergent Formulations
In this Example, various detergent formulations which find use with ASP and/or ASP variants are provided. It is understood that the test methods provided in this section must be used to determine the respective values of the parameters of the present invention.
In the exemplified detergent compositions, the enzymes levels are expressed by pure enzyme by weight of the total composition and unless otherwise specified, the detergent ingredients are expressed by weight of the total compositions. The abbreviated component identifications therein have the following meanings:







TABLE 36-1





Definitions Used in this Example
















LAS
Sodium linear C11−13 alkyl benzene sulfonate.


TAS
Sodium tallow alkyl sulphate.


CxyAS
Sodium C1x-C1y alkyl sulfate.


CxyEz
C1x-C1y predominantly linear primary alcohol 



condensed with an average of z moles of ethylene 



oxide.


CxyAEzS
C1x-C1y sodium alkyl sulfate condensed with an 



average of z moles of ethylene oxide. Added 



molecule name in the examples.


Nonionic
Mixed ethoxylated/propoxylated fatty alcohol e.g. 



Plurafac LF404 being an alcohol with an average 



degree of ethoxylation of 3.8 and an average degree 



of propoxylation of 4.5.


QAS
R2•N+(CH3)2(C2H4OH) with R2 = C12-C14.


Silicate
Amorphous Sodium Silicate 



(SiO2:Na2O ratio = 1.6-3.2:1).


Metasilicate
Sodium metasilicate (SiO2:Na2O ratio = 1.0).


Zeolite A
Hydrated Aluminosilicate of formula 



Na12(A1O2SiO2)12•27H2O


SKS-6
Crystalline layered silicate of formula δ-Na2Si2O5.


Sulfate
Anhydrous sodium sulphate.


STPP
Sodium Tripolyphosphate.


MA/AA
Random copolymer of 4:1 acrylate/maleate, average



molecular weight about 70,000-80,000.


AA
Sodium polyacrylate polymer of average molecular 



weight 4,500.


Polycarboxylate
Copolymer comprising mixture of carboxylated 



monomers such as acrylate, maleate and 



methyacrylate with a MW ranging between 



2,000-80,000 such as Sokolan commercially 



available from BASF, being a copolymer 



of acrylic acid, MW4,500.


BB1
3-(3,4-Dihydroisoquinolinium)propane sulfonate


BB2
1-(3,4-dihydroisoquinolinium)-decane-2-sulfate


PB1
Sodium perborate monohydrate.


PB4
Sodium perborate tetrahydrate of nominal formula



NaBO3•4H2O.


Percarbonate
Sodium percarbonate of nominal formula 



2Na2CO3•3H2O2.


TAED
Tetraacetyl ethylene diamine.


NOBS
Nonanoyloxybenzene sulfonate in the form of the 



sodium salt.


DTPA
Diethylene triamine pentaacetic acid.


HEDP
1,1-hydroxyethane diphosphonic acid.


DETPMP
Diethyltriamine penta (methylene) phosphonate, 



marketed by Monsanto under the Trade name



Dequest 2060.


EDDS
Ethylenediamine-N,N′-disuccinic acid, (S,S) 



isomer in the form of its sodium salt


Diamine
Dimethyl aminopropyl amine; 1,6-hezane diamine; 



1,3-propane diamine; 2-methyl-1,5-pentane diamine; 



1,3-pentanediamine; 1-methyl-diaminopropane.


DETBCHD:
5,12-diethyl-1,5,8,12-tetraazabicyclo[6,6,2] 



hexadecane, dichloride, Mn(II) salt


PAAC
Pentaamine acetate cobalt (III) salt.


Paraffin
Paraffin oil sold under the tradename Winog 70 



by Wintershall.


Paraffin Sulfonate
A Paraffin oil or wax in which some of the hydrogen 



atoms have been replaced by sulfonate groups.


Aldose oxidase
Oxidase enzyme sold under the tradename Aldose 



Oxidase by Novozymes A/S


Galactose oxidase
Galactose oxidase from Sigma


Protease:
Proteolytic enzyme sold under the tradename 



Savinase, Alcalase, Everlase by Novo Nordisk A/S, 



and the following from Genencor International, Inc:



“Protease A” described in U.S. RE 34,606 in



FIGS. 1A, 1B, and 7, and at column 11, lines



11-37; “Protease B” described in U.S. Pat. 



