# Patent

## Title
Method of converting a biomass substrate to a fermentable sugar using a beta-glucosidase variant enzyme

## Abstract

The invention provides variants of the Azospirillum irakense CelA β-glucosidase that have improve β-glucosidase activity, particularly improved thermoactivity, compared to the wild type enzyme. The invention further provides related polynucleotides, vectors, host cell, and methods for making and using the variants.

## Background
This application is a Divisional of co-pending U.S. patent application Ser. No. 12/816,989, filed Jun. 16, 2010, which claims the benefit of provisional applications U.S. Ser. No. 61/187,565, filed Jun. 16, 2009, and U.S. Ser. No. 61/218,020, filed Jun. 17, 2009, pursuant 35 U.S.C. §119(e), all of which are incorporated by reference in their entireties.


FIELD OF THE INVENTION
The present invention relates, inter alia, to novel β-glucosidase polypeptide variants having altered properties relative to Azospirillum irakense β-glucosidase (CelA), the polynucleotides that encode the variants, methods of producing the variants, enzyme compositions comprising the variants, and methods for using the variants in industrial applications.
REFERENCE TO SEQUENCE LISTING
The “Sequence Listing” submitted electronically concurrently herewith pursuant 37 C.F.R. §1.821 in a computer readable form (CRF) via EFS-Web as file name CX3-016US1_ST25.txt is incorporated herein by reference. The electronic copy of the Sequence Listing was created on Jun. 16, 2010, and the size on disk is 93 Kbytes.
BACKGROUND OF THE INVENTION
Cellulosic biomass is a significant renewable resource for the generation of sugars. Fermentation of these sugars can yield numerous end-products such as fuels and chemicals that are currently derived from petroleum. While the fermentation of sugars to fuels such as ethanol is relatively straightforward, the hydrolytic conversion of cellulosic biomass to fermentable sugars such as glucose is difficult because of the crystalline structure of cellulose and its close association with lignin. Ladisch, et al., Enzyme Microb. Technol. 5:82 (1983). Pretreatment, by means including but not limited to, mechanical and solvent means, increases the susceptibility of cellulose to hydrolysis. Pretreatment may be followed by the enzymatic conversion of cellulose to glucose, cellobiose, cello-oligosaccharides and the like using enzymes that specialize in breaking down the β-1-4 glycosidic bonds of cellulose. These enzymes are collectively referred to as “cellulases”.
Cellulases are divided into three sub-categories of enzymes: 1,4-β-D-glucan glucanohydrolase (“endoglucanase” or “EG”); 1,4-β-D-glucan cellobiohydrolase (“exoglucanase”, “cellobiohydrolase”, or “CBH”); and β-D-glucoside-glucohydrolase (“β-glucosidase”, “cellobiase” or “BG”). Endoglucanases randomly attack the interior parts and mainly the amorphous regions of cellulose, mostly yielding glucose, cellobiose, and cellotriose. Exoglucanases incrementally shorten the glucan molecules by binding to the glucan ends and releasing mainly cellobiose units from the ends of the cellulose polymer. β-glucosidases split the cellobiose, a water-soluble β-1,4-linked dimer of glucose, into two units of glucose.
There are several types of microorganisms that produce cellulases. These include fungi, actinomycetes, and bacteria. Cellulases from strains of the filamentous fungi Trichoderma sp. and Chrysosporium sp. have been particularly productive in hydrolyzing cellulose. Trichoderma sp. and other strains typically produce all three types of cellulases described above (e.g., a whole cellulase system). However, one of the major drawbacks of Trichoderma cellulases and other cellulases obtained from filamentous fungi is the low level of β-glucosidase activity, and this low level of activity leads to incomplete conversion of cellobiose to glucose in the cellulose hydrolysis process. Additionally, cellobiose and glucose have been reported to be inhibitors of the cellulase enzyme system; for example it is known that cellobiase is inhibited by glucose. Ait, N., et al., J. Gen. Microbiol. 128:569-577 (1982). Poor glucose yields, whether due to deficiencies in the inherent activities of certain cellulase activities or due to the effect of end product inhibition, are impediments to commercially viable processes for producing sugars and end-products (e.g., alcohols) from biomass.
In order to maximize the hydrolysis of cellulosic substrates it would be highly desirable to develop new cellulases and particularly new β-glucosidase enzymes that could facilitate the implementation of these commercial processes.
SUMMARY OF THE INVENTION
In one embodiment, the present invention provides a β-glucosidase polypeptide variant comprising:
(a) an amino acid sequence that is at least about 56% identical to wild type Azospirillum irakense β-glucosidase (SEQ ID NO: 4) and having at least one substitution or deletion of an amino acid residue at a position selected from the group consisting of T2, A3, I4, A5, Q6, E7, G8, A9, A10, P11, A12, A13, I14, L15, P17, E18, K19, W20, P21, P23, A24, T25, Q26, I29, D30, E34, K35, A39, L41, K42, Q43, L44, E47, V46, G51, Q52, V53, G56, G59, T60, I61, E64, L66, R67, K68, P70, S73, N79, N83, G84, D85, R87, A88, P89, K91, E92, A97, A98, L105, K107, P109, G110, H111, T112, P113, I114, F118, I120, G127, N128, I134, F135, L141, A143, T144, H145, D146, P147, E148, L150, R151, R152, I153, G154, E155, A158, V159, M161, A162, A163, G165, I166, W168, T169, A173, V177, D180, G188, S190, I195, A197, A198, A201, A202, I203, V204, E205, G206, V207, F211, G212, S213, K214, D215, F216, M217, A218, P219, G220, I222, S225, A226, F229, G233, D236, Q237, G238, D243, R245, I246, S247, E248, E250, R253, N256, A257, D264, A272, F274, Q278, I280, H282, H285, Q287, G295, M297, G298, F299, N300, V304, D311, Q312, P314, G315, F319, N320, T323, S324, I326, M331, A335, K339, Q340, Y342, E343, T345, A347, V349, K350, V351, T353, I354, M356, A357, R358, D360, A362, I366, V369, V371, L372, A373, E377, K378, P379, P381, K382, D383, G386, L387, L390, S395, P396, A400, G402, R403, K408, K417, S423, A426, D433, Q418, T419, R425, A426, D436, G439, K440, G444, T452, G453, R455, D456, E458, A460, G461, T463, G467, R470, A474, D475, A476, G478, S479, E481, F482, V484, A485, Q487, Y488, T490, K491, A495, R501, E502, F507, Q508, V511, E512, L514, Q517, P518, D519, Q520, Q522, L524, A525, K528, K529, K531, D532, Q533, G534, I535, A539, W548, P551, L553, S556, D557, A562, W563, L564, T567, G570, A573, V575, F577, K580, K583, Q585, A589, H586, G590, L592, Y594, S595, P597, T599, A600, A601, T603, T604, D609, D611, N613, A617, T623, Y624, K625, K627, K629, L633, P634, E635, E636, S637, G638, V639, P640, A641, E642, A643, R644, Q645, N646, A647, G648, I649, Y650, F651, R652, A653, G654, A655, L656, R657, L658, P659, G660, R661, F662, and L663, wherein amino acid position is determined by optimal alignment with SEQ ID NO: 4, or a C-terminally truncated variant thereof; and

(b) an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions over substantially the entire length of a nucleic acid corresponding to a sequence selected from the group consisting of (i) a polynucleotide sequence that is complementary to a polynucleotide that encodes a polypeptide having the amino acid sequence of SEQ ID NO: 4; and (ii) a polynucleotide that is complementary to a polynucleotide that encodes a C-terminally truncated variant of SEQ ID NO:4, wherein the encoded polypeptide has one or more substitutions or deletions at a position selected from the group consisting of T2, A3, I4, A5, Q6, E7, G8, A9, A10, P11, A12, A13, I14, L15, P17, E18, K19, W20, P21, P23, A24, T25, Q26, I29, D30, E34, K35, A39, L41, K42, Q43, L44, E47, V46, G51, Q52, V53, G56, G59, T60, I61, E64, L66, R67, K68, P70, S73, N79, N83, G84, D85, R87, A88, P89, K91, E92, A97, A98, L105, K107, P109, G110, H111, T112, P113, I114, F118, I120, G127, N128, I134, F135, L141, A143, T144, H145, D146, P147, E148, L150, R151, R152, I153, G154, E155, A158, V159, M161, A162, A163, G165, I166, W168, T169, A173, V177, D180, G188, S190, I195, A197, A198, A201, A202, I203, V204, E205, G206, V207, F211, G212, S213, K214, D215, F216, M217, A218, P219, G220, I222, S225, A226, F229, G233, D236, Q237, G238, D243, R245, I246, S247, E248, E250, R253, N256, A257, D264, A272, F274, Q278, I280, H282, H285, Q287, G295, M297, G298, F299, N300, V304, D311, Q312, P314, G315, F319, N320, T323, S324, I326, M331, A335, K339, Q340, Y342, E343, T345, A347, V349, K350, V351, T353, I354, M356, A357, R358, D360, A362, I366, V369, V371, L372, A373, E377, K378, P379, P381, K382, D383, G386, L387, L390, S395, P396, A400, G402, R403, K408, K417, S423, A426, D433, Q418, T419, R425, A426, D436, G439, K440, G444, T452, G453, R455, D456, E458, A460, G461, T463, G467, R470, A474, D475, A476, G478, S479, E481, F482, V484, A485, Q487, Y488, T490, K491, A495, R501, E502, F507, Q508, V511, E512, L514, Q517, P518, D519, Q520, Q522, L524, A525, K528, K529, K531, D532, Q533, G534, I535, A539, W548, P551, L553, S556, D557, A562, W563, L564, T567, G570, A573, V575, F577, K580, K583, Q585, A589, H586, G590, L592, Y594, S595, P597, T599, A600, A601, T603, T604, D609, D611, N613, A617, T623, Y624, K625, K627, K629, L633, P634, E635, E636, S637, G638, V639, P640, A641, E642, A643, R644, Q645, N646, A647, G648, I649, Y650, F651, R652, A653, G654, A655, L656, R657, L658, P659, G660, R661, F662, and L663, wherein amino acid position is determined by optimal alignment with SEQ ID NO: 4.

In another embodiment, the present invention provides a β-glucosidase polypeptide variant having at least one substitution selected from the group consisting of T2A, A3L/N/P/R/G, I4P/Q/R/S/T, A5L/N/T/Y, Q6A/D/G/N/P/S/T, E7A/G/H/L/P, G8A/C/D/P/Q/R/S/Y, A9E/G/I/K/T, A10G/N/P/S, P11A/E/L/R/S, A12E/F/N/R/S/Y/-, A13PN, I14H/L/M/N/R/T/K, L15I/S, P17R, E18F/G/N/R, K19R, W20T, P21I/S, P23L, A24V, T25P, Q26P/R, A24V, T25A, I29V, D30E, E34D/K, K35E/P/Q/R, A39V, L41F, K42R, Q43P, L44S, E47K, V46F, G51P, Q52P, V53T, G56P, G59E/R/S, T60H/Y, I61V, E64S, L66Q, R67C/H, K68E, P70S, S73A, N79D, N83H, G84A/E/Q, D85N, R87D, A88T, P89S, K91Q, E92D/G/S/V, A97G/T, A99E/K/R/S, L105Y, K107R, P109D/N, G110S, H111D, T112A/I/N, P113A/K/S/V, I114T/V, F118S/L, I120V, G127A/N/S, N128H/K, I134F/N, F135L, L141I, A143IM/Q/T, T144S, H145R, D146C/S, P147I/K/L/T/W/R, E148D/G/K, L150M, R151P/W, R152S, I153T, G154V, E155A/D/K/M/P/Q/W/G, A158T, V159E/I/L/A/Q/R, M161T/V, A162S/T/V, A163T, G165E, I166T/V, W168R, T169N, A173S/C, V177P, D180C, G188D, S190Y, I195L, A197D/M/N, A198C/E/L/M/N/Q/S/T/W/D, A201P/S/G, A202F/K/L/N/P/T/Y/S, I203F/H/Y, V204I, E205X (where “X” refers to any amino acid residue), G206S, V207A/E/F/I/L/Y, F211C/V/Y/W/Q, G212C/R/V/T, S213C/H/P/V, K214P/Y, D215K/L/N/S/G, F216L, M217L/T/V, A218K/P, P219C/E/I/L/M/T/Q/V, G220S/V, I222A/C/G/I/S/V, S225C/F/N/S/T, A226G, F229I, G233P, D236G/Y, Q237R, G238R, D243G, R245K, I246C/V, S247P, E248K, E250G, R253K/Q, N256L/V, A257P/R, D264G, A272V/L, F274A/K/Q/S/T/Y/N, Q278N/R, I280V, H282N/D, H285D/N, Q287E/L/R, D291G, G295A/Q, M297I, G298R, F299S, N300D, V304L, A309G, D311E/G, Q312L, P314L/S, G315E, F319V, N320E/K/Q/S, T323A/D/G, S324V, I326S, M331L, A335P, K339E/R, Q340R, Y342C, E343A/G, T345S, A347G/K/M/V, V349A, K350F/L/T/Y/E/R, D351E, T353M/N/V/S/Y, I354T, M356K/Q/T, A357E/S/T, R358H, D360G, A362S, I366T, V369A, V371D/E/L/M/Y, L372S/W, A373T, E377D, K378R, P379G/V/Y, P381S, K382R, D383N/G, G386C/E/L/W, L387R, L390I/P, S395G/Q/K, P396N/S, A400K/T, G402S, R403S, K408I, K417R/S, S423D/N, A426S, D433G, Q418D, T419V, R425H, A426Q/S, D436N, G439P, K440N, G444P, T452A, G453R, R455K/P/S/T, D457H/E, E458N/D, A460S, G461K, T463P, G467K/Q, R470K, A474Q, D475K/S/E, A476K, G478P, S479A/H/V, E481G, F482Y, V484D, A485P/K, Q487D/K/N/R/L, Y488N, T490I, K491R, A495T, R501Q, E502G/K/N, F507G/S, Q508R/E, V511L, E512G, L514Q, Q517L, P518Q, D519G/N/K, Q520N/T/G/K, Q522K/R, L524W, A525K/S/T/M/G, K528R, K529R/E, K531E/R, D532G/R, Q533H/L, G534E, I535V/M, A539T/V, W548L, P551R, L553M, S556T, D557G, A562P, W563P, L564P, T567A, G570P, L372S, A573S/V, V575A, F577L, K580N/T, K583N/R/Q, Q585R, H586Y, A589R, G590P, L592F, Y594H/F, S595G, P597A, T599A, A600V, A601V, T603A/F/Y, T604P, D609E, D611C/E, N613D, A617D/P/V, T623S, Y624H, K625Q, K627R, K629C/R, L633D, P634S, E635D/-, E636D/G/-, S637-, G638-, V639-, P640-, A641-, E642A/-, A643P/-, R644-, Q645/-, N646K/-, Q645-, N646- A647-, G648-, I649T/-, Y650-, F651-, R652L/-, A653-, G654-, A655T/-, L656-, R657-, L658-, P659-, G660-, R661-, F662L/-, and L663P/Q/-, wherein amino acid position is determined by optimal alignment with SEQ ID NO: 4.
The present invention further provides β-glucosidase polypeptide variants that exhibit improved thermoactivity as well as improved low pH tolerance as compared to the wildtype Azospirillum irakense β-glucosidase.
In certain embodiments, the present invention provides β-glucosidase polypeptide variants that exhibit good tolerance to glucose.
In further embodiments, the present invention provides a polynucleotide encoding the β-glucosidase polypeptide variants of the present invention, vectors containing the polynucleotides, and host cells transformed with the vectors of the present invention.
In a still further embodiment, the present invention provides a method of producing a β-glucosidase polypeptide variant of the present invention, said method comprising culturing a host cell transformed with a β-glucosidase polynucleotide of the present invention under conditions suitable for the expression of the β-glucosidase polypeptide variant.
In another embodiment, the present invention provides compositions containing a β-glucosidase polypeptide of the present invention and another cellulase enzyme.
In other embodiments, the present invention provides methods of using the β-glucosidase polypeptide variants of the present invention. These methods include a method of converting an optionally pretreated biomass substrate to a fermentable sugar, the method comprising contacting a β-glucosidase polypeptide variant of the present invention with the biomass substrate under conditions suitable for the production of the fermentable sugar, and optionally further contacting the fermentable sugar with a fermentable microorganism to produce an alcohol.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1A depicts a polynucleotide sequence encoding Azospirillum irakense β-glucosidase (CelA) (SEQ ID NO: 1). The polynucleotide sequence has been codon optimized for expression in both Bacillus megaterium and Escherichia coli and includes a polynucleotide sequence encoding the Bacillus megaterium penicillin G acylase signal peptide sequence (nucleotides −72 to −1). Nucleotides +1 through +9 encode part of the Bacillus megaterium penicillin G acylase cleavage site and an engineered SpeI restriction site. Nucleotides +10 through +2001 encode the wildtype Azospirillum irakense β-glucosidase CelA.
FIG. 1B depicts the amino acid (SEQ ID NO: 2) encoded by the polynucleotide sequence of FIG. 1A and comprises the sequence encoding wildtype Azospirillum irakense β-glucosidase (CelA). The amino acid sequence includes the Bacillus megaterium penicillin G acylase signal peptide (amino acid residues −24 to −1). The amino acid sequence corresponding to the Bacillus megaterium penicillin G acylase signal peptide is underlined. Cleavage of the signal peptide from the mature CelA occurs between residues −1 and +1. The amino acid residues at positions 2 and 3, threonine and serine, respectively, are encoded by nucleotides that correspond to an engineered SpeI restriction site. Amino acid residues 4 through 666 encode the wildtype Azospirillum irakense β-glucosidase (CelA).
FIG. 2A depicts a codon-optimized (for expression in both B. megaterium and E. coli) polynucleotide sequence encoding the mature form of the catalytic domain of wildtype A. irakense β-glucosidase (CelA) (SEQ ID NO: 3).
FIG. 2B depicts the amino acid sequence encoding the mature form of the catalytic domain of wildtype A. irakense β-glucosidase (CelA) (SEQ ID NO: 4).
FIG. 3 depicts the amino acid sequence encoding an improved β-glucosidase polypeptide variant of the present invention, [H145R] CelA (SEQ ID NO: 5).
FIG. 4 depicts the amino acid sequence encoding an improved β-glucosidase polypeptide variant of the present invention, [N79D+A143M+H145R+V159E+A198S+F211Y] CelA (SEQ ID NO: 6).
FIG. 5 depicts the amino acid sequence encoding an improved β-glucosidase polypeptide variant of the present invention, [T2A+I14M+N79D+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T] CelA-des[A647-L663] (SEQ ID NO: 7).
FIG. 6 provides the activity profile for wildtype Azospirillum irakense CelA at temperatures 40° C.-55° C. and at pH 5.0-8.0 using cellobiose (10 g/L) as a substrate. The experimental procedures is described in Example 6.
FIG. 7 provides the activity profile for CelA Variant No. 264 (T2A+I14M+N79D+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T]CelA-des[A647-L663] (SEQ ID NO: 7, FIG. 5)) at temperatures 50° C.-70° C. and at pH 4.0-7.0 using cellobiose (10 g/L) as a substrate. The experimental procedure is described in Example 14.
FIG. 8 provides the activity profile for CelA Variant No. 366 (T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T-des[A647-L663] (SEQ ID NO: 9)) at temperatures 50° C.-70° C. and at pH 4.0-7.0 using cellobiose (10 g/L) as a substrate. The experimental procedure is described in Example 14.
FIG. 9 provides the activity profile for CelA Variant No. 391 (T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525-des[A647-L663] (SEQ ID NO: 11)) at temperatures 50° C.-70° C. and at pH 4.0-7.0 using cellobiose (10 g/L) as a substrate. The experimental procedure is described in Example 14.
FIG. 10 provides the activity profile for CelA Variant No. 463 (T2A+A3R+A5T+I14M+S73A+N79D+G127N+A143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F211Y+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T-des[A647-L663] (SEQ ID NO: 13)) at temperatures 60° C.-80° C. and at pH 4.1-7.9 using cellobiose (10 g/L) as a substrate. The experimental procedure is described in Example 14.
FIG. 11 provides the activity profile for CelA Variant No. 529 (T2A+A3R+A5T+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F=F211Y+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+T60H+A335P+D475E+Y594F-des[A647-L663])) at temperatures 45° C.-80° C. and at pH 4.0-7.0. The experimental procedure is described in Example 14.
FIG. 12 illustrates the impact of supplementing a commercially available Trichoderma reesei whole cellulose (“TRWC”) mixture with a CelA variant of the present invention. Compositions containing TRWC were supplemented with β-glucosdiases CelA Variant No. 264 (“Cel var1”), CelA Variant No. 366 (“Cel var2”), and a commercially available Aspergillus niger β-glucosidase (“ANBG”) in a ratio of 3:1 TRWC to β-glucosidase. The effectiveness of each composition at hydrolyzing microcrystalline cellulose was evaluated as described in Example 15. A plot of glucose and cellobiose concentration (g/L) vs. cellulase composition is depicted. The control composition was TRWC with no added β-glucosidase.



