Patent Application: US-70669991-A

Abstract:
a maltopentaose producing amylase , and its derivatives modified by gene manipulation , can be expressed in e . col . these amylases facilitate the production of maltopentaose .

Description:
soil samples from various regions of the earth were collected . 0 . 1 - 0 . 2 g of each sample was suspended in 1 ml of sterile physiological saline in sterile vessels . after sedimentation of the coarse fractions , in each case , 0 . 1 ml was plated on a starch / agar plate ( 10 g / l soluble starch ; 5 g / l peptone ; 5 g / l yeast extract ; 1 g / l kh 2 po 4 ; 0 . 2 g / l mgso 4 x 7h 2 o ; 10 g / l na 2 co 3 ; 15 g / l agar ; ph 10 . 4 ). the agar plates were incubated at 30 ° c . for 2 - 3 days . colonies of starch - degrading bacteria showed a cloudy halo produced by retrogradation of low molecular weight starch molecules . the colonies were isolated and purified twice on starch / agar plates . this was followed by culturing in 2 ml of liquid medium of the above composition . after incubation at 30 ° c . for 48 hours , the cells were spun down , and the supernatant was assayed for amylase activity . 200 μl of each supernatant were incubated with 200 μl of a 10 % starch solution in 20 mm tris / cl ph 9 . 0 ; 5 mm cacl 2 at 40 ° c . for 1 - 5 hours . the enzyme assay was stopped by adding 600 μl of methanol ; the supernatant was centrifuged and then analyzed by hplc . out of a large number of isolates , only the strain 163 - 26 showed the g5 - producing enzyme activity . ______________________________________feature isolate 163 - 26______________________________________cell form : rod - like , single cells , dimers and short chainscell size : 1 - 1 . 6 μm × 0 . 2 - 0 . 3 μmmotility : almost all the cells are motile in the log . growth phase ; almost all the cells are non - motile in the stat . growth phaseendospores : no endospores occur in any growth phasegrowth parameters : temperature : optimal growth between 30 ° c . and 37 ° c . ph : optimal between ph 8 . 0 and 9 . 0nacl tolerance : 8 % nacl still toleratedquinones : no quinones occur either aerobically or anaerobicallygram characteristics : 30 %- 70 % of the cells are gram - positive in the log . growth phasefatty acid types : straight - chain and iso -, anteiso - branched fatty acidsmurein type : a 1 γgc content : 41 . 5 ± 0 . 5 mol % ______________________________________ isolate 163 - 26 was cultured in 40 1 of m3 / 1 medium ( 5 g / l noredux 150b ; 5 g / l peptone from casein ; 5 g / l yeast extract ; 5 g / l nacl ; 3 . 5 g / l na 2 co 3 ; 1 g / l kh 2 po 4 ; 0 . 2 g / l mgso 4 ) aerobically at 37 ° c . for 20 hours . after 20 hours , the culture was rapidly cooled to 4 ° c . by adding ice . the cells were removed from the culture broth by cross - flow microfiltration in a millipore filter cassette ( pore size 0 . 2 μm ). the proteins in the cell - free culture supernatant were concentrated to a volume of 1 1 by ultrafiltration through a filtron filter cassette ( separation limit 10 kda ). the filtrate was brought to 60 percent saturation by the addition of powdered ammonium sulfate . the proteins which were precipitated were collected by centrifugation , dissolved in 50 ml of tc buffer ( 20 mm tris / cl ph 7 . 2 ; 5 mm cacl 2 ), and dialyzed against tc buffer . the amylolytic enzymes in the solution were purified by adsorption to starch . for this purpose , the protein solution after the dialysis was brought to 20 % ammonium sulfate saturation , and 3 % soluble starch was added . the mixture was stirred at 4 ° c . for 3 hours and then centrifuged . the precipitate was suspended in half the initial volume of washing buffer ( 20 % saturated with ammonium sulfate , 1m nacl in tc buffer ), stirred at 4 ° c . for 10 minutes , and centrifuged again . the precipitate resulting from this is suspended in 1 initial volume of elution buffer ( 3m nacl ; 0 . 