No. 5,955,340 and U.S. Pat. No. 5,700,676



in FIGS. 1A, 1B and 5, as well as Table 1; and



“Protease C” described in U.S. Pat. No. 6,312,936



and U.S. Pat. No. 6,482,628 in FIGS. 1-3 [SEQ ID 3], 



and at column 25, line 12, “Protease D” being the 



variant 101G/103A/104I/159D/232V/236H/245R/



248D/252K (BPN' numbering) described in 



WO 99/20723.


Amylase
Amylolytic enzyme sold under the tradename 



Purafect ® Ox Am described in WO 94/18314,



WO96/05295 sold by Genencor; Natalase ®,



Termamyl ®, Fungamyl ® and Duramyl ®, 



all available from Novozymes A/S.


Lipase
Lipolytic enzyme sold under the tradename 



Lipolase Lipolase Ultra by Novozymes A/S



and Lipomax by Gist-Brocades.


Cellulase
Cellulytic enzyme sold under the tradename 



Carezyme, Celluzyme and/or Endolase by



Novozymes A/S.


Pectin Lyase
Pectaway ® and Pectawash ® available from 



Novozymes A/S.


PVP
Polyvinylpyrrolidone with an average molecular 



weight of 60,000


PVNO
Polyvinylpyridine-N-Oxide, with an average 



molecular weight of 50,000.


PVPVI
Copolymer of vinylimidazole and vinylpyrrolidone, 



with an average molecular weight of 20,000.


Brightener 1
Disodium 4,4′-bis(2-sulphostyryl)biphenyl.


Silicone antifoam:
Polydimethylsiloxane foam controller with 



siloxane-oxyalkylene copolymer as dispersing 



agent with a ratio of said foam controller to



said dispersing agent of 10:1 to 100:1.


Suds Suppressor
12% Silicone/silica, 18% stearyl alcohol, 



70% starch in granular form.


SRP 1
Anionically end capped poly esters.


PEG X
Polyethylene glycol, of a molecular weight of x.


PVP K60 ®
Vinylpyrrolidone homopolymer (average MW 



160,000)


Jeffamine ® 
Capped polyethylene glycol from Huntsman


ED-2001



Isachem ® AS
A branched alcohol alkyl sulphate from Enichem


MME PEG (2000)
Monomethyl ether polyethylene glycol (MW 2000) 



from Fluka Chemie AG.


DC3225C
Silicone suds suppresser, mixture of Silicone oil 



and Silica from Dow Corning.


TEPAE
Tetreaethylenepentaamine ethoxylate.


BTA
Benzotriazole.


Betaine
(CH3)3N+CH2COO−


Sugar
Industry grade D-glucose or food grade sugar


CFAA
C12-C14 alkyl N-methyl glucamide


TPKFA
C12-C14 topped whole cut fatty acids.


Clay
A hydrated aluminumu silicate in a general formula



Al2O3SiO2•xH2O. Types: Kaolinite, montmorillonite, 



atapulgite, illite, bentonite, halloysite.


pH
Measured as a 1% solution in distilled water 



at 20° C.









The following Table (Table 36-2) provides liquid laundry detergent compositions that are prepared.







TABLE 36-2







Liquid Laundry Detergent Compositions












Component
I
II
III
IV
V















LAS
24.0
32.0
6.0
8.0
6.0


C12-C15 AE1.8S
—
—
8.0
11.0
5.0


C8-C10 amido 
2.0
2.0
2.0
2.0
1.0


propyl 







dimethyl amine







C12-C14 alkyl 
—
—
—
—
2.0


dimethyl 







amine oxide







C12-C15 AS 
—
—
17.0
7.0
8.0


CFAA
—
5.0
4.0
4.0
3.0


C12-C14 Fatty 
12.0
6.0
1.0
1.0
1.0


alcohol







ethoxylate







C12-C18 Fatty 
3.0
—
4.0
4.0
3.0


acid







Citric acid 
6.0
5.0
3.0
3.0
2.0


(anhydrous)