DETAILED DESCRIPTION OF THE INVENTION
Definitions
As used herein, the following terms are intended to have the following meanings.
The term “cellulase” refers to a category of enzymes capable of hydrolyzing cellulose (β-1,4-glucan or β-D-glucosidic linkages) to shorter oligosaccharides, cellobiose and/or glucose. 1,4-β-D-glucan glucanohydrolase (“endoglucanase” or “EG”); 1,4-β-D-glucan cellobiohydrolase (“exoglucanase”, “cellobiohydrolase”, or “CBH”); and β-D-glucoside-glucohydrolase (“β-glucosidase”, “cellobiase” or “BG”) are cellulase enzymes.
The term “β-glucosidase” or “cellobiase” used interchangeably herein means a β-D-glucoside glucohydrolase which catalyzes the hydrolysis of a sugar dimer, including but not limited to cellobiose with the release of a corresponding sugar monomer. In one embodiment, a β-glucosidase is a β-glucosidase glucohydrolase of the classification E.C. 3.2.1.21 which catalyzes the hydrolysis of cellobiose to glucose. Some of the β-glucosidases have the ability to also hydrolyze β-D-galactosides, β-L-arabinosides and/or β-D-fucosides and further some β-glucosidases can act on α-1,4-substrates such as starch. β-glucosidase activity may be measured by methods well known in the art (e.g., HPLC). Illustrative assays are described in Examples 5 and 7 using either p-nitrophenyl-β-D-glucopyranoside (pNPG) or cellobiose as a substrate.
The term “β-glucosidase polypeptide” refers herein to a polypeptide having β-glucosidase activity.
The term “β-glucosidase polynucleotide” refers to a polynucleotide encoding a polypeptide having β-glucosidase activity.
“Cellulolytic activity” encompasses exoglucanase activity (CBH), endoglucanase (EG) activity and/or β-glucosidase activity.
The term “exoglucanase”, “exo-cellobiohydrolase” or “CBH” refers to a group of cellulase enzymes classified as E.C. 3.2.1.91. These enzymes hydrolyze cellobiose from the reducing or non-reducing end of cellulose.
The term “endoglucanase” or “EG” refers to a group of cellulase enzymes classified as E.C. 3.2.1.4. These enzymes hydrolyze internal β-1,4 glucosidic bonds of cellulose.
As used herein, the term “isolated” refers to a nucleic acid, polynucleotide, polypeptide, protein, or other component that is partially or completely separated from components with which it is normally associated (other proteins, nucleic acids, cells, synthetic reagents, etc.).
The term “wildtype” as applied to a polypeptide (protein) or polynucleotide means a polypeptide (protein) or polynucleotide expressed by a naturally occurring microorganism such as bacteria or filamentous fungus found in nature.
A “variant” as used herein means an β-glucosidase polypeptide or polynucleotide encoding a β-glucosidase comprising one or more modifications relative to wildtype Azospirillum irakense β-glucosidase (CelA) or the wildtype polynucleotide such as substitutions, insertions, deletions and/or truncations of one or more specific amino acid residues or of one or more specific nucleotides or codons in the polypeptide or polynucleotide.
A “reference β-glucosidase sequence” refers to a defined sequence used as a basis for a sequence comparison. A reference β-glucosidase sequence may be a subset of a larger sequence. Generally a reference sequence is at least 25 amino acid residues in length, at least 50 residues in length, at least 100 residues in length, at least 150 residues in length at least 200 residues in length, at least 300 residues in length, at least 350 residues in length or the full length of the polypeptide. For instance, a reference sequence based on SEQ ID NO: 4 having at the residue corresponding to E64 a valine, refers to a reference sequence in which the corresponding residue at E64 in SEQ ID NO: 4 has been changed to a valine.
A nucleic acid (such as a polynucleotide) or a polypeptide is “recombinant” when it is artificial or engineered, or derived from an artificial or engineered protein or nucleic acid. For example, a polynucleotide that is inserted into a vector or any other heterologous location, e.g., in a genome of a recombinant organism, such that it is not associated with nucleotide sequences that normally flank the polynucleotide as it is found in nature is a recombinant polynucleotide. A protein expressed in vitro or in vivo from a recombinant polynucleotide is an example of a recombinant polypeptide. Likewise, a polynucleotide sequence that does not appear in nature, for example a variant of a naturally occurring gene, is recombinant.
An “improved property” refers to a β-glucosidase polypeptide that exhibits an improvement in any property as compared to the wildtype Azospirillum irakense β-glucosidase (CelA) (SEQ ID NO: 4). Improved properties may include increased protein expression, thermostability, thermoactivity, pH activity, pH stability, product specificity, increased activity, increased specific activity, substrate specificity, increased resistance to substrate or end-product inhibition, altered temperature profile, and chemical stability.
The term “improved thermoactivity” issued herein to refer to a variant that displays greater catalytic activity and/or greater thermostability relative to a reference enzyme, such as the wildtype Azospirillum irakense β-glucosidase. Greater catalytic activity is demonstrated by a greater rate of hydrolysis and concomitant shorter period of time required and/or lower enzyme concentration required for hydrolysis as compared to the reference (e.g., the wildtype Azospirillum irakense β-glucosidase enzyme), where the hydrolysis reaction is carried out at a temperature higher than the temperature optimum of the reference enzyme (e.g., the wildtype Azospirillum irakense β-glucosidase enzyme). The term “improved thermoactivity” also refers to a variant having improved thermostability relative to the wildtype Azospirillum irakense β-glucosidase. Alternatively a variant with a reduced thermoactivity will catalyze a hydrolysis reaction at a temperature lower than the temperature optimum of the reference enzyme (i.e., wildtype Azospirillum irakense β-glucosidase) as defined by the temperature dependent activity profile of the reference enzyme (i.e., wildtype Azospirillum irakense β-glucosidase).
The term “improved thermostability” as used herein means a variant enzyme displays greater “residual activity” relative to a reference enzyme, e.g., the wildtype enzyme. Residual activity is determined by exposing the enzyme to stress conditions of elevated temperature for a period of time and then determining the β-glucosidase activity. The β-glucosidase activity of the enzyme exposed to stress conditions (“a”) is compared to that of a control in which the enzyme is not exposed to the stress conditions (“b”), and residual activity is equal to the ratio a/b. Exemplary conditions for determining thermostability are provided in Examples 9 and 13. In one embodiment the enzymes are exposed to stress conditions of 50° C. at pH 5.5 or 55° C. at pH 5.0 or 55° C. at pH 5.5, or 65° C. at pH 5.0 for about 1 hour, and assayed at 30° C., pH 7, for about 1 hour.
The terms “percent identity,” “% identity,” “percent identical,” and “% identical” are used interchangeably herein to refer to the percent amino acid sequence identity that is obtained by ClustalW analysis (version W 1.8 available from European Bioinformatics Institute, Cambridge, UK), counting the number of identical matches in the alignment and dividing such number of identical matches by the length of the reference sequence, and using the following default ClustalW parameters to achieve slow/accurate pairwise optimal alignments—Gap Open Penalty: 10; Gap Extension Penalty: 0.10; Protein weight matrix: Gonnet series; DNA weight matrix: IUB; Toggle Slow/Fast pairwise alignments=SLOW or FULL Alignment.
Two sequences are “optimally aligned” when they are aligned for similarity scoring using a defined amino acid substitution matrix (e.g., BLOSUM62), gap existence penalty and gap extension penalty so as to arrive at the highest score possible for that pair of sequences Amino acid substitution matrices and their use in quantifying the similarity between two sequences are well-known in the art. See e.g., Dayhoff et al. (1978), “A model of evolutionary change in proteins”; “Atlas of Protein Sequence and Structure,” Vol. 5, Suppl. 3 (Ed. M. O. Dayhoff), pp. 345-352, Natl. Biomed. Res. Round., Washington, D.C.; and Henikoff et al. (1992) Proc. Natl. Acad. Sci. USA, 89:10915-10919, both of which are incorporated herein by reference. The BLOSUM62 matrix is often used as a default scoring substitution matrix in sequence alignment protocols such as Gapped BLAST 2.0. The gap existence penalty is imposed for the introduction of a single amino acid gap in one of the aligned sequences, and the gap extension penalty is imposed for each additional empty amino acid position inserted into an already opened gap. The alignment is defined by the amino acid position of each sequence at which the alignment begins and ends, and optionally by the insertion of a gap or multiple gaps in one or both sequences so as to arrive at the highest possible score. While optimal alignment and scoring can be accomplished manually, the process is facilitated by the use of a computer-implemented alignment algorithm, e.g., gapped BLAST 2.0, described in Altschul, et al. (1997) Nucleic Acids Res., 25:3389-3402 (incorporated herein by reference), and made available to the public at the National Center for Biotechnology Information Website. Optimal alignments, including multiple alignments can be prepared using readily available programs such as PSI-BLAST, which is described by Altschul, et al. (1997) Nucleic Acids Res., 25:3389-3402 and which is incorporated herein by reference.
“Corresponding to”, “reference to” “or relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence.
The “position” is denoted by a number that sequentially identifies each amino acid in the reference sequence based on its position relative to the N-terminus Owing to deletions, insertions, truncations, fusions, and the like that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N-terminal will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where there is a deletion in an aligned test sequence, there will be no amino acid that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to any amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.
Nucleic acids “hybridize” when they associate, typically in solution. Nucleic acids hybridize due to a variety of well-characterized physico-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like. As used herein, the term “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments, such as Southern and Northern hybridizations, are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) “Laboratory Techniques in biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes,” Part I, Chapter 2 (Elsevier, New York), which is incorporated herein by reference.
For polynucleotides of at least 100 nucleotides in length, low to very high stringency conditions are defined as follows: prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and either 25% formamide for low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures. For polynucleotides of at least 100 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at least at 50° C. (low stringency), at least at 55° C. (medium stringency), at least at 60° C. (medium-high stringency), at least at 65° C. (high stringency), and at least at 70° C. (very high stringency).
In describing the various variants of the present invention, the nomenclature described below is adapted for ease of reference. In all cases the accepted IUPAC single letter or triple letter amino acid abbreviations are employed. For amino acid substitutions the following nomenclature is used: Original amino acid, position, substituted amino acid. Accordingly the substitution of serine with glycine at position 34 is designated “Ser34Gly” or “S34G”. A deletion is represented by “−”. Thus, for example, “Ser34−” or “S34−” refers to a deletion at position 34. A truncation is designated by “des”. For example, “CelA-des[A647-L663]” or “des[A647-L663] refers to a carboxy (C)-terminal truncation of the amino acid residues from the alanine at position 647 to the leucine at position 663 The designation “des[L663]” refers to a deletion/truncation of the terminal leucine at position 663 of SEQ ID NO: 4. Azospirillum irakense CelA variants of the present invention having a combination of substitutions and/or a truncation may be designated by identifying both the mutations and the truncation. For example, the variant, T2A+I14M+N79D+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T CelA des[A647-L663] (which may also be referred to as T2A+I14M+N79D+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T-des[A647-L663]) is a variant of the wildtype CelA (SEQ ID NO: 4) that has the combination of substitutions indicated, and in addition, is C-terminally truncated from positions A647 to L663.
The term “culturing” or “cultivation” refers to growing a population of microbial cells under suitable conditions in a liquid or solid medium. In some embodiments, culturing refers to fermentative bioconversion of a cellulosic substrate to an end-product.
The term “contacting” refers to the placing of a respective enzyme in sufficiently close proximity to a respective substrate to enable the enzyme to convert the substrate to a product. Those skilled in the art will recognize that mixing solution of the enzyme with the respective substrate will effect contacting.
As used herein the term “transformed” or “transformation” used in reference to a cell means a cell has a non-native nucleic acid sequence integrated into its genome or as an episomal plasmid that is maintained through multiple generations.
The term “introduced” in the context of inserting a nucleic acid sequence into a cell means transfected, transduced or transformed (collectively “transformed”) and wherein the nucleic acid is incorporated into the genome of the cell.
β-Glucosidase Polypeptide Variants
The present invention provides novel enzymes that are variants of the catalytic domain of Azospirillum irakense β-glucosidase (CelA). The β-glucosidase of Azospirillum irakense belongs to glycosyl hydrolase family 3 (GHF3) and preferentially hydrolyzes cellobiose, releasing glucose units from the C3, C4, and C5 oligosaccharides. Faure, et al., App. Env. Microbiol. (May 2001) 67(5):2380-2383. β-glucosidase polypeptide variants of the present invention are variants of CelA that exhibit β-glucosidase activity. The present invention further includes β-glucosidase polypeptide variants that exhibit greater β-glucosidase activity as compared to wildtype Azospirillum irakense β-glucosidase (CelA). Also included are β-glucosidase polypeptide variants that exhibit greater stability under conditions relevant to commercial saccharification processes. In particular, variants of the present invention exhibit improved thermoactivity as compared to the wildtype Azospirillum irakense β-glucosidase. β-glucosidase polypeptide variants of the present invention also exhibit greater low pH tolerance as compared to the wildtype Azospirillum irakense β-glucosidase. These variants exhibit greater β-glucosidase activity as compared to the wildtype Azospirillum irakense β-glucosidase at a pH that is typically greater than 4.5 and less than 6.0, and more typically in the range of 5.0-5.5 inclusive, at a temperature of 50° C. or 55° C. or 60° C. or 65° C.
More specifically, the present invention provides a β-glucosidase polypeptide variant (e.g., an isolated and/or recombinant variant) comprising an amino acid sequence that is at least about 56% identical to wildtype Azospirillum irakense β-glucosidase (CelA) (SEQ ID NO: 4) (FIG. 2B) and that has at least one substitution or deletion of an amino acid residue at a position selected from the group consisting of T2, A3, I4, A5, Q6, E7, G8, A9, A10, P11, A12, A13, I14, L15, P17, E18, K19, W20, P21, P23, A24, T25, Q26, I29, D30, E34, K35, A39, L41, K42, Q43, L44, E47, V46, G51, Q52, V53, G56, G59, T60, I61, E64, L66, R67, K68, P70, S73, N79, N83, G84, D85, R87, A88, P89, K91, E92, A97, A98, L105, K107, P109, G110, H111, T112, P113, I114, F118, I120, G127, N128, I134, F135, L141, A143, T144, H145, D146, P147, E148, L150, R151, R152, I153, G154, E155, A158, V159, M161, A162, A163, G165, I166, W168, T169, A173, V177, D180, G188, S190, I195, A197, A198, A201, A202, I203, V204, E205, G206, V207, F211, G212, S213, K214, D215, F216, M217, A218, P219, G220, I222, S225, A226, F229, G233, D236, Q237, G238, D243, R245, I246, S247, E248, E250, R253, N256, A257, D264, A272, F274, Q278, I280, H282, H285, Q287, G295, M297, G298, F299, N300, V304, D311, Q312, P314, G315, F319, N320, T323, S324, I326, M331, A335, K339, Q340, Y342, E343, T345, A347, V349, K350, V351, T353, I354, M356, A357, R358, D360, A362, I366, V369, V371, L372, A373, E377, K378, P379, P381, K382, D383, G386, L387, L390, S395, P396, A400, G402, R403, K408, K417, S423, A426, D433, Q418, T419, R425, A426, D436, G439, K440, G444, T452, G453, R455, D456, E458, A460, G461, T463, G467, R470, A474, D475, A476, G478, S479, E481, F482, V484, A485, Q487, Y488, T490, K491, A495, R501, E502, F507, Q508, V511, E512, L514, Q517, P518, D519, Q520, Q522, L524, A525, K528, K529, K531, D532, Q533, G534, I535, A539, W548, P551, L553, S556, D557, A562, W563, L564, T567, G570, A573, V575, F577, K580, K583, Q585, A589, H586, G590, L592, Y594, S595, P597, T599, A600, A601, T603, T604, D609, D611, N613, A617, T623, Y624, K625, K627, K629, L633, P634, E635, E636, S637, G638, V639, P640, A641, E642, A643, R644, Q645, N646, A647, G648, I649, Y650, F651, R652, A653, G654, A655, L656, R657, L658, P659, G660, R661, F662, and L663 (wherein amino acid position is determined by optimal alignment with SEQ ID NO: 4) or a C-terminally truncated variant thereof. The amino acid sequence encoding the mature form of wildtype Azospirillum irakense β-glucosidase is shown in FIG. 2B (SEQ ID NO: 4). As explained above, variants of the present invention exhibit improved thermoactivity as compared to the wildtype Azospirillum irakense β-glucosidase. Typically, β-glucosidase variants of the present invention exhibit improved thermoactivity and low pH tolerance relative to wildtype Azospirillum irakense β-glucosidase at 55° C. and pH 5.5. In some embodiments, variants of the present invention exhibit improved thermoactivity and low pH tolerance relative to wildtype Azospirillum irakense β-glucosidase at 55° C. and pH 5.0 or at 65° C. and pH 5.0. Relative thermoactivity can be readily determined using the methods described in Example 7. Illustrative examples of variants having improved thermoactivity relative to the wildtype Azospirillum irakense β-glucosidase are provided in Examples 8-13.
In some embodiments, variants of the present invention exhibit increased thermostability as compared to wildtype Azospirillum irakense β-glucosidase under conditions of, for example, 55° C. and pH 5.5 or 55° C. and pH 5.0 or 65° C. and pH 5.0 for a time period in the range of 10 minutes, 1 hour, 4 hours, 5, hours or 48 hours using the methods described in Examples 9 and 13.
In some embodiments, variants of the present invention exhibit good tolerance to glucose. Glucose tolerance can be determined using the method described in Example 16 where an indication of the level of glucose tolerance is the IC50 for glucose. The IC50 for glucose is the glucose concentration at which activity is 50% of the activity under the same conditions with no glucose present. In certain embodiments, variants of the present invention exhibit an IC50 for glucose of at least about 60 g/L, at least about 70 g/L, at least about 80 g/L, at least about 90 g/L, or at least about 100 g/L under conditions of 55° C. and a pH of 5.
β-glucosidase polypeptides encompassed by the invention include those having an amino acid sequence that is at least about 57% identical to SEQ ID NO: 4 (FIG. 2B) and having one or more of the above-identified substitutions. Certain of these β-glucosidase variants may be at least about 58% identical, at least about 59% identical, at least about 60% identical, at least about 61% identical, at least about 62% identical, at least about 63% identical, at least about 64% identical, at least about 65% identical, at least about 66% identical, at least about 67% identical, at least about 68% identical, at least about 69% identical, at least about 70% identical, at least about 71% identical, at least about 72% identical, at least about 73% identical, at least about 73% identical, at least about 74% identical, at least about 75% identical, at least about 76% identical, at least about 77% identical, at least about 78% identical, at least about 79% identical, at least about 80% identical, at least about 81% identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, at least about 89% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical or at least about 99% identical to SEQ ID NO: 4 (FIG. 2B).
The present invention further provides a β-glucosidase polypeptide variant (e.g., an isolated and/or recombinant variant) having an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions over substantially the entire length of a nucleic acid corresponding to a sequence selected from the group consisting of (i) a polynucleotide that os complementary to a polynucleotide that encodes a β-glucosidase polypeptide having the amino acid sequence of SEQ ID NO 4 (e.g., SEQ ID NO: 3 (FIG. 2A)); and (ii) a polynucleotide that is complementary to a polynucleotide that encodes a C-terminally truncated variant of SEQ ID NO:4, wherein the encoded polypeptide has at least one or more substitutions or deletions at a position selected from the group consisting of T2, A3, I4, A5, Q6, E7, G8, A9, A10, P11, A12, A13, I14, L15, P17, E18, K19, W20, P21, P23, A24, T25, Q26, I29, D30, E34, K35, A39, L41, K42, Q43, L44, E47, V46, G51, Q52, V53, G56, G59, T60, I61, E64, L66, R67, K68, P70, S73, N79, N83, G84, D85, R87, A88, P89, K91, E92, A97, A98, L105, K107, P109, G110, H111, T112, P113, I114, F118, I120, G127, N128, I134, F135, L141, A143, T144, H145, D146, P147, E148, L150, R151, R152, I153, G154, E155, A158, V159, M161, A162, A163, G165, I166, W168, T169, A173, V177, D180, G188, S190, I195, A197, A198, A201, A202, I203, V204, E205, G206, V207, F211, G212, S213, K214, D215, F216, M217, A218, P219, G220, I222, S225, A226, F229, G233, D236, Q237, G238, D243, R245, I246, S247, E248, E250, R253, N256, A257, D264, A272, F274, Q278, I280, H282, H285, Q287, G295, M297, G298, F299, N300, V304, D311, Q312, P314, G315, F319, N320, T323, S324, I326, M331, A335, K339, Q340, Y342, E343, T345, A347, V349, K350, D351, T353, I354, M356, A357, R358, D360, A362, I366, V369, V371, L372, A373, E377, K378, P379, P381, K382, D383, G386, L387, L390, S395, P396, A400, G402, R403, K408, K417, S423, A426, D433, Q418, T419, R425, A426, D436, G439, K440, G444, T452, G453, R455, D457, E458, A460, G461, T463, G467, R470, A474, D475, A476, G478, S479, E481, F482, V484, A485, Q487, Y488, T490, K491, A495, R501, E502, F507, Q508, V511, E512, L514, Q517, P518, D519, Q520, Q522, L524, A525, K528, K529, K531, D532, Q533, G534, I535, A539, W548, P551, L553, S556, D557, A562, W563, L564, T567, G570, A573, V575, F577, K580, K583, Q585, H586, A589, G590, L592, Y594, S595, P597, T599, A600, A601, T603, T604, D609, D611, N613, A617, T623, Y624, K625, K627, K629, L633, P634, E635, E636, S637, G638, V639, P640, A641, E642, A643, R644, Q645, N646, A647, G648, I649, Y650, F651, R652, A653, G654, A655, L656, R657, L658, P659, G660, R661, F662, and L663, wherein amino acid position is determined by optimal alignment with SEQ ID NO: 4.
The C-terminally truncated variant of SEQ ID NO: 4 is typically truncated by from 1 to 40 amino acid residues from the C-terminus more typically from 1 to 30 or 1 to 20 amino acid residues, and often by 16 amino acid residues. Exemplary C-terminally truncated variants of SEQ ID NO: 4 are provided in Examples 8 and 10-12. Illustrative polynucleotide sequences encoding a C-terminally truncated variant of SEQ ID NO: 4 are provided as SEQ ID NOs: 8, 10, and 12.
In some embodiments, the polynucleotide that hybridizes to the complement of a polynucleotide which encodes a polypeptide having the amino acid sequence of SEQ ID NO: 4 or C-terminally truncated variant thereof, does so under high or very high stringency conditions to the complement of a polynucleotide sequence that encodes a polypeptide having the sequence of SEQ ID NO: 4 or C-terminally truncated variant thereof.
In some embodiments, the β-glucosidase polypeptide variant of the present invention has at least one substitution selected from the group consisting of T2A, A3L/N/P/R/G, I4P/Q/R/S/T, A5L/N/T/Y, Q6A/D/G/N/P/S/T, E7A/G/H/L/P, G8A/C/D/P/Q/R/S/Y, A9E/G/I/K/T, A10G/N/P/S, P11A/E/L/R/S, A12E/F/N/R/S/Y/-, A13PN, I14H/L/M/N/R/T/K, L15I/S, P17R, E18F/G/N/R, K19R, W20T, P21I/S, P23L, A24V, T25P, Q26P/R, A24V, T25A, I29V, D30E, E34D/K, K35E/P/Q/R, A39V, L41F, K42R, Q43P, L44S, E47K, V46F, G51P, Q52P, V53T, G56P, G59E/R/S, T60H/Y, I61V, E645, L66Q, R67C/H, K68E, P70S, S73A, N79D, N83H, G84A/E/Q, D85N, R87D, A88T, P89S, K91Q, E92D/G/S/V, A97G/T, A99E/K/R/S, L105Y, K107R, P109D/N, G110S, H111D, T112A/I/N, P113A/K/S/V, I114T/V, F118S/L, I120V, G127A/N/S, N128H/K, I134F/N, F135L, L141I, A143IM/Q/T, T144S, H145R, D146C/S, P147I/K/L/T/W/R, E148D/G/K, L150M, R151P/W, R152S, I153T, G154V, E155A/D/K/M/P/Q/W/G, A158T, V159E/I/L/A/Q/R, M161T/V, A162S/T/V, A163T, G165E, I166T/V, W168R, T169N, A173S/C, V177P, D180C, G188D, S190Y, I195L, A197D/M/N, A198C/E/L/M/N/Q/S/T/W/D, A201P/S/G, A202F/K/L/N/P/T/Y/S, 1203F/H/Y, V204I, E205X (where “X” refers to any amino acid residue), G206S, V207A/E/F/I/L/Y, F211C/V/Y/W/Q, G212C/R/V/T, S213C/H/P/V, K214P/Y, D215K/L/N/S/G, F216L, M217L/T/V, A218K/P, P219C/E/I/L/M/T/Q/V, G220S/V, I222A/C/G/I/S/V, S225C/F/N/S/T, A226G, F229I, G233P, D236G/Y, Q237R, G238R, D243G, R245K, I246C/V, S247P, E248K, E250G, R253K/Q, N256L/V, A257P/R, D264G, A272V/L, F274A/K/Q/S/T/Y/N, Q278N/R, I280V, H282N/D, H285D/N, Q287E/L/R, D291G, G295A/Q, M297I, G298R, F299S, N300D, V304L, A309G, D311E/G, Q312L, P314L/S, G315E, F319V, N320E/K/Q/S, T323A/D/G, S324V, I326S, M331L, A335P, K339E/R, Q340R, Y342C, E343A/G, T345S, A347G/K/M/V, V349A, K350F/L/T/Y/E/R, D351E, T353M/N/V/S/Y, I354T, M356K/Q/T, A357E/S/T, R358H, D360G, A362S, I366T, V369A, V371D/E/L/M/Y, L372S/W, A373T, E377D, K378R, P379G/V/Y, P381S, K382R, D383N/G, G386C/E/L/W, L387R, L390I/P, S395G/Q/K, P396N/S, A400K/T, G402S, R403S, K408I, K417R/S, S423D/N, A426S, D433G, Q418D, T419V, R425H, A426Q/S, D436N, G439P, K440N, G444P, T452A, G453R, R455K/P/S/T, D457H/E, E458N/D, A460S, G461K, T463P, G467K/Q, R470K, A474Q, D475K/S/E, A476K, G478P, S479A/H/V, E481G, F482Y, V484D, A485P/K, Q487D/K/N/R/L, Y488N, T490I, K491R, A495T, R501Q, E502G/K/N, F507G/S, Q508R/E, V511L, E512G, L514Q, Q517L, P518Q, D519G/N/K, Q520N/T/G/K, Q522K/R, L524W, A525K/S/T/M/G, K528R, K529R/E, K531E/R, D532G/R, Q533H/L, G534E, I535V/M, A539T/V, W548L, P551R, L553M, S556T, D557G, A562P, W563P, L564P, T567A, G570P, L372S, A573S/V, V575A, F577L, K580N/T, K583N/R/Q, Q585R, H586Y, A589R, G590P, L592F, Y594H/F, S595G, P597A, T599A, A600V, A601V, T603A/F/Y, T604P, D609E, D611C/E, N613D, A617D/P/V, T623S, Y624H, K625Q, K627R, K629C/R, L633D, P634S, E635D/-, E636D/G/-, S637-, G638-, V639-, P640-, A641-, E642A/-, A643P/-, R644-, Q645/-, N646K/-, Q645-, N646- A647-, G648-, I649T/-, Y650-, F651-, R652L/-, A653-, G654-, A655T/-, L656-, R657-, L658-, P659-, G660-, R661-, F662L/-, and L663P/Q/-, wherein amino acid position is determined by optimal alignment with SEQ ID NO: 4.
Particularly suitable are certain substitutions that were identified in variants that performed well with respect to the property of improved thermoactivity relative to the wildtype Azospirillum irakense β-glucosidase at pHs below the pH optimum for the wildtype enzyme (e.g., at a temperature and pH of 50° C. and pH 5.5, at a temperature of 50° C. and pH 5.0, at a temperature of 65° C. and pH 5.0, and the like). In addition, sequence-activity analyses indicated that certain of the above-described mutations (substitutions/deletions) appeared particularly favorable with respect to increasing thermoactivity relative to wildtype Azospirillum irakense β-glucosidase (SEQ ID NO: 4). Sequence-activity analysis was performed in accordance with the methods described in WO 03/075129, U.S. Ser. No. 10/379,378 filed Mar. 3, 2003, and R. Fox et al., “Optimizing the search algorithm for protein engineering by directed evolution,” Protein Eng. 16(8):589-597 (2003), both of which are incorporated herein by reference. See also R. Fox et al., “Directed molecular evolution by machine learning and the influence of nonlinear interactions,” J. Theor. Biol. 234(2):187-199 (2005), which is incorporated herein by reference. The analysis identified substitutions in the following positions as being particularly beneficial for improved thermoactivity and low pH tolerance relative to the wildtype enzyme: A5, A9, I14, L41, N79, A88, P89, P109, G127, N128, M143, V159, A162, T169, V177, A198, A201, A202, I203, V207, F211, I222, S225, A272, N300, A309, D311, A335, D475, Q508, A525, Y594, and K625.
In certain embodiments, therefore, β-glucosidase variants of the present invention have an amino acid sequence that comprises substitutions in one or more positions selected from the group consisting of A5, A9, I14, L41, N79, A88, P89, P109, G127, N128, M143, V159, A162, T169, V177, A198, A201, A202, I203, V207, F211, I222, S225, A272, N300, A309, D311, A335, D475, Q508, A525, Y594, and K625. Typically, these β-glucosidase variants comprise one or more substitutions selected from the group consisting of A5T, A9G, I14M, L41F, N79D, A88T, P89S, P109D/N, G127N/S, N128K, M143T, V159E/Q, A162T, T169N, V177P, A198S, A201P, A202P, I203Y, V207Y, F211Y, I222A/S/V, S225C, A272L, N300D, A309G, D311G, A335P, D475E, Q508R, A525T, Y594F, and K625Q. All of these specific substitutions were identified as being particularly beneficial to improved thermoactivity and low pH tolerance relative to the wildtype Azospirillum irakense β-glucosidase.
In some embodiments, β-glucosidase variants of the present invention have an amino acid sequence that comprises substitutions in one or more positions identified by these analyses, i.e., T2, A3, I4, A5, A9, I14, K35R, L41, S73, N79, A88, P78, N79, P109, G127, N128, A143, H145, P147, V159, M161, A162, T169, V177, A197, A198, A201, A202, I203, V204, V207, F211, I222, S225, A272, H285, Q287, N300, A309, D311, A335, M356, D475, R501, Q508, V511, E512, A525, K529, T567, Y594, Y594, K625, and N646 (wherein amino acid position is determined by optimal alignment with SEQ ID NO: 4). Substitutions in these positions were either identified in variants having particularly improved thermostability relative to the wildtype Azospirillum irakense β-glucosidase or were identified from a sequence-activity analysis performed as described above.
In a specific embodiment, the variant comprises an amino acid sequence that has one or more substitutions selected from the group consisting of T2A, A3L/N/P/R/GR, I4P/Q/R/S/T, A5L/N/T/Y, A9E/G/I/K/T, I14H/L/M/N/R/T/K, K35E/P/Q/R, L41F, S73A, P78S, N79D, A88T, P109D/N, G127A/N/S, N128H/K, A143I/M/Q/T, H145R, P147I/K/L/T, V158E, V159E/I/L/A/Q/R, M161T/V, A162S/T/V, T169N, V177P, A197D/M/N, A198C/E/L/M/N/Q/S/T/W/D, A201P/S/G, A202F/K/L/N/P/T/Y/S, I203YF/H/Y, V204I, V207A/E/F/I/L/Y, F211C/V/Y/W/Q, I222A/C/G/I/S/V, S225C/V/N/S/T, A272V/L, H285D/N, Q287N/R, N300D, A309G, D311E/G, A335P, M356K/Q/T, D475K/S/E, R501Q, Q508R/E, V511L, E512G, A525K/S/T/M/G, K529R/E, T567A, Y594H/F, K625Q, Y594H/F, and N646K (wherein amino acid position is determined by optimal alignment with SEQ ID NO: 4).
In certain embodiments, the variant comprises an amino acid sequence that has one or more substitutions selected from the group consisting of T2A, A3R, I4P/Q/R/S/T, A5T, A9G, I14M, K35E/P/Q/R, L41F, S73A, P78S, N79D, A88T, P109D, G127N, N128K, A143M/T, H145R, P147I/K/L/T, V158E, V159E, M161T/V, A162T, T169N, V177P, A197D/M/N, A198S, A201, A202P, I203Y, V204I, V207Y/F, F211Y, I222A/S/V, S225C, A272L, H285D/N, Q287R, N300D, A309G, D311G, A335P, M356K/Q/T, D475E, R501Q, Q508R, V511L, E512G, A525T, K529R/E, T567A, Y594F, K625Q, Y594H/F, and N646K (wherein amino acid position is determined by optimal alignment with SEQ ID NO: 4).
β-glucosidase variants of the present invention often have one or more substitutions that are selected from the group consisting of T2A, A3L/N/P/R/G, A5L/N/T/Y, I14H/L/M/R/T/K, T60H, S73A, N79D, G127A/N/S, N128H/K, A143I/M/Q/T, H145R, P147I/K/L/T/W/R, V159E/I/L/A/Q/R, M161T/V, T169N, V177P, Y186C, A197D/M/N, A198C/E/L/M/N/Q/S/T/W/D, A202F/K/L/N/P/T/Y, I203F/H/Y, V204I, V207A/E/F/I/L/Y, F211C/V/Y/W/Q, I222A/C/G/I/S/V, S225C/F/N/S/T, A272V/L, Q287E/L/R, D311E/G, A335P, M356K/Q/T, D475K/S/E, R501Q, Q508R/E, E512P, A525K/S/T/M/G, Y594H/F, and N646K. Often, variants of the present invention have at least one substitution selected from the group consisting of T2A, A5T, I14M, N79D, G127N, A142M, H145R, V158E, A197S, V207F, F210Y, I222A, S225C, Q508R, and A525T.