1m maltose in tc buffer ) and stirred at 4 ° c . for 2 hours . the starch is then spun down , and the supernatant is dialyzed against tc buffer . after the dialysis the proteins in the solution are precipitated by adding ammonium sulfate ( 60 % saturation ), dissolved in tc buffer and dialyzed again . the resulting solution now contains only the α - amylases a - 60 formed by the isolate 163 - 26 and the maltopentaose producing amylase a - 180 . the two enzymes can be separated from one another by gel filtrations on a tsk sw3000g ( lkb ) molecular sieve column . the mw determination by sds polyacrylamide gel electrophoresis ( page ) revealed an mw of about 180 kda for the amylase a - 180 . the isoelectric point of the purified enzyme was found to be 4 . 65 by isoelectric focusing . the kinetics of product formation on hydrolysis of starch revealed an initially very high g5 specificity for the amylase a - 180 . a - 180 has a biphasic ph optimum at ph values 6 . 0 and 8 . 5 . irreversible inactivation of a - 180 takes place only at ph values below 5 . 5 or above 11 . 0 . the optimal temperature for hydrolysis of starch is 55 ° c ., although the enzyme is slightly unstable at this temperature , so that a temperature of 45 ° c . is used to produce g5 . γ - cyclodextrin cannot be hydrolyzed by amylase a - 180 . this result , together with the finding of high g5 specificity , shows that a - 180 is an exo - maltopentaohydrolase . cloning -- in order to obtain an a - 180 specific probe which can be used to identify the structural gene , initially , the n - terminal amino - acid sequence of the purified amylase a - 180 was determined by automated edman degradation ( gas phase sequenator ). the amino - acid sequence obtained by the sequencing is : ( seq id no : 1 ) it was possible to deduce , by reverse translation , from a part of this sequence ( seq id no : 2 ) a nucleotide sequence which is 17 bases long and must be present in the a - 180 structural gene . the exact sequence of this oligonucleotide is : ( seq id no : 3 ), wherein y is c or t and n is a , t , c or g . this oligonucleotide sequence ( a 32 - fold degenerate 17 - mer ) was prepared using a dna synthesizer and radiolabeled with 32 p - γ - atp . chromosomal dna of isolate 163 - 26 was cut with various restriction enzymes , fractionated by electrophoresis in a 0 . 8 % agarose gel , and transferred to a nylon membrane ( southern blot ). it was possible to use the radioactive oligonucleotide mixture in hybridization studies to label a 2 . 7 kb clai fragment which codes for the n - terminal region of a - 180 . the clai fragment was isolated , ligated into the vector pbr322 cut with clai , and transformed into e . coli hb 101 . clones which contained the correct insert were identified by hybridization of their plasmid dna with the radioactive oligonucleotide mixture . it was possible , using the cloned dna fragment which was now labeled and was used as hybridization probe , to clone the entire a - 180 structural gene . sequencing -- to determine the nucleotide sequence of the a - 180 structural gene , overlapping fragments of the gene were sub - cloned into the plasmid puc19 . the sequence of the subclones was determined by the dideoxy chain termination method using universal or internal sequencing primers . a printout of the complete a - 180 nucleotide sequence and the derived amino - acid sequence together with the 5 &# 39 ; and 3 &# 39 ; flanking regions of the gene is represented below . ( seq id no : 4 ) the open reading frame which codes for a - 180 comprises 5052 nucleotides , corresponding to 1684 amino acids . the derived mw of 186 . 