DETPMP
—
—
1.0
1.0
0.5


Monoeth-
#
#
5.0
5.0
2.0


anolamine







Sodium 
—
—
2.5
1.0
1.5


hydroxide







Propanediol
12.7
14.5
13.1
10.
8.0


Ethanol
1.8
2.4
4.7
5.4
1.0


DTPA
0.5
0.4
0.3
0.4
0.5


Pectin Lyase
—
—
—
0.005
—


Amylase
0.001
0.002
—

—


Cellulase
—
—
0.0002
—
0.0001


Lipase
0.1
—
0.1
—
0.1


ASP
0.05
0.3
0.08
0.5
0.2


Protease A
—
—
—
—
0.1


Aldose Oxidase
—
—
0.3
—
0.003


DETBCHD
—
—
0.02
0.01
—


SRP1
0.5
0.5
—
0.3
0.3


Boric acid
2.4
2.4
2.8
2.8
2.4


Sodium xylene 
—
—
3.0
—
—


sulfonate







DC 3225C
1.0
1.0
1.0
1.0
1.0


2-butyl-octanol
0.03
0.04
0.04
0.03
0.03


Brightener 1
0.12
0.10
0.18
0.08
0.10







Balance to 100% perfume/dye and/or water





# added to product to adjust the neat pH of the product to about 4.2 for (I) and about 3.8 for (II).






The following Table (36-3) provides hand dish liquid detergent compositions that are prepared.







TABLE 36-3







Hand Dish Liquid Detergent Compositions













Component
I
II
III
IV
V
VI
















C12-C15AE1.8S
30.0
28.0
25.0
—
15.0
10.0


LAS
—
—
—
5.0
15.0
12.0


Paraffin Sulfonate
—
—
—
20.0
—
—


C10-C18 Alkyl Dimethyl
5.0
3.0
7.0
—
—
—


Amine Oxide








Betaine
3.0
—
1.0
3.0
1.0
—


C12 poly-OH fatty acid
—
—
—
3.0
—
1.0


amide








C14 poly-OH fatty acid
—
1.5
—
—
—
—


amide








C11E9
2.0
—
4.0
—
—
20.0


DTPA
—
—
—
—
0.2
—


Tri-sodium Citrate dihydrate
0.25
—
—
0.7
—
—


Diamine
1.0
5.0
7.0
1.0
5.0
7.0


MgCl2
0.25
—
—
1.0
—
—


ASP
0.02
0.01
0.03
0.01
0.02
0.05


Protease A
—
0.01
—
—
—
—


Amylase
0.001
—
—
0.002
—
0.001


Aldose Oxidase
0.03
—
0.02
—
0.05
—


Sodium Cumene
—
—
—
2.0
1.5
3.0


Sulphonate








PAAC
0.01
0.01
0.02
—
—
—


DETBCHD
—
—
—
0.01
0.02
0.01







Balance to 100% perfume/dye and/or water.









The pH of these compositions is about 8 to about 11
Table 36-4 provides liquid automatic dishwashing detergent compositions that are prepared.







TABLE 36-4







Liquid Automatic Dishwashing Detergent Compositions












Component
I
II
Ill
IV
V















STPP
16
16
18
16
16


Potassium Sulfate
—
10
8
—
10 


1,2 propanediol
6.0
0.5
2.0
6.0
0.5


Boric Acid
4.0
3.0
3.0
4.0
3.0


CaCl2 dihydrate
0.04
0.04
0.04
0.04
0.04


Nonionic
0.5
0.5
0.5
0.5
0.5


ASP
0.1
0.03
0.05
0.03
0.06


Protease B
—
—
—
0.01
—


Amylase
0.02
—
0.02
0.02
—


Aldose Oxidase
—
0.15
0.02
 
0.01


Galactose Oxidase
—
—
0.01
 
0.01


PAAC
0.01
—
—
0.01
 


DETBCHD
—
0.01
—
—
0.01







Balance to 100% perfume/dye and/or water









Table 36-5 provides laundry compositions which may be prepared in the form of granules or tablets that are prepared.