In certain embodiments, the variant exhibits greater thermoactivity relative to the wildtype Azospirillum irakense β-glucosidase at a temperature of 50° C. and pH of 6.5. Typically, these variants comprise an amino acid sequence that has a substitution in one or more amino acid positions selected from the group consisting of T2, A3, A5, I14, S73, N79, G127, A143, H145, V159, T169, V177, A198, A202, I203, V207, F211, I222, S225, A272, Q287, D311, Q508, E512, and A525, wherein amino acid position is determined by optimal alignment with SEQ ID NO: 4. In certain embodiments, these variants comprise an amino acid sequence that has one or more substitutions selected from the group consisting of T2A, A3R, A5T, I14M, S73A, N79D, G127N, A143M, H145R, V159E, T169N, V177P, A198S, A202P, I203Y, V207F, F211Y, I222A, S225C, A272L, Q287R, D311G, Q508R, E512G, and A525T.
The amino acid sequences of the β-glucosidase polypeptide variants described herein may have any combination of one or more substitutions at the following amino acid positions: T2, A3, I4, A5, Q6, E7, G8, A9, A10, P11, A12, A13, I14, L15, P17, E18, K19, W20, P21, P23, A24, T25, Q26, I29, D30, E34, K35, A39, L41, K42, Q43, L44, E47, V46, G51, Q52, V53, G56, G59, T60, I61, E64, L66, R67, K68, P70, S73, N79, N83, G84, D85, R87, A88, P89, K91, E92, A97, A98, L105, K107, P109, G110, H111, T112, P113, I114, F118, I120, G127, N128, I134, F135, L141, A143, T144, H145, D146, P147, E148, L150, R151, R152, I153, G154, E155, A158, V159, M161, A162, A163, G165, I166, W168, T169, A173, V177, D180, G188, S190, I195, A197, A198, A201, A202, I203, V204, E205, G206, V207, F211, G212, S213, K214, D215, F216, M217, A218, P219, G220, I222, S225, A226, F229, G233, D236, Q237, G238, D243, R245, I246, S247, E248, E250, R253, N256, A257, D264, A272, F274, Q278, I280, H282, H285, Q287, G295, M297, G298, F299, N300, V304, D311, Q312, P314, G315, F319, N320, T323, S324, I326, M331, A335, K339, Q340, Y342, E343, T345, A347, V349, K350, V351, T353, I354, M356, A357, R358, D360, A362, I366, V369, V371, L372, A373, E377, K378, P379, P381, K382, D383, G386, L387, L390, S395, P396, A400, G402, R403, K408, K417, S423, A426, D433, Q418, T419, R425, A426, D436, G439, K440, G444, T452, G453, R455, D456, E458, A460, G461, T463, G467, R470, A474, D475, A476, G478, S479, E481, F482, V484, A485, Q487, Y488, T490, K491, A495, R501, E502, F507, Q508, V511, E512, L514, Q517, P518, D519, Q520, Q522, L524, A525, K528, K529, K531, D532, Q533, G534, I535, A539, W548, P551, L553, S556, D557, A562, W563, L564, T567, G570, A573, V575, F577, K580, K583, Q585, A589, H586, G590, L592, Y594, S595, P597, T599, A600, A601, T603, T604, D609, D611, N613, A617, T623, Y624, K625, K627, K629, L633, P634, E635, E636, S637, G638, V639, P640, A641, E642, A643, R644, Q645, N646, A647, G648, I649, Y650, F651, R652, A653, G654, A655, L656, R657, L658, P659, G660, R661, F662, and L663, wherein amino acid position is determined by optimal alignment with SEQ ID NO: 4. Suitable combinations include any combination of substitutions at any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more of the above-identified positions, up to a combination of substitutions at all 289 positions.
In certain embodiments, the variant exhibits greater thermoactivity relative to the wildtype Azospirillum irakense β-glucosidase at a temperature of 55° C. and pH of 5.3. Typically, these variants comprise an amino acid sequence that has one or more substitutions in a position selected from the group consisting of N79, A143, H145, V159, A98, and F211, wherein amino acid position is determined by optimal alignment with SEQ ID NO: 4. In some embodiments, these variants have an amino acid sequence that comprises one or more substitutions selected from the group consisting of N79D, A143M, H145R, V159E, A98S and F211Y. Typically, these variants have an amino acid sequence the comprises the substitutions N79D+A143M+H145R+V159E+A98S+F211Y. Exemplary variants are provided in Table 2C of Example 8.
In some embodiments, the variant exhibits greater thermoactivity relative to the wildtype Azospirillum irakense β-glucosidase at a temperature of 55° C. and pH 5.2. Typically, these variants comprise an amino acid sequence that has one or more substitutions in a position selected from the group consisting of T2, I14, N79, A143, H145, V159, F211, I222, S225, Q508, and A525, wherein amino acid position is determined by optimal alignment with SEQ ID NO: 4. In some embodiments, these variants have an amino acid sequence that comprises one or more substitutions selected from the group consisting of T2A, I14M, N79D, A143M, H145R, V159E, A198S, F211Y, I222A, S225C, Q508C, and A525T. Typically, these variants have an amino acid sequence that comprises the substitutions T2A+I14M+N79D+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508C+A525T. Exemplary variants are provided in Tables 2D and 3 of Example 8.
In some embodiments, the variant exhibits greater thermoactivity relative to the wildtype Azospirillum irakense β-glucosidase at a temperature of 65° C. and pH 5. Typically, these variants comprise an amino acid sequence that has one or more substitutions in a position selected from the group consisting of T2, A5, I14, N79, G127, A143, H145, V159, A198, V207, F211, I222, S225, Q508, and A525, wherein amino acid position is determined by optimal alignment with SEQ ID NO: 4. In some embodiments, these variants have an amino acid sequence that comprises one or more substitutions selected from the group consisting of T2A, A5T, I14M, N79D, G127N, A143M, H145R, V159E, A198S, V207F, F211Y, I222A, S225C, Q508R, and A525T. Typically these variants have an amino acid sequence that comprises the substitutions T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T. Exemplary variants are provided in Table 5 of Example 10.
In some embodiments, the variant exhibits greater thermoactivity relative to the wildtype Azospirillum irakense β-glucosidase at a temperature of 67° C. and pH 5. Typically, these variants comprise an amino acid sequence that has one or more substitutions in a position selected from the group consisting of T2, A3, A5, I14, S73, N79, G127, A143, H145, V159, V177, A198, I203, V207, F211, I222, S225, Q508, and A525, wherein amino acid position is determined by optimal alignment with SEQ ID NO: 4. In some embodiments, these variants have an amino acid sequence that comprises one or more substitutions selected from the group consisting of T2A, A5T, I14M, S73A, N79D, G127N, A143M, H145R, V159E, V177P, A198S, I203&, V207F, F211Y, I222A, S225C, Q508R, and A525T. Typically, these variants have an amino acid sequence that comprises the substitutions T2A+A5T+I14M+S73A+N79D+G127N+A143M+H145R+V159E+V177P+A198S+I203Y+V207F+F211Y+I222A+S225C+Q508R+A525T. Exemplary variants are provided in Table 6 of Example 11.
In some embodiments, the variant exhibits greater themoactivity relative to the wildtype Azospirillum irakense β-glucosidase at a temperature of 72° C. and pH5. Typically, these variants comprise an amino acid sequence that has one or more substitutions in a position selected from the group consisting of T2, A3, A5, I14, S73, N79, G127, A143, H145, V159, T169, V177, A198, A202, I203, V207, F211, I222, S225, A272, Q287, D311, Q508, E512, A525, wherein amino acid position is determined by optimal alignment with SEQ ID NO: 4. In some embodiments, these variants have an amino acid sequence that comprises one or more substitutions selected from the group consisting of T2A, A3R, A5T, A14M, S73A, N79D, G127N, A143M, H145R, V159E, T169N, V177P, A198S, A202P, I203Y, V207F, F211Y, I222A, S225C, A272L, Q287R, D311G, Q508R, D311G, Q5084, E512G, and A525T. Typically these variants have an amino acid sequence that comprises the substitutions T2A+A3R+A5T+A14M+S73A+N79D+G127N+A143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F211Y+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T. Exemplary variants are provided in Table 7 of Example 12.
β-glucosidase variants of the present invention may further have an amino acid sequence wherein no substitution is made at positions D309 and/or E509, i.e., the amino acid sequences have an aspartic acid residue at position 309 and/or glutamic acid residue at position 509.
Variants of the present invention may comprise any of the following exemplary combinations of substitutions relative to SEQ ID NO: 4: D311G+D532G+T599A; V46F+I222A; N128K+H145R+A201P+S225C; H145R+I222V; N128K+H145R; H145R+A201P+S225C; H145R+S225C; N128K+H145R+S225C; N128K+H145R+A162T+S225C; E18R+P23L+E34K+E47K+P70S+H145R+S225C; N128K+H145R+D146S+I222A+S225C; H145R+I222A+S225C+A525T; N128K+H145R+I222A; N79D+D85N+H145R+F211Y; A143M+H145R+A198S+P219M; N79D+A143M+H145R+A198S+F211Y; N79D+H145R; N79D+H145R+A198S+P219V; N79D+H145R+F211Y; N79D+A143M+H145R; A143M+H145R; H145R+F211Y; N79D+A143M+H145R+V159E+A198S+F211Y; H145R+V159Q+A198S+F211Y; A143M+H145R+V159E+F211Y; A143M+H145R+F211Y+E642A+A643P; T2A+H145R+A162T+A201P+I222A; N128K+H145R+I222S+S225C; N128K+H145R+A201P+I222S+S225C; H145R+A162T+I222A+S225C; H145R+A162T+S225C+A573S; N79D+N128K+A143M+H145R+V159E+A173C; N128K+A143M+H145R+V159Q+A201P+F211Y+S225C; N79D+A97T+N128K+A143M+H145R+V159E+A173C+F211Y+Q508R; N79D+N128K+H145R+A162T+A173C+A201P+F211Y+S225C+Q487R+A562P; N79D+N128K+H145R+A162T+F211Y+S225C+K625Q; N79D+N128K+H145R+A201P+F211Y; N79D+N128K+A143M+H145R+A162T+A198S+F211Y+I222S+S225C; H145R+V159Q+A201P+F211Y+S225C; N79D+H145R+A162T+A198S+L663P; H145R+D146S+A162T+A173C+A201P; N79D+N128K+H145R+V159E+A201P+F211Y+P219V+S225C; N79D+N128K+A143M+H145R+A162T+A201P+F211Y+P219V; N79D+H145R+D146S+V159E+A201P+F211Y+S225C+K339R; H145R+A201P+I222S+S225C; N79D+A143M+H145R+A162T+A201P+S225C+Q585R; N79D+H145R+D146S+A201P+F211Y+I222S+S225C; N79D+N128K+A143M+H145R+V159E+A201P+F211Y+S225C; N79D+H145R+V159E+A162T+A201P+S225C+I535V; A24V+A143M+H145R+V159E+A201P+F211Y+I222S+S225C; N128K+H145R+V159E+A173C; N79D+K91Q+N128K+H145R+D146S+A201P+F211Y+I222A+S225C; N79D+N128K+F135L+A143M+H145R+A162T+A173C+A198S+P219V; N128K+A143M+H145R+V159E+A173C+A201P+F211Y+P219V; N79D+H145R+V159Q+F211Y+S225C; N128K+A143M+H145R+V159Q+A198S+F211Y+I222A+S225C+M2971+Q487R; N79D+N128K+H145R+A198S+F211Y+P219V+S225C; N79D+N128K+H145R+D146S+V159E+F211Y+I222S+S225C; A143M+H145R+A162T+F211Y+S225C; N68D+A143M+H145R+V159Q+A201P+F211Y+P219V+S225C; A143M+H145R+F211Y+P219V+S225C; N79D+N128K+H145R+A201P+P219V+K491R; A143M+H145R+A198S+F211Y+S225C; N128K+H145R+A162T+A198S+F211Y+P219V+S225C; N79D+H145R+D146S+V159E+A198S+F211Y+S225C; N79D+A143M+H145R+V159Q+A201P+S225C+K378R; N79D+N128K+H145R+A173C+A198S+F211Y+I222S; N79D+H145R+V159E+A198S+S225C; N79D+N128K+H145R+D146S+V159Q+A201P+F211Y+S225C; N79D+I114V+N128K+H145R+A162T+A198S+F211Y+S225C; A143M+H145R+A162T+A173C+A198S+F211Y+L514Q; N79D+H145R+V159Q+A201P+F211Y+I222A+S225C; N79D+E92V+N128K+A143M+H145R+A201P+F211Y+I222S+S225C; A143M+H145R+V159Q+A201P+F211Y+I222A+S225C+F229I; N79D+H145R+A198S+S225C; N79D+N128K+A143M+H145R+A162T+A198S+I222S+S225C+Q237R; N79D+G84A+N128K+H145R+A162T+A198S+F211Y+I222A+S225C; N79D+H145R+I166T+A198S+F211Y+S225C; N79D+H145R+A162T+A198S+S225C; H145R+D146S+V159Q+A201P+F11Y+I222A+S225C; A143M+H145R+A198S+F211Y+I222A+S225C+Q290R+D612E; H148R+A176C+A204P+F214Y+P222V; N79D+N128K+H145R+D146S+A162T+A173C+A198S+F211Y; N79D+N128K+H145R+D146S+S225C; A143M+H145R+A201P+S225C; N79D+H145R+G154V+F211Y+S225C; N79D+N128K+H145R; N128K+H145R+V159Q+A201P+F211Y+I222S+S225C; N79D+H145R+V159Q+A162T+A201P+F211Y+S225C; N128K+H145R+D146S+V159E+A201P+F211Y+S225C+A573V; N79D+H145R+V159E+A198S+F211Y+P219V; H145R+V159Q+F211Y+I222S+S225C+G534E; I14M+N79D+N128K+H145R+A162T+A201P+F211Y+S225C+Q487R; E34D+N79D+N128K+A143M+H145R+V159E+A201P+F211Y; N79D+H145R+D146S+A198S+F211Y+S225C; E7G+N79D+H145R+D146S+A198S+F211Y+I222S+S225C; N128K+A143T+H145R+V159Q+A201P+F211Y+I222S+S225C+E502G; H145R+A198S+F211Y+I222A+S225C+A373T; N79D+N128K+H145R+A173C+A198S+F211Y; N79D+N128K+H145R+D146S+S225C+A655T; N128K+A143M+H143R+V159E+F211Y+S225C; N79D+I114T+N128K+H145R+V159Q+A173C+A198S+F211Y+P219V; N79D+H145R+D146S+A162T+A198S+F211Y+I222A+S225C+D311G+N320K+R358H+F662L; N79D+N128K+H145R+D146S+V159Q+A201P+F211Y+P219V+I280V; H145R+V159Q+A198S+F211Y+I222A+S225C+P381S; N79D+N128K+I134N+H145R+V159Q+S225C; N79D+N128K+H145R+V159Q+A198S+I222A+S225C+K628R; N79D+N128K+H145R+D146S+A162T+A198S+F211Y; H145R+F274K; H145R+F274Q; H145R+F274A+D436N; H145R+F274A; H145R+I326S; N79D+A143M+H145R+V159E+A198S+A257P+A485P; N79D+A143M+H145R+V159E+A198S+I222S+A257P+T604P; N79D+A143M+H145R+V159E+A198S+G570P+T604P; N79D+A143M+H145R+V159E+A198S+A485P+T604P; N79D+A143M+H145R+V159E+A198S+S213P+A485P; N79D+A143M+H145R+V159E+A198S+S213P+G220V; N79D+A143M+H145R+V159E+A198S; N79D+A143M+H145R+V159E+A198S+A539V; N79D+A143M+H145R+V159E+A198S+G570P; N79D+A143M+H145R+V159E+A198S+A485P; Q26R+N79D+A143M+H145R+V159E+A198S+T604P; N79D+A143M+H145R+V159E+A198S+S479A; N79D+A143M+H145R+V159E+A199S+S213P; N79D+A143M+H145R+V159E+A198S+T604P; G59R+N79D+A143M+H145R+V159E+A198S; N79D+A143M+H145R+V159E+A198S+M331L+A485P; N79D+I120V+A143M+H145R+V159E+A198S+Q517L+T604P; A12R+N79D+A143M+H145R+V159E+A198S+F211Y; A13P+N79D+A143M+H145R+V159E+A198S+F211Y; A10G+N79D+A143M+H145R+V159E+A198S+F211Y; A3L+N79D+A143M+H145R+V159E+A198S+F211Y; A10P+N79D+A143M+H145R+V159E+A198S+F211Y; I14T+N79D+A143M+H145R+V159E+A198S+F211Y; A10S+N79D+A143M+H145R+V159E+A198S+F211Y; A3N+N79D+A143M+H145R+V159E+A198S+F211Y; G8P+N79D+A143M+H145R+V159E+A198S+F211Y; A10N+N79D+A143M+H145R+V159E+A198S+F211Y; A9T+N79D+A143M+H145R+V159E+A198S+F211Y; A5T+N79D+A143M+H145R+V159E+A198S+F211Y; Q6D+N79D+A143M+H145R+V159E+A198S+F211Y; I4P+N79D+A143M+H145R+V159E+A198S+F211Y; G8Y+N79D+A143M+H145R+V159E+A198S+F211Y; E7H+N79D+A143M+H145R+V159E+A198S+F211Y; G8S+N79D+A143M+H145R+V159E+A198S+F211Y; G8R+N79D+A143M+H145R+V159E+A198S+F211Y; Q6T+N79D+A143M+H145R+V159E+A198S+F211Y; A12N+N79D+A143M+H145R+V159E+A198S+F211Y; P11E+N79D+A143M+H145R+V159E+A198S+F211Y; A3P+N79D+A143M+H145R+V159E+A198S+F211Y; A5N+N79D+A143M+H145R+V159E+A198S+F211Y; Q6P+N79D+A143M+H145R+V159E+A198S+F211Y; A3R+N79D+A143M+H145R+V159E+A198S+F211Y; Q6S+N79D+A143M+H145R+V159E+A198S+F211Y; A12Y+N79D+A143M+H145R+V159E+A198S+F211Y; Q6G+N79D+A143M+H145R+V159E+A198S+F211Y; A9I+N79D+A143M+H145R+V159E+A198S+F211Y; G8A+N79D+A143M+H145R+V159E+A198S+F211Y; Q6A+N79D+A143M+H145R+V159E+A198S+F211Y; E7P+N79D+A143M+H145R+V159E+A198S+F211Y; A5L+N79D+A143M+H145R+V159E+A198S+F211Y; P11A+N79D+A143M+H145R+V159E+A198S+F211Y; I14R+N79D+A143M+H145R+V159E+A198S+F211Y; I4R+N79D+A143M+H145R+V159E+A198S+F211Y; A9G+N79D+A143M+H145R+V159E+A198S+F211Y; Q6N+N79D+A143M+H145R+V159E+A198S+F211Y; A9K+N79D+A143M+H145R+V159E+A198S+F211Y; A5Y+N79D+A143M+H145R+V159E+A198S+F211Y; I14M+N79D+K91Q+H145R+G154V+V159E+A198S+A201P+F211Y+S225C+A525T+K627R; I14M+N79D+K91Q+A143M+H145R+V159E+A198S+F211Y+I222A+Q508R+A525T+K627R; I14M+N79D+K91Q+A143M+H145R+V159E+A198S+F211Y+Q508R; N79D+K91Q+H145R+V159E+A198S+A201P+F211Y+I222S+Q508R+A525T; I14M+N79D+H145R+V159E+A198S+A201P+F211Y+S225C+A525T+K583R+K628R+L663P; T2A+I14M+N79D+K91Q+A143M+H145R+V159E+A198S+A201P+F211Y+S225C+A525T; I14M+N79D+K91Q+A143M+H145R+G154V+V159E+A198S+A201P+F211Y+S225C+Q508R+A525T; T2A+I14M+N79D+K91Q+A143M+H145R+V159E+A198S+A201P+F211Y+I222A+S225C+A525T+Y594H+K627R; I14M+N79D+K91Q+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+K627R; T2A+I14M+N79D+K91Q+A143M+H145R+V159E+A198S+A201P+F211Y+S225C+L663P; I14M+N79D+A143M+H145R+V159E+A198S+F211Y+I14M+N79D+K91Q+H145R+V159E+A162S+A198S+A201P+F211Y+I222S+S225C+Q508R+A525T+K628R+L663P; T2A+I14M+N79D+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+N79D+K91Q+H145R+V159E+A198S+F211Y+S225C+Q508R+L663P; T2A+I14M+N79D+K91Q+H145R+V159E+A198S+A201P+F211Y+I222A+S225C+L514Q+K628R; T2A+I14M+N79D+A143M+H145R+V159E+A198S+A201P+F211Y+I222A+S225C; I14M+N79D+K91Q+H145R+V159E+A198S+F211Y+S225C+Q508R; N79D+A143M+H145R+V159E+A198S+V207Y+F211Y; N79D+G128N+A143M+H145R+V159E+A198S+F211Y; N79D+A143M+H145R+V159E+A198S+V207F+F211Y; N79D+A143M+H145R+V159E+A198S+V207L+F211Y; N79D+P109D+A143M+H145R+V159E+A198S+F211Y; N79D+A143M+H145R+V159E+M161T+A198S+F211Y; I14M+N79D+K91Q+H145R+V159E+A198S+F211Y+I222S+S225C+Q508R+A525T+L663P; T2A+I14M+N79D+H145R+V159E+A198S+A201P+V207I+F211Y+S225C+Q508R+K627R+L663P; I14M+N79D+K91Q+N128K+H145R+V159E+A198S+F211Y+I222A+Q508R; I14M+N79D+A143M+H145R+V159E+A198S+A201P+F211Y+Q508R+A525T+K627R; I14M+N79D+K91Q+A143M+H145R+V159E+A198S+F211Y+A525T+K627R; T2A+N79D+K91Q+H145R+V159E+A198S+A201P+F211Y+S225C+D236G+K627R; N79D+K91Q+H145R+V159E+A198S+F211Y+S225C+Q508R+A525T; N79D+A143M+H145R+V159E+A198S+F211Y+I246C; N79D+A143M+H145R+V159E+A198S+F211Y+V371M; N79D+A143M+H145R+V159E+A198S+F211Y+G298R+D311E; N79D+A143M+H145R+V159E+A198S+F211Y+G386E; N79D+A143M+H145R+V159E+A198S+F211Y+A358S; N79D+A143M+H145R+V159E+A198S+F211Y+D311G; N79D+A143M+H145R+V159E+A198S+F211Y+L372W; N79D+A143M+H145R+V159E+A198S+F211Y+P379G; T2A+I14M+N79D+A143M+H145R+P147K+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+I14M+N79D+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+E377D+Q508R+A525T; T2A+I14M+N79D+A143M+H145R+P147T+V159E+A198S+F211Y+I223A+S225C+Q508R+A525T; T2A+I14M+N79D+A143M+H145R+V159E+A198S+F211Y+M217L+I222A+S225C+Q508R+A525T; T2A+I14M+N79D+A99K+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+I14M+N79D+A99R+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+I14M+D30E+N79D+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+I14M+N79D+A143M+H145R+P147L+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+I14M+N79D+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+A347K+Q508R+A525T; T2A+I14M+N79D+N83H+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+I14M+N79D+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+R455T+Q508R+A525T; T2A+I14M+N79D+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+E502N+Q508R+A525T; T2A+I14M+N79D+N128H+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+I14M+S73A+N79D+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+I14M+N79D+A143M+H145R+V159E+A198S+I203Y+F211Y+I222A+S225C+Q508R+A525T; T2A+I14M+N79D+A143M+H145R+V159E+A198S+I203H+F211Y+I222A+S225C+Q508R+A525T; T2A+I14M+N79D+A143M+H145R+V159E+A198S+V204I+F211Y+I222A+S225C+Q508R+A525T; T2A+I14M+N79D+A143M+H145R+V159E+A198S+I203F+F211Y+I222A+S225C+Q508R+A525T; T2A+I14M+N79D+A143M+H145R+V159E+V177P+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+I14M+N79D+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T+T603F; T2A+I14M+N79D+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T+T603Y; T2A+I14M+N79D+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T+L553M; T2A+E7P+A9G+A10N+I14M+N79D+N128K+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+A9G+A10N+I14M+N79D+N128K+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+Q6P+E7P+A9G+I14M+N79D+N128K+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+Q6P+A9K+A10N+I14M+N79D+N128K+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+Q6P+A10N+I14M+N79D+N128K+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+Q6P+I14M+N79D+N128K+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+Q6P+E7P+I14M+N79D+N128K+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+E7P+A9K+A10N+I14M+N79D+N128K+A143M+H145R+R152S+V159E+A298S+F211Y+I222A+S225C+Q508R+A525T; T2A+A9G+I14M+N79D+N128K+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+I14M+N79D+N128K+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+Q6P+I14M+N79D+A143M+H145R+V159E+A198S+F211Y+M217V+I222A+S225C+D311G+Q508R+A525T; T2A+I14M+I61V+N79D+G127N+A143M+H145R+V159E+A198T+F211Y+I222A+S225C+Q508R; T2A+A5T+A13P+I14M+N79D+N128K+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+I14M+N79D+N128K+A143M+H145R+V159E+M161V+A198S+V207L+F211Y+S225C+Q508R+A525T+I535V+N646K; T2A+A13P+I14M+N79D+A143M+H145R+V159E+A198S+V208F+F211Y+I222A+S225C+Q508R+A525T; T2A+I14M+N79D+G128N+A143M+H145R+V159E+A198S+V208I+F211Y+I222A+S225C+Y342C+Q508R+A525T+K583N+P634S; T2A+A5T+I14M+N79D+G128N+A143M+H145R+V159E+M161V+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+I14M+N79D+A143M+H145R+V159E+M161V+A198S+V207Y+F211Y+S225C+I354T+Q508R; T2A+I14M+N79D+A143M+H145R+V159E+A198S+V207Y+F211Y+I222A+S225C+Q508R+A525T; T2A+A5T+A13P+I14M+N79D+G127N+N128K+A143M+H145R+V159E+M161V+A198S+F211Y+S225C+Q508R+A525T+F577L; T2A+I4S+I14M+N79D+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T; T2A+14S+I14M+N79D+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T; T2A+A9E+I14M+L66Q+N79D+A143M+H145R+V159E+M161V+A198S+V207L+F211Y+S225C+Q508R+A525T; T2A+A9G+I14M+N79D+P109D+G127N+A143M+H145R+G154V+V159E+A198S+V207F+F211Y+I222A+S225C+A525T; T2A+I14M+N79D+G127N+N128K+A143M+H145R+V159E+M161V+A198S+V207L+F211Y+S225C+Q508R+A525T; T2A+I14M+N79D+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R; T2A+A5T+A13P+I14M+N79D+P109D+A143M+H145R+V159E+A198S+V207L+F211Y+I222A+S225C+Q508R; T2A+A3R+I14M+N79D+P109D+G127N+A143M+H145R+V159E+M161V+A198S+V207F+F211Y+I222A+S225C+Q508R; T2A+A9G+I14M+N79D+G127N+N128K+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+Q533L+E636D; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+A3R+A9G+I14M+N79D+A143M+H145R+V159E+M161V+A198S+V207Y+F211Y+S225C+Q508R+A525T; T2A+I14M+N79D+N128K+A143M+H145R+V159E+A198S+V207Y+F211Y+I222A+S225C+Q508R+A525T+K583N; T2A+I14M+N79D+F119S+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+L390P+Q508R+A525T; T2A+I14M+N79D+A143M+H145R+V159E+M161V+A198S+V207Y+F211Y+I222A+S225C+A525T+N646K; T2A+A13P+I14M+N79D+G127N+N128K+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T; T2A+I14M+N79D+G127N+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+I14M+N79D+A143M+H145R+V159E+M161V+A198S+V207L+F211Y+I222A+S225C+Q508R; T2A+A5T+A9G+I14M+N79D+P89S+P109D+G127N+A143M+H145R+V159E+A198S+V207Y+F211Y+I222A+S225C+Q508R+A525T; T2A+A9G+A13P+I14M+N79D+G127N+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+A525S; T2A+A9G+I14M+N79D+G127N+A143M+H145R+V159E+A2198S+V207Y+F211Y+I222A+S225C+Q508R+A525T; T2A+A5T+A9G+I14M+N79D+G127N+N128K+A143M+H145R+V159E+A198S+V207Y+F211Y+I222A+S225C+Q508R+A525T; T2A+I14M+N79D+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T; T2A+A13P+I14M+N79D+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+N646K; T2A+I14M+N79D+A143M+H145R+V159E+M161V+A198S+F211Y+S225C+Q508R; T2A+I14M+N79D+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T+Q585R; T2A+A9K+I14M+N79D+K107R+N128K+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+A9K+A10N+I14M+N79D+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+E7P+A10N+I14M+N79D+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+M356T+Q508R+A525T+H586Y; T2A+E7P+A9G+A10N+I14M+N79D+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+I14M+N79D+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+Q6P+E7P+A9G+A10N+I14M+N79D+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+I14M+N79D+G127N+N128K+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+I366T+Q508R; T2A+A3R+A5T+A9G+A13P+I14M+L44S+N79D+G127N+A143M+H145R+V159E+A198S+V207Y+F211Y+I222A+S225C+Q508R+P518Q+D519N+A525T; T2A+I14M+N79D+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A600V; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T; T2A+I14M+N79D+G127N+A143M+H145R+G154V+V159E+A198S+V207Y+F211Y+I222A+S225C+Q508R+A525T; T2A+I14M+N79D+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+F274S+Q508R; T2A+A9G+I14M+N79D+G127S+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T; T2A+A9G+A13P+I14M+N79D+P109D+N128K+A143M+H145R+V159E+A198S+V207I+F211Y+S225C+S247P+Q508R+A525S; T2A+I14M+N79D+A143M+H145R+V159E+A198S+V207Y+F211Y+I222A+S225C+Q508R+A525T+D611E; T2A+I14M+N79D+A143M+H145R+V159E+M161V+A198S+V207Y+F211Y+I222A+S225C+Q508R+A525T; T2A+A5T+I14M+N79D+P109N+A143M+H145R+V159E+M161V+A198S+V207Y+F211Y+I222A+Q508R+A525T; and T2A+A9G+I14M+N79D+A143M+H145R+V1S9E+A198S+V207F+F211Y+I222A+S225C+A525T.
Some variant polypeptides of the present invention may have the following exemplary combinations of substitutions: T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P109D+V177P+Q287R+A600V; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+I4S+I61V+V177P+I535V; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+I4S+I61V+V177P+I203Y+I535V; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+I4S+V177P+N320S+K350E; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+V177P+I535V; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P89S+V177P+D236Y; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+I4S+V177P+A600V; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+I4S+P89S+V177P+I535V+Q585R; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+I4S+I61V+S73A+V177P+N613D+A617V; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+V177P+I535V; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+S73A+V177P+I203Y+A600V; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+I4S+P89S+V177P+A600V; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P89S+V177P; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+K35Q+S73A+I203Y; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+I203Y+I535V; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P89S+V177P+I203Y+Y594F; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+S73A+1203Y; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+I4S+V177P; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+I4S+S73A+V177P+I535M; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+I61V+V177P; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+I4S+V177P+MOW; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+M161V+I203Y+A222I+D383G; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+V177P+S213T; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+P109D+V177P+M356T; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+I203Y+M356T+N646K; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T25A+N128H+V177P+I203Y+T525S; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+A13P+P147T+V177P+I203Y+T525S+H586Y; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A13P+P109D+V177P; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A13P+P109D+N128K+V177P+P597A; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A13P+V177P+A400T+H586Y; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+A13P+P147T+E159G+V177P; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P109D+P 147T+V177P+E502N+H586Y; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A13P+P147T+V177P+A226G+H586Y; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P 109D+P147T+V177P+T525S; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+V177P+E502N+T525S; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+V177P+L372S; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+V177P+E502N+R508Q; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+A13P+P147T+V177P+E502N+H586Y; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+I203Y+A400T+E502N+T525S; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A13P+N128H+ V177P+E502N+R508Q; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P109D+V177P+D351E+H586Y; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+V177P+R508Q; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+A13P+I203Y+A400T; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+P109D+P147T+V177P+H586Y; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A13P+N128H+ V177P; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P109D+V177P+A400T+E502N+H586Y; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A13P+P147T+V177P+A400T+E502N+R508Q; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P109D+V177P+A400T; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A13P+V177P+V204I+D291G; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+P147T+V177P+A400T+E502N; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+V177P+A400T; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+N128H+ V177P+E502N+H586Y; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+V177P+A400T+R508Q; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A 13P+P 147T+I203Y+H586Y; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P109D+V177P; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A13P+P109D+V177P+T525S; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+P147T+V177P+I203Y+E502N+H586Y; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+V177P; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A13P+I203Y+A400T+E502N; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+A13P+V177P+A400T+H586Y; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+V177P+E502N; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+P147T+I203Y+E502N; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+T25A+V177P+A400T+R508Q; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+V177P+A400T; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+M14K+N128H+V177P+R508Q; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A13P+P147T+E502N; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+V177P; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+N128H+V177P; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+N128H+ P147T+V177P+T603A; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P147T+I203Y+H586Y; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T169N; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+R508E; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+F274K; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+F274N; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+F274S; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+L41F+A335P; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A272L+N300D; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A309G; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+V304L; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T60H+H285N; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+S198W+T525M; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+S198A+T525G; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+D311G+D475E; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T169N+Q287R+A309G+D311G; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+L41F+A309G+D311G+A335P; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+D311G+K529E; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+Y211Q; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T169N+A202P+A272L+Q287R+D311G+E512G; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T169N+A202P+N300D A309G+D311G+Y594F; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+R67H+T169N+A202P+A335P; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+V304L+A335P; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A272L+A335P+A357S; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A272L; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+L41F+T169N+A272L+N300D+D311G+A335P+D475E+Y594F; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A202P+N300D+A309G+D311G+A335P+K350R+Q487L; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T169N+D311G+A335P+V349A+T452A; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T169N+A335P+Y594F; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+L41F+A309G+D311G+A335P+E343G; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+D311G; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A335P; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T169N+A335P; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+N300D+A309G+A335P; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A335P; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A202P+A335P+Y594F; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+V511L; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T169N+A335P; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+D264G+A272L+A309G+A335P; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A202P+N300D+D311G+A335P; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T169N+D311G+K531R; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+L41F+Q287R+A335P; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T169N+A202P+D215G+A272L+A309G+A335P+Y594F; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A202P+A335P-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+L41F+T169N+A202P+A272L+D311G+K339R+Y594F; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+H282N; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+F118L+T169N+A272L+A335P; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P219Q; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+D519G; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+Q522K; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+G59S; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P147W+D475E; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+Q522R; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A309G; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A226G+E502N; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+E502N+R508Q+N646K; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+Q520T; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+Q520G; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T5A+A589R; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+Q522K; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A202P; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A485K; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+D519K; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+S198D; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+Q520N; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+S198N+T525A; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+N83H+ D457E; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+E155G; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+R3G; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+S395K+D519G; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P 147R+E502N+N646K; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+Q520K; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P219E; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+E502N; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+G386W; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+E502N+N646K; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+D532R; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A226G+T525S; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+K35R+E502N+N646K; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P147R+E502N+R508Q; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+E92D; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P109D+H282D+L372S+E458D+E502N; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T60H; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+T60H+A335P+D475E+V511L+Y594F; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+T60H+A309G+D475E+Y594F; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+T60H+A335P+D475E+Y594F; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+A335P+D475E; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+A309G+A335P+D475E+Y594F; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+1203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+A309G+A335P+T567A; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+A309G+A335P+D475E+Y594F; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+T60H+H285N+A335P+Y594F; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+T60H+A335P+K529E+Y594F; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+1143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+T60H+A309G; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+1203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+T60H+A335P+K529E+Y594F; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+T60H+A309G+A335P; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+1143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+T60H+A335P+Y594F; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+K35R+T60H+A335P+Y594F+K627R; and T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+1143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+T60H+A309G+A335P+V511L. Exemplary sequences comprising these combinations are provided in the Examples hereinbelow.