5 kda corresponds to the 180 kda determined by sds - page . three mutations were necessary to modify the cloned a - 180 structural gene in such a way that massive production , coupled with export and proteolytic stability of the g5 - specific amylase , takes place in suitable e . coli strains . in order to obtain massive expression of the a - 180 structural gene and , thus , extensive amylase production , which can also be controlled by simple methods ( i . e ., induction / repression ) the a - 180 structural gene was placed under the control of a new promoter . for this , the a - 180 structural gene was isolated from the plasmid paca1 ( fig1 ) and cloned downstream of the tac promoter in the polylinker of the expression plasmid pjf118u ( gene 48 , 199 - 131 , 1986 1 ); a derivative of pkk 223 ( the latter is obtainable from pharmacia , freiburg ). this promoter is repressed by the laci q gene product ( which is likewise encoded on pjf118u ) until inducers such as lactose or analogous compounds , for example , iptg , are added to the medium . although this mutation made massive production of a - 180 possible , the recombinant gene product was 100 % located in the cytoplasm of e . coli and was extensively degraded there . in order to achieve export of the produced amylase a - 180 into the culture supernatant , the 37 n - terminal amino acids of a - 180 , which represent the signal peptide necessary for export , were deleted and replaced by the signal peptide of the cgtase from klebsiella oxytoca which is exported in e . coli [ gene 47 , 269 - 277 , ( 1986 )]. the recombinant plasmid is called pex1051 ( fig2 ). expression of the recombinant gene continued via the &# 34 ; tac &# 34 ; promoter . replacement of the signal peptide resulted in no alteration in the export behavior of a - 180 . the massively produced enzyme continues to be located in the cytoplasm and is extensively degraded . the g5 specificity is retained , despite the signal peptide exchange . the third mutation comprised truncating the a - 180 structural gene by 3792 nucleotides at the 3 &# 39 ; end . the deletion of these nucleotides and the integration of a stop triplet in their place truncates the amylase on the c terminus by exactly 1264 amino acids [ plasmid pex21 ( fig2 )]. the remaining amylase residue is , like the entire a - 180 structural gene , massively expressed under the control of the tac promoter after lactose induction . in contrast to the mutated completed amylase , however , the product which is formed is now exported into the periplasm or the culture supernatant or suitable e . coli strains . the exported protein is stable , that is to say it is not degraded , and its enzymic properties are identical in terms of product specificity with those of the complete amylase a - 180 . hence , this gene product meets all the requirements necessary for the production of g5 . expression and secretion of the amylase a - 180 and of the a - 180 derivative ( a - 180d ) in various e . coli strains the e . coli strains hb101 and wcm100 are used for expression of the amylase a - 180 or of the g5 - specific 63 kda a - 180 derivative a - 180d . hb101 is deposited at the deutsche sammlung von mikroorganismen ( dsm 1007 ), and wcm100 can be obtained by the process described in european patent application no . 338 , 410 . it can be replaced for the expression and secretion of the amylases by other strains obtainable by the process disclosed in european patent application no . 338 , 410 . the e . coli strains contain the expression plasmid pex1051 for the expression of a - 180 , and the expression and secretion plasmid pex21 for the expression of a - 180d . 1 , 000 ml of nutrient medium ( 10 g / l peptone from casein , 5 g / l yeast extract , 10 g / l nacl , 5 g / l lactose and 0 . 