TABLE 36-5







Laundry Compositions












Base Product
I
II
III
IV
V















C14-C15AS or 
8.0
5.0
3.0
3.0
3.0


TAS







LAS
8.0
—
8.0
—
7.0


C12-C15AE3S
0.5
2.0
1.0
—
—


C12-C15E5 or E3
2.0
—
5.0
2.0
2.0


QAS
—
—
—
1.0
1.0


Zeolite A
20.0
18.0
11.0
—
10.0


SKS-6 (dry add)
—
—
9.0
—
—


MA/AA
2.0
2.0
2.0
—
—


AA
—
—
—
—
4.0


3Na Citrate 
—
2.0
—
—
—


2H2O







Citric Acid 
2.0
—
1.5
2.0
—


(Anhydrous)







DTPA
0.2
0.2
—
—
—


EDDS
—
—
0.5
0.1
—


HEDP
—
—
0.2
0.1
—


PB1
3.0
4.8
—
—
4.0


Percarbonate
—
—
3.8
5.2
—


NOBS
1.9
—
—
—
—


NACA OBS
—
—
2.0
—
—


TAED
0.5
2.0
2.0
5.0
1.00


BB1
0.06
—
0.34
—
0.14


BB2
—
0.14
—
0.20
—


Anhydrous Na 
15.0
18.0
8.0
15.0
15.0


Carbonate







Sulfate
5.0
12.0
2.0
17.0
3.0


Silicate
—
1.0
—
—
8.0


ASP
0.03
0.05
1.0
0.06
0.1


Protease B
—
0.01
—
—
—


Protease C
—
—
—
0.01
—


Lipase
—
0.008
—
—
—


Amylase
0.001
—
—
—
0.001


Cellulase
—
0.0014
—
—
—


Pectin Lyase
0.001
0.001
0.001
0.001
0.001


Aldose Oxidase
0.03
—
0.05
—
—


PAAC
—
0.01
—
—
0.05







Balance to 100% Moisture and/or Minors*





*Perfume, Dye, Brightener/SRP1/Na Carboxymethylcellulose/Photobleach/MgSO4/PVPVI/Suds suppressor/High Molecular PEG/Clay.






Table 36-6 provides liquid laundry detergent formulations which are prepared.







TABLE 36-6







Liquid Laundry Detergent Formulations













Component
I
I
II
III
IV
V
















LAS
11.5
11.5
9.0
—
4.0
—


C12-C15AE2.85S
—
—
3.0
18.0
—
16.0


C14-C15E2.5S
11.5
11.5
3.0
—
16.0
—


C12-C13E9
—
—
3.0
2.0
2.0
1.0


C12-C13E7
3.2
3.2
—
—
—
—


CFAA
—
—
—
5.0
—
3.0


TPKFA
2.0
2.0
—
2.0
0.5
2.0


Citric Acid
3.2
3.2
0.5
1.2
2.0
1.2


(Anhydrous)








Ca formate
0.1
0.1
0.06
0.1
—
—


Na formate
0.5
0.5
0.06
0.1
0.05
0.05


Na Culmene
4.0
4.0
1.0 
3.0
1.2
—


Sulfonate








Borate
0.6
0.6
—
3.0
2.0
3.0


Na Hydroxide
6.0
6.0
2.0
3.5
4.0
3.0


Ethanol
2.0
2.0
1.0
4.0
4.0
3.0


1,2 Propanediol
3.0
3.0
2.0
8.0
8.0
5.0


Mono-
3.0
3.0
1.5
1.0
2.5
1.0


ethanolamine








TEPAE
2.0
2.0
—
1.0
1.0
1.0


ASP
0.03
0.05
0.01
0.03
0.08
0.02


Protease A
—
—
0.01
—
—
—


Lipase
—
—
—
0.002
—
—


Amylase
—
—
—
—
0.002
—


Cellulase
—
—
—
—
—
0.0001


Pectin Lyase
0.005
0.005
—

—
—


Aldose Oxidase
0.05
—
—
0.05
—
0.02


Galactose 
—
0.04






oxidase








PAAC
0.03
0.03
0.02
—
—
—


DETBCHD
—
—
—
0.02
0.01
—


SRP 1
0.2
0.2
—
0.1
—
—


DTPA
—
—
—
0.3
—
—


PVNO
—
—
—
0.3
—
0.2


Brightener 1
0.2
0.2
0.07
0.1
—
—


Silicone 
0.04
0.04
0.02
0.1
0.1
0.1


antifoam













Balance to 100% perfume/dye and/or water









Table 36-7 provides compact high density dishwashing detergents that are prepared.