In accordance with the present invention, β-glucosidase activity can be determined by methods known in the art. Preferred assays for determining activity include the assays of Examples 5 and 7 for β-glucosidase activity using either pNPG or cellobiose as a substrate.
In some embodiments, β-glucosidase polypeptide variants of the present invention include those having improved (e.g., greater) β-glucosidase activity relative to wildtype Azospirillum irakense β-glucosidase (SEQ ID NO: 4). Improved β-glucosidase activity may be measured by the assays described in either Example 5 or Example 7. For example, β-glucosidase polypeptides of the present invention often have β-glucosidase activity that is at least about 1-fold, at least about 2-fold, up to about 3-fold or greater β-glucosidase activity as compared to wildtype Azospirillum irakense β-glucosidase (SEQ ID NO: 4), as measured for example in the assays described in either Example 5 or Example 7. Exemplary β-glucosidase polypeptide variants having improved β-glucosidase activity relative to wildtype Azospirillum irakense β-glucosidase are identified in the Tables in Examples 8, 9, 10, 11, and 12.
The control β-glucosidase utilized in conjunction with the assays conducted on the variants listed in Tables 2B-D, 3, 5, 6, and 7 were other improved variants (i.e., not wildtype A. irakense CelA, which exhibited poor activity compared to the variants used as controls). Many of the β-glucosidase polypeptide variants of the present invention exhibit at least about 1.1 to about 6-fold and up to about 15-fold or greater β-glucosidase activity as compared to Variant No. 5 [H145R]CelA (SEQ ID NO: 5, described hereinbelow in Table 2A of Example 8) which itself exhibited greater β-glucosidase activity as compared to wildtype A. irakense β-glucosidase in the assay of Example 7 under conditions of 55° C. and pH 6.0. β-glucosidase polypeptide variants of the present invention exhibit even further improved activity, as demonstrated in Tables 2C and 2D of Example 8, hereinbelow. The present invention therefore provides β-glucosidase polypeptide variants that have at least about 1.1-fold to about 1.5-fold, and up to about 3-fold or greater β-glucosidase activity as compared to Variant No. 94 [N79D+A143M+H145R+V159E+A198S+F211Y]CelA (SEQ ID NO: 6, described hereinbelow in Table 2C of Example 8) under conditions of 55° C. and pH 5.3, and at least about 1.1-fold to about 3-fold and up to about 12-fold or greater β-glucosidase activity as compared to Variant No. 264 [T2A+I14M+N79D+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T]CelA-des[A647-L663]] (SEQ ID NO: 7, described hereinbelow in Table 2D of Example 8) as measured in the assay of Example 7 under conditions of 55° C. and pH 5.2. Variant Nos. 5, 94, and 264 all exhibited improved activity (e.g., thermoactivity) over the wildtype A. irakense β-glucosidase.
The present invention further provides β-glucosidase polypeptide variants that exhibit at least about 1.2 to 2.0 fold and from about 2.1 to about 3.0 fold greater β-glucosidase activity as compared to Variant No. 366 [T2A+A5T+I14M+N79D+G127N+A143M+h145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T]CelA-des[A647-L664] (SEQ ID NO: 9, described hereinbelow in Table 5 of Example 10) under conditions of 65° C. and pH5. In other embodiments, β-glucosidase polypeptide variants of the present invention exhibit at least about 0.5 to about 1.0 fold, about 1.1 to about 2.0 fold, about 2.1 to about 3.0 fold, about 3.1 to about 4.1 fold greater β-glucosidase activity as compared to Variant No. 391 [T2A+A5T+I14M+S73A+N79D+G127N+A143M+H145R+V159E+V177P+A198S+I203Y+V207F+F211Y+I222A+S225C+Q508R+A525T]CelA-des[A647-L664] (SEQ ID NO: 11, described hereinbelow in Table 6 of Example 11) under conditions of 67° C. and pH 5. In certain embodiments β-glucosidase polypeptide variants of the present invention exhibit at least about 1.0 to 2.0 fold or greater-glucosidase activity as compared to Variant No. 463 [T2A+A3R+A5T+I14M+S73A+N79D+G127N+A143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F211Y+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T]CelA-des[A147-L663] (SEQ ID NO: 13, described hereinbelow in Table 7 of Example 12) under conditions of 72° C. and pH 5. Variant Nos. 366, 391, and 463 all exhibited improved activity (e.g., thermoactivity) over the wildtype A. irakense β-glucosidase, as demonstrated indirectly by showing improvement over a chain of controls, one of which, Variant No. 5 has been directly compared to the wildtype enzyme.
The variants of the present invention will, in some instances, produce at least about 2-times up to at least about 3 times more glucose as compared to the amount of glucose produced from the hydrolysis of a cellobiose substrate by the wildtype A. irakense β-glucosidase (SEQ ID NO: 4) under substantially the same conditions. Some of the variants of the present invention will produce at least about 1.1 to 6 times and up to 15 times more glucose as compared to Variant No. 5 [H145R]CelA; at least about 1.1 times to about 1.5 times, and up to about 3 times or more glucose as compared to Variant No. 94 [N79D+A143M+H145R+V159E+A198S+F211Y]CelA; and at least about 1.1 times to about 3 times and up to about 12 times more glucose as compared to Variant No. 264 [T2A+I14M+N79D+A143M+H145R+V159E+A198S+F211y+I222A+S225C+Q508R+A525T]CelA-des[A647-L663].
The present invention further provides an isolated or recombinant β-glucosidase polypeptide variant having an amino acid sequence that has a substitution, deletion, and/or insertion of from one to forty amino acid residues in SEQ ID NO: 4, wherein the variant exhibits at least about 2-fold greater β-glucosidase activity than wild type A. irakense β-glucosidase (SEQ ID NO: 4), as measured in the assay of for example, Examples 5 or 7 (using either pNPG or cellobiose as substrate). These β-glucosidase polypeptides may have a substitution, deletion, and/or insertion of from 1 to 2, or from 1 or 2 to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and up to 40 residues.
Typically, these β-glucosidases exhibit β-glucosidase activity that is at least about 2-fold up to at least about 3-fold greater than that of wildtype A. irakense CelA (SEQ ID NO: 4) and/or at least about 1.1 to about 6-fold and up to about 15-fold or greater β-glucosidase activity as compared to Variant No. 5: [H145A] CelA (under conditions of 55° C. and pH 6.0) and/or at least about 1.1-fold to about 1.5-fold and up to about 3-fold or greater β-glucosidase activity as compared to Variant No. 94, [N79D+A143M+H145R+V159E+A198S+F211Y] and/or at least about 1.1-fold to about 3-fold and up to about 12-fold or greater β-glucosidase activity as compared to Variant No. 264: [T2A+I14M+N79D+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T]CelA des[A647-L663], as measured in the assays described in either Example 5 or 7.
In another embodiment, the present invention also provides a fragment of the β-glucosidase polypeptide variants described herein having β-glucosidase activity such as those detected for example in the assays of either Example 5 or 7. These fragments are referred to herein as “β-glucosidase fragments”. As used herein, the term “fragment” refers to a polypeptide having a deletion of from 1 to 50 amino acid residues from the carboxy (C-) terminus, the amino (N-) terminus, or both (i.e., a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid residues from either or both the N- or C-terminus). In certain embodiments, the deletion will be from 1 to 15 amino acid residues from the —N-terminus and from 1 to 40 amino acid residues from the C-terminus These β-glucosidase fragments are also referred to herein as N-terminally truncated and C-terminally truncated β-glucosidase polypeptide variants, respectively. In some embodiments, the deletion may be from 1 to 30, or 1 to 20, or 1 to 10 residues, or 1 to 5 residues from the C-terminus, the N-terminus, or both. Exemplary C-terminally truncated β-glucosidase variants are provided in Examples 8 and 10-12. The C-terminal truncation of 16 amino acid residues appeared particularly beneficial for expression and secretion.
β-glucosidase fragments of the present invention include those that have at least about 2-fold up to at least about 3-fold greater β-glucosidase activity as compared to wildtype A. irakense CelA (SEQ ID NO: 4) (under conditions of 50° C. and pH 6.5) and/or at least about 1.1 to about 6-fold and up to about 15-fold or greater β-glucosidase activity as compared to Variant No. 5 [N79D+A143M+H145R+V159E+A198S+F211Y]CelA and/or at least about 1.1-fold to about 1.5-fold and up to about 3-fold or greater β-glucosidase activity as compared to Variant No. 94 [N79D+A143M+H145R+V159E+A198S+F211Y] (under conditions of 55° C. and pH 5.3) and/or at least about 1.1-fold to about 3-fold and up to about 12-fold or greater β-glucosidase activity as compared to Variant No. 264 [T2A+I14M+N79D+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T]CelA des[A647-L663] (under conditions of 55° C. and pH 5.2), as measured in the assay described in Example 7. β-glucosidase fragments of the present invention may have any of the substitutions or combinations thereof described herein.
Particularly useful variants include those having C-terminal truncations. C-terminally truncated CelA variants may further have any one or combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 and/or more of the substitutions described herein.
Exemplary C-terminally truncated variants having various combinations of the above-described substitutions include: T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P109D+V177P+Q287R+A600V-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+I4S+I61V+V177P+I535V-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+I4S+I61V+V177P+I203Y+I535V-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+I4S+V177P+N320S+K350E-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+V177P+I535V-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P89S+V177P+D236Y-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+I4S+V177P+A600V-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+I4S+P89S+V177P+I535V+Q585R-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+I4S+I61V+S73A+V177P+N613D+A617V-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+V177P+I535V-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+S73A+V177P+I203Y+A600V-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+I4S+P89S+V177P+A600V-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P89S+V177P-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+K35Q+S73A+I203Y-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+I203Y+I535V-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P89S+V177P+I203Y+Y594F-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+S73A+I203Y-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+I4S+V177P-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+I4S+S73A+V177P+I535M-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+I61V+V177P-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+I4S+V177P+A601V-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+M161V+I203Y+A222I+D383G-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+V177P+S213T-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+P109D+V177P+M356T-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+I203Y+M356T+N646K-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T25A+N128H+V177P+I203Y+T525S-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+A13P+P147T+V177P+I203Y+T525S+H586Y-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A13P+P109D+V177P-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A13P+P109D+N128K+V177P+P597A-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A13P+V177P+A400T+H586Y-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+A13P+P147T+E159G+V177P-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P109D+P147T+V177P+E502N+H586Y-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A13P+P147T+V177P+A226G+H586Y-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P109D+P147T+V177P+T525S-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+V177P+E502N+T525S-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+V177P+L372S-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+V177P+E502N+R508Q-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+A13P+P147T+V177P+E502N+H586Y-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+I203Y+A400T+E502N+T525S-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A13P+N128H+V177P+E502N+R508Q-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P109D+V177P+D351E+H586Y-des[A647A-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+V177P+R508Q-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+A13P+I203Y+A400T-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+P109D+P147T+V177P+H586Y-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A13P+N128H+ V177P-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P109D+V177P+A400T+E502N+H586Y-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A13P+P147T+V177P+A400T+E502N+R508Q-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P109D+V177P+A400T-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A13P+V177P+V204I+D291G-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+P 147T+V177P+A400T+E502N-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+V177P+A400T-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+N128H+ V177P+E502N+H586Y-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+V177P+A400T+R508Q-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A13P+P147T+I203Y+H586Y-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P109D+V177P-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A13P+P109D+V177P+T525S-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+P147T+V177P+I203Y+E502N+H586Y-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+V177P-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A13P+I203Y+A400T+E502N-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+A13P+V177P+A400T+H586Y-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+V177P+E502N-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+P147T+I203Y+E502N-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+T25A+V177P+A400T+R508Q-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+V177P+A400T-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+M14K+N128H+V177P+R508Q-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A13P+P147T+E502N-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+V177P-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A3R+N128H+ V177P-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+N128H+ P147T+V177P+T603A-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P 147T+I203Y+H586Y-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T169N-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+R508E-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+F274K-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+F274N-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+F274S-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+L41F+A335P-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A272L+N300D-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A309G-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+V304L-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T60H+H285N-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+S 198W+T525M-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+S198A+T525G-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+D311G+D475E-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T169N+Q287R+A309G+D311G-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+L41F+A309G+D311G+A335P-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+D311G+K529E-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+Y211Q-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T169N+A202P+A272L+Q287R+D311G+E512G-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T169N+A202P+N300D A309G+D311G+Y594F-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+R67H+T169N+A202P+A335P-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+V304L+A335P-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A272L+A335P+A357S-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A272L-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+L41F+T169N+A272L+N300D+D311G+A335P+D475E+Y594F-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A202P+N300D+A309G+D311G+A335P+K350R+Q487L-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T169N+D311G+A335P+V349A+T452A-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T169N+A335P+Y594F-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+L41F+A309G+D311G+A335P+E343G-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+D311G-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A335P-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T169N+A335P-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+N300D+A309G+A335P-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A335P-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A202P+A335P+Y594F-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+V511L-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T169N+A335P-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+D264G+A272L+A309G+A335P-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A202P+N300D+D311G+A335P-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T169N+D311G+1(531R-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+L41F+Q287R+A335P-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T169N+A202P+D215G+A272L+A309G+A335P+Y594F-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A202P+A335P-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+L41F+T169N+A202P+A272L+D311G+K339R+Y594F-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+H282N-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+F118L+T169N+A272L+A335P-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P219Q-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+D519G-des[A647-L663T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+Q522K-des[A647-L663T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+G59S-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P147W+D475E-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+Q522R-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A309G-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A226G+E502N-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+E502N+R508Q+N646K-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+Q520T-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+Q520G-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T5A+A589R-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+Q522K-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A202P-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A485K-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+D519K-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+S198D-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+Q520N-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+S198N+T525A-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+N83H+ D457E-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+E155G-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+R3G-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+S395K+D519G-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P 147R+E502N+N646K-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+Q520K-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P219E-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+E502N-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+G386W-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+E502N+N646K-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+D532R-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+A226G+T525S-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+K35R+E502N+N646K-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P147R+E502N+R508Q-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+E92D-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+P109D+H282D+L372S+E458D+E502N-des[A647-L663]; T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T+T60H-des[A647-L663]; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+T60H+A335P+D475E+V511L+Y594F-des[A647-L663]; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+1222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+T60H+A309G+D475E+Y594F-des[A647-L663]; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+T60H+A335P+D475E+Y594F-des[A647-L663]; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+A335P+D475E-des[A647-L663]; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+A309G+A335P+D475E+Y594F-des[A647-L663]; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+A309G+A335P+T567A-des[A647-L663]; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+A309G+A335P+D475E+Y594F-des[A647-L663]; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+T60H+H285N+A335P+Y594F-des[A647-L663]; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+T60H+A335P+K529E+Y594F-des[A647-L663]; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+T60H+A309G-des[A647-L663]; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+T60H+A335P+K529E+Y594F-des[A647-L663]; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+T60H+A309G+A335P ̃des[A647-L663]; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+T60H+A335P+Y594F-des[A647-L663]; T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+K35R+T60H+A335P+Y594F+K627R ̃des[A647-L663]; and T2A+A3R+A5T+I14M+S73A+N79D+G127N+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F2117+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T+T60H+A309G+A335P+V511L-des[A647-L663], where amino acid position is determined by optimal alignment with SEQ ID NO: 4. Other illustrative C-terminally truncated β-glucosidase polypeptide variants are provided in the Tables of Examples 8-12.
The present invention also provides β-glucosidase polypeptide variants having improved thermoactivity, including improved thermostability, and/or improved stability at low and high pHs, particularly low pHs (typically greater than 4.5 and less than 6.0, more typically in the range of from 5.0 to 5.5) relative to wildtype A. irakense β-glucosidase. β-glucosidase polypeptide variants of the present invention may exhibit a half life at a pH of about 6 or less (such as, for example, about 5.5, about 5, about 4.5 etc.) and a temperature of about 60° C. or more (such as, for example, 65° C., 70° C., 75° C., 80° C., etc.) of at least about 24 hours, at least about 36 hours, at least about 48 hours, up to at least about 72 hours or more as measured using the assay of Example 5A. β-glucosidase polypeptide variants of the present invention may exhibit a half life at a pH of about 8 or more (such as, for example, about 8.5, about 9, etc.) and a temperature of about 60° C. or more (such as, for example, 65° C., 70° C., etc.) of at least about 24 hours, at least about 36 hours, at least about 48 hours, up to at least about 72 hours or more as measured using the assay of Example 5A.
In some embodiments, β-glucosidase polypeptide variants of the present invention exhibit a percent residual activity of at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, and at least about 90% after 48 hours at 55 C, pH 5.0, using, for example the method of Example 5A.
The present invention includes conservatively modified variants of the β-glucosidases described herein. These variants have conservative substitutions made in their amino acid sequences. Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagines), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine, proline, cysteine and methionine). Amino acid substitutions that do not generally alter the specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, in “The Proteins,” Academic Press, New York, which is incorporated herein by reference. The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly as well as these in reverse.
Conservatively substituted variations of the β-glucosidase polypeptide variants of the present invention include substitutions of a small percentage, typically less than 5%, more typically less than 2%, and often less than 1% of the amino acids of the polypeptide sequence, with a conservatively selected amino acid of the same conservative substitution group. The addition of sequences which do not alter the encoded activity of a β-glucosidase, such as the addition of a non-functional or non-coding sequence, is considered a conservative variation of the β-glucosidase polynucleotide.
The amino acid and polynucleotide sequences of β-glucosidase polypeptides not specifically described herein can be readily generated and identified using methods that are well known to those having ordinary skill in the art. Libraries of these β-glucosidase polypeptide variants may be generated and screened using the high throughput screen for presence of β-glucosidase activity described in either Example 5 or Example 7.
Methods for generating variant libraries are well known in the art. For example, mutagenesis and directed evolution methods can be readily applied to polynucleotides (such as, for example, wildtype Azospirillum irakense β-glucosidase encoding polynucleotides (e.g., SEQ ID NO: 3, FIG. 2A) or the polynucleotides of the present invention (described hereinbelow) to generate variant libraries that can be expressed, screened, and assayed using the methods described herein. Mutagenesis and directed evolution methods are well known in the art. See, e.g., Ling, et al., “Approaches to DNA mutagenesis: an overview,” Anal. Biochem., 254(2):157-78 (1997); Dale, et al., “Oligonucleotide-directed random mutagenesis using the phosphorothioate method,” Methods Mol. Biol., 57:369-74 (1996); Smith, “In vitro mutagenesis,” Ann. Rev. Genet., 19:423-462 (1985); Botstein, et al., “Strategies and applications of in vitro mutagenesis,” Science, 229:1193-1201 (1985); Carter, “Site-directed mutagenesis,” Biochem. J., 237:1-7 (1986); Kramer, et al., “Point Mismatch Repair,” Cell, 38:879-887 (1984); Wells, et al., “Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites,” Gene, 34:315-323 (1985); Minshull, et al., “Protein evolution by molecular breeding,” Current Opinion in Chemical Biology, 3:284-290 (1999); Christians, et al., “Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling,” Nature Biotechnology, 17:259-264 (1999); Crameri, et al., “DNA shuffling of a family of genes from diverse species accelerates directed evolution,” Nature, 391:288-291; Crameri, et al., “Molecular evolution of an arsenate detoxification pathway by DNA shuffling,” Nature Biotechnology, 15:436-438 (1997); Zhang, et al., “Directed evolution of an effective fucosidase from a galactosidase by DNA shuffling and screening,” Proceedings of the National Academy of Sciences, U.S.A., 94:45-4-4509; Crameri, et al., “Improved green fluorescent protein by molecular evolution using DNA shuffling,” Nature Biotechnology, 14:315-319 (1996); Stemmer, “Rapid evolution of a protein in vitro by DNA shuffling,” Nature, 370:389-391 (1994); Stemmer, “DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution,” Proceedings of the National Academy of Sciences, U.S.A., 91:10747-10751 (1994); WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767; US 2009/0312196; U.S. Ser. No. 12/562,988, filed Sep. 18, 2009; and WO 2009/152336, all of which are incorporated herein by reference.
Exemplary β-glucosidase polypeptide variants of the invention include those described in the Tables of Example 8-12. These variants exhibited improved thermoactivity and low pH tolerance relative to the wildtype Azospirillum irakense β-glucosidase.
The present invention also provides β-glucosidase variant fusion polypeptides, wherein the fusion polypeptide comprises an amino acid sequence encoding a β-glucosidase variant polypeptide of the present invention or fragment thereof, linked either directly or indirectly through the N- or C-terminus of the β-glucosidase variant polypeptide to an amino acid sequence encoding at least a second (additional) polypeptide. The β-glucosidase variant fusion polypeptide may further include amino acid sequence encoding a third, fourth, fifth, or additional polypeptides. Typically, each additional polypeptide has a biological activity, or alternatively, is a portion of a polypeptide that has a biological activity, wherein the portion has the effect of improving expression and/or secretion of the fusion polypeptide from the desired expression host. These sequences may be fused, either directly or indirectly, to the N- or C-terminus of the β-glucosidase variant polypeptide or fragment thereof, or alternatively, to the N- or C-terminus of the additional polypeptides having biological activity.
Typically, the additional polypeptide(s) encode an enzyme or active fragment thereof, and/or a polypeptide that improves expression and/or secretion of the fusion polypeptide from the desired expression host cell. More typically, the additional polypeptide(s) encode(s) a cellulase (for example, a β-glucosidase having a different amino acid sequence from the β-glucosidase variant polypeptide in the fusion polypeptide (e.g., a wildtype β-glucosidase or a variant thereof, including a different CelA β-glucosidase variant polypeptide), or a polypeptide exhibiting cellobiohydrolase or endoglucanse activity) and/or a polypeptide that improves expression and secretion from the desired host cell, such as, for example, a polypeptide that is normally expressed and secreted from the desired expression host, such as a secreted polypeptide normally expressed from filamentous fungi. These include glucoamylase, α-amylase and aspartyl proteases from Aspergillus niger, Aspergillus niger var. awamori, and Aspergillus oryzae, cellobiohydrolase I, cellobiohydrolase II, endoglucanase I and endoglucase III from Trichoderma and glucoamylase from Neurospora and Humicola sp. See WO 98/31821, which is incorporated herein by reference.
The polypeptide components of the fusion polypeptide may be linked to each other indirectly via a linker. Linkers suitable for use in the practice of the present invention are described in WO 2007/075899, which is incorporated herein by reference. Exemplary linkers include peptide linkers of from 1 to about 40 amino acid residues in length, including those from about 1 to about 20 amino acid residues in length, and those from about 1 to about 10 amino acid residues in length. In some embodiments, the linkers may be made up of a single amino acid residue, such as, for example, a Gly, Ser, Ala, or Thr residue or combinations thereof, particularly Gly and Ser. Linkers employed in the practice of the present invention may be cleavable. Suitable cleavable linkers may contain a cleavage site, such as a protease recognition site. Exemplary protease recognition sites are well known in the art and include, for example, Lys-Arg (the KEX2 protease recognition site, which can be cleaved by a native Aspergillus KEX2-like protease), and Lys and Arg (the trypsin protease recognition sites). See, for example, WO 2007/075899, which is incorporated herein by reference.
β-Glucosidase Polynucleotides
The present invention provides isolated or recombinant polynucleotides that encode any of the above-described β-glucosidase polypeptide variants.
Those having ordinary skill in the art will readily appreciate that due to the degeneracy of the genetic code, a multitude of nucleotide sequences encoding β-glucosidase polypeptides of the present invention exist. Table 1 is a Codon Table that provides the synonymous codons for each amino acid. For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine. Thus, at every position in the nucleic acids of the invention where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described above without altering the encoded polypeptide. It is understood that U in an RNA sequence corresponds to T in a DNA sequence.







