# Patent

## Title
Recombinant β-glucosidase variants for production of soluble sugars from cellulosic biomass

## Abstract

The invention relates to recombinant expression of a variant form of a fungal C1 strain β-glucosidase. The invention also relates to the generation of fermentable sugars from biomass and the production of biofuels by fermentation of the sugars using genetically modified organisms expressing the β-glucosidase variant. The invention provides methods for producing a fermentable sugar, such as glucose, from cellobiose by contacting cellobiose with a recombinant β-glucosidase variant protein, such as a variant protein secreted by a recombinant host cell into culture medium. Methods of the invention may be used for conversion of a biomass substrate to a fermentable sugar, and ultimately to ethanol or other biofuel.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This applications is a continuation of application Ser. No. 12/954,447, filed Nov. 24, 2012, which claims benefit of U.S. provisional application 61/264,608, filed Nov. 25, 2009 and U.S. provisional application No. 61/355,511, filed Jun. 16, 2010, each of which is herein incorporated by reference in its entirety for all purposes.


REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE
The Sequence Listing written in file SEQTXT—90834-834121—002022US, created on Mar. 9, 2012, 121,568 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference in its entirety and for all purposes.
FIELD OF THE INVENTION
The invention relates to expression of recombinant β-glucosidase variants and their use in the production of soluble sugars from cellulosic biomass.
BACKGROUND OF THE INVENTION
Cellulosic biomass is a significant renewable resource for the generation of soluble sugars. These sugars can be used as reactants in various metabolic processes, including fermentation, to produce biofuels, chemical compounds, and other commercially valuable products. While the fermentation of simple sugars such as glucose to ethanol is relatively straightforward, the efficient conversion of cellulosic biomass to soluble sugars is challenging (see, e.g., Ladisch et al., 1983, Enzyme Microb. Technol. 5:82). Cellulose may be pretreated chemically, mechanically, enzymatically or in other ways to increase the susceptibility of cellulose to hydrolysis. Such pretreatment may be followed by the enzymatic conversion of cellulose to cellobiose, cello-oligosaccharides, glucose, and other sugars and sugar polymers, using enzymes that break 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 “BGL”). Endoglucanases break internal bonds and disrupt the crystalline structure of cellulose, exposing individual cellulose polysaccharide chains (“glucans”). Cellobiohydrolases incrementally shorten the glucan molecules, releasing mainly cellobiose units (a water-soluble β-1,4-linked dimer of glucose) as well as glucose, cellotriose, and cellotetraose. β-Glucosidases split cellobiose into glucose monomers.
Cellulases with improved properties for use in processing cellulosic biomass would reduce costs and increase the efficiency of production of biofuels and other commercially valuable compounds.
SUMMARY OF THE INVENTION
In one aspect, the invention provides an isolated or recombinant β-glucosidase variant comprising an amino acid sequence that is at least about 60% identical, sometimes at least about 65% identical and often at least about 70% identical to residues 20-870 of SEQ ID NO:2 (wild-type C1 β-glucosidase), or which is encoded by a nucleic acid that hybridizes under stringent conditions to SEQ ID NO:1 or the exact complement of SEQ ID NO:1, and which has at least one substitution, relative to SEQ ID NO:2, of an amino acid residue described herein, where the variant has greater β-glucosidase activity than the wild-type protein and/or is more thermostable than the wild-type protein. Also provided are polynucleotides encoding the β-glucosidase variants, expression vectors comprising said polynucleotides, and host cells transformed with the expression vectors.
The invention also provides a method of producing a β-glucosidase variant by culturing a host cell transformed with a polynucleotide encoding a β-glucosidase variant under conditions suitable for the expression of the β-glucosidase. In some embodiments, the β-glucosidase polypeptide is recovered from the culture medium or from the transformed and cultured cells.
The invention also provides an enzyme composition comprising an isolated or recombinant C1 β-glucosidase variant. Optionally the enzyme composition also includes at least one additional cellulase enzyme.
In a related and/or other aspect, the invention provides a method of converting a biomass substrate, such as cellobiose, to a fermentable sugar by contacting a β-glucosidase variant with the biomass substrate under conditions suitable for the production of the fermentable sugar. In one embodiment the biomass substrate is maintained in a medium containing cells expressing a β-glucosidase variant. In one embodiment the recombinant host cell expressing a β-glucosidase variant also expresses at least one other recombinant cellulase and/or other enzyme. Optionally the biomass substrate is pretreated before contacting the substrate with a β-glucosidase polypeptide variant.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. (A) Thermostabilities of improved C1 Bgl1 variants produced in the C1 strain; Residual enzyme activity after 6 hr incubation at pH 5, 65° C. was determined by pNPG assay at pH 5, 50° C. for 20 mins. N=6-8; Error bars represent±1 standard deviation. (B) Thermostabilities of improved C1 Bgl1 variants produced in the C1 strain; Residual enzyme activity after 24 hr incubation at pH 5, 65° C. was determined by pNPG assay at pH 5, 50° C. for 20 mins. N=6-30; Error bars represent±1 standard deviation. (C) C1 Bgl1 thermostability correlation between yeast and C1 hosts.
FIG. 2. (A) The thermostabilities of improved C1 Bgl1 variants 481, 269, and 3 were compared to that of the native C1 Bgl1 enzyme. The residual enzyme activity after 24 hr incubation at pH 4.5, 65° C. was measured using pNPG as substrate at pH 5, 50° C. for 20 mins.N=6-16; Error bars represent±1 standard deviation. (B) The thermostability of improved C1 Bgl1 Variant 664 was compared to that of Variant 481. The residual enzyme activity after 4 hr incubation at pH 4, 65° C. was measured using pNPG as substrate at pH 5, 50° C. for 20 mins. N=6-24; Error bars represent±1 standard deviation.
FIG. 3. The thermostabilities of improved C1 Bgl1 variants 3, 481, 664, 916, 885, and 871 were compared to that of the native C1 Bgl1 enzyme. The residual enzyme activity after 72 hr incubation at pH 4.5, 65° C. was measured using pNPG as substrate at pH 5, 50° C. for 20 mins. N=6-10; Error bars represent±1 standard deviation.
FIG. 4. The specific activity of native C1 Bgl1 was compared to that of variant 871 in a cellbiose assay. Assay conditions: pH 4.5-5, temperatures 55° C.-75° C.; 8 g/L cellobiose, 18 hr reaction. Protein concentration was normalized in reactions. N+2; Error bars represent±1 standard deviation.
FIG. 5 shows an alignment of native (wildtype), C1 Bgl1 amino acid sequence (SEQ ID NO:2) with the amino acid sequences of Variants 3 (SEQ ID NO:5), 269 (SEQ ID NO:7), 481 (SEQ ID NO:9), 647 (SEQ ID NO:15), 664 (SEQ ID NO:13), 871 (SEQ ID NO:17), 885 (SEQ ID NO:19), and 916 (SEQ ID NO:21).



DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
The following definitions are provided to assist the reader. Unless otherwise defined, all terms of art are intended to have the meanings commonly understood by those of skill in the molecular biology and microbiology arts. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over the definition of the term as generally understood in the art.
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.
The terms “β-glucosidase” and “cellobiase” are used interchangeably and refer to 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, including the assays described hereinbelow.
The term “β-glucosidase polypeptide” refers 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 terms “exoglucanase”, “exo-cellobiohydrolase” or “CBH” refer 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 terms “endoglucanase” or “EG” refer 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, etc.).
The term “wildtype” as applied to a polypeptide (protein) means a polypeptide (protein) expressed by a naturally occurring microorganism such as bacteria or filamentous fungus. As applied to a microorganism, the term “wildtype” refers to the native, non-recombinant micro-organism. With reference to C1 β-glucosidase, wild-type Bgl1 refers to the mature (secreted) form of the protein having the sequence of residues 20-870 of SEQ ID NO:2.
A “variant” as used herein means a β-glucosidase polypeptide or polynucleotide encoding a β-glucosidase comprising one or more modifications relative to wildtype C1 β-glucosidase (Bgl1) 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, such as SEQ ID NO:2. 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, at least 500 residues in length, at least 600 residues in length, at least 700 residues in length, or the full length of the polypeptide.
A nucleic acid (such as a polynucleotide), a polypeptide, or a cell is “recombinant” when it is artificial or engineered, or derived from or contains 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 C1 β-glucosidase (Bgl1) (residues 20-870 of SEQ ID NO:2). Improved properties may include increased protein expression, thermoactivity, thermostability, pH activity, pH stability, product specificity, increased specific activity, substrate specificity, increased resistance to substrate or end-product inhibition, altered pH/temperature profile, and chemical stability.
A variant with “improved β-glucosidase activity,” as the term is used herein, means a variant displaying an increase, relative to a reference sequence, in the amount of substrate hydrolysis that occurs in a specified time under specified reaction conditions. β-glucosidase activity can be measured using a variety of methods known in the art, such as the cellobiose assays described hereinbelow. To compare β-glucosidase activity of two recombinantly expressed proteins, the specific activity (activity per mole enzyme or activity per gram enzyme) can be compared. Alternatively, cells expressing and secreting the recombinant proteins can be cultured under the same conditions and the β-glucosidase activity per volume culture medium can be compared.
The term “improved thermostability” as used herein means a variant enzyme displays an increase in “residual activity” relative to the wildtype enzyme. Residual activity is determined by exposing the enzyme (variant or reference, e.g., wild-type) to stress conditions of elevated temperature for a period of time and then determining β-glucosidase activity under conditions in which the reference (e.g., wild-type) enzyme normally has 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. A variant with increased thermostability will have greater residual activity than the reference (e.g., wild-type) enzyme. Exemplary conditions for determining thermostability are provided in the Examples, infra. In one embodiment the enzymes are exposed to stress conditions of 65° C. at pH 5 for 6 hrs, and assayed at 50° C., pH 5, for 1.5 hrs.
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 and multiple alignments can be prepared using readily available programs such as PSI-BLAST, which is described by Altschul, et al. (1997), supra. One useful alignment tool is “T-Coffee” (Notredame et al., 2000, J. Mol. Bio. 302:205-17). T-Coffee alignments may be carried out using default parameters (T-Coffee Technical Documentation, Version 8.01, July 2009, WorldWideWeb.tcoffee.org).
“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.
An amino acid or nucleotide base “position” is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5′-end). Due 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-terminus will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where a variant has a deletion relative to an aligned reference sequence, there will be no amino acid in the variant 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 a numbered 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 to form double-stranded structures, 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).
The terms “culturing” or “cultivation” refer to growing a population of microbial cells under suitable conditions in a liquid or solid medium.
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. For example, combining a solution containing the enzyme with the respective substrate will effect contacting. Such contacting also includes incubating a cell secreting an enzyme in a medium containing an enzyme substrate.
As used herein, reference to a cell “metabolizing” a soluble sugar or other substrate to produce an end product means the sugar serves as a carbon source and/or energy source for a metabolic reaction in the cell. Typically the cell is a microbial cell such as a fungal cell or bacterial cell.
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”) or otherwise incorporated into the genome of, or maintained as an episome in, the cell.
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.
A promoter sequence, signal peptide, or other sequence is “heterologous”, when it is operably linked to a nucleic acid or protein sequence with which the promoter, signal peptide or other sequence is not associated in nature.
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.
A β-glucosidase variant polypeptide is “enzymatically active” when it has β-glucosidase activity.
The term “pre-protein” refers to a protein including an amino-terminal signal peptide (or leader sequence) region attached. The signal peptide is cleaved from the pre-protein by a signal peptidase prior to secretion to result in the “mature” or “secreted” protein.
As used herein, a “start codon” is the ATG codon that encodes the first amino acid residue (methionine) of a protein.
In the context of sequence identity, a reference to “at least x % sequence identity” in this specification is intended, unless otherwise specified, to refer to “x % sequence identity” as well as to alternative embodiments in which % sequence identity is defined by each integer from (x+1) % to 99% identity, just as if each alternative embodiment was explicitly listed. For example, reference to “at least 70% sequence identity to SEQ ID NO:2”, or “amino acid residues 20-870 of SEQ ID NO:2”, refers to alternative embodiments with at least 71% sequence identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to amino acid SEQ ID NO:2, or residues 20-870 of SEQ ID NO:2. For an alignment that extends for the entire 851 amino acid length of residues 20-870 of SEQ ID NO:2, there may be at least 596 identities between a variant sequence and SEQ ID NO:2, sometimes at least 604, at least 613, at least 622, at least 630, at least 638, at least 647, at least 655, at least 664, at least 672, at least 681, at least 723, at least 766, or at least 808 identities.
As used herein, “C1” refers to a fungal strain described by Garg, A., 1966, “An addition to the genus Chrysosporium Corda” Mycopathologia 30: 3-4. “Chrysosporium lucknowense” includes the strains described in U.S. Pat. Nos. 6,015,707, 5,811,381 and 6,573,086; US Pat. Pub. Nos. 2007/0238155, US 2008/0194005, US 2009/0099079; International Pat. Pub. Nos., WO 2008/073914 and WO 98/15633, and include, without limitation, Chrysosporium lucknowense Garg 27K, VKM-F 3500 D (Accession No. VKM F-3500-D), C1 strain UVβ-6 (Accession No. VKM F-3632 D), C1 strain NG7C-19 (Accession No. VKM F-3633 D), and C1 strain UV18-25 (VKM F-3631 D), all of which have been deposited at the All-Russian Collection of Microorganisms of Russian Academy of Sciences (VKM), Bakhurhina St. 8, Moscow, Russia, 113184, and any derivatives thereof. Although initially described as Chrysosporium lucknowense, C1 may currently be considered a strain of Myceliophthora thermophilia. Exemplary C1 derivatives include modified organisms in which one or more endogenous genes or sequences has been deleted or modified and/or one or more heterologous genes or sequences has been introduced. Derivatives include UV18#100fΔalpl, UV18#100f Δpyr5 Aalpl, UV18#100.f Δalpl Δpep4 Δalp2, UV18#100.f Δpyr5 Δalpl Δpep4 Δalp2 and UV18#100.f Δpyr4 Δpyr5 Δa1p 1 Δpep4 Δalp2. as described in WO2008073914, incorporated herein by reference.
The following nomenclature may by used to describe substitutions in a polypeptide or polynucleotide sequence relative to a reference sequence: “R-#-V,” where # refers to the position in the reference sequence, R refers to the amino acid (or base) at that position in the reference sequence, and V refers to the amino acid (or base) at the corresponding position in the variant sequence. For example, for a variant polypeptide described with reference to SEQ ID NO: 2, “D369L” indicates that in the variant polypeptide, the aspartic acid at position 369 of the reference sequence is replaced by leucine, with amino acid position being determined by optimal alignment of the variant sequence with SEQ ID NO:2. Similarly, “S182L/W” describes two variants: a variant in which the serine at position 182 is replaced by leucine, and a variant in which the serine at position 182 is replaced by tryptophan.
The following conventions are used to describe amino acid positions in Bgl1 variants. Amino acid positions are numbered in relation the reference sequence SEQ ID NO: 2, which is the sequence of the wild-type C1 Bgl1 pre-protein (including the signal peptide). Alignments to determine a degree of sequence identity (e.g., “at least 70% identity”) or to describe deletions (e.g., deletions of residues at the c-terminus of the protein) are in relation to the secreted form of the wild-type Bgl1 protein, residues 20-870 of SEQ ID NO:2. Nucleotide base substitutions are numbered relative to SEQ ID NO:1. Substitutions in the signal peptide (residues 1-19 of SEQ ID NO: 2) refer to the sequence of the Bgl1 pre-protein.
II. Introduction
The fungus C1 produces a variety of cellulases and other enzymes that act in concert to catalyze decrystallization and hydrolysis of cellulose to yield soluble sugars. C1 was described by Garg, 1966, “An addition to the genus Chrysosporium corda” Mycopathologia 30: 3-4. Also see U.S. Pat. Nos. 6,015,707 and 6,573,086, which are incorporated herein by reference for all purposes.
The C1 genome has been at least partially sequenced, as indicated in U.S. patent publications US 2007/0238155, US 2008/0194005, and US 2009/0099079, incorporated herein by reference for all purposes. The sequence of the C1 β-glucosidase 1 (bgl1) gene and the encoded protein (Bgl1) are set forth herein as SEQ ID NOs:1 and 2 (also see copending application Nos. 61/247,379 and PCT/US10/50982, incorporated herein by reference). Note that these sequences differs from the Bgl1 sequences of patent publication US 2007/0238155.
The C1 β-glucosidase variants described herein are particularly useful for production of soluble sugars from cellulosic biomass. In one aspect the invention relates to a method of producing glucose by contacting a composition comprising cellobiose with a recombinantly expressed C1 β-glucosidase variant under conditions in which the cellobiose is enzymatically converted to glucose. In one aspect, recombinant host cells expressing a β-glucosidase variant may be combined with cellobiose under conditions in which the β-glucosidase is expressed (and in some embodiments, secreted) by the cells. Alternatively, purified or partially purified recombinant β-glucosidase protein may be contacted with cellobiose. In one aspect of the present invention, contacting comprises culturing a recombinant host cell in a medium that contains cellobiose produced from a cellulosic feedstock. For example, the C1 β-glucosidase variants described herein demonstrate benefit in saccharification reactions in conjunction with other cellulases, such as T. reesei cellulases (e.g., T. reesei CBH1, CBH2, and/or EG1 or variants thereof, and/or T. reesei broth) and C1 cellulases (see U.S. Pat. Nos. 6,015,707, 5,811,381 and 6,573,086; US Pat. Pub. Nos. 2007/0238155, US 2008/0194005, US 2009/0099079; International Pat. Pub. Nos. WO 2008/073914 and WO 98/15633, all incorporated herein by reference).
Various aspects of the invention are described in the following sections.
III. Properties of β-Glucosidase Proteins for Use in Methods of the Invention
In one aspect, the invention provides a method for expressing a β-glucosidase protein by culturing a host cell comprising a vector or episomal plasmid comprising a nucleic acid sequence encoding a C1 Bgl1 variant, or which has a nucleic acid sequence encoding a C1 Bgl1 variant integrated into its genome, under conditions in which the β-glucosidase protein or an enzymatically active fragment thereof is expressed. Generally the expressed protein comprises a signal peptide which is removed by the cell as the enzyme is secreted. In one embodiment, transcription of the sequence encoding the C1 Bgl1 variant is controlled by an operably linked heterologous promoter.
β-Glucosidase Polypeptide Variants
The present invention provides novel enzymes that are C1 β-glucosidase (Bgl1) variants. β-glucosidase polypeptide variants of the present invention are variants of Bgl1 that exhibit β-glucosidase activity, typically greater β-glucosidase activity than the wild-type C1 β-glucosidase (residues 20-870 of SEQ ID NO:2) especially under conditions relevant to commercial saccharification processes. Also included are β-glucosidase polypeptide variants that exhibit greater stability under conditions relevant to commercial saccharification processes (e.g., increased thermostability relative to wild-type β-glucosidase).
The present invention provides Bgl1 variants having greater activity and/or thermostability than the wild-type (WT) C1 Bgl1 protein and having at least one of the substitutions found in an increased-activity and/or increased-thermostability variant described herein. As is discussed in more detail below, a polynucleotide encoding the wild-type (WT) C1 Bgl1 protein (SEQ ID NO: 2) was prepared. The polynucleotide was inserted into an expression vector as described in Example 1, infra, libraries of polynucleotides encoding variant Bgl1 proteins were prepared by mutagenesis and directed evolution, and the properties (e.g., β-glucosidase activity and thermostability) of individual Bgl1 variants were assessed (See Tables 2-7 and Examples 2-9, infra). A number of amino acid substitutions and combinations of substitutions were identified in variants with greater than wild-type activity and/or greater than wild-type thermostability. See Table 2. A variant with elevated activity and thermostability was selected and subjected to further mutagenesis and screening. See Table 3. A variant from this round of mutagenesis and screening was selected and subjected to further mutagenesis and screening. See Table 4. A variant from this further round of screening was selected and subjected to additional mutagenesis and screening. See Table 5. Two variants from this round of screening were selected and subjected to further mutagenesis and screening. See Tables 6 and 7.
More specifically, the present invention provides an isolated and/or recombinant β-glucosidase polypeptide variant having greater activity and/or thermostability than the wild-type C1 protein, and which comprises an amino acid sequence that is at least about 70% identical to wild-type C1 β-glucosidase (Bgl1) (residues 20-870 of SEQ ID NO:2) and has at least one substitution of an amino acid residue at a position selected from the group consisting of K57; A88; I106; N112; Q119; T120; A123; R132; Y135; A136; A141; K142; L149; G158; P161; P172; T177; I179; G180; M181; S182; E183; K186; A197; G202; Y219; N220; S222; T224; 1229; M234; F242; A243; V246; Q258; D274; V286; Q291; Q313; V318; A343; T354; T357; D358; E360; D369; P374; I375; A378; Q381; E385; S388; V390; A394; N398; E402; K406; I428; S434; N437; E449; Q474; A475; T482; S489; Y491; K530; N536; T540; T565; V674; R682; 1867; E868; P870 (wherein amino acid position is determined by optimal alignment with SEQ ID NO:2). “Substitution,” in this context, means that the residue in the variant protein is other then the residue shown. For example, “A88” denotes a variant comprising an amino acid other than alanine at position 88 (i.e., one of the other 19 naturally occurring amino acids). In some embodiments, the amino acid in the variant protein is neither the wild-type residue nor a residue that is a conservative substitute for the wild-type residue. As discussed below, in this context, a conservative substitute for a residue is another residue in the same group identified in Table 1.
The present invention additionally provides an isolated and/or recombinant β-glucosidase polypeptide variant having greater activity and/or thermostability than the (WT) C1 Bgl1 protein, wherein the variant comprises an amino acid sequence that is at least about 70% identical to wildtype C1 (Bgl1) (residues 20-870 of SEQ ID NO:2); has a substitution at each of positions Q291, D369 and E402; and has at least one substitution of an amino acid residue at a position selected from the group consisting of I106, A109, Q119, T120, R132, Y135, M181, Q215, A243, V246, Q258, A265, D274, N278, Q313, F314, G332, T357, D358, P374, E385, S434, N437, A475, S489, Y491, E493, K495, K497, S501. A503E N504, A505, N521, K530, N536. T540, D566, T591, A601, K610, T611, R612, S614, L620, G628, T635, V638, V648, D650, S652, N670, R672, V674, S676, T685, T687, A689, Q690, T699, D703, K708, Y715, A732, E742, L757, V775, T777, and D781; (wherein amino acid position is determined by optimal alignment with SEQ ID NO:2). In some embodiments, the amino acid in the variant protein is neither the wild-type residue nor a residue that is a conservative substitute for the wild-type residue. As discussed below, in this context, a conservative substitute for a residue is another residue in the same group identified in Table 1.
The present invention additionally provides an isolated and/or recombinant β-glucosidase polypeptide variant having greater activity and/or thermostability than the wild-type C1 Bgl1 protein, wherein the variant comprises an amino acid sequence that is at least about 70% identical to wild-type C1 β-glucosidase (Bgl1) (residues 20-870 of SEQ ID NO:2); has a substitution at each of positions Q258, Q291, Q313, D369, E402, S434, A475, K495, and G628; and has at least one substitution of an amino acid residue at a position selected from the group consisting of A4, A15, E21, S22, K24, V25, H26, Q27, P29, L30, N45, D47, Q55, S58, A79, Q85, I106, A109, Q119, Y135, A136, P161, V175, G180, G202, G216, I221, L237, D244, V253, V260, L275, D311, F314, A343, D358, E360, Q381, E385, A394, A404, A405, K421, I428, P436, N437, M454, D470, R476, A477, Q479, T482, Y491, E493, E494, T496, S501, A505, N536, V559, V562, N588, S604, R612, G616, A617, G626, N627, F634, D646, D650, S652, R672, V673, T685, T687, A689, Q690, K708, Y715, Q716, A732, A748, D751, L757K, S764, R769, V775, T777, T783, T785, S787, S799, P806, K807, R817, E819, E822, T823, V840, V846, I847, S848, Y850, K866, and P870, (wherein amino acid position is determined by optimal alignment with SEQ ID NO:2). In some embodiments, the amino acid in the variant protein is neither the wild-type residue nor a residue that is a conservative substitute for the wild-type residue. As discussed below, in this context, a conservative substitute for a residue is another residue in the same group identified in Table 1.
The present invention additionally provides an isolated and/or recombinant β-glucosidase polypeptide variant having greater activity and/or thermostability than the wild-type C1 Bgl1 protein, wherein the variant comprises an amino acid sequence that is at least about 70% identical to wild-type C1 β-glucosidase (Bgl1) (residues 20-870 of SEQ ID NO:2); has a substitution at each of positions Q258, Q291, Q313, D369, E402, S434, A475, K495, G628, A689, and Y715; and has at least one substitution of an amino acid residue at a position selected from the group consisting of C8, L9, K24, V25, D47, S58, A79, Q85, 1106, A109, A136, V175, L237, A243, V253, V260, D274, L275, F314, A343, Q381, E385, P436, N437, Q474, R476, A505, S550, N588, S604, G616, G626, D650, D651, S652, V674, T687, Q690, D709, E710, A732, D733, Y736N, L757, S764, T777, T783, T785, K807, M816, D844, V846, L869, and P870 (wherein amino acid position is determined by optimal alignment with SEQ ID NO:2). In some embodiments, the amino acid in the variant protein is neither the wild-type residue nor a residue that is a conservative substitute for the wild-type residue. As discussed below, in this context, a conservative substitute for a residue is another residue in the same group identified in Table 1.
The present invention additionally provides an isolated and/or recombinant β-glucosidase polypeptide variant having greater activity and/or thermostability than the wild-type C1 Bgl1 protein, wherein the variant comprises an amino acid sequence that is at least about 70% identical to wild-type C1 β-glucosidase (Bgl1) (residues 20-870 of SEQ ID NO:2); has a substitution at each of positions D47, Q258, Q291, Q313, A343, D369, E402, S434, A475, K495, G628, T687, A689, and Y715; and has at least one substitution of an amino acid residue at a position selected from the group consisting of A79, Q85, V260, L275, F314, D395, P439, A505, D646, T693, N723, A730, A732, S764, R769, T827, and Y855, (wherein amino acid position is determined by optimal alignment with SEQ ID NO:2). In some embodiments, the amino acid in the variant protein is neither the wild-type residue nor a residue that is a conservative substitute for the wild-type residue. As discussed below, in this context, a conservative substitute for a residue is another residue in the same group identified in Table 1.
The present invention additionally provides an isolated and/or recombinant β-glucosidase polypeptide variant having greater activity and/or thermostability than the wild-type C1 Bgl1 protein, wherein the variant comprises an amino acid sequence that is at least about 70% identical to wild-type C1 β-glucosidase (Bgl1) (residues 20-870 of SEQ ID NO:2); has a substitution at each of positions 1106, Q258, V260, Q291W, Q313, F314, D369, E402, S434, K495, G628, A689, Y715, and A732; and has at least one substitution of an amino acid residue at a position selected from the group consisting of A109, A343, A475, A505, A689, A79, C8, D47, L275, N315, Q85, T591, and T687, (wherein amino acid position is determined by optimal alignment with SEQ ID NO:2). In some embodiments, the amino acid in the variant protein is neither the wild-type residue nor a residue that is a conservative substitute for the wild-type residue. As discussed below, in this context, a conservative substitute for a residue is another residue in the same group identified in Table 1.