TABLE 36-7







Compact High Density Dishwashing Detergents













Component
I
II
III
IV
V
VI
















STPP
—
45.0
45.0
—
—
40.0


3Na Citrate 2H2O
17.0
—
—
50.0
40.2
—


Na Carbonate
17.5
14.0
20.0
—
8.0
33.6


Bicarbonate
—
—
—
26.0
—
—


Silicate
15.0
15.0
8.0
—
25.0
3.6


Metasilicate
2.5
4.5
4.5
—
—
—


PB1
—
—
4.5
—
—
—


PB4
—
—
—
5.0
—
—


Percarbonate
—
—
—
—
—
4.8


BB1
—
0.1
0.1
—
0.5
—


BB2
0.2
0.05
—
0.1
—
0.6


Nonionic
2.0
1.5
1.5
3.0
1.9
5.9


HEDP
1.0
—
—
—
—
—


DETPMP
0.6
—
—
—
—
—


PAAC
0.03
0.05
0.02
—
—
—


Paraffin
0.5
0.4
0.4
0.6
—
—


ASP
0.072
0.053
0.053
0.026
0.059
0.01


Protease B
—
—
—
—
—
0.01


Amylase
0.012
—
0.012
—
0.021
0.006


Lipase
—
0.001
—
0.005
—
—


Pectin Lyase
0.001
0.001
0.001
—
—
—


Aldose Oxidase
0.05
0.05
0.03
0.01
0.02
0.01


BTA
0.3
0.2
0.2
0.3
0.3
0.3


Polycarboxylate 
6.0
—
—
—
4.0
0.9


Perfume
0.2
0.1
0.1
0.2
0.2
0.2







Balance to 100% Moisture and/or Minors*





*Brightener/Dye/SRP1/Na Carboxymethylcellulose/Photobleach/MgSO4/PVPVI/Suds suppressor/High Molecular PEG/Clay.






The pH of the above compositions is from about 9.6 to about 11.3.

Table 36-8 provides tablet detergent compositions of the present invention that are prepared by compression of a granular dishwashing detergent composition at a pressure of 13KN/cm2 using a standard 12 head rotary press:







TABLE 36-8







Tablet Detergent Compositions















Component
I
II
III
IV
V
VI
VII
VIII


















STPP
—
48.8
44.7
38.2
—
42.4
46.1
36.0


3Na Citrate 2H2O
20.0
—
—
—
35.9
—
—
—


Na Carbonate
20.0
5.0
14.0
15.4
8.0
23.0
20.0
28.0


Silicate
15.0
14.8
15.0
12.6
23.4
2.9
4:3
4.2


Lipase
0.001
—
0.01
—
0.02
—
—
—


Protease B
0.01
—
—
—
—
—
—
—


Protease C
—
—
—
—
—
0.01
—
—


ASP
0.01
0.08
0.05
0.04
0.052
0.023
0.023
0.029


Amylase
0.012
0.012
0.012
—
0.015
—
0.017
0.002


Pectin Lyase
0.005
—
—
0.002
—
—
—
—


Aldose Oxidase
—
0.03
—
0.02
0.02
—
0.03
—


PB1
—
—
3.8
—
7.8
—
—
8.5


Percarbonate
6.0
—
—
6.0
—
5.0
—
—


BB1
0.2
—
0.5
—
0.3
0.2
—
—


BB2
—
0.2
—
0.5
—
—
0.1
0.2


Nonionic
1.5
2.0
2.0
2.2
1.0
4.2
4.0
6.5


PAAC
0.01
0.01
0.02
—
—
—
—
—


DETBCHD
—
—
—
0.02
0.02
—
—
—


TAED
—
—
—
—
—
2.1
—
1.6


HEDP
1.0
—
—
0.9
—
0.4
0.2
—


DETPMP
0.7
—
—
—
—
—
—
—


Paraffin
0.4
0.5
0.5
0.5
—
—
0.5
—


BTA
0.2
0.3
0.3
0.3
0.3
0.3
0.3
—


Polycarboxylate
4.0
—
—
—
4.9
0.6
0.8
—


PEG 400-30,000
—
—
—
—
—
2.0
—
2.0


Glycerol
—
—
—
—
—
0.4
—
0.5


Perfume
—
—
—
0.05
0.2
0.2
0.2
0.2







Balance to 100% Moisture and/or Minors*





*Brightener/SRP1/Na Carboxymethylcellulose/Photobleach/MgSO4/PVPVI/Suds suppressor/High Molecular PEG/Clay.






The pH of these compositions is from about 10 to about 11.5. The tablet weight of these compositions is from about 20 grams to about 30 grams.

Table 36-9 provides liquid hard surface cleaning detergent compositions of the present invention that are prepared.







TABLE 36-9







Liquid Hard Surface Cleaning Detergent Compositions














Component
I
II
III
IV
V
VI
VII

















C9-C11E5
2.4
1.9
2.5
2.5
2.5
2.4
2.5


C12-C14E5
3.6
2.9
2.5
2.5
2.5
3.6
2.5


C7-C9E6
—
—
—
—
8.0
—
—


C12-C14E21
1.0
0.8
4.0
2.0
2.0
1.0
2.0


LAS
—
—
—
0.8
0.8
—
0.8


Sodium culmene sulfonate
1.5
2.6
—
1.5
1.5
1.5
1.5


Isachem ® AS
0.6
0.6
—
—
—
0.6
—


Na2CO3
0.6
0.13
0.6
0.1
0.2
0.6
0.2


3Na Citrate 2H2O
0.5
0.56
0.5
0.6
0.75
0.5
0.75


NaOH
0.3
0.33
0.3
0.3
0.5
0.3
0.5


Fatty Acid
0.6
0.13
0.6
0.1
0.4
0.6
0.4


2-butyl octanol
0.3
0.3
—
0.3
0.3
0.3
0.3


PEG DME-2000 ®
0.4
—
0.3
0.35
0.5
—
—


PVP
0.3
0.4
0.6
0.3
0.5
—
—


MME PEG (2000) ®
—
—
—
—
—
0.5
0.5


Jeffamine ® ED-2001
—
0.4
—
—
0.5
—
—


PAAC
—
—
—
0.03
0.03
0.03
 


DETBCHD
0.03
0.05
0.05
—
—
—
—


ASP
0.07
0.05
0.08
0.03
0.06
0.01 
0.04


Protease B
—
—
—
—
—
0.01
—


Amylase
0.12
0.01
0.01
—
0.02
—
0.01


Lipase
—
0.001
—
0.005
—
0.005
—


Pectin Lyase
0.001
—
0.001
—
—
—
0.002


PB1
—
4.6
—
3.8
—
—
—


Aldose Oxidase
0.05
—
0.03
—
0.02
0.02
0.05







Balance to 100% perfume/dye and/or water





The pH of these compositions is from about 7.4 to about 9.5.






Example 37
Animal Feed Comprising ASP
The present invention also provides animal feed compositions comprising ASP and/or ASP variants. In this Example, one such feed, suitable for poultry is provided. However, it is not intended that the present invention be limited to this specific formulation, as the proteases of the present invention find use with numerous other feed formulations. It is further intended that the feeds of the present invention be suitable for administration to any animal, including but not limited to livestock (e.g., cattle, pigs, sheep, etc.), as well as companion animals (e.g., dogs, cats, horses, rodents, etc.). The following Table provides a formulation for a mash, namely a maize-based starter feed suitable for administration to turkey poults up to 3 weeks of age.