TABLE 1







Codon Table








Amino acids
Codon













Alanine
Ala
A
GCA GCC GCG GCU


 


Cysteine
Cys
C
UGC UGU


 


Aspartic acid
Asp
D
GAC GAU


 


Glutamic acid
Glu
E
GAA GAG


 


Phenylalanine
Phe
F
UUC UUU


 


Glycine
Gly
G
GGA GGC GGG GGU


 


Histidine
His
H
CAC CAU


 


Isoleucine
Ile
I
AUA AUC AUU


 


Lysine
Lys
K
AAA AAG


 


Leucine
Leu
L
UUA UUG CUA CUC CUG CUU


 


Methionine
Met
M
AUG


 


Asparagine
Asn
N
AAC AAU


 


Proline
Pro
P
CCA CCC CCG CCU


 


Glutamine
Gln
Q
CAA CAG


 


Arginine
Arg
R
AGA AGG CGA CGC CGG CGU


 


Serine
Ser
S
AGC AGU UCA UCC UCG UCU


 


Threonine
Thr
T
ACA ACC ACG ACU


 


Valine
Val
V
GUA GUC GUG GUU


 


Tryptophan
Trp
W
UGG


 


Tyrosine
Tyr
Y
UAC UAU









Such “silent variations” are one species of “conservative” variation. One of ordinary skill in the art will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified by standard techniques to encode a functionally identical polypeptide. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in any described sequence. The invention contemplates and provides each and every possible variation of nucleic acid sequence encoding a polypeptide of the invention that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code (set forth in Table 1), as applied to the polynucleotide sequences of the present invention.
A group of two or more different codons that, when translated in the same context, all encode the same amino acid, are referred to herein as “synonymous codons.” β-glucosidase polynucleotides of the present invention may be codon optimized for expression in a particular host organism by modifying the polynucleotides to conform with the optimum codon usage of the desired host organism. Those having ordinary skill in the art will recognize that tables and other references providing preference information for a wide range of organisms are readily available See e.g., Henaut and Danchin in “Escherichia coli and Salmonella,” Neidhardt, et al. Eds., ASM Pres, Washington D.C. (1996), pp. 2047-2066, which is incorporated herein by reference.
The terms “conservatively modified variations” and “conservative variations” are used interchangeably herein to refer to those nucleic acids that encode identical or essentially identical amino acid sequences, or in the situation where the nucleic acids are not coding sequences, the term refers to nucleic acids that are identical. One of ordinary skill in the art will recognize that individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence are considered conservatively modified variations where the alterations result in one or more of the following: the deletion of an amino acid, addition of an amino acid, or substitution of an amino acid with a chemically similar amino acid. When more than one amino acid is affected, the percentage is typically less than 5% of amino acid residues over the length of the encoded sequence, and more typically less than 2%. References providing amino acids that are considered conservative substitutions for one another are well known in the art.
An exemplary β-glucosidase polynucleotide sequence of the present invention is provided as SEQ ID NO: 3, which is a polynucleotide sequence that encodes wild type Azospirillum irakense β-glucosidase (SEQ ID NO: 4), but which has been codon optimized to express well in both Bacillus megaterium and Escherichia coli, as described in Example 1, hereinbelow. Other specific changes have been identified in polynucleotides of the present invention that differ from the corresponding wild type Azospirillum irakense β-glucosidase polynucleotide sequence. The present invention further provides an isolated or recombinant β-glucosidase polynucleotide having a polynucleotide sequence comprising one or more substitutions selected from the group consisting of t3c, c12t, a24t, a27t/g, t30c, g33a, c36t, t43c, t69c, g72a, g93n, g93a, a96g, t99g/c, a102g, a104g, t111a/c, g120t, t135c, t138g, a147g, a150g, t171c, a186t, c195t, c199a, t216c, t222c, t249c, a252g, c259a, a270g, t282c, t291c, c303t, a309g, g324a, c330t, g348a, t351c, a366g, t408c, t417c, a426g, a429g, a441t/g, a450t/g, t462a/c, a468g, t489c, g492t, a495g, t501c, g513a, t516c, t528c, a543t/g, a555g, a570g, t576c, t585c, t591c, a600t/g, a606g, t609a, g612t, a615g, a621t, g654c, t666a, a675t, a678g, t688c, t693c, a702g, a714t, a726g, t729c, a732g/t, c735t, a741g, a744g, c747t, t756a, t756c, t762a, t771a, t783c, g786a, t789a, a798g, g804t, t819a, c828t, t849c, a861g, t873c, a882g, a885g, g894t, t897c, a903g, g912t, g915a, t924c, c933t, a939t, t951c, t963c, a969g, g981a, a987g, c1002t/g, t1008c, c1011a, g1017a, a1029g, a1044g, a1050g, t1053c, t1065c, t1036c, a1044g, t1062a, a1071g, a1077g/t, c1086t, a1092g, t1114c, t1137c, a1173g, a1176g, a1179g, a1185g, t1188c, a1197g, a1203g, c1206t, t1218c, t1220c, a1221g, c1227t, t1233c, a1230g, t1254c, t1260c, a1269g, t1290c, a1293, t1296c, a1302t, a1305g, t1324a, g1329a, a1350g, a1353g, t1356c, a1359g, t1371c, t1377c, t1386c, a1398g, t1401a, c1413t, t1425c, a1428t, a1431g, g1434t/a, c1437t, a1443g, t1446c, a1473g, t1476c, t1494c, t1497c, c1500t, a1506g, t1530c, g1536t/a, a1539g, a1545g, a1554g, t1569c, t1575g, t1575c, c1581t, c1588t, a1602c, t1617c, t1620c, t1626c, t1629c, a1635g, a1656g, t1650c, c1668t, t1674a, a1683g, c1698a, a1704g, t1707c, a1725g, a1734g, c1737t, a1749g, a1767g, a1770g, g1776a, t1782c, g1791a/t, t1794c, a1812g, t1821g, t1839c, g1851a, t1854c, t1857c, t1864c, t1878c, t1896c, a1899g, t1902c, a1905g, t1911c, a1914g, t1923g, c1930a, and a1932c, wherein nucleotide position is determined by optimal alignment with SEQ ID NO: 3. Illustrative variants having these silent mutations are provide in Examples 8-12, hereinbelow.
β-glucosidase polynucleotides of the present invention may further comprise a polynucleotide encoding a signal peptide as described in more detail below under the heading “Vectors, Promoters, and Expression Systems”.
Polynucleotides of the present invention can be prepared using methods that are well known in the art. Typically, oligonucleotides of up to about 40 bases are individually synthesized, then joined (e.g., by enzymatic or chemical ligation methods, or polymerase-mediated methods) to form essentially any desired continuous sequence. For example, polynucleotides of the present invention can be prepared by chemical synthesis using, for example, the classical phosphoramidite method described by Beaucage, et al. (1981) Tetrahedron Letters, 22:1859-69, or the method described by Matthes, et al. (1984) EMBO J., 3:801-05., both of which are incorporated herein by reference. These methods are typically practiced in automated synthetic methods. According to the phosphoramidite method, oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.
In addition, essentially any nucleic acid can be custom ordered from any of a variety of commercial sources, such as The Midland Certified Reagent Company (Midland, Tex.), The Great American Gene Company (Ramona, Calif.), ExpressGen Inc. (Chicago, Ill.), Operon Technologies Inc. (Alameda, Calif.), and many others.
Polynucleotides may also be synthesized by well-known techniques as described in the technical literature. See, e.g., Carruthers, et al., Cold Spring Harbor Symp. Quant. Biol., 47:411-418 (1982) and Adams, et al., J. Am. Chem. Soc., 105:661 (1983), both of which are incorporated herein by reference. Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
General texts that describe molecular biological techniques which are useful herein, including the use of vectors, promoters and many other relevant topics, include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 (“Sambrook”) and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 1999) (“Ausubel”), all of which are incorporated herein by reference. Examples of protocols sufficient to direct persons of skill through in vitro amplification methods, including the polymerase chain reaction (PCR) and the ligase chain reaction (LCR). Reference is made to Berger, Sambrook, and Ausubel, as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al. eds) Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3, 81-94; (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J. Clin. Chem 35, 1826; Landegren et al., (1988) Science 241, 1077-1080; Van Brunt (1990) Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene 4, 560; Barringer et al. (1990) Gene 89, 117, and Sooknanan and Malek (1995) Biotechnology 13: 563-564, all of which are incorporated herein by reference. Improved methods for cloning in vitro amplified nucleic acids are described in Wallace et al., U.S. Pat. No. 5,426,039, which is incorporated herein by reference.
Vectors, Promoters, and Expression Systems
The present invention also includes recombinant constructs comprising one or more of the β-glucosidase polynucleotide sequences as broadly described above. The term “construct”, “DNA construct”, or “nucleic acid construct” refers herein to a nucleic acid, either single- or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature. The term “nucleic acid construct” is synonymous with the term “expression cassette” when the nucleic acid construct contains the control sequences required for expression of a β-glucosidase coding sequence of the present invention.
The present invention also provides an expression vector comprising a β-glucosidase polynucleotide of the present invention operably linked to a promoter. Example 1 provides a description of how to make constructs for expression of β-glucosidase. However, one skilled in the art is aware of means for making DNA constructs. The term “control sequences” refers herein to all the components that are necessary or advantageous for the expression of a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter and transcriptional and translational stop signals. In some embodiments, the control sequence may include a polyadenylation sequence. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding a polypeptide.
The term “operably linked” refers herein to a configuration in which a control sequence is appropriately placed at a position relative to the coding sequence of the DNA sequence such that the control sequence influences the expression of a polypeptide. When used herein, the term “coding sequence” is intended to cover a nucleotide sequence, which directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon. The coding sequence typically includes a DNA, cDNA, and/or recombinant nucleotide sequence.
As used herein, the term “expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
The term “expression vector” refers herein to a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of the invention, and which is operably linked to additional segments that provide for its transcription.
Nucleic acid constructs of the present invention comprise a vector, such as, a plasmid, a cosmid, a phage, a virus, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), and the like, into which a nucleic acid sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available.
Polynucleotides of the present invention can be incorporated into any one of a variety of expression vectors suitable for expressing a polypeptide. Suitable vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, pseudorabies, adenovirus, adeno-associated virus, retroviruses and many others. Any vector that transduces genetic material into a cell, and, if replication is desired, which is replicable and viable in the relevant host can be used.
When incorporated into an expression vector, a β-glucosidase polynucleotide of the invention is operatively linked to an appropriate transcription control sequence (promoter) to direct mRNA synthesis, e.g., T5 promoter. Examples of such transcription control sequences particularly suited for use in transgenic plants include the cauliflower mosaic virus (CaMV) and figwort mosaic virus (FMV). Other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses and which can be used in some embodiments of the invention include SV40 promoter, E. coli lac or trp promoter, phage lambda PL promoter, tac promoter, T7 promoter, and the like. Examples of suitable promoters useful for directing the transcription of the nucleotide constructs of the present invention in a filamentous fungal host cell are promoters such as cbh1, cbh2, egl1, egl2, pepA, hfb1, hfb2, xyn1, amy, and glaA (Nunberg et al., Mol. Cell Biol., 4:2306-2315 (1984), Boel et al., EMBO J. 3:1581-1585 ((1984) and EPA 137280, which are incorporated herein by reference). In bacterial host cells, suitable promoters include the promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), a gene from a Bacillus sp., such as, for example, the Bacillus subtilis levansucranse gene (sacB), the Bacillus licheniformis alpha-amylase gene (amyl), the Bacillus megaterium InhA gene (which is described in U.S. Ser. No. 61/169,848, filed Apr. 16, 2009 and U.S. Ser. No. 12/760,827, filed Apr. 15, 2010, both of which are incorporated herein by reference), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus subtilis xylA and xylB genes, the xylose promoter (Pxyl) from Bacillus megaterium, and the promoter obtained from the prokaryotic beta-lactamase gene.
An expression vector optionally contains a ribosome binding site for translation initiation, and a transcription terminator, such as PinII. The vector also optionally includes appropriate sequences for amplifying expression, e.g., an enhancer.
The vector or DNA construct may also generally include a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and which directs the encoded polypeptide into the cells secretory pathway. Signal peptides that are suitable for use in the practice of the present invention include the Bacillus megaterium penicillin G acylase signal peptide sequence (amino acid residues −1 to −24 of SEQ ID NO: 2, as shown in FIG. 1B, encoded by nucleic acids −1 to −72 of SEQ ID NO: 1, as depicted in FIG. 1A).
Variants of the Bacillus megaterium penicillin G acylase signal peptide that are effective at directing the β-glucosidase to the secretory pathway of Bacillus megaterium are also suitable. Exemplary variants are described in Tables 3, 5, 6, and 7 in Examples 8 (e.g., F-8E L-19Q, and F-10T), 10 (e.g., F-10T, K-21R, N-5H/S, and I-14V), 11 (e.g., N-5D and I-15V), and 12 (e.g., F-10T), respectively. The numbering of amino acid substitutions in the signal sequence is indicated in FIG. 1B.
Other effective signal peptide coding regions for bacterial host cells may be obtained from the genes of Bacillus NCIB 11837 maltogenic amylase, B. stearothermophilus alpha-amylase, B. licheniformis subtilisin, B. licheniformis beta-lactamase, B. stearothermophilus neutral proteases (nprT, nprS, nprM) and B. subtilis prsS. Further signal sequences are described in Simonen and Palva (1993), Microbiological Reviews 57:109-137, which is incorporated herein by reference. Effective signal peptide coding regions for filamentous fungal host cells include but are not limited to the signal peptide coding regions obtained from Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei asparatic proteinase, Humicola insolens cellulase and Humicola lanuginosa lipase. Variants of these signal peptides and other signal peptides are suitable, as well as expression mutants thereof having one or more silent mutations. An exemplary Bacillus megaterium penicillin G acylase signal peptide having a silent mutation is described in Table 3, with the mutation c-46g relative to SEQ ID NO: 1 (depicted in FIG. 1A). Additional illustrative silent mutations in the Bacillus megaterium penicillin G acylase signal peptide are provided in Table 6: g-67a, g-61a, c-57t, c-46t, a-43t, and c-39t.
In addition, expression vectors of the present invention optionally contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells. Suitable marker genes include those coding for antibiotic resistance such as, ampicillin, kanamycin, chloramphenicol, or tetracycline resistance. Further examples include the antibiotic spectinomycin or streptomycin (e.g., the aada gene), the streptomycin phosphotransferase (SPT) gene coding for streptomycin resistance, the neomycin phosphotransferase (NPTII) gene encoding kanamycin or geneticin resistance, the hygromycin phosphotransferase (HPT) gene coding for hygromycin resistance. Additional selectable marker genes include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance in E. coli. 
The vector may further contain genetic elements that facilitate integration by either homologous or non-homologous recombination. Genetic elements that facilitate integration by homologous recombination have sequence homology to targeted integration sites in the genomic sequence of the desired expression host cell. Genetic elements or techniques which facilitate integration by non-homologous recombination include restriction enzyme-mediated integration (REMI) (see Manivasakam et al., Mol. Cell Biol. (1998) 18(3):1736-1745, which is incorporated herein by reference), transposon-mediated integration, and other elements and methods that are well known in the art.
An exemplary expression vector for the expression of β-glucosidase polypeptides of the present invention is described in Example 1, hereinbelow. Vectors of the present invention can be employed to transform an appropriate host to permit the host to express an invention protein or polypeptide.
β-glucosidase polynucleotides of the invention can also be fused, for example, in-frame to nucleic acids encoding a secretion/localization sequence, to target polypeptide expression to a desired cellular compartment, membrane, or organelle of a cell, or to direct polypeptide secretion to the periplasmic space or into the cell culture media. Such sequences are known to those of skill, and include secretion leader peptides, organelle targeting sequences (e.g., nuclear localization sequences, endoplasmic reticulum (ER) retention signals, mitochondrial transit sequences, peroxisomal transit sequences, and chloroplast transit sequences), membrane localization/anchor sequences (e.g., stop transfer sequences, GPI anchor sequences), and the like.
Expression Hosts
The present invention also provides engineered (recombinant) host cells that are transformed with a vector or DNA construct of the invention (e.g., an invention cloning vector or an invention expression vector), as well as the production of β-glucosidase polypeptide variants of the invention. Thus, the present invention is directed to a (non-human) host cell comprising any β-glucosidase polynucleotide of the present invention that is described hereinabove. As used herein, a genetically modified or recombinant host cell includes the progeny of said host cell that comprises a β-glucosidase polynucleotide which encodes a variant polypeptide of the invention.
In some embodiments, the genetically modified or recombinant host cell is a eukaryotic cell. Suitable eukaryotic host cells include, but are not limited to, fungal cells, algal cells, insect cells, and plant cells. Suitable fungal host cells include, but are not limited to, Ascomycota, Basidiomycota, Deuteromycota, Zygomycota, Fungi imperfecti. Particularly preferred fungal host cells are yeast cells and filamentous fungal cells. The filamentous fungi host cells of the present invention include all filamentous forms of the subdivision Eumycotina and Oomycota. (see, for example, Hawksworth et al., In Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK, which is incorporated herein by reference). Filamentous fungi are characterized by a vegetative mycelium with a cell wall composed of chitin, cellulose and other complex polysaccharides. The filamentous fungi host cells of the present invention are morphologically distinct from yeast.
In the present invention a filamentous fungal host cell may be a cell of a species of, but not limited to Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora (e.g., Myceliophthora thermophila), Mucor, Neurospora, Penicillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia, Trametes, Tolypocladium, Trichoderma, Verticillium, Volvariella, or teleomorphs, or anamorphs, and synonyms or taxonomic equivalents thereof.
In some embodiments of the invention, the filamentous fungal host cell is of the, Aspergillus species, Ceriporiopsis species, Chrysosporium species, Corynascus species, Fusarium species, Humicola species, Neurospora species, Penicillium species, Tolypocladium species, Tramates species, or Trichoderma species.
In some embodiments of the invention, the filamentous fungal host cell is of the Trichoderma species, e.g., T. longibrachiatum, T. viride (e.g., ATCC 32098 and 32086), Hypocrea jecorina or T. reesei (NRRL 15709, ATTC 13631, 56764, 56765, 56466, 56767 and RL-P37 and derivatives thereof—See Sheir-Neiss et al., Appl. Microbiol. Biotechnology, 20 (1984) pp 46-53), T. koningii, and T. harzianum. In addition, the term “Trichoderma” refers to any fungal strain that was previously classified as Trichoderma or currently classified as Trichoderma. 
In some embodiments of the invention, the filamentous fungal host cell is of the Aspergillus species, e.g., A. awamori, A. funigatus, A. japonicus, A. nidulans, A. niger, A. aculeatus, A. foetidus, A. oryzae, A. sojae, and A. kawachi. (Reference is made to Kelly and Hynes (1985) EMBO J. 4,475479; NRRL 3112, ATCC 11490, 22342, 44733, and 14331; Yelton M., et al., (1984) Proc. Natl. Acad. Sci. USA, 81, 1470-1474; Tilburn et al., (1982) Gene 26, 205-221; and Johnston, I. L. et al. (1985) EMBO J. 4, 1307-1311, all of which are incorporated herein by reference).
In some embodiments of the invention, the filamentous fungal host cell is of the Chrysosporium species, e.g., C. lucknowense, C. keratinophilum, C. tropicum, C. merdarium, C. Mops, C. pannicola, and C. zonatum. 
In some embodiments of the invention, the filamentous fungal host cell is of the Fusarium species, e.g., F. bactridioides, F. cerealis, F. crookwellense, F. culmorum, F. graminearum, F. graminum. F. oxysporum, F. roseum, and F. venenatum. In some embodiments of the invention, the filamentous fungal host cell is of the Neurospora species, e.g., N. crassa. Reference is made to Case, M. E. et al., (1979) Proc. Natl. Acad. Sci. USA, 76, 5259-5263; U.S. Pat. No. 4,486,553; and Kinsey, J. A. and J. A. Rambosek (1984) Molecular and Cellular Biology 4, 117-122, all of which are incorporated herein by reference. In some embodiments of the invention, the filamentous fungal host cell is of the Humicola species, e.g., H. insolens, H. grisea, and H. lanuginosa. In some embodiments of the invention, the filamentous fungal host cell is of the Mucor species, e.g., M. miehei and M. circinelloides. In some embodiments of the invention, the filamentous fungal host cell is of the Rhizopus species, e.g., R. oryzae and R. niveus. In some embodiments of the invention, the filamentous fungal host cell is of the Penicillum species, e.g., P. purpurogenum, P. chrysogenum, and P. verruculosum. In some embodiments of the invention, the filamentous fungal host cell is of the Thielavia species, e.g., T. terrestris. In some embodiments of the invention, the filamentous fungal host cell is of the Tolypocladium species, e.g., T. inflatum and T. geodes or of the Trichoderma species, e.g., T. reesei. In some embodiments of the invention, the filamentous fungal host cell is of the Trametes species, e.g., T. villosa and T. versicolor. 
In the present invention a yeast host cell may be a cell of a species of, but not limited to Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, and Yarrowia. In some embodiments of the invention, the yeast cell is Hansenula polymorpha, Saccharomyces cerevisiae, Saccaromyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia kodamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans, and Yarrowia lipolytica. 
In some embodiments on the invention, the host cell is an algal such as, Chlamydomonas (e.g., C. Reinhardtii) and Phormidium (P. sp. ATCC29409).
In other embodiments, the host cell is a prokaryotic cell. Suitable prokaryotic cells include gram positive, gram negative and gram-variable bacterial cells. The host cell may be a species of, but not limited to Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter, Acidothemus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium, Brevibacterium, Butyrivibrio, Buchnera, Campestris, Camplyobacter, Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia, Enterococcus, Enterobacter, Erwinia, Fusobacterium, Faecalibacterium, Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Hyobacter, Micrococcus, Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium, Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus, Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia, Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces, Streptococcus, Synecoccus, Saccharomonospora, Staphylococcus, Serratia, Salmonella, Shigella, Thermoanaerobacterium, Tropheryma, Tularensis, Temecula, Thermosynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella, Yersinia and Zymomonas. 
In some embodiments, the host cell is a species of Agrobacterium, Acinetobacter, Azobacter, Bacillus, Bifidobacterium, Buchnera, Geobacillus, Campylobacter, Clostridium, Corynebacterium, Escherichia, Enterococcus, Erwinia, Flavobacterium, Lactobacillus, Lactococcus, Pantoea, Pseudomonas, Staphylococcus, Salmonella, Streptococcus, Streptomyces, and Zymomonas. 
In yet other embodiments, the bacterial host strain is non-pathogenic to humans. In some embodiments the bacterial host strain is an industrial strain. Numerous bacterial industrial strains are known and suitable in the present invention.
In some embodiments of the invention the bacterial host cell is of the Agrobacterium species, e.g., A. radiobacter, A. rhizogenes, and A. rubi. In some embodiments of the invention the bacterial host cell is of the Arthrobacter species, e.g., A. aurescens, A. citreus, A. globformis, A. hydrocarboglutamicus, A. mysorens, A. nicotianae, A. paraffineus, A. protophonniae, A. roseoparqffinus, A. sulfureus, and A. ureafaciens. In some embodiments of the invention the bacterial host cell is of the Bacillus species, e.g., B. thuringiensis, B. anthracis, B. megaterium, B. subtilis, B. lentus, B. circulans, B. pumilus, B. lautus, B. coagulans, B. brevis, B. firmus, B. alkaophius, B. licheniformis, B. clausii, B. stearothermophilus, B. halodurans and B. amyloliquefaciens. In particular embodiments, the host cell will be an industrial Bacillus strain including but not limited to B. subtilis, B. pumilus, B. licheniformis, B. megaterium, B. clausii, B. stearothermophilus and B. amyloliquefaciens. Some preferred embodiments of a Bacillus host cell include B. subtilis, B. licheniformis, B. megaterium, B. stearothermophilus and B. amyloliquefaciens. In some embodiments the bacterial host cell is of the Clostridium species, e.g., C. acetobutylicum, C. tetani E88, C. lituseburense, C. saccharobutylicum, C. perfringens, and C. beijerinckii. In some embodiments the bacterial host cell is of the Corynebacterium species e.g., C. glutamicum and C. acetoacidophilum. In some embodiments the bacterial host cell is of the Escherichia species, e.g., E. coli. In some embodiments the bacterial host cell is of the Erwinia species, e.g., E. uredovora, E. carotovora, E. ananas, E. herbicola, E. punctata, and E. terreus. In some embodiments the bacterial host cell is of the Pantoea species, e.g., P. citrea, and P. agglomerans. In some embodiments the bacterial host cell is of the Pseudomonas species, e.g., P. putida, P. aeruginosa, P. mevalonii, and P. sp. D-01 10. In some embodiments the bacterial host cell is of the Streptococcus species, e.g., S. equisimiles, S. pyogenes, and S. uberis. In some embodiments the bacterial host cell is of the Streptomyces species, e.g., S. ambofaciens, S. achromogenes, S. avermitilis, S. coelicolor, S. aureofaciens, S. aureus, S. fungicidicus, S. griseus, and S. lividans. In some embodiments the bacterial host cell is of the Zymomonas species, e.g., Z. mobilis, and Z. lipolytica. 
Strains that may be used in the practice of the invention including both prokaryotic and eukaryotic strains, are readily accessible to the public from a number of culture collections such as American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).
Introduction of a vector or DNA construct into a host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, electroporation, or other common techniques (See Davis, L., Dibner, M. and Battey, I. (1986) Basic Methods in Molecular Biology, which is incorporated herein by reference). The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the β-glucosidase polynucleotide. Culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art and in the references cited herein, including, for example, Sambrook, Ausubel and Berger, as well as, for example, Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley-Liss, New York; Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla., all of which are incorporated herein by reference.
Production and Recovery of β-Glucosidase Polypeptide Variants
The present invention is directed to a method of making a polypeptide having β-glucosidase activity, the method comprising providing a host cell transformed with any one of the described β-glucosidase polynucleotides of the present invention; culturing the transformed host cell in a culture medium under conditions that cause said polynucleotide to express the encoded β-glucosidase polypeptide variant; and optionally recovering or isolating the expressed β-glucosidase polypeptide variant, or recovering or isolating the culture medium containing the expressed β-glucosidase polypeptide variant. The method further provides optionally lysing the transformed host cells after expressing the encoded β-glucosidase polypeptide variant and optionally recovering or isolating the expressed β-glucosidase polypeptide variant from the cell lysate. The present invention further provides a method of making a β-glucosidase polypeptide variant, said method comprising cultivating a host cell transformed with a β-glucosidase polynucleotide under conditions suitable for the production of the β-glucosidase polypeptide variant and recovering the β-glucosidase polypeptide variant.
Typically, recovery or isolation of the β-glucosidase polypeptide variant is from the host cell culture medium, the host cell or both, using protein recovery techniques that are well known in the art, including those described herein.
Following transformation of a suitable host strain and growth (cultivating or culturing) of the host strain to an appropriate cell density, the selected promoter may be induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract may be retained for further purification. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, or other methods, which are well known to those skilled in the art.
As noted, many references are available for the culture and production of many cells, including cells of bacterial, plant, animal (especially mammalian) and archebacterial origin. See e.g., Sambrook, Ausubel, and Berger (all supra), as well as Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley-Liss, New York and the references cited therein; Doyle and Griffiths (1997) Mammalian Cell Culture: Essential Techniques John Wiley and Sons, NY; Humason (1979) Animal Tissue Techniques, fourth edition W.H. Freeman and Company; and Ricciardelli, et al., (1989) In vitro Cell Dev. Biol. 25:1016-1024, all of which are incorporated herein by reference. For plant cell culture and regeneration, Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York); Jones, ed. (1984) Plant Gene Transfer and Expression Protocols, Humana Press, Totowa, N.J. and Plant Molecular Biology (1993) R. R. D. Croy, Ed. Bios Scientific Publishers, Oxford, U.K. ISBN 0 12 198370 6, all of which are incorporated herein by reference. Cell culture media in general are set forth in Atlas and Parks (eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla., which is incorporated herein by reference. Additional information for cell culture is found in available commercial literature such as the Life Science Research Cell Culture Catalogue (1998) from Sigma-Aldrich, Inc (St Louis, Mo.) (“Sigma-LSRCCC”) and, for example, The Plant Culture Catalogue and supplement (1997) also from Sigma-Aldrich, Inc (St Louis, Mo.) (“Sigma-PCCS”), all of which are incorporated herein by reference.
In some embodiments, cells expressing the β-glucosidase polypeptide variants of the invention are grown under batch or continuous fermentations conditions. Classical batch fermentation is a closed system, wherein the compositions of the medium is set at the beginning of the fermentation and is not subject to artificial alternations during the fermentation. A variation of the batch system is a fed-batch fermentation which also finds use in the present invention. In this variation, the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression is likely to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium. Batch and fed-batch fermentations are common and well known in the art. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth. Continuous fermentation systems strive to maintain steady sate growth conditions. Methods for modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology.
The resulting polypeptide may be recovered/isolated and optionally purified by any of a number of methods known in the art. For example, the polypeptide may be isolated from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), or precipitation. Protein refolding steps can be used, as desired, in completing the configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed in the final purification steps. In addition to the references noted supra, a variety of purification methods are well known in the art, including, for example, those set forth in Sandana (1997) Bioseparation of Proteins, Academic Press, Inc.; Bollag et al. (1996) Protein Methods, 2nd Edition, Wiley-Liss, NY; Walker (1996) The Protein Protocols Handbook Humana Press, NJ; Harris and Angal (1990) Protein Purification Applications: A Practical Approach, IRL Press at Oxford, Oxford, England; Harris and Angal Protein Purification Methods: A Practical Approach, IRL Press at Oxford, Oxford, England; Scopes (1993) Protein Purification: Principles and Practice 3rd Edition, Springer Verlag, NY; Janson and Ryden (1998) Protein Purification: Principles, High Resolution Methods and Applications, Second Edition, Wiley-VCH, NY; and Walker (1998) Protein Protocols on CD-ROM, Humana Press, NJ, all of which are incorporated herein by reference. An exemplary procedure for producing β-glucosidases is provided in Example 4, hereinbelow. The skilled artisan will readily appreciate that this procedure can be used to produce the β-glycosidase polypeptide variants of the present invention.
Cell-free transcription/translation systems can also be employed to produce β-glucosidase polypeptides using the polynucleotides of the present invention. Several such systems are commercially available. A general guide to in vitro transcription and translation protocols is found in Tymms (1995) In vitro Transcription and Translation Protocols: Methods in Molecular Biology, Volume 37, Garland Publishing, NY, which is incorporated herein by reference.
Methods of Using β-Glucosidase Polypeptides and Related Compositions
As described supra, β-glucosidase polypeptide variants of the present invention can be used to catalyze the hydrolysis of a sugar dimer with the release of the corresponding sugar monomer, for example, the conversion of cellobiose with the release of glucose. Thus, the present invention provides a method for producing glucose, said method comprising: (a) providing a cellobiose; and (b) contacting the cellobiose with a β-glucosidase polypeptide variant of the invention under conditions sufficient to form a reaction mixture for converting the cellobiose to glucose. The β-glucosidase polypeptide variant may be utilized in such methods in either isolated form or as part of a composition, such as any of those described herein. The β-glucosidase polypeptide variant may also be provided in cell culturing media or in a cell lysate. For example, after producing the β-glucosidase polypeptide variant by culturing a host cell transformed with a β-glucosidase polynucleotide or vector of the present invention, the β-glucosidase need not be isolated from the culture medium (i.e., if the β-glucosidase is secreted into the culture medium) or cell lysate (i.e., if the β-glucosidase is not secreted into the culture medium) or used in purified form to be useful in further methods of using the β-glucosidase polypeptide variant. Any composition, cell culture medium, or cell lysate containing a β-glucosidase polypeptide variant of the present invention may be suitable for using in methods that utilize a β-glucosidase. Therefore, the present invention further provides a method for producing glucose, the method comprising: (a) providing a cellobiose; and (b) contacting the cellobiose with a culture medium or cell lysate or composition comprising a β-glucosidase polypeptide variant of the present invention under conditions sufficient to form a reaction mixture for converting the cellobiose to glucose.
The present invention further provides compositions that are useful for the enzymatic conversion of cellobiose to glucose. For example, one or more β-glucosidase polypeptide variants of the present invention may be combined with another enzyme and/or an agent that alters the bulk material handling properties or further processability of the β-glucosidase(s) (e.g., a flow aid agent, water, buffer, a surfactant, and the like) or that improves the efficiency of the conversion of cellobiose to glucose, as described in more detail hereinbelow. The other enzyme may be a different β-glucosidase or another cellulase enzyme. For example, in some embodiments, the β-glucosidase is combined with other cellulases to form a cellulase mixture. The cellulase mixture may include cellulases selected from CBH and EG cellulases (e.g., cellulases from Trichoderma reesei (e.g., C2730 Cellulase from Trichoderma reesei ATCC No. 25921, Sigma-Aldrich, Inc.), C9870 ACCELLERASETM 1500, Genencor, Inc., and the like), Acidothermus cellulolyticus, Thermobifida fusca, Humicola grisea and Chrysosporium sp.). The enzymes of the cellulase mixture work together resulting in decrystallization and hydrolysis of the cellulose from a biomass substrate to yield soluble sugars, such as but not limited to glucose (See Brigham et al., (1995) in Handbook on Bioethanol (C. Wyman ed.) pp 119-141, Taylor and Francis, Washington D.C., which is incorporated herein by reference).
β-glucosidase polypeptide variants of the present invention may be used in combination with other optional ingredients such as water, a buffer, a surfactant, and/or a scouring agent. A buffer may be used with a β-glucosidase polypeptide variant of the present invention (optionally combined with other cellulases, including another β-glucosidase) to maintain a desired pH within the solution in which the β-glucosidase is employed. The exact concentration of buffer employed will depend on several factors which the skilled artisan can determine Suitable buffers are well known in the art. A surfactant may further be used in combination with the cellulases of the present invention. Suitable surfactants include any surfactant compatible with the β-glucosidase and optional other cellulases being utilized. Exemplary surfactants include an anionic, a non-ionic, and ampholytic surfactants.
Suitable anionic surfactants include, but are not limited to, linear or branched alkylbenzenesulfonates; alkyl or alkenyl ether sulfates having linear or branched alkyl groups or alkenyl groups; alkyl or alkenyl sulfates; olefinsulfonates; alkanesulfonates, and the like. Suitable counter ions for anionic surfactants include, for example, alkali metal ions, such as sodium and potassium; alkaline earth metal ions, such as calcium and magnesium; ammonium ion; and alkanolamines having from 1 to 3 alkanol groups of carbon number 2 or 3. Ampholytic surfactants suitable for use in the practice of the present invention include, for example, quaternary ammonium salt sulfonates, betaine-type ampholytic surfactants, and the like. Suitable nonionic surfactants generally include polyoxalkylene ethers, as well as higher fatty acid alkanolamides or alkylene oxide adduct thereof, fatty acid glycerine monoesters, and the like. Mixtures of surfactants can also be employed as is known in the art.
β-glucosidase polypeptide variants of the present invention, as well as any composition, culture medium, or cell lysate comprising such variants, may be used in the production of monosaccharides, disaccharides, or oligomers of a mono- or di-saccharide as chemical or fermentation feedstock from biomass. As used herein, the term “biomass” refers to living or dead biological material that contains a polysaccharide substrate, such as, for example, cellulose, starch, and the like. Therefore, the present invention provides a method of converting a biomass substrate to a fermentable sugar, the method comprising contacting a culture medium or cell lysate containing a β-glucosidase polypeptide variant according to the invention, with the biomass substrate under conditions suitable for the production of the fermentable sugar. The present invention further provides a method of converting a biomass substrate to a fermentable sugar, the method comprising: (a) pretreating a cellulose substrate to increase its susceptibility to hydrolysis; (b) contacting the pretreated cellulose substrate of step (a) with a composition, culture medium or cell lysate containing a β-glucosidase polypeptide variant of the present invention under conditions suitable for the production of the fermentable sugar.
In some embodiments, the biomass includes cellulosic substrates including but not limited to, wood, wood pulp, paper pulp, corn stover, corn fiber, rice, paper and pulp processing waste, woody or herbaceous plants, fruit or vegetable pulp, distillers grain, grasses, rice hulls, wheat straw, cotton, hemp, flax, sisal, corn cobs, sugar cane bagasse, switch grass and mixtures thereof. The biomass may optionally be pretreated to increase the susceptibility of cellulose to hydrolysis using methods known in the art such as chemical, physical and biological pretreatments (e.g., steam explosion, pulping, grinding, acid hydrolysis, solvent exposure, and the like, as well as combinations thereof).
In some embodiments, the β-glucosidase polypeptide variants and β-glucosidase polypeptide variant-containing compositions, cell culture media, and cell lysates may be reacted with the biomass or pretreated biomass at a temperature in the range of about 25° C. to about 100° C., about 30° C. to about 90° C., about 30° C. to about 80° C., about 40° C. to about 80° C. and about 35° C. to about 75° C. Also the biomass may be reacted with the β-glucosidase polypeptide variants and β-glucosidase polypeptide variant-containing compositions, cell culture media, and cell lysates at a temperature about 25° C., at about 30° C., at about 35° C., at about 40° C., at about 45° C., at about 50° C., at about 55° C., at about 60° C., at about 65° C., at about 70° C., at about 75° C., at about 80° C., at about 85° C., at about 90° C., at about 95° C. and at about 100° C. In addition to the temperatures described above, conditions suitable for converting a biomass substrate to a fermentable sugar that employ a β-glucosidase polypeptide variant of the present invention (optionally in a composition, cell culture medium, or cell lysate) include carrying out the process at a pH in a range from about pH 3.0 to about 8.5, about pH 3.5 to about 8.5, about pH 4.0 to about 7.5, about pH 4.0 to about 7.0, about pH 4.0 to about 6.5, about pH 5.0 to about 6.0, and about pH 5.0 to about 5.5. Those having ordinary skill in the art will appreciate that the reaction times for converting a particular biomass substrate to a fermentable sugar may vary but the optimal reaction time can be readily determined. Exemplary reaction times may be in the range of from about 1.0 to about 240 hours, from about 5.0 to about 180 hrs and from about 10.0 to about 150 hrs. For example, the incubation time may be at least 1 hr, at least 5 hrs, at least 10 hrs, at least 15 hrs, at least 25 hrs, at least 50 hr, at least 100 hrs, at least 180 and the like.
Reaction of the β-glucosidase with biomass substrate or pretreated biomass substrate under these conditions may result in the release of substantial amounts of the soluble sugars from the substrate. For example at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more soluble sugar may be available as compared to the release of sugar by the wildtype Azospirillum irakense CelA (SEQ ID NO: 4). In some embodiments, the soluble sugars will comprise glucose.
The soluble sugars produced by the methods of the present invention may be used to produce an alcohol (such as, for example, ethanol, butanol, and the like). The present invention therefore provides a method of producing an alcohol, where the method comprises (a) providing a fermentable sugar, such as one produced using a β-glucosidase polypeptide variant of the present invention in the methods described supra; (b) contacting the fermentable sugar with a fermenting microorganism to produce the alcohol; and (c) recovering the alcohol.
In some embodiments, the β-glucosidase polypeptide variant of the present invention, or composition, cell culture medium, or cell lysate containing such variant(s) may be used to catalyze the hydrolysis of a biomass substrate to a fermentable sugar in the presence of a fermenting microorganism such as a yeast (e.g., Saccharomyces sp., such as, for example, S. cerevisiae, Pichia sp., and the like) or other C5 or C6 fermenting microorganisms that are well known in the art, to produce an end-product such as ethanol. In this simultaneous saccharification and fermentation (SSF) process, the fermentable sugars (e.g., glucose and/or xylose) are removed from the system by the fermentation process.
The soluble sugars produced by the use of a β-glucosidase variant polypeptide of the present invention may also be used in the production of other end-products. such as, for example, acetone, an amino acid (e.g., glycine, lysine, and the like), an organic acid (e.g., lactic acid, and the like), glycerol, a diol (e.g., 1,3 propanediol, butanediol, and the like) and animal feeds.
One of skill in the art will readily appreciate that the β-glucosidase polypeptide variant compositions of the present invention may be used in the form of an aqueous solution or a solid concentrate. When aqueous solutions are employed, the β-glucosidase solution can easily be diluted to allow accurate concentrations. A concentrate can be in any form recognized in the art including, for example, liquids, emulsions, suspensions, gel, pastes, granules, powders, an agglomerate, a solid disk, as well as other forms that are well known in the art. Other materials can also be used with or included in the β-glucosidase composition of the present invention as desired, including stones, pumice, fillers, solvents, enzyme activators, and anti-redeposition agents depending on the intended use of the composition.
β-glucosidase polypeptide variants and compositions thereof may also be used in the food and beverage industry for example in the process of wine making for the efficient release of monoterpenols (see, for example, Yanai and Sato (1999) Am. J. Enol. Eitic., 50:231-235, which is incorporated herein by reference) and for the preparation of glycon isoflavone-enriched tofu (see, for example, Mase et al., (2004) J. Appl. Glycosci., 51:211-216, which is incorporated herein by reference). β-glucosidase polypeptide variants of the present invention may also be employed in detergent compositions for improved cleaning performance (see, for example, U.S. Pat. No. 7,244,605; U.S. Pat. No. 5,648,263 and WO 2004/048592, which are incorporated herein by reference).
The foregoing and other aspects of the invention may be better understood in connection with the following non-limiting examples.
EXAMPLES
The following examples are offered to illustrate, but not to limit the claimed invention.
Example 1
Wild Type Azospirillum irakense CelA Gene Acquisition and Construction of Expression Vectors
A gene coding for Azospirillum irakense CelA was codon optimized for expression in B. megaterium and E. coli based on the reported amino acid sequence (AAG43575.1) and a codon optimization algorithm incorporated as described in Example 1 of PCT publication WO2008/042876, which is incorporated herein by reference. The gene was synthesized by GenScript Corporation (GenScript Corporation, 120 Centennial Ave., Piscataway, N.J. 08854, USA) and the DNA sequence verified. The gene was cloned behind a nucleotide sequence encoding the Bacillus megaterium penicillin G acylase signal peptide plus a spacer region (encoding the signal peptide cleavage site and nucleotides corresponding to a SpeI restriction site) into an E. coli/B. megaterium shuttle vector pSSBm28 using the BsrGI/NgoMIV cloning sites. The polynucleotide sequence encoding wildtype A. irakense CelA is shown in FIG. 1A (SEQ ID NO: 1). Nucleotides −72 to −1 encode the B. megaterium penicillin G acylase signal peptide. Nucleotides 1-3 encode part of the signal peptide cleavage site. Nucleotides 4-9 correspond to a SpeI restriction site. The vector pSSBm28 is a modified vector based on the Bacillus megaterium shuttle vector pMM1525 (Boca Scientific Inc., Boca Raton, Fla.). pSSBm28 differs from pMM1525 in that it has the penicillin G acylase signal peptide instead of the LipA signal peptide that pMM1625 has. In both vectors, the signal peptide and gene are under the control of and xylose promoter (Pxyl) regulated by the xylose repressor gene (xylR) present on the shuttle vector. The vector contained the ‘rep U’ origin of replication for Bacillus and a tetracycline ampicillin resistance marker. The vector also contained the pBR322 origin of replication and an ampicillin resistance marker for maintenance in E. coli. The resulting plasmid (pSSBm28-CelA) was transformed by a standard PEG-mediated method of DNA transfer into B. megaterium protoplasts. The CelA sequence from the transformants was verified. The amino acid sequence of the encoded polypeptide is shown in FIG. 1B (SEQ ID NO: 2). Amino acid residues −1 to −24 correspond to the B. megaterium penicillin G acylase signal peptide. The signal peptide is cleaved between amino acid residues −1 and +1. The amino acid residue at position +1, glycine, was engineered into the polypeptide to signal peptide processing in B. megaterium. The amino acid residues at positions 2 and 3, threonine and serine, respectively, are encoded by a nucleotide sequence that corresponds to a SpeI restriction site. The wildtype CelA catalytic domain is encoded by amino acid residues +4 to +666 of SEQ ID NO: 2 (using the numbering convention depicted in FIG. 1B) and also amino acid residues 1-663 of SEQ ID NO: 4 (FIG. 2B) and is encoded by the polynucleotide sequence defined by nucleic acids 10-2001 of SEQ ID NO: 1 (using the numbering convention depicted in FIG. 1A) and nucleic acids 1-1992 of SEQ ID NO: 3 (FIG. 2A).
Example 2
Shake Flask Procedure for Producing CelA
A single microbial colony of Bacillus megaterium containing the vector prepared in Example 1 with the CelA gene was inoculated into 3 ml Luria-Bertani (LB) Broth (0.01 g/L Peptone from casein, 0.005 g/L yeast extract, 0.01 g/L sodium chloride) containing 10 μg/mL tetracycline. Cells were grown overnight (at least 16 hrs) in an incubator at 37° C. with shaking at 250 rpm. 0.5 mls of this culture was then diluted into 50 mL A5 media (2 g/L (NH4)2SO4, 3.5 g/L KH2HPO4, 7.3 g/L Na2HPO4, 1 g/L yeast extract, pH to 6.8), 50 μL of trace elements solution (49 g/L MnCl2.4H2O, 45 g/L CaCl2, 2.5 g/L (NH4)Mo7.O24.H2O, 2.5 g/L CoCl2.6H2O), 750 μL of 20% glucose, 1.25 ml of 20% xylose, 75 μL of 1M MgSO4, 50 μL of 10 mg/mL tetracycline, 50 μL of 2.5 g/L FeSO4.7H2O in a 250 ml flask. It was allowed to grow at 37° C. for 24 hours. Cells were pelleted by centrifugation (4000 rpm, 15 min, 4° C.). The clear media supernatant containing the secreted CelA enzyme was collected and stored at 4° C. The activity of wild-type CelA was confirmed using pNPG (p-nitrophenyl-β-D-glucopyranoside) as substrate as described by Breves et al. (1997) Appl. Environmental Microbiol. 63:3902, which is incorporated herein by reference.
Example 3
Inoculation Shake Flask Procedure for Producing CelA
A single microbial colony of B. megaterium containing a vector coding for CelA was inoculated into 250 ml A5 broth (2.0 g/L ammonium sulfate, 7.26 g/L of disodium monohydrogen phosphate, 3.52 g/L of potassium dihydrogen phosphate, 1.0 g/L of Tastone-154 yeast extract, 1.5 ml/L of 1M magnesium sulfate solution, 1.0 ml of 2.5 g/L iron sulfate septahydrate solution, and 1.0 ml/L of trace element solution containing 45.0 g/L of calcium chloride, 49.0 g/L manganese chloride tetrahydrate, 2.5 g/L cobalt chloride hexahydrate, and 2.5 g/L ammonium molybdate hydrate) containing 10 μg/ml tetracycline and 0.5% glucose. The vector that was utilized was the same as that described in Example 1 except that the promoter from Bacillus megaterium InhA was used. This promoter is described in U.S. Ser. No. 61/169,848, filed Apr. 16, 2009 and U.S. Ser. No. 12/760,827, filed Apr. 15, 2010, both of which are incorporated herein by reference. Cells were grown overnight (at least 12 hrs) in an incubator at 30° C. with shaking at 250 rpm. When the OD600 of the culture was 3.0 to 5.0, the cells were removed from the incubator and used immediately for inoculating the fermentor, or stored at 4° C. until used.
Example 4
Reference Cellobiase Expression
Fermentation Procedure
In an aerated agitated 15 L fermentor, 6.0 L of growth medium containing 0.88 g/L ammonium sulfate, 1.0 g/L of sodium citrate, 12.5 g/L of dipotassium monohydrogen phosphate trihydrate, 6.25 g/L of potassium dihydrogen phosphate, 3.3 g/L of Tastone-154 yeast extract, 2.0 g/L of Phytone peptone, and 1.0 ml/L of trace element solution containing 45.0 g/L of calcium chloride, 49.0 g/L manganese chloride tetrahydrate, 2.5 g/L cobalt chloride hexahydrate, and 2.5 g/L ammonium molybdate hydrate was sterilized and brought to a temperature of 37° C. 120.0 mL of a feed solution containing 500 g/L glucose monohydrate, 12 g/L ammonium chloride and 5.0 g/L magnesium sulfate anhydrous was added. 0.083 g/L ferric ammonium citrate and 10 μg/mL tetracycline were added. The fermentor was inoculated with a late exponential culture of B. megaterium, containing a vector coding for CelA, grown in a shake flask as described in Example 3 to a starting OD600 of 3.0 to 5.0. The vector that was utilized was the same as that described in Example 1 except that the promoter from Bacillus megaterium InhA was used. This promoter is described in U.S. Ser. No. 61/169,848, filed Apr. 16, 2009 and U.S. Ser. No. 12/760,827, filed Apr. 16, 2010, both of, which are incorporated herein by reference. The fermentor was agitated at 500-1200 rpm and air was supplied to the fermentation vessel at 0.6-25.0 L/min to maintain dissolved oxygen level of 50% saturation. The pH of the culture was controlled at 7.0 by addition of 28% v/v ammonium hydroxide. Growth of the culture was maintained by the addition of a feed solution containing 500 g/L glucose monohydrate, 12 g/L ammonium chloride and 5.0 g/L magnesium sulfate anhydrous. After the culture reached an OD600 of 70±10, the expression of CelA was induced by the addition of xylose to obtain and maintain a concentration of 0.5%. The culture was grown for another 12 hours. The culture was then chilled to 4° C. and maintained at 4° C. until harvested. Media supernatant was harvested by centrifugation at 5000G for 30 minutes in a Sorval RC12BP centrifuge at 4° C.
The clear supernatant was decanted and concentrated ten-fold using a polyethersulfone polymer ultrafiltration membrane with a molecular weight cut off of 10 kDa. The concentrate was diafiltered using at least 3 volumes of 100 mM sodium phosphate buffer pH 6.5. The final concentrate was stored at 4° C.
Example 5
High Throughput Assays to Determine β-Glucosidase and Cellobiase Activity
Beta-glucosidase activity may be determined either by a para-nitrophenyl-β-D-glucopyranoside (pNPG) assay, or a cellobiose assay.
A. Para-nitrophenyl glucopyranoside (pNPG) Assay
A colorimetric pNPG (p-nitrophenyl-β-D-glucopyranoside)-based assay was used for measuring β-glucosidase activity. In a total volume of 100 μL, 20 μL clear media supernatant containing β-glucosidase enzyme was added to 4 mM pNPG (Sigma-Aldrich, Inc. St. Louis, Mo.) solution in 50 mM sodium phosphate buffer at pH6.5. The reactions were incubated at pH 6.5, 45° C. for 1 hour. The reaction mixture was quenched with 100 μL of 1M sodium carbonate pH 11 solution. The absorbance of the solution was measured at 405 nm to determine the conversion of pNPG to p-nitrophenol. The release of p-nitrophenol (ε=17,700 M-1 cm-1) was measured at 405 nm to calculate β-glucosidase activity. Detectable β-glucosidase activity was observed under high throughput screening conditions (pH 7, 50° C.).
B. Cellobiose Assay
β-glucosidase activity was also determined using a cellobiose assay, which used cellobiose as substrate. In a total volume of 100 μL, 25 μL clear media supernatant containing CelA enzyme was added to 10 g/L cellobiose (Fluka Cat. No. 22150, Sigma-Aldrich, Inc., St. Louis, Mo.) in 100 mM sodium phosphate buffer (pH 6-7) or sodium acetate buffer (pH 5-5.5). The reaction was incubated at 45-70° C. for an appropriate time (25 minutes to overnight depending on the enzyme concentration) while shaking. Glucose production was determined using an enzymatic glucose assay (K-GLUC, Megazyme, Ireland). 10 μl of each reaction was added to 190 μl GOPOD reagent (supplied as part of the K-GLUC assay kit). The reaction was incubated at 45° C. for 20 minutes and the absorbance of the solution was measured at 510 nm. The GOPOD reagent contains 50 mM Potassium phosphate buffer pH7.4, 0.011M p-hydroxybenzoic acid, 0.008% w/v sodium azide, glucose oxidase (>12,000 U/L), peroxidase (>650 U/L) and 80 mg/L 4-aminoantipyrine. The glucose oxidase enzyme in the reagent reacts with any glucose present in the sample and produces hydrogen peroxide which then reacts with the 4-aminoantipyrine to produce a quinoneimine dye in quantities proportionate with the amount of glucose present and can be measured spectrophotometrically at 510 nm. Detectable β-glucosidase activity was observed under high throughput screening conditions (i.e., pH 7, 50° C.).
Example 6
Evaluation of Optimal CelA Activity
The native CelA activity profile was investigated at different temperatures (40-55° C.) and pH (5.0-8.0) using cellobiose (10 g/L) as a substrate. The experimental and analytical procedures are described in Example 5. CelA exhibited optimum activity at pH 6.0 and 47° C., and detectable CelA activity was observed at pH 4 and 70° C. as shown in FIG. 6.
Example 7
High Throughput Assays to Identify Improved CelA Variants
Plasmid libraries containing variant CelA genes were transformed into B. megaterium and plated on Luria-Bertani (LB) agar plates containing 3 μg/mL tetracycline with a DM3 regeneration media overlay (400 mM sodium succinate dibasic, pH 7.3, 0.5% casamino acids, 0.5% yeast extract, 0.4% K2HPO4, 0.2% KH2PO4, 20 mM MgCl2, 0.5% glucose and 0.2% BSA). After incubation for at least 18 hours at 30° C., colonies were picked using a Q-bot® robotic colony picker (Genetix USA, Inc., Beaverton, Oreg.) into shallow, 96-well well microtiter plates containing 180 μL LB and 10 μg/mL tetracycline. Cells were grown overnight at 37° C. with shaking at 200 rpm and 85% humidity. 10 μL of this culture was then transferred into 96-well microtiter plates (deep well) containing 390 μL A5-glucose-xylose medium and 10 μg/mL tetracycline as described in example 2. The plates were then incubated at 37° C. with shaking at 250 rpm and 85% humidity overnight ( ̃18-24 hours). The deep plates were centrifuged at 4000 rpm for 15 minutes and the clear media supernatant containing the secreted CelA enzyme was used for the high throughput pNPG or cellobiose assay of Example 5.
The CelA libraries were screened in high throughput using the cellobiose assay of Example 5 (Substrate: cellobiose; pH: 5-7; temperature: 45-72° C.; time: 2-24 hrs) for the identification of improved variants.
In shallow, 96-well microtiter plates 25 μL of media supernatant was added to 75 μL of 10 g/l cellobiose in 150 mM sodium acetate buffer pH 5-5.5 or 150 mM sodium phosphate buffer pH6-7. After sealing with aluminum/polypropylene laminate heat seal tape (Velocity 11 (Menlo Park, Calif.), Cat#06643-001), the plates were shaken at 45-65° C. for up to 24 hrs. The plates were centrifuged for 5 minutes at 4000 rpm. In shallow well clear microtiter plates, 10 μL of the reaction mixture was added to 190 μL of GOPOD reagent (as in example 5B) per well. The solutions were incubated at 45° C. for 1 hour and absorbance was measured at 510 nm for the identification of active CelA variants.
Example 8
Improved β-Glucosidase Activities of Engineered CelA Variants
Improved CelA variants were identified from the high throughput screening of various CelA variant libraries as described in Example 7, using the cellobiose assay of Example 5 using 3.3 g/l cellobiose at temperatures from 50°-55° C. at a pH in the range of 5.3-6.5. Tables 2A, 2B, 2C, and 2D and Table 3 depict the improvement in activities of CelA variants encompassed by the invention. The assay conditions are indicated in each table. Each subsequent table provides variants that are improved over variants in a previous table (and therefore the wildtype CelA) with respect to thermoactivity. Assay conditions were selected outside of the temperature and pH optimum of the reference enzyme (i.e., wildtype CelA in Table 2A; Variant No. 5 in Table 2B; Variant No. 94 in Table 2C; and Variant No. 264 in Tables 2D and Table 3). Table 2A provides the fold improvement in cellobiase activity for illustrative variants relative to wildtype CelA. The wildtype CelA and variants were prepared in accordance with the method of Example 1. Tables 2B, 2C, and 2D report fold improvement in cellobiase activity for further improved variants relative to Variant 5 ([H145R]CelA, reported in Table 2A), Variant 94 ([N79D+A143M+H145R+V159E+A198S+F211Y]CelA, reported in Table 2B), and Variant 264 ([T2A+I14M+N79D+A143M+H145R+V159E+A198S+F211Y+I223A+S225C+Q508R+A525T]CelA-des[A647-L663], reported in Table 2C), respectively. The native B. megaterium penicillin G acylase signal sequence, amino acid residues −1 to −24 of SEQ ID NO: 2, was used in connection with the expression of wildtype CelA and the variants listed in the Tables. The mutations listed in the tables are indicated relative to SEQ ID NO: 4, the wildtype CelA. All of the sequences described in the tables below, including the wildtype CelA, included residues GTS prior to the N-terminus of SEQ ID NO: 4 (prepared as described in Example 1, and shown as amino acid residues 1-3 in SEQ ID NO: 2), unless otherwise noted.