TABLE 1







basic amino acids
arginine, lysine, histidine


acidic amino acids
glutamic acid, aspartic acid


polar amino acids
glutamine, asparagine


hydrophobic amino acids
leucine, isoleucine, valine


aromatic amino acids
phenylalanine, tryptophan, tyrosine


small amino acids
glycine, alanine, serine, threonine, proline,



cysteine, methionine









In some embodiments, the amino acid in the variant protein is neither the wild-type residue nor a residue that is a residue commonly exchanged with the wild-type residue as defined by the following pairs: 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 summarized in Table 2, in certain embodiments, the present invention provides an isolated and/or recombinant β-glucosidase polypeptide variant comprising an amino acid sequence that has greater activity and/or thermostability than the wild-type wild-type C1 Bgl1 protein, is at least about 70% identical to wild-type C1 β-glucosidase (Bgl1) (residues 20-870 of SEQ ID NO:2), and which has at least one substitution of an amino acid residue selected from the group consisting of K57R; A88S; I106V; N112V; Q119E/L; T120M/Y/V/H; A123N; R132K/G/W; Y135I/Q/M; A136E; A141F; K142R; L149Q/M; G158D; P161S; P172A; T177I; I179M; G180E; M181Y; S182L/W; E183G/M/Q/K; K186R; A197V; G202M/V; Y219V; N220Y/S/L; S222Q/E; T224N; 1229M; M234E/I; F242L; A243V; V246L; Q258N; D274Y/N; V2861; Q291W/A/F; Q313M; V318E; A343V/T; T354Q; T357L/P; D358K/N; E360R/A/D; P374Y; I375V; A378K/T; Q381V/D/I/L; D369Q/L/Y/C/A/I/P/E/K/R/F/M/H/V; E385L; S388W/C; V390I; A394G/V/L/P/Q; N398G; E402N; K406D; I428V; S434P; N437F/Y/D/L/W; E449Q; Q474I; A475F/Y/H/W/C; T482A; S489L; Y491H/F; K530M/G; N536K; T540K; T565A/G/P; V6741; R682W; I867M; E868R; P870S (wherein amino acid position is determined by optimal alignment with SEQ ID NO:2). Beneficial combinations of the above-listed substitutions 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, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or more of the above-identified positions.
As summarized in Table 3, in certain embodiments, the present invention provides an isolated and/or recombinant β-glucosidase polypeptide variant comprising an amino acid sequence that has greater activity and/or thermostability than the wild-type C1 Bgl1 protein, wherein the variant is at least about 70% identical to wild-type C1 (Bgl1) (residues 20-870 of SEQ ID NO:2); has the substitutions Q291W, D369H/L/R/Y, and E402N; and has at least one substitution of an amino acid residue at a position selected from the group consisting of A109S, A243V, A265S, A475C/F/L/W, A503E, A505C, A601T, A689I, A732G/M, D274Y, D358E/K/N, D566G, D650F/V, D703K, D781N, E385L, E493A/V/Y, E742G, F314L/V, G332D, G628L/V/W, I106V, K495F/H/I/N/Q/V, K497R, K530C/D/E/I/M/N/V, K610S, K708F, L620M, L757K, M181Y, N278Y, N437D/F/I/K/L/V/W/Y, N504Y, N521C, N536K, N670D, P374Y, Q119E, Q215E/M, Q258H/N, Q313M, Q690A/K/R, R132H, R612H/P, R672A/D/F/G/I/S/T/V, S434P, S489I/L/N/T, S501H/N/R, S614A/C/D/H/L/R/V/Y, S652D, S676C, S764F, T120H/Y, T357A, T540K, T591A/C/R, T611A/H/Q, T635A/I/V, T685V. T687C/F/K/L/M/W/Y, T699L, T777N, T779S, V2461, V638E/R/S, V648W, V674M, V775C, Y135Q, Y491F/H/L, and Y715P; (wherein amino acid position is determined by optimal alignment with SEQ ID NO:2). Beneficial combinations of the above-listed substitutions 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, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70, or more of the above-identified positions.
As summarized in Table 4, in certain embodiments, the present invention provides an isolated and/or recombinant β-glucosidase polypeptide variant comprising an amino acid sequence that has greater activity and/or thermostability than the wild-type C1 Bgl1 protein, is at least about 70% identical to wild-type C1 β-glucosidase (Bgl1) (residues 20-870 of SEQ ID NO:2), has the substitutions Q258N, Q291W, Q313M, D369R, E402N, S434P, A475L, K495N, and G628W; and has at least one substitution selected from the group consisting of A4V, A15V, A109S/T, A136L, A343C, A394G, A404S, A405T, A477G, A505C, A617V, A689I, A732G/M/S, A748T, A79E/G/M, D244H, D311N, D358K, D470N, D47I, D646N, D650F/N/Y, D751N, E21Q/R, E360D, E385L, E493A/V/Y, E494K, E819A/L/V, E822A/G/K/M, F314L/V, F634A, G180E, G202M, G216L, G616D, G626D, H26R, I106V, I221V, I428V, 1847T, K24G/L/T, K421R, K708F, K807R, K866I/Q, L237Y, L275Y, L30K, L757K, M454E, N437W, N45H, N536K, N588F, N627H, P161S, P29M/Q/R, P436E/Q, P806L, P870S, Q119E, Q27H/R, Q381V, Q479R, Q55R, Q690A/H/K, Q716R, Q85N, R476Q, R612H, R672G, R769H, R817P, S22L/R, S501C/R, S58G, S6041, S652D, S764F, S787G, S799N, S848N, T482A, T496A, T685V, T687C/K/M, T777N, T783H/Q, T785L, T823K, V175A, V253F, V25A/G/R, V260G/L, V559T, V562C/L, V673A, V775C, V8401, V846F, Y135M/Q, Y491F, Y715P, and Y850H/Q, (wherein amino acid position is determined by optimal alignment with SEQ ID NO:2). In some embodiments, the Bgl1 polypeptide does not have a substitution at position A475. Beneficial combinations of the above-listed substitutions 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, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more of the above-identified positions.
As summarized in Table 5, in certain embodiments, the present invention provides an isolated and/or recombinant β-glucosidase polypeptide variant comprising an amino acid sequence that has greater activity and/or thermostability than the wild-type C1 Bgl1 protein, is at least about 70% identical to wild-type C1 β-glucosidase (Bgl1) (residues 20-870 of SEQ ID NO:2), has the substitutions Q258N, Q291W, Q313M, D369R, E402N, S434P, A475L, K495N, G628W, A689I, and Y715P; and has at least one substitution selected from the group consisting of: C8A, L9F, K24G/T, V25A, D471/N, S58G, A79E/G/M, Q85H/N, I106V, A109S/T, A136L, V175A, L237Y, A243G, V253F, V260G, D274Y, L275F/Y, F314L/V, A343C/G, Q381V, E385L, P436Q, N437D/K, Q474I/L, R476G, A505C, S550C, N588F, S604A/C/I/V, G616D, G626D, D650N/Y, D651E, S652D, V6741, T687C/K/W, Q690H/K, D709E, E710G, A732G/M/V, D733G, Y736N, L7571/K, S764F, T777N, T783A, T785L, K807R, M816L, D844G, V846F/L/Q, L869R, and P870S (wherein amino acid position is determined by optimal alignment with SEQ ID NO:2). In some embodiments, the Bgl1 polypeptide variant does not have a substitution at position A475. Beneficial combinations of the above-listed substitutions 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, 25, 30, 35, 40, 45, 50, or more of the above-identified positions.
As summarized in Table 6, in certain embodiments, the present invention provides an isolated and/or recombinant β-glucosidase polypeptide variant comprising an amino acid sequence that has greater activity and/or thermostability than the wild-type C1 Bgl1 protein, is at least about 70% identical to wild-type C1 β-glucosidase (Bgl1) (residues 20-870 of SEQ ID NO:2), has the substitutions D47I, Q258N, Q291W, Q313M, A343C, D369R, E402N, S434P, A475L, K495N, G628W, T687K, A689I, Y715P; and has at least one substitution selected from the group consisting of: A79E/G/M, Q85N, V260G, L275Y, F314L/V, D395N, P439S, A505C, D646N, T693A/E, N723G, A730S, A732G/M/V, S764Y, R769H, T827I, and Y855, (wherein amino acid position is determined by optimal alignment with SEQ ID NO:2). In some embodiments, the Bgl1 variant polypeptide has the substitution T687K or T687W in place of T687K. In some embodiments, the Bgl variant polypeptide does not have a substitution at position A475. Beneficial combinations of the above-listed substitutions include any combination of substitutions at any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or more of the above-identified positions.
As summarized in Table 7, in certain embodiments, the present invention provides an isolated and/or recombinant β-glucosidase polypeptide variant comprising an amino acid sequence that has greater activity and/or thermostability than the wild-type C1 Bgl1 protein, is at least about 70% identical to wild-type C1 β-glucosidase (Bgl1) (residues 20-870 of SEQ ID NO:2), has the substitutions I106V, Q258N, V260G, Q291W, Q313M, F314L/V, D369R, E402N, S434P, K495N, G628W, A689I, Y715P, and A732G; and has at least one substitution selected from the group consisting of: A109S/T, A343C/G, A475L, A505C, A79E/G/M, C8G, D47I, L275F/Y, N315D, Q85H/N, and T591I, T687C/K/W, (wherein amino acid position is determined by optimal alignment with SEQ ID NO:2). In some embodiments, the Bgl1 protein additionally has the substitution F314V. Beneficial combinations of the above-listed substitutions include any combination of substitutions at any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more of the above-identified positions.
The present invention further provides an isolated and/or recombinant β-glucosidase polypeptide variant having greater activity and/or thermostability than the wild-type C1 Bgl1 protein and having an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO:1 (e.g., over substantially the entire length of a nucleic acid exactly complementary to SEQ ID NO:1) wherein the encoded polypeptide has at least one substitution of an amino acid residue selected from the group consisting of K57; A88; I106; N112; Q119; T120; A123; R132; Y135; A136; A141; K142; L149; G158; P161; P172; T177; I179; G180; M181; S182; E183; K186; A197; G202; Y219; N220; S222; T224; 1229; M234; F242; A243; V246; Q258; D274; V2861; Q291; Q313; V318; A343; T354; T357; D358; E360; P374; I375; A378; Q381; D369; E385L; S388; V390I; A394; N398; E402; K406; I428; S434; N437; E449Q; Q474I; A475; T482; S489; Y491; K530; N536; T540; T565; V674; R682; I867; E868; P870 (wherein amino acid position is determined by optimal alignment with SEQ ID NO:2). Beneficial combinations of the above-listed substitutions 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 or more of the above-identified positions.
The present invention further provides an isolated and/or recombinant β-glucosidase polypeptide variant having greater activity and/or thermostability than the wild-type C1 Bgl1 protein and having an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO:1 (e.g., over substantially the entire length of a nucleic acid exactly complementary to SEQ ID NO:1); wherein the encoded polypeptide has a substitution at each of positions Q291, D369 and E402; and has at least one substitution of an amino acid residue at a position selected from the group consisting of I106, A109, Q119, T120, R132, Y135, M181, Q215, A243, V246, Q258, A265, D274, N278, Q313, F314, G332, T357, D358, P374, E385, S434, N437, A475, S489, Y491, E493, K495, K497, S501. A503E N504, A505, N521, K530, N536. T540, D566, T591, A601, K610, T611, R612, S614, L620, G628, T635, V638, V648, D650, S652, N670, R672, V674, S676, T685, T687, A689, Q690, T699, D703, K708, Y715, A732, E742, L757, V775, T777, and D781; (wherein amino acid position is determined by optimal alignment with SEQ ID NO:2). Beneficial combinations of the above-listed substitutions 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, or more of the above-identified positions.
The present invention further provides an isolated and/or recombinant β-glucosidase polypeptide variant having greater activity and/or thermostability than the WT C1 Bgl1 protein and having an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO:1 (e.g., over substantially the entire length of a nucleic acid exactly complementary to SEQ ID NO:1); wherein the encoded polypeptide has substitutions at each of positions Q258, Q291, Q313, D369, E402, S434, A475, K495, and G628; and has at least one substitution of an amino acid residue at a position selected from the group consisting of A4, A15, E21, S22, K24, V25, H26, Q27, P29, L30, N45, D47, Q55, S58, A79, Q85, I106, A109, Q119, Y135, A136, P161, V175, G180, G202, G216, I221, L237, D244, V253, V260, L275, D311, F314, A343, D358, E360, Q381, E385, A394, A404, A405, K421, I428, P436, N437, M454, D470, R476, A477, Q479, T482, Y491, E493, E494, T496, S501, A505, N536, V559, V562, N588, S604, R612, G616, A617, G626, N627, F634, D646, D650, S652, R672, V673, T685, T687, A689, Q690, K708, Y715, Q716, A732, A748, D751, L757K, S764, R769, V775, T777, T783, T785, S787, S799, P806, K807, R817, E819, E822, T823, V840, V846, I847, S848, Y850, K866, and P870; (wherein amino acid position is determined by optimal alignment with SEQ ID NO:2). Beneficial combinations of the above-listed substitutions 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, or more of the above-identified positions.
The present invention further provides an isolated and/or recombinant β-glucosidase polypeptide variant having greater activity and/or thermostability than the wild-type C1 Bgl1 protein and having an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO:1 (e.g., over substantially the entire length of a nucleic acid exactly complementary to SEQ ID NO:1) wherein the encoded polypeptide has at least one substitution of an amino acid residue selected from the group consisting of K57R; A88S; I106V; N112V; Q119E/L; T120M/Y/V/H; A123N; R132K/G/W; Y1351/Q/M; A136E; A141F; K142R; L149Q/M; G158D; P161S; P172A; T1771; 1179M; G180E; M181Y; S182L/W; E183G/M/Q/K; K186R; A197V; G202M/V; Y219V; N220Y/S/L; S222Q/E; T224N; I229M; M234E/I; F242L; A243V; V246L; Q258N; D274Y/N; V2861; Q291W/A/F; Q313M; V318E; A343V/T; T354Q; T357L/P; D358K/N; E360R/A/D; P374Y; V; A378K/T; Q381V/D/I/L; D369Q/L/Y/C/A/I/P/E/K/R/F/M/H/V; E385L; S388W/C; V390I; A394G/V/L/P/Q; N398G; E402N; K406D; I428V; S434P; N437F/Y/D/L/W; E449Q; Q474I; A475F/Y/H/W/C; T482A; S489L; Y491H/F; K530M/G; N536K; T540K; T565A/G/P; V674I; R682W; I867M; E868R; P870S (wherein amino acid position is determined by optimal alignment with SEQ ID NO:2). Beneficial combinations of the above-listed substitutions 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 or more of the above-identified positions.
The present invention further provides an isolated and/or recombinant β-glucosidase polypeptide variant having greater activity and/or thermostability than the wild-type C1 Bgl1 protein and having an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO:1 (e.g., over substantially the entire length of a nucleic acid exactly complementary to SEQ ID NO:1) wherein the encoded polypeptide has a substitution Q291W, D369H/L/R/Y, and E402N; and has at least one substitution of an amino acid residue selected from the group consisting of A109S, A243V, A265S, A475C/F/L/W, A503E, A505C, A601T, A689I, A732G/M, D274Y, D358E/K/N, D369H/L/R/Y, D566G D650F/V, D703K, D781N, E385L, E402N, E493A/V/Y, E742G, F314V, G332D, G628L/V/W, I106V, K495F/H/I/N/Q/V, K497R, K530C/D/E/I/M/N/V, K610S, K708F, L620M, L757K, M181Y, N278Y, N437D/F/I/K/L/V/W/Y, N504Y, N521C, N536K, N670D, P374Y, Q119E, Q215E/M, Q258H/N, Q291W, Q313M, Q690A/K/R, R132H, R612H/P, R672A/D/F/G/I/S/T/V, S434P, S489I/L/N/T, S501H/N/R, S614A/C/D/H/L/R/V/Y, S652D, S676C S764F, T120H/Y, T357A, T540K, T591A/C/R, T611A/H/Q, T635A/I/V, T685V. T687C/F/K/L/M/W/Y, T699L, T777N, T779S, V2461, V638E/R/S, V648W, V674M, V775C, Y135Q, Y491F/H/L, and Y715P (wherein amino acid position is determined by optimal alignment with SEQ ID NO:2). Beneficial combinations of the above-listed substitutions 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 or more of the above-identified positions.
The present invention further provides an isolated and/or recombinant β-glucosidase polypeptide variant having greater activity and/or thermostability than the wild-type C1 Bgl1 protein and having an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO:1 (e.g., over substantially the entire length of a nucleic acid exactly complementary to SEQ ID NO:1) wherein the encoded polypeptide has a substitution at position Q258N, Q291W, Q313M, D369R, E402N, S434P, A475L, K495N, and G628W; and has at least one substitution selected from the group consisting of A4V, A15V, A109S/T, A136L, A343C, A394G, A404S, A405T, A477G, A505C, A617V, A689I, A732G/M/S, A748T, A79E/G/M, D244H, D311N, D358K, D470N, D47I, D646N, D650F/N/Y, D751N, E21Q/R, E360D, E385L, E493A/V/Y, E494K, E819A/L/V, E822A/G/K/M, F314L/V, F634A, G180E, G202M, G216L, G616D, G626D, H26R, I106V, 1221V, I428V, I847T, K24G/L/T, K421R, K708F, K807R, K866I/Q, L237Y, L275Y, L30K, L757K, M454E, N437W, N45H, N536K, N588F, N627H, P161S, P29M/Q/R, P436E/Q, P806L, P870S, Q119E, Q27H/R, Q381V, Q479R, Q55R, Q690A/H/K, Q716R, Q85N, R476Q, R612H, R672G, R769H, R817P, S22L/R, S501C/R, S58G, S6041, S652D, S764F, S787G, S799N, S848N, T482A, T496A, T685V, T687C/K/M, T777N, T783H/Q, T785L, T823K, V175A, V253F, V25A/G/R, V260G/L, V559T, V562C/L, V673A, V775C, V8401, V846F, Y135M/Q, Y491F, Y715P, Y850H/Q, (wherein amino acid position is determined by optimal alignment with SEQ ID NO:2). Beneficial combinations of the above-listed substitutions 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 or more of the above-identified positions. In certain embodiments, the C1 Bgl1 protein has the substitutions Q258N, Q291W, Q313M, D369L, E402N, S434P, A475L, K495N, and G628W.
It will be appreciated that Bgl1 variants of the invention may encompass additional amino acid substitutions beyond those listed above (such as additional conservative substitutions) and may be less-than-full length compared to wild-type C1 Bgl1 protein. Thus, Bgl1 variants of the invention may comprise insertions or deletions (e.g., truncation at the amino- and/or carboxy-termini) relative to residues 20-870 of SEQ ID NO:2. The wild-type secreted form of C1 Bgl1 protein is about 851 amino acids in length; variants of the present invention may be longer or shorter than wild-type. For illustration and not limitation, in some embodiments the variant may be longer or shorter by up to 10% of the wild-type length, sometimes up to 5%, sometimes up to 4%, sometimes up to 3%, sometimes up to 2%, sometimes up to 1%.
Sequence-activity analysis of variants was performed in accordance with the methods described in WO 03/075129, U.S. Ser. No. 10/379,378 filed Mar. 3, 2003; R. Fox et al., 2003, “Optimizing the search algorithm for protein engineering by directed evolution,” Protein Eng. 16(8):589-597, and R. Fox et al., 2005, “Directed molecular evolution by machine learning and the influence of nonlinear interactions,” J. Theor. Biol. 234(2):187-199, all of which are incorporated herein by reference, to identify substitutions with likely the most significant effects on activity.
Certain β-glucosidase variants of the present invention have an amino acid sequence that includes at least one substitution of an amino acid residue selected from the group consisting of Q119E; R132H; Y135Q; G180E; G202M/V; Q258N; D274Y; Q291W/A/F; D358K/N; D369L/Y/V/A/H/R/F/E/M/I/K/C/P/Q; P374Y; E385L; A394L; E402N; S434P; N437F/D/L/W/Y; Q474I; A475F/Y/H/C/W; T482A; S489L; K530M; N536K; T540K, which are predicted to be highly beneficial substitutions for increasing β-glucosidase activity.