TABLE 37-1







Animal Feed Composition










Ingredient Amount
(wt. %)














Maize
36.65



Soybean meal (45.6% CP)
55.4



Animal-vegetable fat
3.2



Dicalcium phosphate
2.3



Limestone
1.5



Mineral premix
0.3



Vitamin premix
0.3



Sodium chloride
0.15



DL methionine
0.2










In some embodiments, this feed formulation is supplemented with various concentrations of the protease(s) of the present invention (e.g., 2,000 units/kg, 4,000 units/kg and 6,000 units/kg).
All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. However, the citation of any publication is not to be construed as an admission that it is prior art with respect to the present invention.
Having described the preferred embodiments of the present invention, it will appear to those ordinarily skilled in the art that various modifications may be made to the disclosed embodiments, and that such modifications are intended to be within the scope of the present invention.
Those of skill in the art readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The compositions and methods described herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. It is readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.
The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

## Claims

1. An isolated serine protease obtained from a member of the suborder Micrococcineae having at least 95% amino acid identity with the amino acid sequence set forth in SEQ ID NO: 8 and having the serine protease activity of the Cellulomonas 69B4 protease set forth by SEQ ID NO: 8.

2. The serine protease of claim 1, wherein said protease is a cellulomonadin.

3. The serine protease of claim 1, wherein said protease is obtained from a Cellulomonas species.

4. The serine protease of claim 3, wherein said protease is obtained from Cellulomonas 69B4.

5. The serine protease of claim 4, wherein said protease comprises the amino acid sequence set forth in SEQ ID NO: 8.

6. A composition comprising an isolated serine protease having immunological cross-reactivity with said serine protease of claim 4.

7. A composition comprising an isolated serine protease having immunological cross-reactivity with said serine protease of claim 1.

8. A cleaning composition comprising the serine protease of claim 1.

9. The cleaning composition of claim 8, wherein said protease is a serine protease obtained from a Cellulomonas organism.

10. The cleaning composition of claim 9, wherein said protease is obtained from Cellulomonas 69B4.

11. The cleaning composition of claim 10, wherein said protease comprises the amino acid sequence set forth in SEQ ID NO: 8.

12. A cleaning composition comprising a serine protease, wherein said serine protease has immunological cross-reactivity with the serine protease of claim 8.

13. A cleaning composition comprising a serine protease, wherein said serine protease has immunological cross-reactivity with the serine protease of claim 10.

14. A composition comprising the serine protease of claim 1 and at least one stabilizing agent.

15. The composition of claim 14, wherein said stabilizing agent is selected from the group consisting of borax, glycerol, and competitive inhibitors.

16. The composition of claim 15, wherein said competitive inhibitors stabilize said serine protease to anionic surfactants.

17. The cleaning composition according to claim 8, said composition comprising a sufficient amount of a pH modifier to provide said composition with a neat pH of from about 3 to about 5, said composition being essentially free of materials that hydrolyze at a pH of from about 3 to about 5.

18. The cleaning composition according to claim 17, wherein said materials that hydrolyze comprise a surfactant material.

19. The cleaning composition according to claim 17, said cleaning composition being a liquid composition.

20. The cleaning composition according to claim 18, wherein said surfactant material comprises a sodium alkyl sulfate surfactant that comprises an ethylene oxide moiety.

21. A cleaning composition that comprises at least one acid stable enzyme, said cleaning composition comprising a sufficient amount of a pH modifier to provide said composition with a neat pH of from about 3 to about 5, said composition being essentially free of materials that hydrolyze at a pH of from about 3 to about 5, and wherein said acid stable enzyme is stable at the neat pH of from about 3 to about 5.

22. The cleaning composition according to claim 21, wherein said materials that hydrolyze comprise a surfactant material.

23. The cleaning composition according to claim 21, said cleaning composition being a liquid composition.

24. The cleaning composition according to claim 21, wherein said surfactant material comprises a sodium alkyl sulfate surfactant that comprises an ethylene oxide moiety.

25. The composition according to claim 17, said composition comprising from about 0.001 to about 0.5 weight % of SEQ ID NO: 8.

26. The composition according to claim 25, said composition comprising from about 0.01 to about 0.1 weight percent of SEQ ID NO: 8.

27. The composition of claim 8, said composition being a granule cleaning composition, powder cleaning composition, or tablet cleaning composition.

28. The composition of claim 27, said composition comprising a bleaching material.

29. The composition of claim 27, said composition being a granule cleaning composition.

30. The composition of claim 27, said composition being a powder cleaning composition.

31. The composition of claim 27, said composition being a tablet cleaning composition.