TABLE 2A







Improved CelA variants derived from the native CelA (SEQ ID NO: 4).


These variants were directly compared to native CelA (SEQ ID NO: 4)


(expressed from codon optimized CelA, SEQ ID NO: 1) in screening.


Assay conditions: 3.3 g/l cellobiose, 50° C. and pH 6.5.










Sample
Amino Acid
Silent
Fold improvement3


No.
Substitutions1
Mutations2
over wildtype CelA





Control





Wildtype


CelA


 1
G206S
a426g
+


 2
N128K

+


 3
Q287E

+


 4
D311G + D532G +

+



T599A


 5
*H145R
t489c
+


 6
A162S

+


 7
F211Y

++


 8
A162T

+


 9
A201G

+


10
A198M

+


11
V159L

+


12
A202N

+


13
A198C

+


14
D146S

++


15
R151P

+


16
A198S

+


17
P219M

+


18
V159A

+


19
A198L

+


20
A198N

+


21
V159Q

+


22
V159I

+


23
A202P

+


24
P219V

+


25
R151P

+


26
A202F

+


27
A202Y

+


28
F211W

++


29
A201P

++


30
E155D

+


31
A198Q

+


32
I222G

+


33
V159R

+


34
I222S

+


35
A202K

+


36
A143M

+


37
I195L

+


38
A173S

+


39
A143Q

+


40
A173C

++


41
S225C

++


42
I222A

+


43
G467Q

+


44
F482Y

+


45
Q487N

+


46
L387R

+


47
D475K

+


48
N79D

++


49
Q487D

+


50
K629C

+


51
D519G

+


52
L141I
c1698a
+


53
I153T
t528c
+


54
L663Q

+


55
S225T

+


56
S225F
c747t
+


57
I222C

+


58
E155M

+


59
E155Q

+


60
E155K

+


61
E155A

+


62
E155W

+


63
R151W

+


64
A162V

+


65
P219Q

+


66
P219E

+


67
P219C

+


68
P219I

+


69
P219T

+


70
P219L

+


71
A202L

+


72
V46F + I222A

+


73
I222V

+


73a
T60Y

+


73b
A202S

+





1Amino acid position determined by optimal alignment with SEQ ID NO: 4.


2Nucleotide position determined by optimal alignment with SEQ ID NO: 3.


3Fold improvement over wildtype CelA (SEQ ID NO: 4) is represented as follows: + = 1.1 to 2.0 fold improvement over native CelA (SEQ ID NO: 4) and ++ = 2.1 to 2.6 fold improvement over native CelA (SEQ ID NO: 4).


*Variant used for comparison of the further improved variants described in Table 2B.













TABLE 2B







Improved CelA variants and comparison to Control #2, Variant No. 5


([H145R]CelA) (SEQ ID NO: 5, FIG. 3). Assay conditions: 3.3 g/l cellobiose, 55° C. and pH 6.0.













Fold improvement3





over Control




Silent
Variant No. 5 from


Variant No.
Amino Acid Substitutions1
Mutations2
Table 2A





 5
H145R




Control #2


 74
N128K + H145R + A201P + S225C
t489c
++


 75
H145R + I222V
t489c
++


 76
N128K + H145R
t489c
++


 77
H145R + A201P + S225C
t489c
++


 78
H145R + S225C
t489c
++


 79
N128K + H145R + S225C
t489c
++


 80
N128K + H145R + A162T + S225C
t489c
+++


 81
E18R + P23L + E34K + E47K + P70S +

++



H145R + S225C


 82
N128K + H145R + D146S + I222A + S225C
a450t + t489c;
+++


 83
H145R + I222A + S225C + A525T
t489c
+++


 84
N128K + H145R + I222A
t489c
+++


 85
N79D + D85N + H145R + F211Y
t489c
++


 86
A143M + H145R + A198S + P219M
t489c
++


 87
N79D + A143M + H145R + A198S + F211Y
t489c
+++


 88
N79D + H145R
t489c
++


 89
N79D + H145R + A198S + P219V
t489c + t666a
++


 90
N79D + H145R + F211Y
t489c
++


 91
N79D + A143M + H145R
t489c
+++


 92
A143M + H145R
t489c
++


 93
H145R + F211Y
t489c
++


 94
**N79D + A143M + H145R + V159E +
t489c
+++



A198S + F211Y


 95
H145R + V159Q + A198S + F211Y
t489c
++


 96
A143M + H145R + V159E + F211Y
t489c + g492t
++


 97
A143M + H145R + F211Y + E642A +
t489c + t1923g +
+++



A643P
a1932c


 98
T2A + H145R + A162T + A201P + I222A
t111a + t489c;
+++




a675t; a1352g


 99
N128K + H145R + I222S + S225C
t489c; c1437t +
+++




a1539g


100
N128K + H145R + A201 + I222S + S225C
t489c + t771a +
+++




t1065c


101
H145R + A162T + I222A + S225C
t489c
+++


102
H145R + A162T + S225C + A573S
t489c
++


103
H145R-des[A647-L663]
t489c
++


104
H145R-des[F651-L663]
t489c
++


105
H145R-des[E635-L663]
t489c
++


106
H145R-des[S637-L663]
t489c
++


107
H145R-des[Q645-L663]
t489c
++


108
H145R-des[A641-L663]
t489c
++


109
H145R-des[L663]
t489c
++


110
H145R-des[A643-L663]
t489c
++


111
H145R-des[A653-L663]
t489c
++


112
N79D + N128K + A143M + H145R +
t489c
+++



V159E + A173C


113
N128K + A143M + H145R + V159Q +
t489c
+++



A201P + F211Y + S225C


114
N79D + A97T + N128K + A143M + H145R +
t249c + t489c
+++



V159E + A173C + F211Y + Q508R


115
N79D + N128K + H145R + A163T + A173C +
t489c +
++



A201P + F211Y + S225C + Q487R +
a1050g



A562P


116
N79D + N128K + H145R + A162T + F211Y +
t489c +
+++



S225C + K625Q
a1221g


117
N79D + N128K + H145R + A201P + F213Y
t489c + a798g
+++


118
N79D + N128K + A143M + H145R +
t489c
+++



A162T + A198S + F211Y + I222S + S225C


119
H145R + V159Q + A201P + F211Y +
t489c
+++



S225C


120
N79D + H145R + A162T + A198S + L663P
t489c + a570g +
+++




t1497c; +




t1878c


121
H145R + D146S + A162T + A173C +
t489c
++



A201P


122
N79D + N128K + H145R + V159E + A201P +
t489c
+++



F211Y + P219V + S225C


123
N79D + N128K + A143M + H145R +
t489c +
++



A162T + A201P + F211Y + P219V
g1851a +




a1905g


124
N79D + H145R + D146S + V159E + A201P +
t489c
+++



F211Y + S225C + K339R


125
H145R + A201P + I222S + S225C
t489c + t1617c
+++


126
N79D + A143M + H145R + A162T +
t489c
++



A201P + S225C + Q585R


127
N79D + H145R + D146S + A201P + F211Y +
t489c
+++



I222S + S225C


128
N79D + N128K + A143M + H145R +
t489c + t756c +
+++



V159E + A201P + F211Y + S225C
t1575c


129
N79D + H145R + V159E + A162T + A201P +
t489c
+++



S225C + I535V


130
A24V + A143M + H145R + V159E +
t489c + a939t
+++



A201P + F211Y + I222S + S225C


131
N128K + H145R + V159E + A173C
t489c
++


132
N79D + K91Q + N128K + H145R + D146S +
t489c +
+++



A201P + F211Y + I222A + S225C
a1899g


133
N79D + N128K + F135L + A143M +
t489c
++



H145R + A162T + A173C + A198S +



P219V


134
N128K + A143M + H145R + V159E +
t489c
++



A173C + A201P + F211Y + P219V


1354
N82D + H148R + V162Q + F214Y + S228C
t489c + t528c +
+++




a1104g


136
N128K + A143M + H145R + V159Q +
t489c
++



A198S + F211Y + I222A + S225C +



M297I + Q487R


137
N79D + N128K + H145R + A298S + F211Y +
t489c
+++



P219V + S225C


138
N79D + N128K + H145R + D146S + V159E +
t489c
+++



F211Y + I222S + S225C


139
A143M + H145R + A162T + F211Y +
t489c
+++



S225C


140
N79D + A143M + H145R + V159Q +
t111c + t489c +
+++



A201P + F211Y + P219V + S225C
t1218c


141
A143M + H145R + F211Y + P219V +
t489c
+++



S225C


142
N79D + N128K + H145R + A201P + P219V +
t489c + a861g
+++



K491R


143
A143M + H145R + A198S + F211Y +
t489c
+++



S225C


144
N128K + H145R + A162T + A198S +
t489c + t1650c
+++



F211Y + P219V + S225C


145
N79D + H145R + D146S + V159E + A198S +
t489c + g981a
+++



F211Y + S225C


146
N79D + A143M + H145R + V159Q +
t489c
+++



A201P + S225C + K378R


147
N79D + N128K + H145R + A173C +
t489c + c1227t +
+++



A198S + F211Y + I222S
a1770g


148
N79D + H145R + V159E + A198S + S225C
a309g + t489c +
+++




a1359g


149
N79D + N128K + H145R + D146S + V159Q +
t489c +
+++



A201P + F211Y + S225C
a1749g


150
N79D + I114V + N128K + H145R + A162T +
t489c +
+++



A198S + F211Y + S225C
a1269g


151
A143M + H145R + A162T + A173C +
t489c
+++



A198S + F211Y + L514Q


152
N79D + H145R + V159Q + A201P + F211Y +
t489c
+++



I222A + S225C


153
N79D + E92V + N128K + A143M + H145R +
g33a + t489c
++



A201P + F211Y + I222S + S225C


154
A143M + H145R + V159Q + A201P +
t489c
++



F211Y + I222A + S225C + F229I


155
N79D + H145R + A198S + S225C
a270g + t498c
+++


156
N79D + N128K + A143M + H145R +
t489c
+++



A162T + A198S + I222S + S225C + Q237R


157
N79D + G84A + N128K + H145R + A162T +
t111c + t489c +
+++



A198S + F211Y + I223A + S225C
a1230g


158
N79D + H145R + I166T + A198S + F211Y +
t489c + t1878c
++



S225C


159
N79D + H145R + A162T + A198S + S225C
t489c
+++


160
H145R + D146S + V159Q + A201P +
t489c
+++



F211Y + I222A + S225C


161
A143M + H145R + A198S + F213Y +
t489c
+++



I222A + S225C + Q287R + D609E


162
H145R + A173C + A201P + F211Y +
a151g + t489c +
+++



P219V
a543t


163
N79D + N128K + H145R + D146S + A162T +
t489c
+++



A173C + A198S + F211Y


164
N79D + N128K + H145R + D146S + S225C
t489c
+++


165
A143M + H145R + A201P + S225C
t489c
+++


166
N79D + H145R + G154V + F211Y + S225C
t489c
+++


167
N79D + N128K + H145R
t489c + t1356c
++


168
N128K + H145R + V159Q + A201P +
t489c + a714t
+++



F211Y + I222S + S225C


169
N79D + H145R + V159Q + A162T + A201P +
t489c
+++



F211Y + S225C


170
N128K + H145R + D146S + V159E +
t462a + t489c +
++



A201P + F211Y + S225C + A573V
a600t


171
N79D + H145R + V159E + A198S + F211Y +
t291c + t489c +
+++



P219V
a1077g


172
H145R + V159Q + F211Y + I222S + S225C +
t489c
+++



G534E


173
I14M + N79D + N128K + H145R + A162T +
t489c
+++



A201P + F211Y + S225C + Q487R


174
E34D + N79D + N128K + A143M + H145R +
t489c + t1114c
++



V159E + A201P + F211Y


175
N79D + H145R + D146S + A198S + F211Y +
t489c + t756a
+++



S225C


176
E7G + N79D + H145R + D146S + A198S +
t489c
+++



F211Y + I223S + S225C


177
N128K + A143T + H145R + V159Q +
t489c + a702g
++



A201P + F211Y + I223S + S225C + E502G


178
H145R + A198S + F211Y + I222A + S225C +
c36t + t489c +
+++



A373T
a744g +




a1071g +




a1179g +




a1602c


179
N79D + N128K + H145R + A173C +
t489c + a615g +
+++



A198S + F211Y
a1071g +




t1188c


180
N79D + N128K + H145R + D146S + S225C +
t489c + t1036c +
+++



A655T
t1857c


181
N128K + A143M + H145R + V159E +
t171c + t489c +
+++



F211Y + S225C
a1767g


182
N79D + I114T + N128K + H145R + V159Q +
t489c
++



A173C + A198S + F211Y + P219V


183
N79D + H145R + D146S + A162T + A198S +
t489c
++



F211Y + I222A + S225C + D311G +



N320K + R358H + F662L


184
N79D + N128K + H145R + D146S + V159Q +
t489c
+++



A201P + F211Y + P219V + I280V


185
H145R + V159Q + A198S + F211Y + I222A +
t489c + a885g +
++



S225C + P381S
t1293c


186
N79D + N128K + I134N + H145R + V159Q +
t489c
+++



S225C


187
N79D + N128K + H145R + V159Q +
t489c
+++



A198S + I223A + S225C + K627R


188
N79D + N128K + H145R + D146S + A162T +
t489c
++



A198S + F211Y


189
H145R + F274K

++


190
H145R + F274Q

++


191
H145R + F274A + D436N

++


192
H145R + F274A

++


193
H145R + I326S

++


194
N79D + A143M + H145R + V159E +
t489c + a882g
++



A198S + A257P + A485P


195
N79D + A143M + H145R + V159E +
t489c
++



A198S + I223S + A257P + T604P


196
N79D + A143M + H145R + V159E +
t489c +
++



A198S + G570P + T604P
a1914g


197
N79D + A143M + H145R + V159E +
t489c
++



A198S + A485P + T604P


198
N79D + A143M + H145R + V159E +
t489c + t1254c
++



A198S + S213P + A485P


199
N79D + A143M + H145R + V159E +
t489c
++



A198S + S213P + G220V


200
N79D + A143M + H145R + V159E +
t498c
+++



A198S


201
N79D + A143M + H145R + V159E +
t489c
++



A198S + A539V


202
N79D + A143M + H145R + V159E +
t489c
++



A198S + G570P


203
N79D + A143M + H145R + V159E +
t489c
++



A198S + A485P


204
Q26R + N79D + A143M + H145R + V159E +
t489c
++



A198S + T604P


205
N79D + A143M + H145R + V159E +
t489c + a732g
++



A198S + S479A


206
N79D + A143M + H145R + V159E +
g93a + t489c
++



A198S + S213P


207
N79D + A143M + H145R + V159E +
t489c
++



A198S + T604P


208
G59R + N79D + A143M + H145R + V159E +
t489c
++



A198S


209
N79D + A143M + H145R + V159E +
t489c
++



A198S + M331L + A485P


210
N79D + I120V + A143M + H145R + V159E +
t489c
++



A198S + Q517L + T604P





1Amino acid position determined by optimal alignment with SEQ ID NO: 4.


2Nucleotide position determined by optimal alignment with SEQ ID NO: 3.


3Fold improvement over Control #2, i.e., Variant No. 2, H145R, (SEQ ID NO: 5, FIG. 3) is represented as follows: ++ = 1.1 to 6 fold improvement over control Variant No. 2 and +++ = 6 to 13 fold improvement over control Variant No. 2.


4This sequence had the sequence GAS preceding the N-terminus (instead of GTS) relative to SEQ ID NO: 4.


**Variant used for comparison of the further improved variants described in Table 2C.













TABLE 2C







Improved CelA variants and comparison to Control #3, Variant 94


([N79D + A143M + H145R + V159E + A198S + F211Y]) (SEQ ID NO: 6, FIG. 4).


Assay conditions: 3.3 g/l cellobiose, 55° C. and pH 5.3 (incubation overnight).













Fold





improvement3





over Control


Variant

Silent
Variant No. 94


No.
Amino Acid Mutations1
Mutations2
from Table 2B





 94
N79D + A143M + H145R + V159E +
t489c



Control #3
A198S + F211Y


211
A12R + N79D + A143M + H145R +
t489c
+++



V159E + A198S + F211Y


212
A13P + N79D + A143M + H145R +
t489c
++++



V159E + A198S + F213Y-



des[P641-L663]


213
A10G + N79D + A143M + H145R +
t489c
++++



V159E + A198S + F211Y-



des[P641-L663]


214
A3L + N79D + A143M + H145R + V159E +
t489c
++++



A198S + F211Y-des[P641-L663]


215
A10P + N79D + A143M + H145R +
t489c
++++



V159E + A198S + F211Y-



des[P641-L663]


216
I14T + N79D + A143M + H145R +
t489c
++++



V159E + A198S + F211Y-



des[P641-L663]


217
A10S + N79D + A143M + H145R +
t489c
++++



V159E + A198S + F211Y-



des[P641-L663]


218
A3N + N79D + A143M + H145R +
t489c
+++



V159E + A198S + F211Y


219
G8P + N79D + A143M + H145R + V159E +
t489c
+++



A198S + F211Y


220
A10N + N79D + A143M + H145R +
t489c
+++



V159E + A198S + F211Y


221
A9T + N79D + A143M + H145R + V159E +
t489c
+++



A198S + F211Y


222
A5T + N79D + A143M + H145R + V159E +
t489c
++++



A198S + F211Y-des[P641-L663]


223
Q6D + N79D + A143M + H145R +
t489c
++++



V159E + A198S + F211Y-



des[P641-L663]


224
I4P + N79D + A143M + H145R + V159E +
t489c
++++



A198S + F211Y


225
G8Y + N79D + A143M + H145R +
t489c
++++



V159E + A198S + F211Y-



des[P641-L663]


226
E7H + N79D + A143M + H145R + V159E +
t489c
+++



A198S + F211Y


227
G8S + N79D + A143M + H145R + V159E +
t489c
++++



A198S + F211Y-des[P641-L663]


228
G8R + N79D + A143M + H145R + V159E +
t489c
++++



A198S + F211Y-des[P641-L663]


229
Q6T + N79D + A143M + H145R + V159E +
t489c
++++



A198S + F211Y-des[P641-L663]


230
A12N + N79D + A143M + H145R +
t489c
++++



V159E + A198S + F211Y-



des[P641-L663]


231
P13E + N79D + A143M + H145R +
t489c
++++



V159E + A198S + F211Y-



des[P641-L663]


232
A3P + N79D + A143M + H145R + V159E +
t489c
++++



A198S + F213Y des[P641-L663]


233
A5N + N79D + A143M + H145R +
t489c
++++



V159E + A198S + F211Y



des[P641-L663]


234
Q6P + N79D + A143M + H145R + V159E +
t489c
++++



A198S + F211Y des[P641-L663]


235
A3R + N79D + A143M + H145R + V159E +
t489c
++++



A198S + F211Y des[P641-L663]


236
Q6S + N79D + A143M + H145R +
t489c
+++



V159E + A198S + F211Y


237
A12Y + N79D + A143M + H145R +
t489c
++++



V159E + A198S + F211Y



des[P641-L663]


238
Q6G + N79D + A143M + H145R +
t489c
+++



V159E + A198S + F211Y


239
A9I + N79D + A143M + H145R +
t489c
++++



V159E + A198S + F213Y



des[P641-L663]


240
G8A + N79D + A143M + H145R +
t489c
++++



V159E + A198S + F211Y



des[P641-L663]


241
Q6A + N79D + A143M + H145R +
t489c
++++



V159E + A198S + F211Y



des[P641-L663]


242
E7P + N79D + A143M + H145R +
t489c
+++



V159E + A198S + F213Y


243
A5L + N79D + A143M + H145R +
t489c
+++



V159E + A198S + F211Y


244
P11A + N79D + A143M + H145R +
t489c
++++



V159E + A198S + F211Y



des[P641-L663]


245
I14R + N79D + A143M + H145R +
t489c
+++



V159E + A198S + F211Y


246
I4R + N79D + A143M + H145R +
t489c
+++



V159E + A198S + F211Y


247
A9G + N79D + A143M + H145R +
t489c
++++



V159E + A198S + F211Y


248
Q6N + N79D + A143M + H145R +
t489c
++++



V159E + A198S + F211Y



des[P641-L663]


249
A9K + N79D + A143M + H145R +
t489c
++++



V159E + A198S + F211Y



des[P641-L663]


250
A5Y + N79D + A143M + H145R +
t489c
++++



V159E + A198S + F211Y



des[P641-L663]


251
I14M + N79D + K91Q + H145R +
a429g + t489c +
+++



G154V + V159E + A198S + A201P +
a555g



F211Y + S225C + A525T + K627R


252
I14M + N79D + K91Q + A143M +
t489c + a555g
+++



H145R + V159E + A198S + F211Y +



I222A + Q508R + A525T + K627R-



des[S637-L663]


253
I14M + N79D + K91Q + A143M +
t489c + 528c +
++++



H145R + V159E + A198S + F211Y +
a555g



Q508R-des[A647-L663]


254
N79D + K91Q + H145R + V159E +
a429g + t489c +
+++



A198S + A201P + F211Y + I222S +
a555g



Q508R + A525T-des[S637-L663]


255
I14M + N79D + H145R + V159E +
a429g + t489c +
++++



A198S + A201P + F211Y + S225C +
a555g



A525T + K583R + K627R + L663P


256
T2A + I14M + N79D + K91Q + A143M +
t489c + a555g +
+++



H145R + V159E + A198S + A201P +
a1473g



F211Y + S225C + A525T-



des[S637-L663]


257
I14M + N79D + K91Q + A143M + H145R +
a96g + t489c +
+++



G154V + V159E + A198S +
a555g



A201P + F211Y + S225C + Q508R +



A525T-des[S637-L663]


258
T2A + I14M + N79D + K91Q + A143M +
t489c + a555g +
+++



H145R + V159E + A198S + A201P +
a1914g



F211Y + I222A + S225C + A525T +



Y594H + K627R-



des[A647-L663]


259
I14M + N79D + K91Q + H145R + V159E +
a429g + t489c +
+++



A198S + F211Y + I222A + S225C +
a555g



Q508R + K627R


260
T2A + I14M + N79D + K91Q + A143M +
t489c + a555g
+++



H145R + V159E + A198S + A201P +



F211Y + S225C + L663P


261
I14M + N79D + A143M + H145R +
t498c + a555g
+++



V159E + A198S + F211Y + K627R +



L663P


262
I14M + N79D + H145R + G154V +
a429g + t489c; +a555g +
+++



V159E + A198S + A201P + F211Y +
g654c



S225C + Q508R + A525T + K627R-



des[S637-L663]


263
T2A + I14M + N79D + K91Q + H145R +
a429g + t489c +
+++



V159E + A162S + A198S + A201P +
a555g



F211Y + I222S + S225C + Q508R +



A525T + K627R + L663P


264
***T2A + I14M + N79D + A143M +
t489c + a555g
+++



H145R + V159E + A198S + F211Y +



I222A + S225C + Q508R + A525T-



des[A647-L663]


265
T2A + N79D + K91Q + H145R + V159E +
a429g + t49c +
++++



A198S + F211Y + S225C + Q508R +
a555g



L663P


266
T2A + I14M + N79D + K91Q + H145R +
a429g + t489c +
+++



V159E + A198S + A201P + F211Y +
a565g



I222A + S225C + L514Q + K627R-



des[S637-L663]


267
T2A + I14M + N79D + A143M + H145R +
t489c + a555g
+++



V159E + A198S + A201P + F211Y +



I222A + S225C-des[A647-L663]


268
I14M + N79D + K91Q + H145R + V159E +
a429g + 489c +
++++



A198S + F211Y + S225C +
a555g



Q508R-des[S637-L663]


269
N79D + A143M + H145R + V159E +
t489c
+++



A198S + V207Y + F211Y


270
N79D + G127N + A143M + H145R +
t489c
+++



V159E + A198S + F211Y


271
N79D + A143M + H145R + V159E +
t4898c
+++



A198S + V207F + F211Y


272
N79D + A143M + H145R + V159E +
t489c
+++



A198S + V207L + F211Y


273
N79D + P109D + A143M + H145R +
t489c
+++



V159E + A198S + F211Y


274
N79D + A143M + H145R + V159E +
t489c
+++



M161T + A198S + F211Y


275
I14M + N79D + K91Q + H145R + V159E +
a419g + t489c +
++++



A198S + F211Y + I223S + S225C +
a555g



Q509R + A525T + L663P


276
T2A + I14M + N79D + H145R + V159E +
a419g + t489c +
++++



A198S + A201P + V207I + F213Y +
a564g



S225C + Q508R + K627R + L663P


277
I14M + N79D + K91Q + N128K + H145R +
a429g + t489c +
++++



V159E + A198S + F211Y + I222A +
a55g



Q508R


278
I14M + N79D + A143M + H145R +
t489c + a555g +
++++



V159E + A198S + A201P + F211Y +
a1812g



Q508R + A525T + K627R-



des[A647-L663]


279
I14M + N79D + K91Q + A143M + H145R +
t489c + a555g
+++



V159E + A198S + F211Y +



A525T + K627R-des[S637-L663]


280
T2A + N79D + K91Q + H145R + V159E +
a429g + t489c +
++++



A198S + A201P + F211Y + S225C +
a555g



D236G + K627R-des[A647-L663]


281
N79D + K91Q + H145R + V159E +
a429g + t489c +
++++



A198S + F211Y + S225C + Q508R +
a555g



A525T


282
N79D + A143M + H145R + V159E +
t489c
++++



A198S + F211Y + I246C


283
N79D + A143M + H145R + V159E +
t489c
++++



A198S + F211Y + V371M-



des[P641-L663]


284
N79D + A143M + H145R + V159E +
t489c
++++



A198S + F211Y + G298R + D311E


285
N79D + A143M + H145R + V159E +
t489c
++++



A198S + F211Y + G386E-



des[P641-L663]


286
N79D + A143M + H145R + V159E +
t489c
+++



A198S + F211Y + A357S


287
N79D + A143M + H145R + V159E +
t489c
++++



A198S + F211Y + D311G


288
N79D + A143M + H145R + V159E +
t489c
+++



A198S + F211Y + L372W


289
N79D + A143M + H145R + V159E +
t489c
+++



A198S + F211Y + P379G





1Amino acid position determined by optimal alignment with SEQ ID NO: 4.