Certain β-glucosidase variants of the present invention have an amino acid sequence that includes the substitutions Q291W+D369L+E402N (as found in Variant 3), or the substitutions Q291W+D369H/L/R/Y+E402N. In some embodiments, the sequence includes at least one substitution of an amino acid residue selected from the group consisting of Q258N/H, D358K/N, I106V, E385L, D274Y, K495H/I/N/Q, Q119E, Y135Q, G628W, R132H, N437I, S501R, P374Y, N437V, S614A, Q313M, L369H, S434P, N437W/D, and Y491F. In some embodiments, the at least one substituted amino acid residue is Q258N. In some embodiments, the at least one substituted amino acid residue is D358K or Q258H. In some embodiments, the at least one substituted amino acid residue is I106V, E385L, D274Y, or K495H. In some embodiments, the at least one substituted amino acid residue is K495I/N, Q119E, Y135Q, or D358N. In some embodiments, the at least one substituted amino acid residue is G628W, R132H, K495Q, N437I, S501R, or P374Y. In some embodiments, the at least one substituted amino acid residue is N437V, S614A, Q313M, L369H, S434P, N437W/D, or Y491F. These substitutions are predicted to be highly beneficial substitutions for increasing β-glucosidase activity.
Certain β-glucosidase variants of the present invention have an amino acid sequence that includes the substitutions Q258N, Q291W, Q313M, D369R, E402N, S434P, A475L, K495N, and G628W (as found in Variant 269), and least one substitution of an amino acid residue selected from the group consisting of Y715P, S764F, E385D/L, A732G/M, A689I, A109T, T687M/C/K, L757K, Q381V/D, A109S, T777N, I106V, T823K, Q716R, V846F, T685V, T687K, D650Y, F314V/L, S652D, and I428V. In some embodiments, the at least one substituted amino acid residue is Y715P. In some embodiments, the at least one substituted amino acid residue is S764F, E385L, A732G, A689I, or A109T, or E285D. In some embodiments, the at least one substituted amino acid residue is T687M, A732M, or L757K. In some embodiments, the at least one substituted amino acid residue is Q381V, A109S, T777N, I106V, T823K, T687C, or Q716R. In some embodiments, the at least one substituted amino acid residue is V846F, T685V, T687K, D650Y, F314V/L, S652D, Q381D, or I428V. These substitutions, are predicted to be highly beneficial substitutions for increasing β-glucosidase activity.
Certain β-glucosidase variants of the present invention have an amino acid sequence that includes the substitutions Q258N, Q291W, Q313M, D369R, E402N, S434P, A475L, K495N, G628W, A689I, and Y715P (as found in Variant 481); or the substitutions 258N, Q291W, Q313M, D369R, E402N, S434P, K495N, G628W, A689I, and Y715P; and least one substitution of an amino acid residue selected from the group consisting of A79E/M/G, Q85N, I106V, A109S/T, V260G, F314L/V, A343C/G, A505C, T687K/W/C, and A732G/M/V. In some embodiments, the at least one substituted amino acid residue is T687W. In some embodiments, the at least one substituted amino acid residue is T687C or T687W, V260G, A343C, A732G, or A109T. In some embodiments, the at least one substituted amino acid residue is F314V/L, A732M/V, A109S, or A343G. In some embodiments, the at least one substituted amino acid residue is A79E, A505C, I106V, or A79M/G. In some embodiments, the variant These substitutions, are predicted to be highly beneficial substitutions for increasing β-glucosidase activity.
Certain β-glucosidase variants of the present invention have an amino acid sequence that includes the substitutions D47I, Q258N, Q291W, Q313M, A343C, D369R, E402N, A475L, S434P, K495N, G628W, T687K, A689I, and Y715P (as found in Variant 647) or the substitutions D47I, Q258N, Q291W, Q313M, A343C, D369R, E402N, S434P, A475L, K495N, G628W, T687W, A689I, and Y715P; or the substitutions D47I, Q258N, Q291W, Q313M, A343C, D369R, E402N, S434P, K495N, G628W, T687K/W/C, A689I, and Y715P; and least one substitution of an amino acid residue selected from the group consisting of A79G/E/M, Q85N, V260G, L275Y, and F314L/V, and A732G/M. In some embodiments, the at least one substituted amino acid residue is A79E, A79M, A79G, A732G, V260G, or F314L. In some embodiments, the at least one substituted amino acid residue is Q85N, F314V, or A732M. These substitutions, are predicted to be highly beneficial substitutions for increasing β-glucosidase activity.
Certain β-glucosidase variants of the present invention have an amino acid sequence that includes the substitutions I106V, Q258N, V260G, Q291W, Q313M, F314L/V, D369R, E402N, S434P, K495N, G628W, A689I, Y715P, and A732G (as found in Variant 664) and least one substitution of an amino acid residue selected from the group consisting of D47I, A79G/E/M, A109T, A505C, A343C/G, T687W/C/K, N315D, and L275F. In some embodiments, the at least one substituted amino acid residue is A79E or A79G. In some embodiments, the at least one substituted amino acid is A109T, A79M, A505C, or A343C. In some embodiments, the at least one substituted amino acid is T687W, T687C, D47I, T687K N315D, or A343G. These substitutions, are predicted to be highly beneficial substitutions for increasing β-glucosidase activity.
Certain β-glucosidase variants of the present invention have an amino acid sequence that includes at least one substitution of an amino acid residue selected from the group consisting of I106V; A123N; G180E; M181Y; M234E; Q258N; Q291W/F/A; T357L/P; D358K; E360D; D369L/V/C/Y/R/K/M/A/H/E/F/Q/P/I; P374Y; E385L; A394G; N437D/L; T482A; S489L; K530M; N536K; T540K, which are predicted to be highly beneficial substitutions for increasing thermostability.
Certain β-glucosidase variants of the present invention have an amino acid sequence that includes the substitutions Q291W, D369H/L/R/Y, and E402N; and at least one substitution of an amino acid residue selected from the group consisting of Q313M, Q258N/H, N536K, Q119E, S489I/N/L/T, T120Y, K495N/V/I/H/Q, I106V, T120H, N437I/D/W/F/K/L/V/Y, E385L, M181Y, D274Y, S434P, S501R, T591C, Y135Q, Y491H/F, T540K, N521C, T591R, G628W, and A475L. In some embodiments, the at least one substituted amino acid residue is Q313M or Q258N. In some embodiments, the at least one substituted amino acid residue is N437I, K495N, Q258H, or N536K. In some embodiments, the at least one substituted amino acid residue is Q119E, S489I, T120Y, K495V, S489L, I106V, T120H, or N437D. In some embodiments, the at least one substituted amino acid residue is N437W, K495I, N437F, E385L, K495H, N437K, N437L, S489T, M181Y, D274YS434P, or N437V. In some embodiments, the at least one substituted amino acid residue is S501R, T591C, Y135q, Y491H, R491F, T540K, N521C, T591R, K495K, G628W, N437Y, S489N, or A475L. These substitutions are predicted to be highly beneficial substitutions for increasing thermostability.
Certain β-glucosidase variants of the present invention have an amino acid sequence that includes the substitutions Q258N, Q291W, Q313M, D369R, Q381V, E385L, E402N, S434P, A475L, K495N, and G628W; and least one substitution of an amino acid residue selected from the group consisting of S764F, Y715P, E385L/D, A732G/M, A109T/S, T687M/C/K, T777N, I106V, L475A, F314V, D650Y, L757K. Q716R, T685V, or F314L. In some embodiments, the at least one substituted amino acid residue is S764F or Y715P. In some embodiments, the at least one substituted amino acid residue is E385L or A732G. In some embodiments, the at least one substituted amino acid residue is A109T or T687M. In some embodiments, the at least one substituted amino acid residue is T777N, I106V, !732M, T687C, T687K, E385D, L475A or A109S. In some embodiments, the at least one substituted amino acid residue is P720P, F314V, D650Y, L757K, Q716R, T685V, or F314L. These substitutions are predicted to be highly beneficial substitutions for increasing thermostability.
Certain β-glucosidase variants of the present invention have an amino acid sequence that includes the substitutions Q258N, Q291W, Q313M, D369R, E402N, S434P, A475L, K495N, G628W, A689I and 715P (as found in Variant 481); and least one substitution of an amino acid residue selected from the group consisting of D47I, A79E, Q85N, I106V, A109S/T, V260G, L275Y, F314L/V, A505C, T687W/C/K, and A732G/M/V. In some embodiments, the at least one substituted amino acid residue is T687K/W/C. In some embodiments, the at least one substituted amino acid is I106V, V260G, F314V, A732G, A109T, F314L, A732M, Q85N, or A109S. In some embodiments, the at least one substituted amino acid is F314, A732V, A505C, D47I, L275Y, or A79E. These substitutions are predicted to be highly beneficial substitutions for increasing thermostability.
Certain β-glucosidase variants of the present invention have an amino acid sequence that includes the substitutions D47I, Q258N, Q291W, Q313M, A343C, D369R, E402N, S434P, A475L, K495N, G628W, T687K/W, A689I, and Y715P (as found in Variant 647); or the substitutions D47I, Q258N, Q291W, Q313M, A343C, D369R, E402N, S434P, K495N, G628W, T687K/W, A689I, and Y715P; and least one substitution of an amino acid residue selected from the group consisting of A79E/G/M, Q85N, V260G, L275Y, F314V/L, D395N, and A732G, which are predicted to be highly beneficial substitutions for increasing thermostability. In some embodiments, the at least one substituted amino acid residue is A79M, F314V, or A79G. In some embodiments, the at least one substituted amino acid residue is A79E, F314L, V260G, A85N, D395N, or A732G.
Certain β-glucosidase variants of the present invention have an amino acid sequence that includes the substitutions I106V, Q258N, V260G, Q291W, Q313M, F314L/V, D369R, E402N, S434P, K495N, G628W, A689I, Y715P, and A732G (as found in Variant 664) and least one substitution of an amino acid residue selected from the group consisting of A109S/T, A79G/E/M, D47I, Q85N/H, L314V, and T5911. In some embodiments, the at least one substituted amino acid residue is A109S, A79G, A109T, or A79E. In some embodiments, the at least one substituted amino acid residue is D47I, A79M, A85N, or A85H. These substitutions, are predicted to be highly beneficial substitutions for increasing thermostability.
Certain β-glucosidase variants of the present invention have an amino acid sequence that includes at least one substitution of an amino acid residue at a position selected from the group consisting of G180E; Q258N; Q291W/A/F; D358K; D369L/Y/V/A/H/R/F/E/M/I/K/C/P/Q; P374Y; E385L; N437D/L; T482A; S489L; K530M; N536K; and T540K, which are predicted to be highly beneficial substitutions for increasing both thermostability and β-glucosidase activity.
Certain β-glucosidase variants of the present invention have an amino acid sequence that includes Q291W, D369H/L/R/Y, and E402N; and least one substitution of an amino acid residue selected from the group consisting of Q258N/H, I106V, E385L, D274Y, K495H/I/N/Q, Q119E, Y135Q, G628W, N437I/V/W/D, S501R, Q313M, S434P, and Y491F, which are predicted to be highly beneficial substitutions for increasing both thermostability and β-glucosidase activity.
Certain β-glucosidase variants of the present invention have an amino acid sequence that includes Q258N, Q291W, Q313M, D369R, E402N, S434P, A475L, K495N, and G628W; or Q258N, Q291W, Q313M, D369R, E402N, S434P, K495N, and G628W; and least one substitution of an amino acid residue selected from the group consisting of S764F, Y715P, E385L/D, A732G/M, A109T/S, T687M/C/K, T777N, I106V, F314V, D650Y, L757K. Q716R, T685V, and F314L. the substitutions are predicted be highly beneficial substitutions for increasing both thermostability and β-glucosidase activity.
Certain β-glucosidase variants of the present invention have an amino acid sequence that includes Q258N, Q291W, Q313M, D369R, E402N, S434P, A475L, K495N, G628W, A689I and 715P (as found in Variant 481); and least one substitution of an amino acid residue selected from the group consisting of A79E, Q85N, T687K/W/C, I106V, A109T/S, V260G, F314V/L, A505C, and A732G/M/V. The substitutions are predicted be highly beneficial substitutions for increasing both thermostability and β-glucosidase activity.
Certain β-glucosidase variants of the present invention have an amino acid sequence that includes D47I, Q258N, Q291W, Q313M, A343C, D369R, E402N, S434P, A475L, K495N, G628W, T687K/W, A689I, and Y715P (as found in Variant 647); or the D47I, Q258N, Q291W, Q313M, A343C, D369R, E402N, S434P, K495N, G628W, T687K/W, A689I, and Y715P; and least one substitution of an amino acid residue selected from the group consisting of A79E/G/M, Q85N, V260G, F314V/L, A732G, and L275Y. In some embodiments, the substituted amino acid residue is A79E, A79M, A79G, Q85N, V260G, F314V, F314L, or A732G. The substitutions are predicted be highly beneficial substitutions for increasing both thermostability and β-glucosidase activity.
Certain β-glucosidase variants of the present invention have an amino acid sequence that includes the substitutions I106V, Q258N, V260G, Q291W, Q313M, F314L/V, D369R, E402N, S434P, K495N, G628W, A689I, Y715P, and A732G (as found in Variant 664) and least one substitution of an amino acid residue selected from the group consisting of D47I, A79E/M/G, and A109T, which are predicted be highly beneficial substitutions for increasing both thermostability and β-glucosidase activity.
Notably, a number of variants with superior activity and/or thermostability comprise substitutions at position 369, with 14 different alternative (i.e., non-aspartic acid) residues appearing in beneficial variants. Substitutions at position 369 are predicted to increase activity and/or thermostability. Thus, in one aspect the invention provides an isolated and/or recombinant β-glucosidase polypeptide variant comprising an amino acid sequence that is at least about 70% identical to wild-type C1 β-glucosidase (Bgl1) (residues 20-870 of SEQ ID NO:2) wherein the amino acid at position 369 is not aspartic acid. In some embodiments the amino acid at position 369 is selected from the group consisting of Q, L, Y, C, A, I, P, E, K, R, F, M, H, and V. In some embodiments the amino acid at position 369 is L (leucine), which appears particularly beneficial for β-glucosidase and thermostability.
A number of variants with superior activity and/or thermostability comprise substitutions at position 291. Substitutions at position 291 are predicted to increase activity and/or thermostability. Thus, in one aspect the invention provides an isolated and/or recombinant β-glucosidase polypeptide variant comprising an amino acid sequence that is at least about 70% identical to wild-type C1 β-glucosidase (Bgl1) (residues 20-870 of SEQ ID NO:2) wherein the amino acid at position 291 is not glutamine. In some embodiments the amino acid at position 291 is W, A, or F. In some embodiments the amino acid at position 369 is W (tryptophan), which appears particularly beneficial for β-glucosidase and thermostability.
A number of variants with superior activity and/or thermostability comprise substitutions at position 402. Substitutions at position 402 are predicted to increase activity and/or thermostability. Thus, in one aspect the invention provides an isolated and/or recombinant β-glucosidase polypeptide variant comprising an amino acid sequence that is at least about 70% identical to wild-type C1 β-glucosidase (Bgl1) (residues 20-870 of SEQ ID NO:2) wherein the amino acid at position 402 is not glutamic acid. In some embodiments, the amino acid is neither glutamic acid nor aspartic acid. In some embodiments, the amino acid is not glutamic acid, aspartic acid, or proline. In some embodiments, the amino acid at position 402 is asparagine, glutamine, histidine, lysine, or arginine. In some embodiments, the amino acid at position 402 is asparagine or glutamine. In some embodiments the amino acid at position 402 is N (asparagine), which appears particularly beneficial for β-glucosidase and thermostability.
In some embodiments, the isolated and/or recombinant β-glucosidase polypeptide variant of the present invention is at least about 70% identical to wild-type C1 Bgl1 (residues 20-870 of SEQ ID NO:2) and comprises a substitution set selected from the group consisting of: M181Y+Q291W+E402N+S434P; R132K+L149M+Q313M+D369L+E385L+N437D; Q291W+D369L+E402N; Y135I+Q258N+Q474I; M181Y+Q291W+E360D+D369V+P374Y+T482A; Q258N+N437F+S489L+Y491H; Q119E+Q258N+T357L+Q474I+S489L; Q258N+D369R+S489L+Y491H; N220Y+Q258N+T357L+D369R+Q474I+Y491F; M234E+V246L+D358K+D369L+N398G+K530M; Y135Q+I229M+F242L+D369L+K530M; D369Q+P374Y+E402N+T540K; Y135Q+P172A+I179M+1229M+Q291A+D358K+D369L+N398G; R132K+D369L+E385L; Y135M+I179M+Q291A+D358K+D369L; Q291W+P374Y+E402N+S434P; Q119E+N220Y+Q258N+T357L+S489L; I179M+Q291A+D358K+D369L+I375V+S388W; Q291W+D369C; R132K+354Q+D369L+N437L; Q291A+D369L+N398G+K530M; Q119E+Q258N+D274Y+S489L; Q119E+N220Y+Q258N+Q474I+S489L; M181Y+D369L; T120M+5222E+Q313M+T354Q+D369L+E385L; A4G+Q258N+D274Y+T357L+N437W+Q474I+Y491H; R132G+D369L+N437D; S182L+Q313M+D369L+E385L; R132G+D369L+E385L; N112V+D358K+D369L+S388W+K530M; I106V+G180E+D369L+Q381V; I179M+Q291A+D369L; I179M+D358K+D369L+S388W; M234E+Q291A+D369L+N398G; I179M+Q291A+D358K+D369L+Q381I; Y135Q+N220L+Q291A+D369L+N398G; Y135I+D369L; S182L+D369L+E385L+N437L; T120Y+R132K+D369L+N437D; M234E+D369L+S388W+N398G+K530M; Y135M+I179M+D369L+V390I+K530M; L149Q+A197V+Q313M+D369L; T120M+R132K+D369L+N437L; T120M+R132W+L149M+Q313M+T354Q+D369L+E385L+N437D; A4G+Y135I+N220Y+Q258N+T357P+N437Y; D358K+D369L+S388W; Q258N+D274Y+N437F; D369L+S434P+T540K; G158D+I179M+Q291A+D358K+D369L+I375V+N398G+K530M; Y135I+D274Y+D369R; Q258N+D369L+Q474I+S489L+Y491F; R132W+S182W+D369L+E385L; S182L+T354Q+D369L+E385L; S222Q+T354Q+D369L+E385L+N437D+T565G; Y135M+P161S+Q291A+A343T+D369L+I375V+K406D; Q291W+D369C+T540K; D369L+P374Y+E402N; Y135I+A343V+D369F+S489L; M234E+D358K+D369L+S388W; T120M+L149Q+D369L+E385L; Q313M+D369L+E385L; T120Y+D369L+E385L; D369L+N437D+T565A; N112V+Q291A+D369L+I375V; R132W+L149Q+Q313M+T354Q+D369L; Y135M+D369L+I375V+N398G; D369L+E385L; T120M+S182L+Q313M+T354Q+D369L+E385L; T177I+Q291W+P374Y+T482A; Q258N+N437F; T120V+R132W+D369L+T565G; N112V+Q291A+D358N+D369E+S388C+K406D; Q291A+D358K+D369L+S388W+K406D; I106V+E360R+D369L+Q381V; M234E+Q291A+D369L+S388W+N398G; L149M+Q313M+D369L; M234I+Q291W+E360D+D369V+T482A; T120V+T354Q+D369L+E385L+T565P; M234I+Q291W+E360D+S434P; D369Y+1867M+E868R; R132W+S182L+Q313M+D369L; M234I+Q291W+P374Y+T482A; Y219V+M234I+D369C+P374Y+S434P; S182L+Q313M+D369L+E385L+N437L; M234E+D358K+D369L+S388W+K530M; R132G+S222E+D369L+E385L; R132K+L149M+S182L+D369L+N437L; T120H+Q313M+D369L; R132W+D369L; D369L+P374Y; N112V+N220L+Q291A+D369L+S388W+N398G; Q291A+D369L+I375V+K530G; R132K+L149M+S182L+T354Q+D369L+N437D+T565G; T120H+ S222E+Q313M+D369L+N437D+T565G; A123N+Q291W+T482A+T540K; S222E+Q313M+T354Q+D369L+E385L; R132W+D369L+T565G; G202M+E360A+D369I+A394L; R132G+S222Q+Q313M+D369L; R132W+L149M+D369L; L149M+Q313M+T354Q+D369L; Q119L+G202M+D369L; M181Y+M234I+D369C; I179M+N220L+Q291A+D369L+I375V; S222E+Q313M+D369L+E385L+N437L; I179M+M234E+D358K+D369L+S388W+N398G; A123N+Y219V+Q291W+E360D+P374Y+S434P; D369L+E402N; D369L; Y219V+M234I+Q291W+T482A; Q291W+E360D+S434P; D369L+S434P; G180E+E360R+D369L; A123N+M181Y+Q291W+D369K+S434P+T540K; Q119E+T357L+D369M+S489L; Q119E+D369F+Y491H; Q119L+D369L; Q119E+N220Y+V286I+S489L; Q291A+D369L+Q381I; A475F; D369L+N536K; Q119E+Y135I+D369H; Q258N+S489L; Q119E+Y135+N437Y; Q119E+Y135I+N437F+Y491F; Q119L+D369L+A394V; E183G+E360A+D369L+I428V; D369L+E449Q+N536K; Q119L+G202M+E360A+A475F; I106V+D369L; Y135I+D369M; M234I+D369C+S434P; D369L+A475Y; Q119E+Y135I+S489L; D369Y+N536K; E360R+D369L; G202V+A475H; D369L+Q381V+N536K; N220Y+Q258N+S489L+Y491F; Q258N+T357L+D369M; I179M+Q291A+D369L+Q381L+S388W+N398G; D369Y+A394G+N536K; Q291W+E360D+D369V+P374Y; T120M+L149Q+T354Q+D369L+E385L; S489L+Y491H; N220S+Q291F+D369L; D369C+S434P+T540K; V318E+D369L+I428V; E183M+G202M+E360A+D369L+A378K+A394V; A394G+N536K; Q291W+T540K; N220L+Q291A+D369L+Q381L+S388W+K530M; T120V+R132W+E385L+N437D; Q119L+E360A+D369L+A378K; D369A+N536K; G202V+D369L+A475H; Q381V+A475Y+N536K; S434P; I106V+G180E+D369Y+A394G; G180E+Q381V+A475H; Q119E+N220Y+Q474I; I106V+D369Q; A475Y+N536K; K142R+Y219V+Q291W+S434P+V6741; L149Q+S182L+Q313M+D369L+N437L; N112V+I179M+M234E+D369L+N398G; E360A+D369L+A378K; E360R+D369Y+N536K; E360R+D369A+Q381V+N536K; D369L+Q381D; N437F+S489L; E183M+G202M+V318E+D369I+A394L+I428V; E360D+D369L+E402N+S434P; Q119L+A141F+G202M+A394L+I428V+A475F; T357L+D369R+S489L+Y491H; Y135I+Q258N+T357L; K142R+Q291W+E360D+D369C+E402N; E183M+A243V+D369L+A378K+A475F; R132K+L149M+E385L; D369Y; M234I+E402N+S434P; N437Y; Q119E+N437F; N536K; Q119L+E183Q+G202M+D369P; N112V+I179M+Q291A+D358K+K406D; A123N+Q291W+T540K; D369I+A394L+1428V; A88S+N536K; Q119E+Y135I+N437F; A141F+G202M+E360A+D369P+A378K; A123N+T482A; Q313M+N437D; E360R+D369Y; E183M+G202M+A475F; Q119L+E360A+A394V+A475F; D369L+A378K; E360R+D369L+A394G; M181Y+D369E+S434P; D3691; N112V+Y135Q+I375V+K406D+K530M+P870S; Q119E+Y135I+N220Y+Q258N; D369P+A394V+I428V; V318E+D369L; K186R+N536K; Q119L+D369L+A378K; M234I+D369K+S434P; N112V+I179M+N220L+Q291A+Q381I+S388W+N398G; E402N+S434P; Q119L+A141F+G202M+A394Q; Q313M+T354Q+N437D; N112V+M234E+D369L+I375V+K406D; Q119E+A136E+N220Y; Q381D+A394G+N536K; Q119L+E183G+D369Q+A378T+V390I; Y135I+T357L+Q474I+S489L+Y491F; D369E+A394P+I428V; N112V+Q291A+Q381I+N398G; E360R+N536K; E360D+D369C; R132W+S182L+E385L; I179M+Q291A+D358K+N398G+K530G; N220Y+Q258N+T357L; T224N+D274Y+T357L+N437F; N437D; M234I+E360D+T482A; A141F+G202M+D274N+V318E+E360A+I428V; D369Q; N112V+N220L+D358K+D369L+I375V+Q381I+N398G; Y135I+D369F; A243V+V318E+E360A+A475W; N220L+D369L+Q381L+S388W+N398G+K530G; S489L; K142R+Y219V; Y135I+N220Y+Y491F; S182W+T354Q+E385L; I179M+D358K+S388W; L149M+Q313M+T565P; R132G+E385L; V318E; Q119L+E183K; R132W+E385L; E183G+V318E+E360A+A394Q+A475C; E183M+G202M+E360A; A394G; D369P; Y135M+Q291A+S388W+N398G; Y219V+D369C; A394V+I428V; Q119L; Y1351; E360R+D369Q+Q381D+N536K; N220L+M234E+Q291A+I375V+K530M; Q119L+G202M+E360A; N220Y+Q258N+D369R; M181Y+M234I+Q291W+E402N; A394V; T120M+L149Q+Q313M; Q119L+V318E+I428V; E360R+Q381V; K57R+G202M+E360A+A394V; I179M+D358K+I375V+Q381L; E360A; I179M+R682W; E360A+I428V; and D369K+P374Y (where amino acid position is determined by optimal alignment with SEQ ID NO:2).