1 g / l ampicillin ) are inoculated with 20 ml of a preculture of the particular strain ( in the same medium ) and incubated aerobically at 20 ° c . ( pex1051 ) or 25 ° c . ( pex21 ). after 48 hours ( pex1051 ) or 24 hours ( pex21 ), the cells are harvested by centrifugation of the culture broth . when the strains hb101 / pex1051 and wcm100 / pex1051 are used , the harvested cells are washed with tc buffer , suspended in 1 / 200 of the culture volume of tc buffer and lyzed using ultrasound ( sonifier ) or pressure ( french press ). the resulting cell lysates are treated with dnase and then centrifuged at 10 , 000 × g for 10 minutes . after this centrifugation the supernatant ( which will hereinafter be referred to as &# 34 ; cytoplasmic fraction &# 34 ;) contains the amylase a - 180 and can be used directly for starch conversion . when strain hp101 / pex21 is used , the amylase a - 180d , which is located in the periplasm , is extracted from the cells by chcl 3 treatment ( ames et al ( 1984 ) j . bact ., 160 ; 1181 - 1183 ). for this , the spun - down cells are suspended in 5 ml of 10 mm tris / hcl , ph 8 . 0 , mixed with 5 ml of chcl 3 and incubated at room temperature for 15 minutes . the suspension is then diluted with 40 ml of tc buffer and centrifuged at 6 , 000 × g for 20 minutes . after centrifugation , the cell pellet is discarded . the supernatant ( periplasmic fraction ) contains 60 % to 70 % of the amylase a - 180d formed . other proteins contained in the plasmic fraction do not inhibit the a - 180d activity so that further purification is not necessary . when strain wcm100 / pex21 is used , the harvested cells are discarded . under the described conditions , the culture supernatant contains 0 . 1 - 0 . 5 g of the recombinant gene product a - 180d , while the inducer lactose has been almost completely consumed by this time . the cell - free culture supernatant can be used directly for starch conversion . starch conversion with maltopentaose producing amylases obtained from isolate 163 - 26 or e . coli starch conversion with amylase a - 180 purified from the culture supernatant from isolate 163 - 26 purified amylase a - 180 is dissolved to a concentration of 50 μg / ml in tc buffer . a 10 % solution of soluble starch in tc buffer is brought to a temperature of 45 ° c . the two solutions are mixed in the ratio 1 : 1 and incubated at 45 ° c . after 1 hour , the reaction is stopped by adding 1 . 5 parts by volume of methanol . the unhydrolyzed residual starch precipitated by the methanol addition is spun down . the hydrolysis products remaining in the solution can be qualitatively and quantitatively investigated by reversed phase column chromatography . in a typical starch conversion in which 1 ml of enzyme solution and 1 ml of substrate solution have been employed , 18 . 5 % of the starch contained in the mixture was hydrolyzed after 1 hour . the resulting products have the following composition : g5 , 82 . 7 %; g4 , 6 . 4 %; g3 , 4 . 2 %; g2 , 3 . 9 %; g1 , 2 . 8 %. starch conversion with amylase a - 180 contained in the cytoplasmic protein fractions from e . coli cells the cytoplasmic protein fractions from e . coli hb101 / pex1051 or e . coli wcm100 / pex1051 are prepared as described in example 6 . the concentration of the proteins is adjusted to 2 mg / ml with tc buffer . 35 ml of a 30 % noredux 150b solution ( in tc buffer ) are equilibrated at 45 ° c . noredux 150b is a starch partially hydrolyzed by acid treatment and supplied by henkel . the substrate is then mixed with 5 ml of the protein solution ( 2 mg / ml ) and incubated at 45 ° c . 4 ml samples of the mixture are removed after 1 , 2 , 3 and 4 hours and mixed with 6 ml methanol and are centrifuged . the qualitative and quantitative composition of the soluble products in each supernatant is determined by hplc analysis . the results of a typical starch conversion with amylase a - 180 contained in the cytoplasmic protein fraction of e . coli hb101 / pex1051 or e . coli wcm100 / pex1051 are shown in the following table : ______________________________________ 1 h 2 h 3 h 4 h______________________________________proportion of 12 . 1 % 19 . 9 % 24 . 95 % 31 . 1 % substrate hydrolyzedproduct composition : maltopentaose : 100 % 79 % 72 % 64 % maltotetraose : 0 % 8 . 5 % 10 . 4 % 12 . 2 % maltotriose : 0 % 7 . 5 % 10 . 4 % 11 . 6 % maltose : 0 % 5 % 6 . 2 % 7 . 4 % glucose : 0 % 0 % 1 % 4 . 