2Nucleotide position determined by optimal alignment with SEQ ID NO: 3.


3Fold improvement over Control #3, Variant No. 94, N79D + A143M + H145R + V159E + A198S + F211Y (SEQ ID NO: 6), is depicted as follows: ++++ = 1.1 to 1.5 fold improvement over Control No. 3, Variant No. 94 and +++++ = 1.6 to 3.0 fold improvement over Control No. 3, Variant No. 94.


***Variant used for comparison of the further improved variants described in Table 2D.













TABLE 2D







Improved CelA truncated variants and comparison to Variant 264


(T2A + I14M + N79D + A143M + H145R + V159E + A198S + F211Y +


I222A + S225C + Q508R + A525T]CelA-des[A647-L663]) (SEQ ID NO: 7, FIG. 5).


All variants in this table were truncated after N646. Assay conditions:


3.3 g/l cellobiose, 55° C. and pH 5.2.













Fold improvement3





over Control


Variant

Silent
Variant No. 264


No.
Amino Acid Mutations1
Mutations2
from Table 1C





264
T2A + I14M + N79D + A143M +
t489c + a555g



Control #4
H145R + V159E + A198S +



F211Y + I222A + S225C +



Q508R + A525T-



des[A647-L663]


290
T2A + I14M + N79D + A143M +
t489c + a555g
++++



H145R + P147K + V159E +



A198S + F211Y + I222A +



S225C + Q508R + A525T-



des-[A647-L663]


291
T2A + I14M + N79D + A143M +
t489c + a555g
++++



H145R + V159E + A198S +



F211Y + I222A + S225C +



E377D + Q508R + A525T-



des-[A647-L663]


292
T2A + I14M + N79D + A143M +
t489c + a555g
++++



H145R + P147T + V159E +



A198S + F211Y + I222A +



S225C + Q508R + A525T-



des-[A647-L663]


293
T2A + I14M + N79D + A143M +
t489c + a555g
++++



H145R + V159E + A198S +



F211Y + M217L + I222A +



S225C + Q508R + A525T-



des-[A647-L663]


294
T2A + I14M + N79D + A99K +
t489c + a555g
++++



A143M + H145R + V159E +



A198S + F211Y + I222A +



S225C + Q508R + A525T-



des-[A647-L663]


295
T2A + I14M + N79D + A99R +
t489c + a555g
++++



A143M + H145R + V159E +



A198S + F211Y + I222A +



S225C + Q508R + A525T-



des-[A647-L663] +


296
T2A + I14M + D30E + N79D +
g99a + t489c +
++++



A143M + H145R + V159E +
a5554g



A198S + F211Y + I222A +



S225C + Q508R + A525T-



des-[A647-L663]


297
T2A + I14M + N79D + A143M +
t489c + a555g
++++



H145R + P147L + V159E +



A198S + F211Y + I222A +



S225C + Q508R + A525T-



des-[A647-L663]


298
T2A + I14M + N79D + A143M +
t489c + a555g
++++



H145R + V159E + A198S +



F211Y + I222A + S225C +



A347K + Q508R + A525T-



des-[A647-L663]


299
T2A + I14M + N79D + N83H +
t489c + a555g
++++



A143M + H145R + V159E +



A198S + F211Y + I223A +



S225C + Q508R + A525T-



des-[A647-L663]


300
T2A + I14M + N79D + A143M +
t489c + a555g
++++



H145R + V159E + A198S +



F211Y + I223A + S225C +



R455T + Q508R + A525T-



des-[A647-L663]


301
T2A + I14M + N79D + A143M +
t489c + a555g
++++



H145R + V159E + A198S +



F211Y + I222A + S225C +



E502N + Q508R + A525T-



des-[A647-L663]


302
T2A + I14M + N79D + N128H +
t489c + a555g
+++++



A143M + H145R + V159E +



A198S + F213Y + I222A +



S225C + Q508R + A525T-



des-[A647-L663]


303
T2A + I14M + S73A + N79D +
t489c + a555g
++++



A143M + H145R + V159E +



A198S + F211Y + I222A +



S225C + Q508R + A525T-



des-[A647-L663]


304
T2A + I14M + N79D + A143M +
t489c + a555g +
++++



H145R + V159E + A298S +
a606g



I203Y + F211Y + I223A +



S225C + Q508R + A525T-



des-[A647-L663]


305
T2A + I14M + N79D + A143M +
t489c + a555g +
++++



H145R + V159E + A198S +
a60tg + a62t



I203H + F211Y + I222A +



S225C + Q508R + A525T-



des-[A647-L663]


306
T2A + I14M + N79D + A143M +
t498c + a564g
++++



H145R + V159E + A198S +



V204I + F211Y + I222A +



S225C + Q508R + A525T-



des-[A647-L663]


307
T2A + I14M + N79D + A143M +
t489c + a555g +
++++



H145R + V159E + A198S +
a606g +



I203F + F211Y + I222A +



S225C + Q508R + A525T-



des-[A647-L663]


308
T2A + I14M + N79D + A143M +
t489c + a555g
++++



H145R + V159E + V177P +



A198S + F211Y + I222A +



S225C + Q508R + A525T-



des-[A647-L663]


309
T2A + I14M + N79D + A143M +
t489c + a555g
++++



H145R + V159E + A198S +



F211Y + I222A + S225C +



Q508R + A525T + T603F-



des-[A647-L663]


310
T2A + I14M + N79D + A143M +
t489c + a555g
++++



H145R + V159E + A198S +



F211Y + I222A + S225C +



Q508R + A525T + T603Y-



des-[A647-L663]


311
T2A + I14M + N79D + A143M +
t489c + a555g
++++



H145R + V159E + A198S +



F211Y + I222A + S225C +



Q508R + A525T + L553M-



des-[A647-L663]


312
T2A + E7P + A9G + A10N +
t135c + t417c +
+++++



I14M + N79D + N128K + A143M +
t489c + a555g



H145R + V159E +



A198S + F211Y + I223A +



S225C + Q508R + A525T-



des-[A647-L663]


313
T2A + A9G + A10N + I14M +
t489c + a555g
+++++



N79D + N128K + A143M +



H145R + V159E + A198S +



F211Y + I222A + S225C +



Q508R + A525T-des-



[A647-L663]


314
T2A + Q6P + E7P + A9G + I14M +
t30c + t222c +
+++++



N79D + N128K + A143M +
t489c + a555g +



H145R + V159E +
t585c



A198S + F211Y + I222A +



S225C + Q508R + A525T-



des-[A647-L663]


315
T2A + Q6P + A9K + A10N +
t489c + a555g
+++++



I14M + N79D + N128K +



A143M + H145R + V159E +



A198S + F211Y + I222A +



S225C + Q508R + A525T-



des-[A647-L663]


316
T2A + Q6P + A10N + I14M +
a27t + t489c +
+++++



N79D + N128K + A143M +
a555g



H145R + V159E + A198S +



F211Y + I222A + S225C +



Q508R + A525T-des-



[A647-L663]


317
T2A + Q6P + I14M + N79D +
a27t + t30c +
++++



N128K + A143M + H145R +
t489c + a555g +



V159E + A198S + F211Y +
a1353g



I223A + S225C + Q508R +



A525T-des-[A647-L663]


318
T2A + Q6P + E7P + I14M +
a30t + t328c +
++++



N79D + N128K + A143M +
t489c + a555g +



H145R + V159E + A198S +
a1506g



F211Y + I222A + S225C +



Q508R + A525T-des-



[A647-L663]


319
T2A + E7P + A9K + A10N +
t489c + a555g
++++



I14M + N79D + N128K + A143M +



H145R + R152S +



V159E + A198S + F211Y +



I222A + S225C + Q508R +



A525T-des-[A647-L663]


320
T2A + A9G + I14M + N79D +
t30c + t489c +
++++



N128K + A143M + H145R +
a555g + a702g



V159E + A198S + F211Y +



I222A + S225C + Q508R +



A525T-des-[A647-L663]


321
T2A + I14M + N79D + N128K +
t489c + a555g
+++++



A143M + H145R + V159E +



A198S + F211Y + I222A +



S225C + Q508R + A525T-



des-[A647-L663]


322
T2A + Q6P + I14M + N79D +
a27t + t33c +
++++



A143M + H145R + V159E +
t489c + a555g +



A198S + F211Y + M217V +
t951c



I222A + S225C + D311G +



Q508R + A525T-des-



[A647-L663]


323
T2A + I14M + I61V + N79D +
t489c + a555g +
+++++



G127N + A143M + H145R +
t1371c +



V159E + A198T + F211Y +
t1575g



I222A + S225C + Q508R-



des-[A647-L663]


324
T2A + A5T + A13P + I14M +
t489c + a555g +
+++++



N79D + N128K + A143M +
t1575g +



H145R + V159E + A198S +
t1623c + t1674a



F211Y + I222A + S225C +



Q508R + A525T-des-



[A647-L663]


325
T2A + I14M + N79D + N128K +
t489c + a555g +
+++++



A143M + H145R + V159E +
t575c



M161V + A198S + V208L +



F211Y + S225C + Q508R +



A525T + I535V + N646K-



des[A647-L663]


326
T2A + A13P + I14M + N79D +
t417c + t489c +
+++++



A143M + H145R + V159E +
a555g + t1062a



A198S + V207F + F211Y +



I222A + S225C + Q508R +



A525T-des-[A647-L663]


327
T2A + I14M + N79D + G128N +
t489c + a555g
++++



A143M + H145R + V159E +



A198S + V207I + F211Y +



I222A + S225C + Y342C +



Q508R + A525T + K583N +



P634S-des-



[A647-L663]


328
T2A + A5T + I14M + N879 +
t489c + a555g +
+++++



G127N + A143M + H145R +
t1821g



V159E + M161V + A198S +



F211Y + I222A + S225C +



Q508R + A525T-



des[A647-L663]


329
T2A + I14M + N79D + A143M +
g99a + t489c +
+++++



H145R + V159E + M161V +
a555g + t1575g



A198S + V207Y +



F211Y + S225C + I354T +



Q508R-des-[A647-L663]


330
T2A + I14M + N79D + A143M +
t489c + a555g
+++++



H145R + V159E + A198S +



V207Y + F211Y + I222A +



S225C + Q508R + A525T-



des-[A647-L663]


331
T2A + A5T + A13P + I14M +
t489c + a555g
++++



N79D + G127N + N128K +



A143M + H145R + V159E +



M161V + A198S + F211Y +



S225C + Q508R + A525T +



F577L-des[A647-L663]


332
T2A + I4S + I14M + N79D +
t489c + a555g +
+++++



A143M + H145R + V159E +
a1431g



A198S + V207F + F211Y +



I222A + S225C + Q508R +



A525T-des[A647-L663]


333
T2A + I4S + I14M + N79D +
t489c + a555g +
+++++



A143M + H145R + V159E +
a1431g



A198S + V207F + F211Y +



I222A + S225C + Q508R +



A525T-des[A647-L663]


334
T2A + A9E + I14M + L66Q +
t4489c + a555g
++++



N79D + A143M + H145R +



V159E + M161V + A198S +



V207L + F211Y + S225C +



Q508R + A525T-



des[A647-L663]


335
T2A + A9G + I14M + N79D +
t489c + a555g +
+++++



P109D + G127N + A143M +
t1356c



H145R + G154V + V159E +



A198S + V207F + F211Y +



I222A + S225C + A525T-



des[A647-L663]


336
T2A + I14M + N79D + G127N +
t489c + g513a +
+++++



N128K + A143M + H145R +
a555g



V159E + M161V +



A198S + V207L + F211Y +



S225C + Q508R + A525T-



des-[A647-L663]


337
T2A + I14M + N79D + A143M +
t282c + t489c +
++++



H145R + V159E + A198S +
a555g + t1575g



F211Y + I222A + S225C +



Q508R-des[A647-L663]


338
T2A + A5T + A13P + I14M +
t489c + a555g +
+++++



N79D + P109D + A143M +
t1575g



H145R + V159E + A198S +



V207L + F211Y + I222A +



S225C + Q508R-



des[A647-L663]


339
T2A + A3R + I14M + N79D +
t489c + a555g +
+++++



P109D + G127N + A143M +
t1575g



H145R + V159E + M161V +



A198S + V207F + F211Y +



I222A + S225C + Q508R-



des[A647-L663]


340
T2A + A9G + I14M + N79D +
t489c + a555g +
+++++



G127N + N128K + A143M +
c1419



H145R + V159E + A198S +



V207F + F211Y + I222A +



S225C + Q508R + A525T +



Q533L + E636D-



des[A647-L663]


341
T2A + A5T + I14M + N79D +
t69c + g72a +
+++++



G127N + A143M + H145R +
t489c + a555g +



V159E + A198S + F211Y +
t1620c



I222A + S225C + Q508R +



A525T-des[A647-L663]


342
T2A + A3R + A9G + I14M +
t489c + a555g
+++++



N79D + A143M + H145R +



V159E + M161V + A198S +



V207Y + F211Y + S225C +



Q508R + A525T-



des[A647-L663]


343
T2A + I14M + N79D + N128K +
t417c + t489c +
+++++



A143M + H145R + V159E +
a2g



A198S + V207Y + F211Y +



I222A + S225C + Q508R +



A525T + K583N-



des[A647-L663]


344
T2A + I14M + N79D + F118S +
c303t + t489c +
++++



A143M + H145R + V159E +
a555g



A198S + F211Y + I222A +



S225C + L390P + Q508R +



A525T-des[A647-L663]


345
T2A + I14M + N79D + A143M +
t489c + a555g
+++++



H145R + V159E + M161V +



A198S + V207Y +



F211Y + I222A + S225C +



A525T + N646K-



des[A647-L663]


346
T2A + A13P + I14M + N79D +
t489c + a555g +
+++++



G127N + N128K + A143M +
a1635g



H145R + V1659 + A198S +



V207F + F211Y + I222A +



S225C + Q508R + A528T-



des[A647-L663]


347
T2A + I14M + N79D + G127N +
t4c + a555g
+++++



A143M + H145R + V159E +



A198S + F211Y + I222A +



S225C + Q508R + A525T-



des[A647-L663]


348
T2A + I14M + N79D + A143M +
t489c + a555g +
+++++



H145R + V159E + M161V +
a1398g +



A198S + V207L + F211Y +
t1575g



I222A + S225C + Q508R-



des[A647-L663]


349
T2A + A5T + A9G + I14M +
t489c + a555g
+++++



N79D + P89S + P109D + G127N +



A143M + H145R +



V159E + A198S + V207Y +



F211Y + I222A + S225C +



Q508R + A525T-



des[A647-L663]


350
T2A + A9G + A13P + I14M +
t489c + a555g
+++++



N79D + G127N + A143M +



H145R + V159E + A198S +



F211Y + I222A + S225C +



A525S-des[A647-L663]


351
T2A + A9G + I14M + N79D +
t489c + a555g
+++++



G127N + A143M + H145R +



V159E + A198S + V207Y +



F211Y + I222A + S225C +



Q508R + A525T-



des[A647-L663]


352
T2A + A5T + A9G + I14M +
t417c + t53c +
+++++



N79D + G127N + N128K +
5666g



A143M + H145R + V159E +



A198S + V207Y + F211Y +



I222A + S225C + Q508R +



A525T-des[A647-L663]


353
T2A + I14M + N79D + A143M +
t489c + a555g +
+++++



H145R + V159E + A198S +
a13433g



V207F + F211Y + I222A +



S225C + Q508R + A525T-



des[A647-L663]


354
T2A + A13P + I14M + N79D +
t489c + a555g
+++++



A143M + H145R + V159E +



A198S + V207F + F211Y +



I222A + S225C + Q508R +



A525T + N646K-



des[A647-L663]


355
T2A + I14M + N79D + A143M +
a366g + t489c +
+++++



H145R + V159E + M161V +
a555g +



A198S + F211Y + S225C +
c735t + t1575g



Q508R-



des[A647-L663]





1Amino acid position determined by optimal alignment with SEQ ID NO: 4.


2Nucleotide position determined by optimal alignment with SEQ ID NO: 3.


3Fold improvement over Control #4, Variant No. 264, T2A + I14M + N79D + A143M + H145R + V159E + A198S + F211Y + I222A + S225C + Q508R + A525T]CelA-des[A647-L663] (SEQ ID NO: 7, FIG. 5), is depicted as follows: ++++ = 1.1 to 3.0 fold improvement over Control #4, Variant 264 and +++++ = 3.1 to 12.0 fold improvement over Control #4, Variant 264.






Further variants were prepared as described using the method of Example 1, except the polynucleotide encoding the CelA variant was cloned behind a variant of the Bacillus megaterium signal peptide. Mutations in the variant signal peptide sequences and the catalytic CelA domain sequences are described below in Table 3 and are indicated relative to FIG. 1B (signal peptide sequence) and SEQ ID NO: 4 (the wildtype CelA catalytic CelA domain), respectively. As with the sequences described in the tables above, the sequences in Table 3 included residues GTS prior to the N-terminus of SEQ ID NO: 4 (prepared as described in Example 1). Fold improvement was determined as described in Example 7 (with assay conditions indicated below), and is reported relative to Variant No. 264, [T2A+I14M+N79D+A143M+H145R+V159E+A198S+F211Y+I222A+S225C+Q508R+A525T] CelA-des[A647-L663] (SEQ ID NO: 7).







TABLE 3







Signal Peptide and CelA Variants. Assay conditions: 3.3


g/l cellobiose, 55° C. and pH 5.2 (incubation overnight).















Fold






Improvement 3


CelA
Mutations


over Control


Variant
in Signal
Amino Acid Mutations in
Silent
Variant No. 264


No.
Peptide
CelA 1
Mutations 2
from Table 2C





356
F-8E 4
T2A + I14M + N79D +
t489c +
++++




A143M + H145R + V159E +
t501c +




A198S + F211Y + I223A +
a555g




S225C + Q508R + A525T +




Q585R-des[A647-L663]


357
L-19Q 4
T2A + A9K + I14M +
t30c +
+++++




N79D + K107R + N128K +
t489c +




A143M + H145R + V159E +
a555g




A198S + F211Y + I222A +




S225C + Q508R +




A525T-des[A647-L663]


358
F-8E 4
T2A + A9K + A10N +
a147g +
++++




I14M + N79D + A143M +
a471g +




H145R + V159E + A198S +
t489c +




F211Y + I222A + S225C +
a555g




Q508R + A525T-des[A647-




L663]


359
c-46g 5
T2A + E7P + A10N + I14M +
a27t +
++++



(silent mutation)
N79D + A143M + H145R +
t489c +




V159E + A198S + F211Y +
a555g




I222A + S225C + M356T +




Q508R + A525T +




H586Y-des[A647-L663]


360
F-8E 4
T2A + E7P + A9G + A10N +
t489c +
++++




I14M + N79D + A143M +
a555g +




H145R + V159E + A198S +
a1173g +




F211Y + I222A + S225C +
g1728a




Q508R + A525T-




des[A647-L663]


361
F-8E 4
T2A + I14M + N79D +
t32c +
++++




A143M + H145R + V159E +
t489c +




A198S + F211Y + I222A +
a555g




S225C + Q508R + A525T -




des[A647-L663]


362
F-8E 4
T2A + Q6P + E7P + A9G +
t489c +
++++




A10N + I14M + N79D +
a555g +




A143M + H145R + V159E +
a1350g




A198S + F211Y + I222A +




S225C + Q508R + A525T -




des[A647-L663]


363
F-10T 4
T2A + I14M + N79D +
t489c +
+++++




G127N + N128K + A143M +
a555g +




H145R + V159E + A198S +
t1575g




V207F + F211Y + I222A +




S225C + I366T + Q508R-




des[A647-L663]


364
F-10T 4
T2A + A3R + A5T + A9G +
t489c +
+++++




A13P + I14M + L44S +
a555g +




N79D + G127N + A143M +
a1899t




H145R + V159E + A198S +




V207Y + F211Y + I222A +




S225C + Q508R + P518Q +




D519N + A525T-des[A647-




L663]


365
F-10T 4
T2A + I14M + N79D +
t489c +
+++++




A143M + H145R + V159E +
a555g




A198S + V207F + F211Y +




I222A + S225C + Q508R +




A525T + A600V-




des[A647-L663]


366
F-10T 4
****T2A + A5T + I14M +
t489c +
+++++




N79D + G127N + A143M +
a555g




H145R + V159E + A198S +




V207F + F211Y + I222A +




S225C + Q508R + A525T-




des[A647-L663]


367
F-10T 4
T2A + I14M + N79D +
t489c +
+++++




G127N + A143M + H145R +
a555g +




G154V + V159E +
a114g




A198S + V207Y + F211Y +




I222A + S225C + Q508R +




A525T-des[A647-L663]


368
F-10T 4
T2A + I14M + N79D +
t489c +
++++




A143M + H145R + V159E +
t510c +




A198S + F211Y + I222A +
a555g +




S225C + F274S +
a741g +




Q508R-des[A647-L663]
t1575g


369
F-10T 4
T2A + A9G + I14M +
t489c +
+++++




N79D + G127S + A143M +
a555g +




H145R + V159E + A198S +
a879g +




F211Y + I222A + S225C +
a1566g




Q508R + A525T-des[A647-




L663]


370
F-10T 4
T2A + A9G + A13P + I14M +
t489c +
++++




N79D + P109D + N128K +
a555g +




A143M + H145R +
t1218c




V159E + A198S + V207I +




F211Y + S225C + S247P +




Q508R + A525S-des[A647-




L663]


371
F-10T 4
T2A + I14M + N79D +
t489c +
+++++




A143M + H145R + V159E +
a555g +




A198S + V207Y + F211Y +
t1653c




I222A + S225C + Q508R +




A525T + D611E-




des[A647-L663]


372
F-10T 4
T2A + I14M + N79D +
t489c +
+++++




A143M + H145R + V159E +
a555g +




M161V + A198S +
a1467g +




V207Y + F211Y + I222A +
a1767g




S225C + Q508R + A525T-




des[A647-L663]


373
F-10T 4
T2A + A5T + I14M +
t489c +
++++




N79D + P109N + A143M +
a555g +




H145R + V159E + M161V +
a675c +




A198S + V208Y + F211Y +
a1242g




I222A + Q508R +




A525T-des[A647-L663]


374 6
F-10T 4
T2A + A9G + I14M +
t489c +
+++++




N79D + A143M + H145R +
a555g +




V159E + A198S + V207F +
t1407c




F211Y + I222A + S225C +




A525T -des-A647-L663]





1 Amino acid position determined by optimal alignment with SEQ ID NO: 4.


2 Nucleotide position determined by optimal alignment with SEQ ID NO: 3.


3 Fold improvement over Control #4, Variant No. 264(SEQ ID NO: 7), is depicted as follows:


++++ = 1.1 to 3.0 fold improvement over Control #4, Variant No. 264 and


+++++ = 3.1 to 12.0 fold improvement over Control #4, Variant No. 264.


4 Amino acid position determined by optimal alignment with SEQ ID NO: 2.


5 Nucleotide position determined by optimal alignment with SEQ ID NO: 1.


6 This sequence had the sequence GAS (instead of GTS) preceding the N-terminus of SEQ ID NO: 4.






Example 9
Characterization of Enzyme Stability
Four CelA variants and wildtype CelA were characterized to determine their stabilities at high temperature (55° C.) and low pH (pH5.5) using the method of Example 5A. The samples containing various CelA variant enzymes were pre-incubated at pH 5.5, 55° C. for 0-6 hrs. The residual enzyme activity after the thermal challenge was measured using pNPG as substrate at pH 7, 30° C. for 1 hr. Table 4 illustrates the residual activity of improved CelA variants at pH 5, 65° C. after pre-incubations for different lengths of time. The mutations listed in the table are indicated relative to SEQ ID NO: 4, the wildtype CelA. These sequences included residues GTS prior to the N-terminus of SEQ ID NO: 4 (prepared as described in Example 1).







TABLE 4







Half-lives of improved CelA variants











% residual
% residual
% residual



activity after
activity after
activity after



10 mins @
1 hour @
6 hours @


Amino Acid Mutations1
pH 5.5, 55° C.
pH 5.5, 55° C.
pH 5.5, 55° C.





CelA wildtype
0
0
0


H145R
0
0
0


(Variant No. 5)


N79D + A143M + H145R +
 3%
0
0


V159E + A198S + F211Y


(Variant No. 94)


T2A + I14M + N79D + A143M +
 95%
 78%
 11%


H145R + V159E + A198S + F211Y +


I222A + S225C + Q508R + A525T -


des[A647-L663]


(Variant No. 264)





1Amino acid position is determined by optimal alignment with SEQ ID NO: 4






The results indicate that certain variants exhibit greater thermo- and pH-stability relative to wildtype Azospirillum irakense CelA.
Example 10
Further Improved β-Glucosidase Activities of Engineered CelA Variants
The CelA variants in Example 8 were further improved by introducing substitutions into CelA variant No. 366 (the variant catalytic CelA domain is provided as SEQ ID NO: 9). The polynucleotide sequence encoding the catalytic CelA domain of Variant No. 366 is provided as SEQ ID NO: 8. As with the variants described in the above examples, the new variants contained the native B. megaterium penicillin G acylase signal sequence, amino acid residues −1 to −24 of SEQ ID NO: 2 (with numbering depicted in FIG. 1B). As explained in Example 1, the signal peptide is cleaved between amino acid residues −1 and +1. The amino acid residue at position +1, glycine, was engineered into the polypeptide to signal peptide processing in the expression host, B. megaterium. The amino acid residues at positions 2 and 3, threonine and serine, respectively, (or in some cases, threonine and arginine, as indicated in the table below), are encoded by a nucleotide sequence that corresponds to a SpeI restriction site. Therefore, the residues GTS/R occur between the C-terminus of the B. megaterium signal sequence and the N-terminus of the CelA catalytic domain in the expression construct.
Improved CelA variants were identified from the high throughput screening as described in Example 7, using the cellobiose assay of Example 5 with 3.3 g/l cellobiose at a temperature of 65° C. and pH of 5, with incubation overnight. Table 5 provides the improvement in activities of the variants generated by mutating the C-terminally truncated CelA variant, Variant No. 366. All of these variants include all of the substitutions of variant No. 366 (i.e., T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525-des[A647-L663], in addition to the substitutions separately listed for each variant in Table 5, except where indicated by footnote that a reversion back to the wildtype residue occurred. The amino acid mutations in the CelA catalytic domain are indicated relative to SEQ ID NO: 4 (the wildtype CelA catalytic domain) and the silent (nucleotide) mutations are indicated relative to SEQ ID NO: 3, which corresponds to the codon optimized polynucleotide sequence that encodes the wildtype CelA catalytic domain Amino acid and silent (nucleotide) mutations in the signal sequence are indicated in Table 4 relative to SEQ ID NOs: 2 and 1, respectively. All of the variants in Table 5 contained the mutation F-10T in the signal sequence (where the numbering of the signal peptide is depicted in FIG. 1B, and the sequence corresponds to amino acid residues 1 through 24 of SEQ ID NO: 2).
Fold improvement is reported relative to Variant No. 366 (substitution in signal sequence: F-10T (with reference to the numbering of amino acid position in the signal sequence depicted in FIG. 1B (SEQ ID NO: 2)); substitutions in CelA catalytic domain: T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525T-des[A647-L663] (relative to SEQ ID NO: 4). The variant catalytic CelA domain of Variant No. 366 is provided as SEQ ID NO: 9. Variant No. 366 contains the substitution F-10T in the signal sequence (where the numbering of amino acid positions in the signal sequence is in accordance with that depicted in FIG. 1B).







TABLE 5







Improved CelA truncated variants and comparison to Variant No. 366 (substitution


in signal sequence: F-10T (refer to numbering of amino acid position in signal


sequence in FIG. 1B (SEQ ID NO: 2)); substitutions in CelA catalytic domain: T2A +


A5T + I14M + N79D + G127N + A143M + H145R + V159E +


A198S + V207F + F211Y + I222A + S225C + Q508R +


A525T-des[A647-L663] (relative to SEQ ID NO: 4). Assay conditions: 3.3 g/l


cellobiose, 65° C. and pH 5 (incubation overnight).