In some embodiments, the isolated and/or recombinant β-glucosidase polypeptide variant of the present invention is at least about 70% identical to wild-type C1 Bgl1 (residues 20-870 of SEQ ID NO:2) and comprises a substitution set selected from the group consisting of: I106V+Q258N+Q291W+D369L+E402N+S434P; I106V+Y135Q+Q258N+Q291W+Q313M+D369H+E402N+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; I106V+Q258N+Q291W+Q313M+D369H+E402N+S434P+A475C+K495I+T540K+G628W; I106V+Q258N+Q291W+D369R+E402N+S434P+K495H+G628L; D274Y+Q291W+D358K+D369L+E385L+E402N+N437I+S489N; Q258N+Q291W+Q313M+D369H+E402N+S434P+T540K; I106V+Q258N+Q291W+D369L+E402N+S434P+A475F+K495H+G628V; Q258N+Q291W+Q313M+D369L+E402N+S434P+A475C+K495I+G628W; I106V+Y135Q+M181Y+Q258N+Q291W+Q313M+D369L+E402N+S434P+K495N; Y135Q+Q258N+Q291W+D369L+E402N+K495N; Q119E+D274Y+Q291W+D358K+D369L+E385L+E402N+N437V+S489N+5614A; Y135Q+Q258N+Q291W+Q313M+D369L+E402N+S434P+A475F+K495N+G628V; R132H+Q258N+Q291W+D369Y+E402N+K495Q; Y135Q+Q258N+Q291W+D369L+E402N+S434P+A475F+K495H; Y135Q+Q258N+Q291W+D369R+E402N+S434P+K495I+G628V; I106V+M181Y+Q258N+Q291W+D369L+E402N; I106V+Q258N+Q291W+D369H+E402N+S434P+A475F+K495F+G628V; Q258N+Q291W+Q313M+D369L+E402N+S434P+G628V; Q258N+Q291W+D369L+E402N+A475F+K495I; Q119E+D274Y+Q291W+D369L+E385L+E402N+N4371; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L; Q258N+Q291W+D369Y+E402N+S434P+K495H+G628W; D274Y+Q291W+D369L+E385L+E402N+N437I+S489N+K530C; I106V+Y135Q+Q258N+Q291W+Q313M+D369Y+E402N+S434P+A475F+K495H+T540K; Q119E+Q291W+D358K+D369L+E385L+E402N; I106V+M181Y+Q258N+Q291W+Q313M+D369H+E402N+S434P+A475L+K495Q+G628V; Q119E+D274Y+Q291W+D358N+D369L+E385L+E402N+N437D; Q258N+Q291W+D369R+E402N+S434P+G628V; Q258N+Q291W+Q313M+D369H+E402N+S434P+G628L; Q258N+Q291W+D369L+E402N; D274Y+Q291W+D358K+D369L+E385L+E402N+N437F+S614D; Q119E+D274Y+Q291W+D358N+D369L+E385L+E402N+N437L+K530V; Q119E+D274Y+Q291W+D358N+D369L+E385L+E402N+N437V+S489L+K530N; Q258N+Q291W+D369L+E402N+S434P+A475F+K495F+T540K; M181Y+Q291W+Q313M+D369Y+E402N; A243V+D274Y+Q291W+D358K+D369L+E402N+N437I+K530C; Q119E+D274Y+Q291W+D358E+D369L+E385L+E402N+N437V+K530V+S614A; Q291W+D358K+D369L+E385L+E402N+N437W+K530C+S614A; Q119E+D274Y+Q291W+D369L+E385L+E402N+N437W; D274Y+Q291W+D358N+D369L+E385L+E402N+N437I+K530N+S614V; M181Y+Q258N+Q291W+Q313M+D369L+E402N+S434P; A109T+Q291W+D369L+E402N; Q119E+Q291W+D369L+E385L+E402N+N437Y+S489I+K530N; Q119E+D274Y+Q291W+D358N+D369L+E402N+N437D; D274Y+Q291W+D358K+D369L+E385L+E402N+N437W+K530V+S614D; Q258N+Q291W+D369R+E402N+A475W+K495H+G628V; A109S+Q291W+D369L+E402N; Q119E+D274Y+Q291W+D358E+D369L+E385L+E402N+N437L+S614A; Q291W+D369L+E402N+E493Y+N504Y+T611A; Q119E+D274Y+Q291W+D358K+D369L+E402N+S614V; D274Y+Q291W+D358E+D369L+E385L+E402N+N437L+K530N+S614A; Q258H+Q291W+Q313M+D369R+E402N+A475F+K495N+G628L; Q258N+Q291W+Q313M+D369Y+E402N+A475W+K495V+T540K+G628W; D274Y+Q291W+D358N+D369L+E385L+E402N+N437W+S614V; I106V+Y135Q+Q291W+Q313M+D369L+E402N+A475L+K495Q; Q258N+Q291W+D369R+E402N+S434P+A475F+G628V; Q119E+D274Y+Q291W+D369L+E385L+E402N+N437V; D274Y+Q291W+D358K+D369L+E385L+E402N+N437Y+S489L+K530V; Q119E+D274Y+Q291W+D358N+D369L+E402N; Q291W+D369L+E402N+K495V+S501R+A503E+K530N+T611H; Q119E+D274Y+Q291W+D358N+D369L+E385L+E402N+N437V+S489I+K530C; Q119E+Q291W+D358K+D369L+E402N+N437L+S489N; D274Y+Q291W+D369L+E385L+E402N+N437W+K530C+S614D; Q258N+Q291W+D369R+E402N+A475F+K495N+T540K+G628V; Q291W+D369L+E402N+E493V+N504Y; Q291W+D358E+D369L+E385*+E402N+S489T; Q258N+Q291W+Q313M+D369R+E402N+A475L+K495V+A601T+G628W; D274Y+Q291W+D358N+D369L+E385L+E402N+S489N+S614C; D274Y+Q291W+D358K+D369L+E402N+N437V+S489L+K530C+S614A; Q119E+D274Y+Q291W+D358E+D369L+E402N+N437D; Q119E+D274Y+Q291W+D358N+D369L+E402N+N437L+S614R; D274Y+Q291W+D358N+D369L+E402N+N437Y+K530V; D274Y+Q291W+D358E+D369L+E402N+N437Y+S489N+K530N+S614V+D781N; Q258H+Q291W+D369*+E402N+S434P+T540K+G628L; Q119E+Q291W+D358E+D369L+E385L+E402N+N437L+S489N+K530V; Q258N+Q291W+D369L+E402N+G628V; Q291W+D358K+D369L+E385L+E402N+S489I+K530N; Q119E+Q291W+D369L+E385L+E402N+S489N+K530V+S614A; I106V+Y135Q+Q291W+D369L+E402N+S434P+A475F+K495H+G628L; Q119E+Q291W+D358K+D369L+E402N+N437V+K497R+S614A; Q119E+D274Y+Q291W+D358E+D369L+E385L+E402N+N437L; D274Y+Q291W+D358K+D369L+E402N+N437L+S489I+K530V+S614A; I106V+Q291W+Q313M+D369L+E402N+S434P+A475W+K495N+G628W; Q291W+D369L+E402N+A505C+L620M+T635I; Q291W+D369L+E402N+E493A+N504Y+A505C+L620M+T635A; D274Y+Q291W+D358N+D369L+E402N+S489N+K530C+S614A; D274Y+Q291W+D358K+D369L+E385L+E402N+N437W+K530D; Q291W+D369L+E402N+S489N+K495H+S501R+K530N; D274Y+Q291W+D358E+D369L+E385L+E402N+N437D+K530C+S614H; Q119E+Q291W+D358E+D369L+E385L+E402N+N437W+S489N+K530E+S614A; Q291W+D369L+E402N+E493Y+N504Y+N521C+T591A+R612P+L620M+T635I; Q291W+D358K+D369L+E385L+E402N+N437I+K530M+S614L; Q291W+D369L+E402N+R6721; D274Y+Q291W+D358E+D369L+E385L+E402N+N437L+S489L; A265S+Q291W+D369L+E402N; Q215M+Q291W+D369L+E402N; Q291W+D369L+E402N+E493V+N504Y+N521C+T591A+L620M+T635I; Q119E+D274Y+Q291W+D369L+E385L+E402N+N437L+S489N+K530M+S614D; Q119E+Q291W+D369L+E385L+E402N; Q291W+D369L+E402N+E493A+N504Y+D566G+R612P+L620M+T635A; Q119E+D274Y+Q291W+D369L+E385L+E402N+S614Y; I106V+Y135Q+M181Y+Q258N+Q291W+D369L+E402N; Q119E+Q291W+D358E+D369L+E385L+E402N+N437W+K530V; Q215E+Q291W+D369L+E402N; Q291W+D369L+E402N+N504Y+N521C+T591A+R612H+L620M+T635I; Q119E+D274Y+Q291W+D358K+D369L+E402N+N437Y+K530I; Q258H+Q291W+D369L+E402N+K495N; Q291W+D358N+D369L+E385L+E402N+N437D+S489N+K530I; Q291W+D369L+P374Y+E402N+Y491L+S501R; Q258N+Q291W+Q313M+G332D+D369H+E402N+S434P; Q291W+D358N+D369L+E385L+E402N+N437F+S489L+K530V; Q291W+D369L+E402N+R672S; Q291W+D369L+E402N+T687M; Q291W+F314V+D369L+E402N; Q258N+Q291W+T357A+D369H+E402N+S434P+K495F; Q291W+D369L+P374Y+E402N+Y491F+S501R+N521C; Q291W+D369L+E402N+Q690K; Q291W+D358E+D369L+E385L+E402N+N437F+K530V+S614A; Q291W+D369L+E402N+R672A; Q291W+D369L+E402N+R672T; Q291W+D369L+E402N+D703K; D274Y+Q291W+D369L+E402N; Q291W+D369L+E402N+R672F; Q291W+D369L+E402N+R672D; Q291W+D369L+E402N+Y491F+S501R+N536K+D566G; Q291W+D369L+E402N+A732G; Q291W+D369L+E402N+E493Y+N504Y+N521C+T591C+R612P+L620M; I106V+Y135Q+Q291W+D369L+E402N+A475F+G628W; Q291W+D369L+E402N+R672G; Q291W+D369L+E402N+T777N; Q291W+Q313M+D369L+E402N+T540K; Q291W+D369L+E402N+K708F; Q291W+D369L+P374Y+E402N+S501H; Q291W+D369L+E402N+Y715P; Q291W+D369L+E402N+A732M; Q291W+D369L+E402N+E493A+N504Y+N521C+D566G+R612P+L620M; Q291W+D369L+E402N+Q690R; D274Y+Q291W+D369L+E385L+E402N+N437V+K530I+S614D; Q291W+D369L+E402N+L757K; Q291W+D369L+E402N+T687Y; Q291W+D369L+E402N+Y491H+S501R+N521C+T591A; Q291W+D369L+E402N+V775C; Q291W+D369L+E402N+R672V; Q291W+D369L+E402N+N670D; Q291W+D369L+E402N+T779S; Q291W+D369L+E402N+V638R; Q291W+D369L+E402N+T687F; Q291W+D369L+E402N+T687L; Q291W+D369L+E402N+K610S; Q291W+D369L+E402N+Y491L+S501R+N521C; D274Y+Q291W+D358E+D369L+E402N+K530V+S614V; I106V+M181Y+Q258N+Q291W+D369R+E402N+S434P+A475W+K495V+T540K+G628V; Q291W+D369L+E402N+N536K; Q291W+D369L+E402N+E493V+N504Y+R612P+L620M; Q291W+D369L+E402N+S676C; Q291W+D369L+E402N+T540K+G628W; Q291W+D369L+E385L+E402N+N437D+S489L+K530C+S614D; Q119E+D274Y+Q291W+D358N+D369L+E402N+N437F+S489N+S614L; Q291W+D369L+E402N+S434P+A475W+K495V; Q291W+D369L+E402N+A6891; Q291W+D369L+E402N+E493A+N504Y+N521C+D566G+R612H+L620M; Q291W+D369L+E402N+V638S; Q291W+D369L+E402N+V648W; Q291W+D369L+E402N+D650V; Q291W+D369L+E402N+V674M; Q291W+D369L+E402N+V638E; I106V+Q291W+D369L+E402N; Y135Q+Q291W+D369L+E402N; Q291W+D369L+E402N+S764F; Q291W+D369L+E402N+E493A+N504Y+A505C+N521C+T591A+R612P; Q291W+D369L+E402N+S489N+K495Q+S501R+K530N+T611Q; Q291W+D369L+E402N+T685V; I106V+Q291W+D369L+E402N+S434P+A475C+K495N+T540K; Q119E+Q291W+D358N+D369L+E402N+N437D+K530N+S614V; Q291W+D369L+E402N+T687K; Q291W+D369L+E402N+S652D; Q291W+D369R+E402N+A475F+K495Q+T540K+G628L; Y135Q+Q291W+D369L+E402N+G628V; Q291W+D369L+E402N+E493Y+A505C+N521C+T591A+L620M; Q291W+D369L+E402N+T687W; Q291W+D369L+E402N+D650F; Q291W+D369L+E402N+T687C; Q291W+D369L+E402N+S434P+K495N+G628V; Q291W+D369L+E402N+S501N; D274Y+Q291W+D369L+E402N+N437K+S489I+K530V+S614L; Q291W+D369L+E402N+T699L; Q119E+V246I+Q291W+D358E+D369L+E402N+S614L; Q291W+D369L+E402N+N504Y+N521C+D566G+L620M+T635A; Q291W+D369L+E402N+E493Y+N504Y+D566G+T591A+R612P+L620M; Q291W+D369L+E402N+N536K+T591A; Q291W+D369L+E402N+Q690A; Q291W+D358K+D369L+E402N+N437L+S489I+K530D; Q119E+D274Y+Q291W+D358E+D369L+E385L+E402N+N437V+S489N+K530M+S614H; Q291W+D369L+P374Y+E402N+Y491L; Q291W+D369L+E402N+S501R; Q291W+D369L+E402N+Y491L+N521C+N536K; Q291W+D369L+E402N+E493Y+N504Y+N521C+T591C+R612P+L620M+T635V; Q119E+Q291W+D358N+D369L+E385L+E402N+N437I+S489I+S614L; T120Y+Q291W+D369L+E402N+S501R+N521C+D566G; Q291W+D369L+E402N+Y491L+N521C; Q291W+D369L+E402N+Y491L+S501N+D566G+T591A; D274Y+N278Y+Q291W+D358E+D369L+E402N+N437Y+S489L+K530E; I106V+Q291W+D369L+E402N+S434P+A475W+K495F+T540K+G628V; Q119E+Q291W+D358N+D369L+E402N+N437Y+E742G; Y135Q+Q291W+D369Y+E402N+A475F+K495F; T120H+Q291W+D369L+E402N+Y491H+S501H+T591C; Q119E+Q291W+D358N+D369L+E402N+N437F+S489N+K530E+S614L; Q119E+D274Y+Q291W+D358N+D369L+E402N+N437I+S489N+K530V+S614Y; T120Y+Q291W+D369L+E402N+N521C+N536K; Q291W+D369L+E402N+Y491F+S501N+N521C+N536K+T591R; Q291W+D369L+E402N+S501N+N521C+D566G+T591R; Q291W+D369L+E402N+Y491L+S501R+N536K+D566G+T591R; and D274Y+Q291W+D358E+D369L+E402N+N437Y+S489N+K530I+S614L; (where amino acid position is determined by optimal alignment with SEQ ID NO:2).
In some embodiments, the isolated and/or recombinant β-glucosidase polypeptide variant of the present invention is at least about 70% identical to WT C1 Bgl1 (residues 20-870 of SEQ ID NO:2) and comprises a substitution set selected from the group consisting of: Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+Y715P+T823K; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+T685V+Y715P; I106V+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+S764F; A109T+Q258N+Q291W+Q313M+D369R+E402N+I428V+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+Y491F+K495N+G628W+Y715P; Q258N+Q291W+Q313M+D369R+Q381V+E402N+S434P+A475L+K495N+S501R+G628W+Y715P; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+S764F; Q258N+Q291W+Q313M+D369R+E385L+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+N627H+G628W+A732G; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A6891; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+T685V+Y715P+T777N; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+D650Y+Q716R+L757K; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+Y715P; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+V562L+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+A505C+G628W+S764F; Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+Y715P+E819V; A109T+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+S652D+V846F; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+Q690K; D47I+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W; E21Q+V175A+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+E360D+D369R+E402N+S434P+A475L+K495N+G628W+L757K; Q258N+Q291W+Q313M+F314L+D369R+E402N+S434P+A475L+K495N+S6041I+N627H+G628W+A732G; A109T+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+V775C; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+S764F; P29Q+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; A136L+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+S764F+P870S; Q258N+Q291W+Q313M+D369R+E385L+E402N+S434P+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+T687M; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+N588F+G628W; Q258N+Q291W+Q313M+D358K+D369R+E402N+S434P+A475L+K495N+G628W; Y135M+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+Y715P; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+T785L; A79G+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; A109T+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+S848N; I106V+Q258N+Q291W+Q313M+F314V+D369R+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A732G; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+L757K; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+N627H+G628W+T687M+E822K; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G616D+G628W; A79M+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; I106V+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+F314V+D369R+E402N+S434P+A475L+K495N+A617V+G628W; Q258N+Q291W+Q313M+F314V+D369R+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+P436Q+A475L+K495N+G626D+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+T687C; Q85N+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; A109S+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+N536K+G628W; S58G+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; E21Q+Q258N+Q291W+Q313M+D369R+Q381V+E402N+S434P+A475L+K495N+G628W; Q258N+L275Y+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+A505C+G628W; E21R+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; V25A+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; H26R+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; L30K+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; N45H+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+E493V+K495N+G628W; P29R+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; P29M+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A732S+A748T+V8401; Q55R+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; K24G+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; G180E+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+Q479R+K495N+S501R+G628W+Y715P+E819L; A79E+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+R672G; Q258N+Q291W+Q313M+F314V+D369R+E402N+S434P+A475L+K495N+A505C+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+T687K; Q27R+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; V253F+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; S22L+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+Q690H; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+S799N; Q258N+Q291W+Q313M+F314V+E360D+D369R+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+E822M; K24T+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+S787G; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+R612H+G628W+L757K+S787G; S22L+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+Q479R+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+T687K+E822M; Q258N+Q291W+Q313M+D369R+E402N+S434P+M454E+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+V562C+G628W; S22R+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; V25G+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+T685V; Q258N+Q291W+Q313M+D369R+E402N+A404S+S434P+A475L+K495N+G628W; G216L+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; V25R+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; V25G+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; Q27H+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; K24L+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+D650F+L757K; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+T482A+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+A617V+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+S501C+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+K495N+N536K+G628W+R817P; G180E+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+R476Q+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+T777N; Q119E+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; A109S+Q258N+Q291W+Q313M+D369R+E402N+S434P+K495N+G628W+S848N; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+Y850H; Q258N+Q291W+Q313M+D369R+E402N+S434P+P436E+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+R769H+E819A; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+T496A+G628W; Q258N+V260L+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; D244H+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+E819V; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+S652D; Q258N+Q291W+D311N+Q313M+D369R+E402N+S434P+A475L+K495N+R612H+G628W+A689I+K866I; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+T783H; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+T823K; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+D650N+S787G; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+K708F; Q258N+Q291W+Q313M+E360D+D369R+E402N+S434P+A475L+K495N+G628W+S652D; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+E822G; L237Y+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+E494K+K495N+G628W; Q258N+Q291W+Q313M+E360D+D369R+E402N+S434P+D470N+A475L+K495N+G628W+L757K; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+S764F; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+D650F; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+S848N; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+T496A+N536K+G628W; Q258N+Q291W+Q313M+D369R+E402N+K421R+S434P+A475L+K495N+G628W+T777N; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+N627H+G628W+E822M; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+F634A; A4V+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+N437W+A475L+K495N+G628W; Y135Q+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; Y135Q+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+V673A+T685V; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+L757K+P806L; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+V559T+G628W; Y135M+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+V775C; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+N627H+G628W; Q258N+Q291W+Q313M+D369R+E402N+A405T+S434P+A475L+K495N+G628W; I221V+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+S501R+G628W+T685V; Q258N+Q291W+Q313M+D369R+A394G+E402N+S434P+A475L+K495N+G628W+L757K; P161S+Q258N+Q291W+Q313M+D358K+D369R+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G616D+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+K807R; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A732M; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+A505C+G628W+E822G; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+E493V+K495N+G628W+S652D; A15V+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+K866Q; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+E493A+K495N+G628W+S652D+Q690A; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+E819L; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+E819V; G180E+Q258N+Q291W+Q313M+D369R+E385L+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+D751N; G202M+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+N627H+G628W+T687K+E822A; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+K866I; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+T783Q; Q258N+Q291W+Q313M+D369L+E402N+S434P+A475L+K495N+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+E493Y+K495N+A505C+G628W; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+T482A+K495N+G628W+D646N; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+A477G+K495N+G628W+S764F+S848N; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+1847T; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+T685V+Y850Q; Y135M+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+S501R+G628W+T685V+T777N; and Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+Y850Q; (where amino acid position is determined by optimal alignment with SEQ ID NO:2).