8 % ______________________________________ starch conversion with amylase a - 180d contained in the periplasmic fraction of e . coli hb101 / pex21 95 ml of 10 % noredux 150b solution are equilibrated at 45 ° c . the solution is then mixed with 5 ml of periplasmic fraction of e . coli hb101 / pex21 ( compare example 6 ) and incubated at 45 ° c . after 1 hour , the reaction is stopped by adding 150 ml of methanol . the mixture is centrifuged ; the product composition in the supernatant is then determined by hplc analysis . under the conditions described , 38 . 4 % of the employed substrate is hydrolyzed after 1 hour . the resulting products have the following composition : g5 , 67 . 7 %; g4 , 11 . 1 %; g3 , 1 . 7 %; g2 , 8 . 7 %; g1 , 10 . 8 %. starch conversion with amylase a - 180d contained in the culture supernatant from e . coli wcm100 / pex21 75 ml of 10 % noredux 150b solution are equilibrated at 45 ° c . the solution is then mixed with 25 ml of culture supernatant from e . coli wcm100 / pex21 ( compare example 6 ) and incubated at 45 ° c . after 1 hour , the reaction is stopped by adding 150 ml of methanol . the mixture is centrifuged ; the product composition in the supernatant is then determined by hplc analysis . after one hour , there was 15 . 8 % composition of the substrate employed . the product composition was : while only two embodiments of the present invention have been shown and described , it is to be understood that many changes and modifications ma be made thereunto without departing from the spirit and scope of the invention as defined in the appended claims . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 4 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 25 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 1 : glnglutyrarggluleu asnglnleugluasnlys1510prophesertrpaspasnalaasnvaltyrpheval1520leu25 ( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 2 : trpaspasnalaasnval15 ( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 17 bases ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 3 : tgggayaayg cnaaygt17 ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 5741 bases ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 4 : ggtaccgcctatctcagtgtgtgaaagctatgcatcaaaataccta ctcc50atgagcgtttcttcaacacgaatctactttatttaatattattcataacg100aaacatcagaaaatattgttattacctaaattccttgtttttgtctttta150atgttggtcaatgttctatggttgtgctaataaaaatgttaacgctttct200caggagg ctatatgagaggggtgatgtctgctaaacaataaggattcatc250aacaccatggttataaaaaattaaagattgaaaggaggaaaaggtaatg299metaagcaacagcttaatcgcgtgataagtatcgtattatgtttaatt344l ysglnglnleuasnargvalileserilevalleucysleuile51015gtcatgctctcggtgtttgaaagtactattatgttattaccaggt389valmetleuservalphegluserthrilemetleuleupr ogly202530tcagtagaggtaaaaggccaagagtatcgagaactaaatcagcta434servalgluvallysglyglnglutyrarggluleuasnglnleu354045gaaaataaacctttttca tgggataatgcaaacgtttactttgtg479gluasnlysprophesertrpaspasnalaasnvaltyrpheval505560ttaaccgatcgtttttacaatggaaatacaagtaatgataattct524leuthras pargphetyrasnglyasnthrserasnaspasnser657075tatgggagaccgcaaatagatgcttggggtacaaacattggtact569tyrglyargproglnileaspalatrpglythrasnileglythr 808590ttccatggcggggacataaaaggattaacaaagaaattggaagaa614phehisglyglyaspilelysglyleuthrlyslysleugluglu95100105ggttactttacagacctaggta caaatgccatatggatatctgct659glytyrphethraspleuglythrasnalailetrpileserala110115120ccatgggaacaaatgcatggctgggttggtgggaaagatggtgat704protrpglug lnmethisglytrpvalglyglylysaspglyasp125130135tttgctcactatggctatcatggttactatggattagattttacg749phealahistyrglytyrhisglytyrtyrglyleuaspphethr 140145150gctatggatcagaatatgggtacaattgatgaaatgcgtgaattt794alametaspglnasnmetglythrileaspglumetarggluphe155160165gttgaccttgcacattcat taggcattagagttgttctcgacatt839valaspleualahisserleuglyileargvalvalleuaspile170175180gttatgaatcacgttggctatccaacgatcgttgacatgcatgaa884valmeta snhisvalglytyrprothrilevalaspmethisglu185190195tttggttttggtgatactggaggacttccaagagattggacacct929pheglypheglyaspthrglyglyleuproargasptrpthrpr 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