Amino Acid Mutations in






CelA catalytic domain, SEQ



Mutations
ID NO: 4, in addition to: T2A +



in Signal
A5T + I14M + N79D + G127N +
Silent Mutations
Fold


CelA
Peptide in
A143M + H145R + V159E + A198S +
(with reference to
Improvement3


Variant
addition to
V207F + F211Y + I222A + S225C +
SEQ ID NO: 3,
over Control


No.
F-10T:
Q508R + A525T-des[A647-L663]:
FIG. 2A)
Variant





375

P109D + V177P + Q287R +

++




A600V


376

I4S + I61V + V177P + I535V
t516c
++


3771

A3R + I4S + I61V + V177P +

++




I203Y + I535V


3781

I4S + V177P + N320S +
g1791a
++




K350E


379

A3R + V177P + I535V
c12t + a1734g
++


380

P89S + V177P + D236Y
a1197g
++


381

I4S + V177P + A600V
a600g + a1044g +
++





a1077g + t1902c


382

I4S + P89S + V177P + I535V +

++




Q585R


3831

I4S + I61V + S73A + V177P +

++




N613D + A617V


384

V177P + I535V
t351c
++


385

A3R + S73A + V177P + I203Y +
c12t + a732t
++




A600V


3861

I4S + P89S + V177P + A600V
a1656g
++


387

P89S + V177P

++


388

K35Q + S73A + I203Y
t693c + a1554g
++


389

I203Y + I535V
c12t + t693c + t873c
++


390

P89S + V177P + I203Y +
c12t + a1173g
++




Y594F


3911

A3R + S73A + V177P + I203Y
c12t
+++


392

S73A + I203Y
t1008c + t1233c
++


393

A3R + I4S + V177P

++


3941

A3R + I4S + S73A + V177P +
t43c + a450g
++




I535M


395

A3R + I61V + V177P
c12t + t585c + t1260c +
++





a1293g


396

I4S + V177P + A601V
t1290c
++


397

A3R + M161V + I203Y +

++




A222I + D383G


398

A3R + V177P + S213T

++


399

A3R + P109D + V177P +
a27g + a1092g
++




M356T


400

I203Y + M356T + N646K

++


401

T25A + N128H + V177P +
g612t + t1782c +
++




I203Y + T525S
t1794c


402
K-21R2
A3R + A13P + P147T + V177P +
g612t + a726g
++




I203Y + T525S + H586Y


403

A13P + P109D + V177P

++


404

A13P + P109D + N128K +

++




V177P + P597A


405

A13P + V177P + A400T +
t1896c
++




H586Y


406

A3R + A13P + P147T + E159G5 +

++




V177P


407

P109D + P147T + V177P +

+++




E502N + H586Y


408

A13P + P147T + V177P +
a741g
+++




A226G + H586Y


409

P109D + P147T + V177P +

+++




T525S


410

V177P + E502N + T525S5
a1305g
+++


411

V177P + L372S

++


412

V177P + E502N + R508Q5

+++


413

A3R + A13P + P147T + V177P +

+++




E502N + H586Y


414

A3R + I203Y + A400T +
g612t
++




E502N + T525S


415

A13P + N128H + V177P +

++




E502N + R508Q5


416

P109D + V177P + D351E +
a252g + t1494c
++




H586Y


417

V177P + R508Q4

++


418
N-5H2
A3R + A13P + I203Y + A400T
a441t + t516c +
++





g612t


419

A3R + P109D + P147T +
t609a + g612t
++




V177P + H586Y


4203

A13P + N128H + V177P
t849c + t1707c
++


421

P109D + V177P + A400T +
t762a
++




E502N + H586Y


422

A13P + P147T + V177P +
t3c + g324a + c330t +
+++




A400T + E502N + R508Q
a468g


423

P109D + V177P + A400T
t849c
++


424

A13P + V177P + V204I +
t609a
++




D291G


425

A3R + P147T + V177P +

+++




A400T + E502N


426

V177P + A400T

++


427

N128H + V177P + E502N +

++




H586Y


428

V177P + A400T + R508Q5
a1176g
++


429

A13P + P147T + I203Y +
g612t + t1377c
++




H586Y


430

P109D + V177P
t849c
++


431

A13P + P109D + V177P +

++




T525S


432
I-14V2
A3R + P147T + V177P +
g612t
++




I203Y + E502N + H586Y


433

A3R + V177P

++


434

A13P + I203Y + A400T +
g612t + t1008c +
++




E502N
t1446c


435

A3R + A13P + V177P +
c195t + t1053c
++




A400T + H586Y


436

A3R + V177P + E502N

++


437
N-5S2
A3R + P147T + I203Y +
g612t
++




E502N


438

A3R + T25A + V177P +

++




A400T + R508Q5


439

A3R + V177P + A400T

++


440

A3R + M14K + N128H +

++




V177P + R508Q5


441

A13P + P147T + E502N

++


442

V177P
t99c
++


443

A3R + N128H + V177P
t819a
++


444

N128H + P147T + V177P +
t1617c
++




T603A


445

P147T + I203Y + H586Y
g612t
++


446

T169N

++


447

R508E

++


448

F274K

++


449

F274N

+++


450

F274S

++


451

L41F + A335P

++


452

A272L + N300D

++


453

A309G
g912t + g915a
++


454

V304L
g915a + a1683g
++





1This sequence had the sequence GTR (instead of GTS) preceding the N-terminus of the catalytic CelA domain (i.e., corresponding to SEQ ID NO: 4).


2Amino acid position determined by optimal alignment with SEQ ID NO: 2.


3This sequence had the sequence ATR (instead of GTS) preceding the N-terminus of the catalytic CelA domain (i.e., corresponding to SEQ ID NO: 4).


4Fold improvement over Control # 5, i.e. Variant No. 366, (SEQ ID NO: 9) is represented as follows:


++ = 1.1 to 2.0 fold improvement over control Variant No. 366 and


+++ = 2.1 to 3.0 fold improvement over control Variant No. 366.


5Represents reversion to the wildtype residue.






Example 11
Further Improved β-Glucosidase Activities of Engineered CelA Variants
Additional variants were prepared by introducing substitutions into CelA variant No. 391 (the variant catalytic domain of Variant No. 391 is provided as SEQ ID NO: 11). The polynucleotide encoding the catalytic CelA domain of Variant No. 391 is provided as SEQ NO: 10. These variants were screened using the high throughput screen of Example 7, using the cellbiose assay of Example 5 with 3.3 g/l cellbiose at a temperature of 67° C. and pH 5, with incubation overnight. These variants had the same construction as the variants described above with the native B. megaterium penicillin G acylase signal sequence, amino acid residues −1 to −24 of SEQ ID NO: 2 (with the numbering of amino acid positions in the signal sequence as depicted in FIG. 1B), glycine at position +1 to facilitate signal peptide processing in the expression host, B. megaterium, and in these variants, threonine and arginine at positions +2 and +3, respectively, followed by a variant CelA catalytic domain. Therefore, the residues GTR occur between the C-terminus of the B. megaterium signal sequence and the N-terminus of the CelA variant catalytic domain in the expression construct.
Table 6 provides the improvement in activities of the variants generated by mutating the C-terminally truncated CelA variant, Variant No. 391. All of these variants include all of the substitutions of variant No. 391 (i.e., T2A+A5T+I14M+N79D+G127N+A143M+H145R+V159E+A198S+V207F+F211Y+I222A+S225C+Q508R+A525-des[A647-L663], in addition to the substitutions separately listed for each variant in Table 6, except where indicated by footnote that a reversion back to the wildtype residue occurred. The amino acid mutations in the CelA catalytic domain are indicated relative to SEQ ID NO: 4 (the wildtype CelA catalytic domain) and the silent (nucleotide) mutations are indicated relative to SEQ ID NO: 3, which corresponds to the codon optimized polynucleotide sequence that encodes the wildtype CelA catalytic domain. Amino acid and silent (nucleotide) mutations in the signal sequence are indicated in Table 6 relative to SEQ ID NOs: 2 and 1, respectively. All of the variants in Table 6 contained the mutation F-10T in the signal sequence (where the numbering of amino acid position in the signal sequence is in accordance with that depicted in FIG. 1B).
Fold improvement is reported relative to Variant No. 391 (substitution in signal sequence: F-10T (refer to numbering of amino acid position in signal sequence in FIG. 1B (SEQ ID NO: 2)); substitutions in CelA catalytic domain: T2A+A3R+A5T+I14M+S73A+N79D+G127N+A143M+H145R+V159E+V177P+A198S+I203Y+V207F+F211Y+I222A+S225C+Q508R+A525T-des[A647-L663]), where the amino acid substitutions are indicated relative to the wildtype CelA catalytic domain (SEQ ID NO: 4)).







TABLE 6







Improved CelA truncated variants and comparison to Variant No. 391 (substitution in signal sequence:


F-10T (refer to numbering of amino acid position in signal sequence in FIG. 1B (SEQ ID NO: 2));


substitutions in CelA catalytic domain: T2A + A3R + A5T + I14M + S73A + N79D + G127N +


A143M + H145R + V159E + V177P + A198S + I203Y + V207F + F211Y + I222A + S225C +


Q508R + A525T-des[A647-L663]), where amino acid substitutions in the CelA catalytic domain


are indicated relative to SEQ ID NO: 4. Assay conditions: 3.3 g/l cellobiose, 67° C. and pH 5.













Amino Acid Mutations in CelA






catalytic domain, SEQ ID NO: 4, in



Mutations
addition to: T2A + A5T + I14M +



in Signal
S73A + N79D + G127N + A143M + H145R +
Silent Mutations
Fold


CelA
Peptide in
V159E + V177P + A198S + I203Y + V207F +
with reference to
Improvement3


Variant
addition to
F211Y + I222A + S225C + Q508R +
SEQ ID NO: 3,
over Control


No.
F-10T:
A525T- des[A647-L663]:
FIG. 2A
Variant





455

T60H + H285N
c195t + c199a +
++++





t1617c


456

S198W + T525M

++++


457

S198A + T525G

++++


458

D311G + D475E
c1413t + a1428t
+++


459

T169N + Q287R + A309G + D311G
a24t + g33a +
+++





a543g + g915a +





t1864c


460

L41F + A309G + D311G + A335P
a24t + g33a +
+++





g915a + c1002t +





g1776a + g1791t


461

D311G + K529E
g786a
+++


462

Y211Q
t1425c
+++


463

T169N + A202P + A272L + Q287R +
t576c + g804t +
+++




D311G + E512G
c828t + g915a


464

T169N + A202P + N300D A309G +
g915a
+++




D311G + Y594F
g1776a


465
g-67a + g-
R67H + T169N + A202P + A335P
c933t + a1353g
+++



61a + c-57t +



c-46t + a-43t +



c-39t1



(silent mutation)


466

V304L + A335P
g915a
+++


467

A272L + A335P + A357S
c1002t + a1077t +
++





c1086t + t1324a +





g1329a


468
g-67a + g-
A272L
a24t + c828t +
++



61a + c-57t +

g912t + g915a +



c-46t + a-43t +

t1324a + g1329a +



c-39t1

g1434t + c1437t +



(silent mutation)

t1629c + g1776a +





g1791t


469

L41F + T169N + A272L + N300D +
t729c + g894t +
++




D311G + A335P + D475E + Y594F
g915a + a1077g +





t1324a + g1329a +





g1776a + g1791t


470
g-67a + g-
A202P + N300D + A309G + D311G +
t789c + g915a +
++



61a + c-57t +
A335P + K350R + Q487L
a1029g



c-46t + a-43t +



c-4739t1



(silent mutation)


471

T169N + D311G + A335P + V349A +
t501c + a987g +
++




T452A
c1002t + c1011a +





g1776a


472

T169N + A335P + Y594F
g912t + g915a +
++





a1545g + g1776a


473

L41F + A309G + D311G + A335P +
g33a + g915a +
++




E343G
t1386c


474

D311G
g915a + g1776a
++


475

A335P
c1011a
++


476

T169N + A335P
a24t + a678g +
++





g912t + g915a


477

N300D + A309G + A335P
g915a + c933t +
++





c1002t + a1077t +





t1324a + g1329a +





t1530c + g1776a +





c1930a


478

A335P
t216c + t462c +
++





c1002t + c1011a +





g1434t + c1437t +





a1899g


479

A202P + A335P + Y594F
g912t + g915a +
++





c1011a + g1776a


480

V511L

++


481

T169N + A335P
g1776a
++


482

D264G + A272L + A309G + A335P
g915a + c1011a
++


483

A202P + N300D + D311G + A335P
g894t + g915a +
++





c1002t + g1329a


484

T169N + D311G + K531R
a24t + g915a +
++





t1324a + g1329a +





a1704g


485

L41F + Q287R + A335P
c933t + a969g +
++





a1185g + t1324a +





g1329a + g1434t +





c1437t


486

T169N + A202P + D215G + A272L +
a24t + g915a +
++




A309G + A335P + Y594F
c933t + c1002t +





t1137c + a1302t +





g1434a + c1437t +





c1737t + g1776a +





g1791t +


487

A202P + A335P
t1324a + g1329a
++


488

L41F + T169N + A202P + A272L +
g120t + a441g +
++




D311G + K339R + Y594F
c828t + g915a +





t1324a + g1329a +





g1776a + t1839c


489

H282N
t924c + t1911c
++


490

F118L + T169N + A272L + A335P
c1011a + t1254c +
++





t1324a + g1329a


491

P219Q

++


492

D519G

++


493

Q522K

++


494

G59S

++


495

P147W + D475E

++


496

Q522R
a732g
++


497

A309G
g915a + c933t +
++





t1324a + g1329a +





g1776a


498
N-5D2
A226G + E502N
c1206t + c1668t
++



g-67a + g-



61a + c-57t +



c-46t + a-43t +



c-39t1



(silent mutation)


499
g-67a + g-
E502N + R508Q4 + N646K
t688c + a1203g +
++



61a + c-57t +

t1324a + g1329a



c-46t + a-43t +



c-39t1



(silent mutation)


500

Q520T

++


501

Q520G

++


502

T5A4 + A589R
t1569c
++


503

Q522K

++


504

A202P

++


505

A485K
t963c
++


506

D519K

++


507

S198D

++


508

Q520N

++


509

S198N + T525A4
t1476c
++


510

N83H + D457E
t216c + c259a +
++





a1767g


511

E155G

++


512
I-15V2
R3G

++


513

S395K + D519G

++


514

P147R + E502N + N646K

++


515

Q520K

++


516

P219E
t1854c
++


517
g-67a + g-
E502N
a24t + g33a +
++



61a1

t1296c + t1324a +



(silent mutation)

g1329a


518

G386W

++


519

E502N + N646K
a186t + t1324a +
++





g1329a


520

D532R
a1605t
++


521
c-46t + a-43t +
A226G + T525S
a615g + t783c +
++



c-39t-1

g912t + g915a +



(silent mutation)

c1581t


522

K35R + E502N + N646K
a24t + a102g +
++





t1324a + g1329a


523

P147R + E502N + R508Q4

+


524

E92D
c259a
+


525

P109D + H282D + L372S + E458D +
a24t + g33a +
+




E502N
g324a + g912t +





g915a + t1324a +





g1329a


526

T60H
c195t + g1017a
+





1Nucleotide position determined by optimal alignment with SEQ ID NO: 1


2Amino acid position determined with reference to the numbering of amino acid position in the signal sequence as depicted in FIG. 1B.


3Fold improvement over Control # 6, i.e. Variant No. 391, (SEQ ID NO: 11) is represented as follows:


+ = 0.5-1.0 fold improvement over control Variant No. 391;


++ = 1.1 to 2.0 fold improvement over control Variant No. 391;


+++ = 2.1 to 3.0 fold improvement over control Variant No. 391; and


++++ = 3.1 to 4.1 fold improvement over control Variant No. 391.


4Represents a reversion to the wildtype residue.






Example 12
Further Improved β-Glucosidase Activities of Engineered CelA Variants
Further variants were prepared by introducing mutations into CelA Variant No. 463 (the variant catalytic domain of Variant No. 463 is provided as SEQ ID NO: 13). The polynucleotide sequence encoding the catalytic CelA domain of Variant No. 463 is provided as SEQ ID NO: 12. These variants were screened using the high throughput screen of Example 7, using the cellbiose assay of Example 5 with 3.3 g/l cellbiose at a temperature of 72° C. and pH 5, with incubation overnight These variants had the same construction as the variants described above with the native B. megaterium penicillin G acylase signal sequence, amino acid residues −1 to −24 of SEQ ID NO: 2 (with the numbering of amino acid positions in the signal sequence as depicted in FIG. 1B), glycine at position +1 to facilitate signal peptide processing in the B. megaterium expression host, and threonine and arginine at positions +2 and +3, respectively, followed by a variant CelA catalytic domain. Therefore, the residues GTR occur between the C-terminus of the B. megaterium signal sequence and the N-terminus of the CelA variant catalytic domain in the pre-protein form of each variant.
Table 7 provides the improvement in activities of the variants generated by mutating the C-terminally truncated CelA variant, Variant No. 463. All of these variants include all of the substitutions of Variant No. 463 (i.e., substitution in signal sequence: F-10T (relative to FIG. 1B, SEQ ID NO: 2); substitutions in the CelA catalytic domain: T2A+A3R+A5T+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F211Y+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T-des[A647-L663] (relative to SEQ ID NO: 4)), in addition to the substitutions separately listed for each variant in Table 7. The amino acid mutations in the CelA catalytic domain are indicated relative to SEQ ID NO: 4 (the wildtype CelA catalytic domain) and the silent (nucleotide) mutations are indicted relative to SEQ ID NO: 3, which corresponds to the codon optimized polynucleotide sequence that encodes the wildtype CelA catalytic domain. Amino acid and silent (nucleotide) mutations in the signal sequence are indicated in Table 7 relative to SEQ ID NOs: 2 and 1, respectively. All of the variants in Table 7 contained the mutation F-10T in the signal sequence (wherein the numbering of amino acid position in the signal sequence is in accordance with that depicted in FIG. 1B).
Fold improvement is reported relative to Variant No. 463 [substitution in signal sequence: F-10T (with reference to the numbering of amino acid position in the signal sequence as depicted in FIG. 1B (SEQ ID NO: 2)); substitutions in CelA catalytic domain: T2A+A3R+A5T+I14M+S73A+N79D+G127N+I143M+H145R+V159E+T169N+V177P+A198S+A202P+I203Y+V207F+F211Y+I222A+S225C+A272L+Q287R+D311G+Q508R+E512G+A525T-des[A647-L663], where the amino acid substitutions are indicated relative to the wildtype CelA catalytic domain (SEQ ID NO: 4)).







TABLE 7







Improved CelA truncated variants and comparison to Variant No. 463 (S03837072) (substitution


in signal sequence: F-10T(refer to numbering of amino acid position in signal sequence in FIG.


1B (SEQ ID NO: 2)); substitutions in CelA catalytic domain: T2A + A3R + A5T +


I14M + S73A + N79D + G127N + A143M + H145R + V159E + T169N +


V177P + A198S + A202P + I203Y + V207F + F211Y + I222A +


S225C + A272L + Q287R + D311G + Q508R + E512G + A525T- des[A647-


L663], where amino acid substitutions in the CelA catalytic domain are indicated relative


to SEQ ID NO: 4. Assay conditions: 3.3 g/l cellobiose, 72° C. and pH 5.













Amino Acid Mutations in CelA






Relative to SEQ ID NO: 4 in




addition to [T2A + A3R + A5T +




I14M + S73A + N79D + G127N + A143M +



Mutations
H145R + V159E + T169N + V177P +



in Signal
A198S + A202P + I203Y + V207F +
Silent Mutations
Fold


CelA
Peptide in
F211Y + I222A + S225C + A272L +
(with reference to
Improvement1


Variant
Addition
Q287R + D311G + Q508R + E512G +
SEQ ID NO: 3,
over Control


No.
to F-10T:
A525T- des[A647-L663]]:
FIG. 2A)
Variant





527

T60H + A335P + D475E + V511L +
a24t + t897c +
++




Y594F
c1002g + g1536t


528

T60H + A309G + D475E + Y594F
a24t + a678g +
++





a903g


529

T60H + A335P + D475E + Y594F
a24t + a678g +
++





c1002g


530

A335P + D475E
g348a + a678g +
++





c1002g


531

A309G + A335P + D475E + Y594F
a24t + g912t +
++





c1002g + a1506g


532

A309G + A335P + T567A
c1002g
++


533

A309G + A335P + D475E + Y594F
a24t + a678g +
++





g912t + c1002g


534

T60H + H285N + A335P + Y594F
a24t + a678g +
++





c1002g + t1626c


535

T60H + A335P + K529E + Y594F
t789a + g912t +
++





c1002g + c1588t +





a1725g


536

T60H + A309G
t591c
++


537

T60H + A335P + K529E + Y594F
a24t + a147g +
++





g912t + c1002g +





c1500t + c1588t


538

T60H + A309G + A335P
a24t + c1002g +
++





t1401a + a1734g


539

T60H + A335P + Y594F
a24t + c1002g
++


540

K35R + T60H + A335P + Y594F +
a24t + a678g +
++




K627R
c1002g + g1536a


541

T60H + A309G + A335P + V511L
a24t + t282c +
++





a495g + c1002g +





t1617c





1Fold improvement over Control #7, i.e., Variant No. 463, (SEQ ID NO: 13) is represented as follows:


++ = 1.0 to 2.0 fold improvement over control Variant No. 463.






Example 13
Characterization of Enzyme Stability
Representative CelA variants from Tables 6 and 7 were characterized to determine their stabilities at high temperatures (55° C. and 65° C.) and low pH (5.0) using the method of Example 5A. The samples containing various CelA variant enzymes were pre-incubated at pH 5.0, 55° C. for 48 hours and at pH 5.0, 65° C. for either 4 or 5 hours. The residual enzyme activity after the thermal challenge was measured using pNPG as substrate at pH 7, 30° C. for approximately 1 hour. The negative control was the B. megaterium-E. coli vector described in Example 1, without any CelA sequence. Tables 8A and 8B list the residual activities of the improved CelA variants at pH5.0, 55° C. The mutations listed in the table are indicated relative to SEQ ID NO: 4, the wildtype CelA.







TABLE 8A







Stability of Enzymes from Table 6.










% residual activity after 48
% residual activity after 4


Variant No.
hours at 55° C., pH 5.0
hours at 65° C., pH 5.0












Negative Control

N/A


391
0.9
0.2


(T2A + A5T + I14M + N79D +


G127N + A143M + H145R +


V159E + A198S + A207F +


F211Y + I222A + S225C +


Q508R + A3R + S73A +


V177P + I2037 - des[A647-


L663])


463
74.2
26.5


(T2A + A5T + I14M + N79D +


G127N + A143M + H145R +


V159E + A198S + V207F +


F211Y + I22A + S225C +


Q508R + A525T + T169N +


A202P + A272L + Q287R +


D311G + E512G - des[A647-


L663])


459
70.2
0.1


(T2A + A5T + I14M + N79D +


G127N + A143M + H145R +


V159E + A198S + V207F +


F211Y + I22A + S225C +


Q508R + A525T + T169N +


Q287R + A309G + D311G-


des[A647-L662])


460
89.2
1.6


(T2A + A5T + I14M + N79D +


G127N + A143M + H145R +


V159E + A198S + V207F +


F211Y + I22A + S225C +


Q508R + A525T + L41F +


A309G + D311G + A335P -


des[A647-L663]
















TABLE 8B







Stability of Enzymes from Table 7.










% residual activity after 48
% residual activity after 5


Variant No.
hours at 55° C., pH 5.0
hours at 65° C., pH 5.0





Negative Control

N/A


463
77.4
23.1


(T2A + A5T + I14M + N79D +


G127N + A143M + H145R +


V159E + A198S + V207F +


F211Y + I22A + S225C +


Q508R + A525T + T169N +


A202P + A272L + Q287R +


D311G + E512G - des[A647-


L663])


533
94.5
71.0


(T2A + A3R + A5T + I14M +


S73A + N79D + G127N +


I143M + H145R + V159E +


T169N + V177P + A198S +


A202P + I203Y + V207F =


F211Y + I222A + S225C +


A272L + Q287R + D311G +


Q508R + E512G + A525T +


A309G + A335P + D475E +


Y594F - des[A647-L663])


541
90.9
79.9


(T2A + A3R + A5T + I14M +


S73A + N79D + G127N +


I143M + H145R + V159E +


T169N + V177P + A198S +


A202P + I203Y + V207F =


F211Y + I222A + S225C +


A272L + Q287R + D311G +


Q508R + E512G + A525T +


T60H + A309G + A335P +


V511L - des[A647-L663])


538
86.4
74.3


(T2A + A3R + A5T + I14M +


S73A + N79D + G127N +


I143M + H145R + V159E +


T169N + V177P + A198S +


A202P + I203Y + V207F =


F211Y + I222A + S225C +


A272L + Q287R + D311G +


Q508R + E512G + A525T +


T60H + A309G + A335P-


des[A647-L663])


539
86.4
57.5


(T2A + A3R + A5T + I14M +


S73A + N79D + G127N +


I143M + H145R + V159E +


T169N + V177P + A198S +


A202P + I203Y + V207F +


F211Y + I222A + S225C +


A272L + Q287R + D311G +


Q508R + E512G + A525T +


T60H + A335P + Y594F -


des[A647-L663])


534
84.8
49.9


(T2A + A3R + A5T + I14M +


S73A + N79D + G127N +


I143M + H145R + V159E +


T169N + V177P + A198S +


A202P + I203Y + V207F =


F211Y + I222A + S225C +


A272L + Q287R + D311G +


Q508R + E512G + A525T +


T60H + H285N + A335P +


Y594F - des{A647-L663])


529
81.8
59.0


(T2A + A3R + A5T + I14M +


S73A + N79D + G127N +


I143M + H145R + V159E +


T169N + V177P + A198S +


A202P + I203Y + V207F =


F211Y + I222A + S225C +


A272L + Q287R + D311G +


Q508R + E512G + A525T +


T60H + A335P + D475E +


Y594F - des[A647-L663])









Example 14
Evaluation of Optimal CelA Activity for Additional Variants
The activity profiles for Variant Nos. 264, 366, 391, 463, and 529 were determined at different temperatures and pH using cellobiose (10 g/L) as a substrate. The experimental and analytical procedures are described in Example 5, incubating the variant with cellobiose at temperatures in the range of 45-80. The results are depicted in FIGS. 7-11. Variant No. 264 exhibited optimum activity at pH 6 and 58° C. The temperature and pH profile for this variant is depicted in FIG. 7. Variant No. 366 exhibited optimum activity at pH 6 and 66° C. The temperature and pH profile for this variant is depicted in FIG. 8. Variant No. 391 exhibited optimum activity at pH 5.5 and 69° C. The temperature and pH profile for this variant is depicted in FIG. 9. Variant No. 463 exhibited optimum activity at pH 6 and 69° C. (with the pH 6 optimum determined by extrapolation). The temperature and pH profile for this variant is depicted in FIG. 10. Variant No. 529 exhibited optimum activity at pH 6 and 69° C. The temperature and pH profile for this variant is depicted in FIG. 11.
Example 15
Evaluation of Addition of CelA Variants to Commercially Available Cellobiase
The impact of adding a CelA variant of the present invention to commercially available cellulose mixtures and microcrystalline cellulose, AvicelTM, was evaluated. In the test reactions, 1 g/L Trichoderma reesei whole cellulose (“TRWC”, Sigma catalog #C85456-10KU (ATCC 26921)) was used to convert 200 g/L of the microcrystalline cellulose AvicelTM (200 mM Sodium acetate; 100 g/L xylose, pH5.5 and 55° C.). The amount of glucose was measured after 48 hours of reaction. As a comparison, 25% of the TRWC was replaced with a β-glucosidase: Variant No. 264 (“Cel var1”), Variant No. 366 (“Cel var2”), or with the commercially available cellobiase, Aspergillus niger beta-glucosidase (“ANBG”, Sigma catalog #49291-1G).
A plot of the results (glucose production vs. cellulase composition) is, depicted in FIG. 11. The results indicate that 1 g/L of TRWC alone in this saccharification reaction yielded 38 g/L of cellobiose and glucose combined, with about equal amounts of each. Substitution of 25% of the TRWC with ANBG yielded 52 g/L with 94% of the sugar being glucose. Substitution of 25% of TRWC with either Variant No. 264 or Variant 366 gave comparable results.
Example 16
The level of glucose inhibition for variant #391 was determined using the cellobiose assay of Example 5B (except the cellobiose consumption was determined by HPLC) and spiking in glucose as well as varying amounts of glucose under conditions of pH 5, 55° C. in separate reactions. Percent residual activity was calculated relative to the activity computed for the reaction with no glucose. This was used to determine the IC50 for glucose (i.e., the concentration of glucose at which enzyme activity is 50% of the activity for the same reaction under conditions of no glucose). The IC50 can be determined from a plot of % residual activity vs. Initial Glucose Concentration of Reaction (g/L) as the glucose concentration where % residual activity is 50%. For variant #391, the IC50 for glucose was greater than 100 g/l.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

## Claims

1. A method of converting a biomass substrate to a fermentable sugar, the method comprising contacting the biomass substrate with a recombinant β-glucosidase polypeptide variant having beta-glucosidase activity, under conditions suitable for the production of the fermentable sugar from said biomass substrate, wherein the recombinant β-glucosidase polypeptide variant comprises:
(a) an amino acid sequence that has at least 85% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein SEQ ID NO: 4 is the amino acid sequence of wild type Azospirillum irakense β-glucosidase and wherein said amino acid sequence comprises substitution or deletion of the amino acid residue F211, wherein amino acid position is numbered with reference to the amino acid sequence of SEQ ID NO:4; or
(b) an amino acid sequence encoded by a nucleic acid that hybridizes under highly stringent conditions with the complementary strand of a polynucleotide encoding the amino acid sequence of SEQ ID NO: 4, wherein the highly stringent conditions comprise prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/mL sheared and denatured salmon sperm DNA and 50% formamide and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C., wherein the encoded amino acid sequence has a substitution or deletion of the amino acid residue F211, wherein amino acid position is numbered with reference to the amino acid sequence of SEQ ID NO: 4, and wherein the nucleic acid encodes a polypeptide having beta-glucosidase activity.

2. A method of converting a biomass substrate to a fermentable sugar, the method comprising contacting the biomass substrate with a recombinant β-glucosidase polypeptide variant having beta-glucosidase activity, under conditions suitable for the production of the fermentable sugar from said biomass substrate, wherein the recombinant β-glucosidase polypeptide variant comprises an amino acid sequence that has at least 85% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein SEQ ID NO: 4 is the amino acid sequence of wild type Azospirillum irakense β-glucosidase and wherein said amino acid sequence comprises substitution or deletion of the amino acid residue F211, wherein amino acid position is numbered with reference to the amino acid sequence of SEQ ID NO:4.

3. A method of converting a biomass substrate to a fermentable sugar, the method comprising contacting the biomass substrate with a recombinant β-glucosidase polypeptide variant having beta-glucosidase activity, under conditions suitable for the production of the fermentable sugar from said biomass substrate, wherein the recombinant β-glucosidase polypeptide variant comprises an amino acid sequence that has at least 85% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein SEQ ID NO: 4 is the amino acid sequence of wild type Azospirillum irakense β-glucosidase and wherein said amino acid sequence comprises substitution of the amino acid residue at F211, wherein amino acid position is numbered with reference to the amino acid sequence of SEQ ID NO:4.

4. A method of converting a biomass substrate to a fermentable sugar, the method comprising contacting the biomass substrate with a recombinant β-glucosidase polypeptide variant having beta-glucosidase activity, under conditions suitable for the production of the fermentable sugar from said biomass substrate, wherein the recombinant β-glucosidase polypeptide variant comprises an amino acid sequence that has at least 85% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein SEQ ID NO: 4 is the amino acid sequence of wild type Azospirillum irakense β-glucosidase and wherein said amino acid sequence comprises deletion of the amino acid residue F211, wherein amino acid position is numbered with reference to the amino acid sequence of SEQ ID NO:4.

5. The method of claim 1, wherein the fermentable sugar comprises glucose and/or xylose.

6. The method of claim 1, further comprising pretreating the biomass substrate before contacting the biomass substrate with said recombinant β-glucosidase polypeptide variant.

7. The method of claim 1, further comprising contacting the biomass substrate with a mixture of the recombinant β-glucosidase polypeptide variant and at least one additional enzyme.

8. The method of claim 7, wherein said at least one additional enzyme is a cellulase.

9. The method of claim 8, wherein said at least one additional enzyme is a beta-glucosidase, cellobiohydrolase, and/or endoglucanase.

10. The method of claim 8, wherein said mixture comprises a beta-glucosidase, cellobiohydrolase, and endoglucanase.

11. The method of claim 7, wherein said at least one additional enzyme is an alpha-amylase, glucoamylase, and/or aspartyl protease.