In some embodiments, the isolated and/or recombinant β-glucosidase polypeptide variant of the present invention is at least about 70% identical to WT C1 Bgl1 (residues 20-870 of SEQ ID NO:2) and comprises a substitution set selected from the group consisting of: Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; D47I+Q258N+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687K+A689I+Y715P; A109S+Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; A109T+Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687W+A689I+Y715P+S764F; D47I+Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687C+A689I+Y715P+S764F; Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; D47I+Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+T687C+A689I+Y715P; D47I+A109T+Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+T687W+A689I+Y715P; Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+S764F; Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+A732G; D47I+A109T+Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+S764F; Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+S764F; D47I+A109T+Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+S764F; D47I+A109T+Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; A109T+Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687C+A689I+Y715P; D47I+I106V+Q258N+V260G+Q291W+Q313M+F314L+D369R+E402N+S434P+A475L+K495N+G628W+A6891+Y715P+A732G; Q258N+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687W+A689I+Y715P; I106V+Q258N+V260G+Q291W+Q313M+F314L+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; Q258N+V260G+Q291W+Q313M+F314L+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; D47I+A109S+Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687W+A689I+Y715P; Q258N+V260G+Q291W+Q313M+F314V+D369R+E402N+S434P+A475L+K495N+G628W+S652D+A689I+Y715P+A732G; Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; I106V+Q258N+V260G+Q291W+Q313M+F314V+D369R+E402N+S434P+A475L+K495N+G628W+S652D+A689I+Y715P+A732G; Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+S764F; D47I+Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; I106V+Q258N+V260G+Q291W+Q313M+F314L+D369R+E402N+S434P+A475L+K495N+G628W+S652D+A689I+Y715P+A732M; A109T+Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+S764F; Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+V260G+Q291W+Q313M+A343G+D369R+E402N+S434P+Q474L+A475L+K495N+G628W+A689I+Y715P; D47I+Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+S764F; D47I+A109T+Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+S764F; Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+S764F; D47I+A109T+Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+S764F; Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; D47I+Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; A109T+Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; I106V+Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+A732G+P870S; Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; I106V+Q258N+V260G+Q291W+Q313M+F314V+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+A732M; Q258N+Q291W+Q313M+F314V+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+A732G; I106V+Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+A732V; Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; D47I+I106V+Q258N+Q291W+Q313M+F314L+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+A732G; Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+K495N+G628W+S652D+A689I+Y715P+A732G; D47I+I106V+Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+K495N+G628W+S652D+A689I+Y715P+A732G; D47I+I106V+Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+K495N+G628W+S652D+A689I+Y715P; Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; C8A+L9F+D47I+Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; D47I+Q258N+V260G+Q291W+Q313M+F314V+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+A732G+D844G; Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; I106V+Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+A732G; D47N+Q258N+V260G+Q291W+Q313M+F314V+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+A732G; Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; D47I+I106V+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+S652D+A689I+Y715P+A732M; A109T+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+T687W+A689I+Y715P; Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; V25A+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; D47I+Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+A732M; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+T687W+A689I+Y715P; D47I+I106V+Q258N+V260G+Q291W+Q313M+F314V+D369R+E402N+S434P+A475L+K495N+G628W+S652D+A689I+Y715P; D47I+A109T+Q258N+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; D47I+Q258N+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+S764F; Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+T687K+A689I+Y715P; A109T+Q258N+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+S764F; Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; D47I+I106V+Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+S764F; Q258N+V260G+Q291W+Q313M+F314L+D369R+E402N+S434P+K495N+G628W+S652D+A689I+Y715P; Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+S764F; Q258N+V260G+Q291W+Q313M+F314V+D369R+E402N+S434P+K495N+G628W+A689I+Y715P; L237Y+Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q85N+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+L757K; Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+S604C+G628W+A689I+Y715P; D47I+Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+R476G+K495N+N588F+G628W+D651E+A689I+Y715P; Q85N+Q258N+Q291W+Q313M+D369R+E402N+S434P+P436Q+A475L+K495N+G628W+A689I+Y715P+L757K; I106V+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+A732M; Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+V260G+Q291W+Q313M+F314L+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+Q291W+Q313M+D369R+E402N+S434P+K495N+G628W+S652D+A689I+Y715P+A732G; A109T+Q258N+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; D47I+Q258N+Q291W+Q313M+F314L+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+Y736N; Q258N+Q291W+Q313M+F314L+D369R+E402N+S434P+A475L+K495N+G628W+S652D+A689I+Y715P+A732G; V25A+Q85N+Q258N+Q291W+Q313M+D369R+E402N+S434P+P436Q+A475L+K495N+G616D+G628W+D650Y+A689I+Y715P+L757K; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+S764F; V253F+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Q690K+D709E+E710G+Y715P; Q85N+V175A+Q258N+L275Y+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Q690K+715P; V25A+Q85N+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G616D+G628W+A689I+Y715P+L757K; Q85N+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; V25A+Q85N+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+T687C+A689I+Y715P; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Q690H+Y715P+M816L; V25A+Q85N+Q258N+L275Y+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G616D+G628W+A689I+Y715P+V846F; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+V846Q; A79E+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G626D+G628W+A689I+Y715P; Q258N+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; A79E+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+A505C+G628W+A689I+Y715P; Q258N+Q291W+Q313M+F314L+D369R+Q381V+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; D47I+I106V+Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A6891+Y715P; Q85N+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; V25A+Q85N+Q258N+Q291W+Q313M+D369R+E402N+S434P+P436Q+A475L+K495N+G628W+A689I+Y715P+L757K+V846Q; V25A+Q85N+Q258N+Q291W+Q313M+D369R+E402N+S434P+P436Q+A475L+K495N+G628W+D650N+A689I+Y715P; D47I+I106V+Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+S550C+G628W+S652D+A689I+Y715P+A732G; A109S+Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+S604A+G628W+A689I+Y715P; V25A+Q85N+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+V846Q; D47I+Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; A109T+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+V260G+Q291W+Q313M+F314L+D369R+E402N+S434P+K495N+N588F+G628W+A689I+Y715P+A732M; Q85N+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G616D+G628W+A689I+Y715P; V25A+Q85N+Q258N+Q291W+Q313M+D369R+E402N+S434P+P436Q+A475L+K495N+G616D+G628W+D650N+A689I+Y715P; A79E+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+A505C+G628W+V674I+A689I+Y715P; V25A+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+L757K; V25A+Q85H+Q258N+L275F+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+D650N+A689I+Y715P+L7571; K24T+A79E+A136L+Q258N+D274Y+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+A505C+G628W+A689I+Y715P; V25A+Q85N+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; K24G+A79E+Q258N+D274Y+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+A505C+G628W+A689I+Y715P; Q85N+V175A+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; V25A+Q85N+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+D650N+A689I+Y715P; V25A+Q85N+Q258N+L275Y+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+L757K; A79M+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+A505C+G628W+A689I+Y715P; Q85N+Q258N+Q291W+Q313M+D369R+E402N+S434P+P436Q+A475L+K495N+G628W+A689I+Y715P+; Q85N+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q85N+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q85N+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; I106V+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; D47I+Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+S604A+G628W+A689I+Y715P; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; V25A+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+Q474I+A475L+K495N+G628W+A689I+Y715P+S764F; Q258N+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; D47I+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+N588F+G628W+A689I+Y715P; S58G+Q258N+Q291W+Q313M+D369R+Q381V+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; V253F+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+T785L+M816L; A243G+Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+S764F; I106V+Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+N588F+G628W+S652D+A689I+Y715P+D733G; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+V846L; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+S604A+G628W+A689I+Y715P; A79G+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+T777N; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; V25A+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P; K24T+A79E+Q258N+D274Y+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+A505C+G628W+A689I+Y715P+T777N; D47I+Q258N+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+S604V+G628W+T687C+A689I+Y715P+S764F+L869R; Q258N+D274Y+Q291W+Q313M+D369R+E385L+E402N+S434P+A475L+K495N+G626D+G628W+A689I+Y715P+T777N; S58G+Q258N+Q291W+Q313M+D369R+Q381V+E402N+S434P+N437D+A475L+K495N+G628W+A689I+Q690H+D709E+E710G+Y715P; D47I+Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+Q474I+A475L+K495N+G628W+A689I+Y715P; Q258N+Q291W+Q313M+D369R+E402N+S434P+N437K+A475L+K495N+G628W+A689I+Y715P+T785L; A109S+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+S604I+G628W+A689I+Y715P; Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+S604V+G628W+A689I+Y715P+K807R; Q258N+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+S604V+G628W+A689I+Y715P; Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+L757K; D47I+Q258N+Q291W+Q313M+D369R+E402N+S434P+K495N+N588F+G628W+S652D+A689I+Y715P; A79M+A136L+Q258N+D274Y+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+A505C+G628W+A689I+Y715P+T783A; D47I+A109T+Q258N+V260G+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+S604V+G628W+A689I+Y715P+S764F; S58G+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+T785L; K24G+A136L+Q258N+D274Y+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+A505C+G628W+A689I+Y715P+T777N; S58G+Q258N+Q291W+Q313M+D369R+Q381V+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+T785L; and S58G+Q258N+Q291W+Q313M+D369R+E402N+S434P+A475L+K495N+G628W+A689I+Y715P+T785L+M816L; (where amino acid position is determined by optimal alignment with SEQ ID NO:2).
In some embodiments, the isolated and/or recombinant β-glucosidase polypeptide variant of the present invention is at least about 70% identical to WT C1 Bgl1 (residues 20-870 of SEQ ID NO:2) and comprises a substitution set selected from the group consisting of: D47I+Q258N+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687K+A689I+Y715P; D47I+A79G+Q85N+Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687K+A689I+Y715P+A732M; D47I+Q258N+V260G+Q291W+Q313M+F314L+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687C+A689I+Y715P+A732G; D47I+A79E+Q85N+Q258N+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+A505C+G628W+T687W+A689I+Y715P; D47I+A79E+Q85N+Q258N+V260G+Q291W+Q313M+F314L+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687K+A689I+Y715P; D47I+A79G+Q85N+Q258N+V260G+L275Y+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+A475L+K495N+A505C+G628W+T687C+A689I+Y715P+S764Y+R769H; D47I+A79G+Q85N+Q258N+V260G+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687K+A689I+Y715P; D47I+A79M+Q85N+Q258N+V260G+L275Y+Q291W+Q313M+F314L+A343C+D369R+E402N+S434P+A475L+K495N+A505C+G628W+T687K+A689I+Y715P; D47I+A79M+Q85N+Q258N+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+A505C+G628W+T687C+A689I+Y715P+A732G; D47I+Q258N+V260G+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+K495N+A505C+G628W+T687C+A689I+Y715P+A732G; D47I+A79M+Q258N+V260G+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+K495N+G628W+T687K+A689I+Y715P+A732G; D47I+Q258N+L275Y+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+K495N+G628W+T687K+A689I+Y715P+A732G; D47I+A79E+Q85N+Q258N+V260G+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+K495N+A505C+G628W+T687W+A689I+Y715P+A732V; D47I+A79M+Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+K495N+A505C+G628W+T687K+A689I+Y715P+A732M; D47I+Q85N+Q258N+V260G+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687C+A689I+Y715P; D47I+A79G+Q258N+V260G+L275Y+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687K+A689I+Y715P+A732G; D47I+Q258N+V260G+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687W+A689I+Y715P+A732G; D47I+Q258N+V260G+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687K+A689I+Y715P+A732M; D47I+A79G+Q85N+Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687K+A689I+Y715P; D47I+A79G+Q85N+Q258N+V260G+L275Y+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687C+A689I+Y715P+A732G; D47I+A79G+Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687K+A689I+Y715P+A732M; D47I+Q85N+Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+K495N+G628W+T687K+A689I+Y715P; D47I+Q85N+Q258N+V260G+L275Y+Q291W+Q313M+A343C+D369R+E402N+S434P+K495N+G628W+T687K+A689I+Y715P+A732G; D47I+A79M+Q85N+Q258N+V260G+L275Y+Q291W+Q313M+A343C+D369R+E402N+S434P+K495N+G628W+T687W+A689I+Y715P; D47I+A79G+Q85N+Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+A505C+G628W+T687K+A689I+Y715P; D47I+Q258N+V260G+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687C+A689I+Y715P+A732G; D47I+A79G+Q85N+Q258N+V260G+L275Y+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687K+A689I+Y715P; D47I+Q85N+Q258N+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687W+A689I+Y715P+A732G; D47I+A79G+Q85N+Q258N+V260G+L275Y+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+A475L+K495N+A505C+G628W+T687C+A689I+Y715P; D47I+Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+A505C+G628W+T687K+A689I+Y715P+A732G; D47I+Q258N+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687W+A689I+Y715P+A732G; D47I+A79G+Q85N+Q258N+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+K495N+G628W+T687K+A689I+Y715P; D47I+A79M+Q85N+Q258N+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687K+A689I+Y715P+A732G; D47I+Q85N+Q258N+V260G+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687K+A689I+Y715P; D47I+Q85N+Q258N+L275Y+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+K495N+G628W+T687C+A689I+Y715P; D47I+A79G+Q85N+Q258N+V260G+L275Y+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687K+A689I+Y715P+A732G; D47I+Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687K+A689I+Y715P; D47I+Q85N+Q258N+V260G+L275Y+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687K+A689I+Y715P; D47I+A79G+Q258N+Q291W+Q313M+F314L+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687K+A689I+Y715P; D47I+Q258N+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+K495N+G628W+T687K+A689I+Y715P; D47I+Q258N+V260G+L275Y+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+A505C+G628W+D646N+T687K+A689I+Y715P+A732G; D47I+Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687K+A689I+Y715P; D47I+Q258N+V260G+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+K495N+G628W+T687K+A689I+Y715P+A732V; D47I+A79G+Q85N+Q258N+Q291W+Q313M+A343C+D369R+E402N+S434P+P439S+A475L+K495N+G628W+T687K+A689I+Y715P; D47I+Q85N+Q258N+Q291W+Q313M+F314V+A343C+D369R+D395N+E402N+S434P+A475L+K495N+G628W+T687K+A689I+Y715P+A732V; D47I+A79G+Q258N+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+A505C+G628W+T687C+A689I+T693A+Y715P+T827I; D47I+Q85N+Q258N+V260G+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+A505C+G628W+T687K+A689I+Y715P+A732V; and D47I+A79G+Q258N+L275Y+Q291W+Q313M+A343C+D369R+E402N+S434P+A475L+K495N+A505C+G628W+T687K+A689I+T693E+N723G+A730S+Y855; (where amino acid position is determined by optimal alignment with SEQ ID NO:2).
In some embodiments, the isolated and/or recombinant β-glucosidase polypeptide variant of the present invention is at least about 70% identical to WT C1 Bgl1 (residues 20-870 of SEQ ID NO:2) and comprises a substitution set selected from the group consisting of: I106V+Q258N+V260G+Q291W+Q313M+F314L+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; D47I+A79E+Q85N+I106V+A109T+Q258N+V260G+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; D47I+A79E+Q85N+I106V+Q258N+V260G+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+K495N+A505C+G628W+A689I+Y715P+A732G; A79M+Q85N+I106V+A109T+Q258N+V260G+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; D47I+A79E+I106V+A109T+Q258N+V260G+Q291W+Q313M+F314L+A343C+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; D47I+A79G+Q85N+I106V+A109T+Q258N+V260G+L275Y+Q291W+Q313M+F314L+A343C+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; A79E+Q85N+I106V+A109T+Q258N+V260G+Q291W+Q313M+F314L+A343C+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; A79G+Q85N+I106V+A109T+Q258N+V260G+L275F+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+K495N+A505C+G628W+T687C+A689I+Y715P+A732G; D47I+I106V+Q258N+V260G+Q291W+Q313M+F314L+A343C+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; D47I+A79E+Q85N+I106V+A109S+Q258N+V260G+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; D47I+A79E+Q85N+I106V+Q258N+V260G+L275Y+Q291W+Q313M+F314L+N315D+D369R+E402N+S434P+K495N+A505C+G628W+T687W+A689I+Y715P+A732G; D47I+Q85N+I106V+Q258N+V260G+L275Y+Q291W+Q313M+F314L+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; A79E+Q85N+I106V+A109S+Q258N+V260G+Q291W+Q313M+F314V+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; A79G+I106V+Q258N+V260G+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+K495N+G628W+T687W+A689I+Y715P+A732G; D47I+A79G+Q85N+I106V+Q258N+V260G+L275Y+Q291W+Q313M+F314L+A343C+D369R+E402N+S434P+K495N+A505C+G628W+A689I+Y715P+A732G; D47I+Q85N+I106V+A109S+Q258N+V260G+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; D47I+A79E+Q85N+I106V+Q258N+V260G+Q291W+Q313M+F314L+A343C+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; D47I+A79M+Q85N+I106V+Q258N+V260G+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; D47I+I106V+A109T+Q258N+V260G+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; D47I+A79G+Q85N+I106V+Q258N+V260G+Q291W+Q313M+F314V+D369R+E402N+S434P+K495N+A505C+G628W+A689I+Y715P+A732G; A79M+I106V+Q258N+V260G+Q291W+Q313M+F314L+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; A79M+Q85N+I106V+Q258N+V260G+Q291W+Q313M+F314L+A343C+D369R+E402N+S434P+K495N+A505C+G628W+A6891+Y715P+A732G; A79M+Q85N+I106V+Q258N+V260G+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; D47I+A79G+Q85N+I106V+A109S+Q258N+V260G+Q291W+Q313M+F314L+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; A79E+Q85H+I106V+Q258N+V260G+Q291W+Q313M+F314L+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; D47I+A79G+Q85N+I106V+Q258N+V260G+Q291W+Q313M+F314L+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; A79G+Q85N+I106V+Q258N+V260G+Q291W+Q313M+F314L+A343C+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; D47I+A79E+Q85N+I106V+Q258N+V260G+L275Y+Q291W+Q313M+F314L+A343C+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; D47I+I106V+A109T+Q258N+V260G+Q291W+Q313M+F314V+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; C8G+D47I+Q85N+I106V+Q258N+V260G+Q291W+Q313M+F314L+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; A79E+Q85N+I106V+Q258N+V260G+Q291W+Q313M+F314L+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; A79G+Q85N+I106V+Q258N+V260G+Q291W+Q313M+F314L+A343C+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; D47I+A79G+Q85N+I106V+Q258N+V260G+Q291W+Q313M+F314L+A343C+D369R+E402N+S434P+K495N+T591I+G628W+A689I+Y715P+A732G; Q85N+I106V+A109T+Q258N+V260G+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; A79M+I106V+Q258N+V260G+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; Q85N+I106V+Q258N+V260G+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; Q85N+I106V+A109S+Q258N+V260G+Q291W+Q313M+F314L+D369R+E402N+S434P+K495N+A505C+G628W+A689I+Y715P+A732G; I106V+Q258N+V260G+Q291W+Q313M+F314L+D369R+E402N+S434P+K495N+A505C+G628W+T687K+A689I+Y715P+A732G; Q85N+I106V+A109T+Q258N+V260G+Q291W+Q313M+F314V+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; D47I+Q85N+I106V+A109T+Q258N+V260G+Q291W+Q313M+F314V+A343G+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; D47I+Q85N+I106V+A109T+Q258N+V260G+Q291W+Q313M+F314L+A343C+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; D47I+Q85N+I106V+A109S+Q258N+V260G+L275Y+Q291W+Q313M+F314L+A343C+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; D47I+I106V+Q258N+V260G+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; I106V+Q258N+V260G+Q291W+Q313M+F314V+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; D47I+I106V+Q258N+V260G+Q291W+Q313M+F314L+A343C+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; D47I+Q85N+I106V+A109S+Q258N+V260G+L275Y+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+A475L+K495N+G628W+T687C+A689I+Y715P+A732G; D47I+A79M+I106V+Q258N+V260G+Q291W+Q313M+F314L+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; A79G+Q85N+I106V+A109S+Q258N+V260G+Q291W+Q313M+F314V+D369R+E402N+S434P+K495N+A505C+G628W+A689I+Y715P+A732G; D47I+A79E+Q85N+I106V+Q258N+V260G+Q291W+Q313M+F314L+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; Q85N+I106V+Q258N+V260G+L275Y+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+K495N+G628W+T687W+A689I+Y715P+A732G; I106V+Q258N+V260G+L275Y+Q291W+Q313M+F314L+A343C+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; D47I+Q85N+I106V+A109T+Q258N+V260G+Q291W+Q313M+F314V+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; Q85N+I106V+A109S+Q258N+V260G+L275Y+Q291W+Q313M+F314V+A343C+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; D47I+A79M+I106V+A109S+Q258N+V260G+Q291W+Q313M+F314L+A343C+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; A79M+Q85N+I106V+Q258N+V260G+Q291W+Q313M+F314L+A343C+D369R+E402N+S434P+K495N+G628W+A689I+Y715P+A732G; and A79M+Q85N+I106V+Q258N+V260G+L275Y+Q291W+Q313M+F314L+A343C+D369R+E402N+S434P+K495N+A505C+G628W+A689I+Y715P+A732G (where amino acid position is determined by optimal alignment with SEQ ID NO:2).
In some embodiments, the variant comprises a substitution set selected from those exemplified above (Table 2, 3, 4, 5, 6, 7) and comprises further substitutions that (a) do not diminish the activity or thermostability of the variant and (b) do not include substitutions at any additional residues in the group consisting of: K57; A88; I106; N112; Q119; T120; A123; R132; Y135; A136; A141; K142; L149; G158; P161; P172; T177; I179; G180; M181; S182; E183; K186; A197; G202; Y219; N220; S222; T224; 1229; M234; F242; A243; V246; Q258; D274; V286; Q291; Q313; V318; A343; T354; T357; D358; E360; D369; P374; I375; A378; Q381; E385; S388; V390; A394; N398; E402; K406; I428; S434; N437; E449; Q474; A475; T482; S489; Y491; K530; N536; T540; T565; V674; R682; 1867; E868; and P870.
In some embodiments, the isolated and/or recombinant β-glucosidase polypeptide variant of the present invention is at least about 70% identical to wild-type C1 Bgl1 (residues 20-870 of SEQ ID NO:2) and comprises a substitution set selected from the group consisting of substitution sets showing at least 6.0 to 6.9 fold improvement in activity over the native C1 Bgl1, as identified in Table 2. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 5.0 to 5.9 fold improvement in activity over the native C1 Bgl1, as identified in Table 2. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 4.0 to 4.9 fold improvement in activity over the native C1 Bgl1, as identified in Table 2. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 3.0 to 3.9 fold improvement in activity over the native C1 Bgl1, as identified in Table 2. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 2.0 to 2.9 fold improvement in thermostability over the native C1 Bgl1, as identified in Table 2. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 1.1 to 1.9 fold improvement in thermostability over the native C1 Bgl1, as identified in Table 2.
In some embodiments, the isolated and/or recombinant β-glucosidase polypeptide variant of the present invention is at least about 70% identical to wild-type C1 Bgl1 (residues 20-870 of SEQ ID NO:2) and comprises a substitution set selected from the group consisting of substitution sets showing at least 6.0 to 6.9 fold improvement in activity over the Variant 3, as identified in Table 3. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 5.0 to 5.9 fold improvement in activity over the Variant 3, as identified in Table 3. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 4.0 to 4.9 fold improvement in activity over the Variant 3, as identified in Table 3. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 3.0 to 3.9 fold improvement in activity over the Variant 3, as identified in Table 3. In some embodiments, the variant a substitution set selected from the group consisting of substitution sets showing at least 2.0 to 2.9 fold improvement in activity over the Variant 3, as identified in Table 3. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 1.1 to 1.9 fold improvement in activity over the Variant 3, as identified in Table 3.
In some embodiments, the isolated and/or recombinant β-glucosidase polypeptide variant of the present invention is at least about 70% identical to wild-type C1 Bgl1 (residues 20-870 of SEQ ID NO:2) and comprises a substitution set selected from the group consisting of substitution sets showing at least 4.0 to 4.9 fold improvement in activity over the Variant 269, as identified in Table 4. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 3.0 to 3.9 fold improvement in activity over the Variant 269, as identified in Table 4. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 2.0 to 2.9 fold improvement in activity over the Variant 269, as identified in Table 4. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 1.1 to 1.9 fold improvement in activity over the Variant 269 as identified in Table 4.
In some embodiments, the isolated and/or recombinant β-glucosidase polypeptide variant of the present invention is at least about 70% identical to wild-type C1 Bgl1 (residues 20-870 of SEQ ID NO:2) and comprises a substitution set selected from the group consisting of substitution sets showing at least 4.0 to 4.9 fold improvement in activity over Variant 481, as identified in Table 5. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 3.0 to 3.9 fold improvement in activity over the Variant 481, as identified in Table 5. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 2.0 to 2.9 fold improvement in activity over the Variant 481, as identified in Table 5. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 1.1 to 1.9 fold improvement in activity over the Variant 481, as identified in Table 5.
In some embodiments, the isolated and/or recombinant β-glucosidase polypeptide variant of the present invention is at least about 70% identical to wild-type C1 Bgl1 (residues 20-870 of SEQ ID NO:2) and comprises a substitution set selected from the group consisting of substitution sets showing at least 4.0 to 4.9 fold improvement in activity over Variant 647, as identified in Table 6. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 3.0 to 3.9 fold improvement in activity over the Variant 647, as identified in Table 6. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 2.0 to 2.9 fold improvement in activity over the Variant 647, as identified in Table 6. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 1.1 to 1.9 fold improvement in activity over the Variant 647, as identified in Table 6. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 0.6 to 1.0 fold improvement in activity over the Variant 647, as identified in Table 6.
In some embodiments, the isolated and/or recombinant β-glucosidase polypeptide variant of the present invention is at least about 70% identical to wild-type C1 Bgl1 (residues 20-870 of SEQ ID NO:2) and comprises a substitution set selected from the group consisting of substitution sets showing at least 3.0 to 3.9 fold improvement in activity over Variant 664, as identified in Table 7. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 2.0 to 2.9 fold improvement in activity over the Variant 664, as identified in Table 7.
In some embodiments, the isolated and/or recombinant β-glucosidase polypeptide variant of the present invention is at least about 70% identical to wild-type C1 Bgl1 (residues 20-870 of SEQ ID NO:2) and comprises a substitution set selected from the group consisting of substitution sets showing at least 4.0 to 4.9 fold improvement in thermostability over the native C1 Bgl1, as identified in Table 2. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 3.0 to 3.9 fold improvement in thermostability over the native C1 Bgl1, as identified in Table 2. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 2.0 to 2.9 fold improvement in thermostability over the native C1 Bgl1, as identified in Table 2. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 1.1 to 1.9 fold improvement in thermostability over the native C1 Bgl1, as identified in Table 2.
In some embodiments, the isolated and/or recombinant β-glucosidase polypeptide variant of the present invention is at least about 70% identical to wild-type C1 Bgl1 (residues 20-870 of SEQ ID NO:2) and comprises a substitution set selected from the group consisting of substitution sets showing at least 3.0 to 3.9 fold improvement in thermostability over the Variant 3, as identified in Table 3. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 2.0 to 2.9 fold improvement in thermostability over the Variant 3, as identified in Table 3. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 1.1 to 1.9 fold improvement in thermostability over the Variant 3, as identified in Table 3.
In some embodiments, the isolated and/or recombinant β-glucosidase polypeptide variant of the present invention is at least about 70% identical to wild-type C1 Bgl1 (residues 20-870 of SEQ ID NO:2) and comprises a substitution set selected from the group consisting of substitution sets showing at least 2.0 to 2.0 fold improvement in thermostability over the Variant 269, as identified in Table 4. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 1.1 to 1.9 fold improvement in thermostability over the Variant 269, as identified in Table 4.
In some embodiments, the isolated and/or recombinant β-glucosidase polypeptide variant of the present invention is at least about 70% identical to wild-type C1 Bgl1 (residues 20-870 of SEQ ID NO:2) and comprises a substitution set selected from the group consisting of substitution sets showing at least 4.0 to 4.9 fold improvement in thermostability over Variant 481, as identified in Table 5. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 2.0 to 2.9 fold improvement in thermostability over the Variant 481, as identified in Table 5. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 1.1 to 1.9 fold improvement in thermostability over the Variant 481, as identified in Table 5. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 0.6 to 1.0 fold improvement in thermostability over the Variant 481, as identified in Table 5.
In some embodiments, the isolated and/or recombinant β-glucosidase polypeptide variant of the present invention is at least about 70% identical to wild-type C1 Bgl1 (residues 20-870 of SEQ ID NO:2) and comprises a substitution set selected from the group consisting of substitution sets showing at least 2.0 to 2.9 fold improvement in thermostability over Variant 647, as identified in Table 6. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 1.1 to 1.9 fold improvement in thermostability over the Variant 647, as identified in Table 6. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 0.6 to 1.0 fold improvement in thermostability over the Variant 647, as identified in Table 6. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 0.2 to 0.5 fold improvement in thermostability over the Variant 647, as identified in Table 6.
In some embodiments, the isolated and/or recombinant β-glucosidase polypeptide variant of the present invention is at least about 70% identical to wild-type C1 Bgl1 (residues 20-870 of SEQ ID NO:2) and comprises a substitution set selected from the group consisting of substitution sets showing at least 1.1 to 1.9 fold improvement in thermostability over Variant 664, as identified in Table 7. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing at least 0.6 to 1.0 fold improvement in thermostability over the Variant 664, as identified in Table 7.
In some embodiments, the isolated and/or recombinant β-glucosidase polypeptide variant of the present invention is at least about 70% identical to wild-type C1 Bgl1 (residues 20-870 of SEQ ID NO:2) and comprises a substitution set selected from the group consisting of substitution sets showing any improvement in activity and/or thermostability over the native C1 Bgl1, as identified in Table 2. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing any improvement in activity and/or thermostability over the Variant 3 as identified in Table 3. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing any improvement in activity and/or thermostability over the Variant 269 as identified in Table 4. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing any improvement in activity and/or thermostability over the Variant 481 as identified in Table 5. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing any improvement in activity and/or thermostability over the Variant 647 as identified in Table 6. In some embodiments, the variant comprises a substitution set selected from the group consisting of substitution sets showing any improvement in activity and/or thermostability over the Variant 664 as identified in Table 7.
As noted above, β-glucosidase polypeptides encompassed by the invention have at least about 70% sequence identity to residues 20-870 of SEQ ID NO:2. In some embodiments, β-glucosidase polypeptides encompassed by the invention include those having an amino acid sequence 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 residues 20-870 of SEQ ID NO:2. Each recitation herein of “70%” should be understood to also include, in the alternative, any of the higher values above.
As noted above, Bgl1 variants of the invention may encompass additional amino acid substitutions beyond those listed above including, for example, variants with one or more additional 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 present invention also provides enzymatically active fragments of the β-glucosidase polypeptide variants described herein having β-glucosidase activity and at least one substitution described herein. It is believed based on prior studies that C1 Bgl1 tolerates truncation (i.e., retains activity). It has been observed that a variant of C1 Bgl1 having a sequence that differed from wild-type at each of the N-terminal 25 amino acid positions retained β-glucosidase activity (not shown). Similarly, truncation at the carboxy termini of 16 amino acid residues is tolerated in Azospirillum irakense β-glucosidase (CelA). See U.S. Ser. No. 61/218,020, which is incorporated herein by reference. Accordingly, the present invention further provides an isolated or recombinant β-glucosidase polypeptide variant having an amino acid sequence 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) with respect to SEQ ID NO:2. In certain embodiments, the deletion will be from 1 to 15 amino acid residues from the N-terminus and/or 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.











TABLE 2









Stability Fold





Activity Fold
improvement





improvement
over WT


Variant

Silent Nucleotide
over WT
(SEQ ID NO:


Number
Amino Acid Changes1
Changes2
(SEQ ID NO: 2)3
2)3



















1
M181Y + Q291W + E402N + S434P

++++++
++


2
R132K + L149M + Q313M + D369L + E385L + N437D

++++++
++


3
Q291W + D369L + E402N

+++++
+++


4
Y135I + Q258N + Q474I

+++++
+


5
M181Y + Q291W + E360D + D369V + P374Y + T482A

+++++
+++


6
Q258N + N437F + S489L + Y491H

+++++
++


7
Q119E + Q258N + T357L + Q474I + S489L

+++++
++


8
Q258N + D369R + S489L + Y491H

+++++
++


9
N220Y + Q258N + T357L + D369R + Q474I + Y491F
g1428a
+++++
++


10
M234E + V246L + D358K + D369L + N398G + K530M

+++++
++


11
Y135Q + I229M + F242L + D369L + K530M

++++
++


12
D369Q + P374Y + E402N + T540K

++++
++


13
Y135Q + P172A + I179M + I229M + Q291A + D358K + D369L + N398G

++++
++


14
R132K + D369L + E385L

++++
++


15
Y135M + I179M + Q291A + D358K + D369L
t573g
++++
++


16
Q291W + P374Y + E402N + S434P

++++
++


17
Q119E + N220Y + Q258N + T357L + S489L

++++
+


18
I179M + Q291A + D358K + D369L + I375V + S388W
c1068a + c1461t
++++
++


19
Q291W + D369C
c2580t
++++
++


20
R132K + T354Q + D369L + N437L

++++
+


21
Q291A + D369L + N398G + K530M

++++
++


22
Q119E + Q258N + D274Y + S489L

++++
++


23
Q119E + N220Y + Q258N + Q474I + S489L

++++
++


24
M181Y + D369L

++++
+


25
T120M + S222E + Q313M + T354Q + D369L + E385L

++++
++


26
A4G + Q258N + D274Y + T357L + N437W + Q474I + Y491H
c27t
++++
++


27
R132G + D369L + N437D

++++
++


28
S182L + Q313M + D369L + E385L

++++
++


29
R132G + D369L + E385L

++++
++


30
N112V + D358K + D369L + S388W + K530M

++++
++


31
I106V + G180E + D369L + Q381V

++++
++


32
I179M + Q291A + D369L
c489t + c1284t
++++
++


33
I179M + D358K + D369L + S388W

++++
++


34
M234E + Q291A + D369L + N398G

++++
++


35
I179M + Q291A + D358K + D369L + Q381I

++++
++


36
Y135Q + N220L + Q291A + D369L + N398G

++++
++


37
Y135I + D369L

++++
+


38
S182L + D369L + E385L + N437L

++++
++


39
T120Y + R132K + D369L + N437D

++++
++


40
M234E + D369L + S388W + N398G + K530M
c1098t
++++
++


41
Y135M + I179M + D369L + V390I + K530M
t573g
++++
++


42
L149Q + A197V + Q313M + D369L
c852t + c1308t
++++
+


43
T120M + R132K + D369L + N437L

++++
++


44
T120M + R132W + L149M + Q313M + T354Q + D369L + E385L + N437D
c540t
++++
++


45
A4G + Y135I + N220Y + Q258N + T357P + N437Y

++++
+


46
D358K + D369L + S388W
t573g + c657t
++++
++


47
Q258N + D274Y + N437F

++++
+


48
D369L + S434P + T540K
c285t
++++
++


49
G158D + I179M + Q291A + D358K + D369L + I375V + N398G + K530M

++++
++


50
Y135I + D274Y + D369R

++++
+


51
Q258N + D369L + Q474I + S489L + Y491F

++++
++


52
R132W + S182W + D369L + E385L

++++
++


53
S182L + T354Q + D369L + E385L

++++
++


54
S222Q + T354Q + D369L + E385L + N437D + T565G

++++
++


55
Y135M + P161S + Q291A + A343T + D369L + I375V + K406D
c1200a
++++
++


56
Q291W + D369C + T540K
c855t
++++
++


57
D369L + P374Y + E402N

++++
+


58
Y135I + A343V + D369F + S489L

++++
+


59
M234E + D358K + D369L + S388W

++++
++


60
T120M + L149Q + D369L + E385L

++++
++


61
Q313M + D369L + E385L

++++
++


62
T120Y + D369L + E385L
c750g
+++
++


63
D369L + N437D + T565A

+++
+


64
N112V + Q291A + D369L + I375V
c1128t
+++
++


65
R132W + L149Q + Q313M + T354Q + D369L
c1305t
+++
+


66
Y135M + D369L + I375V + N398G

+++
++


67
D369L + E385L

+++
++


68
T120M + S182L + Q313M + T354Q + D369L + E385L

+++
++


69
T177I + Q291W + P374Y + T482A

+++
+


70
Q258N + N437F

+++
+


71
T120V + R132W + D369L + T565G

+++
+


72
N112V + Q291A + D358N + D369E + S388C + K406D

+++
+


73
Q291A + D358K + D369L + S388W + K406D
t573g
+++
++


74
I106V + E360R + D369L + Q381V

+++
+


75
M234E + Q291A + D369L + S388W + N398G

+++
++


76
L149M + Q313M + D369L

+++
++


77
M234I + Q291W + E360D + D369V + T482A

+++
+++


78
T120V + T354Q + D369L + E385L + T565P

+++
++


79
M234I + Q291W + E360D + S434P
t42n
+++
++


80
D369Y + I867M + E868R

+++
+


81
R132W + S182L + Q313M + D369L
c285t + c1092t + c1095t
+++
+


82
M234I + Q291W + P374Y + T482A

+++
++


83
Y219V + M234I + D369C + P374Y + S434P

+++
++


84
S182L + Q313M + D369L + E385L + N437L
c1044t
+++
++


85
M234E + D358K + D369L + S388W + K530M

+++
++


86
R132G + S222E + D369L + E385L
a51g
+++
++


87
R132K + L149M + S182L + D369L + N437L

+++
+


88
T120H + Q313M + D369L

+++
++


89
R132W + D369L
c1047t
+++
+


90
D369L + P374Y

+++
++


91
N112V + N220L + Q291A + D369L + S388W + N398G

+++
++


92
Q291A + D369L + I375V + K530G

+++
++


93
R132K + L149M + S182L + T354Q + D369L + N437D + T565G

+++
+


94
T120H + S222E + Q313M + D369L + N437D + T565G

+++
++


95
A123N + Q291W + T482A + T540K

+++
++


96
S222E + Q313M + T354Q + D369L + E385L

+++
++


97
R132W + D369L + T565G

+++
+


98
G202M + E360A + D369I + A394L

+++
*


99
R132G + S222Q + Q313M + D369L

+++
++


100
R132W + L149M + D369L

+++
+


101
L149M + Q313M + T354Q + D369L

+++
+


102
Q119L + G202M + D369L

+++
+


103
M181Y + M234I + D369C

+++
+


104
I179M + N220L + Q291A + D369L + I375V

+++
++


105
S222E + Q313M + D369L + E385L + N437L

+++
++


106
I179M + M234E + D358K + D369L + S388W + N398G

+++
++


107
A123N + Y219V + Q291W + E360D + P374Y + S434P

+++
++


108
D369L + E402N
c1092t
+++
+


109
D369L

+++
++


110
Y219V + M234I + Q291W + T482A

+++
+


111
Q291W + E360D + S434P

+++
++


112
D369L + S434P

+++
++


113
G180E + E360R + D369L

+++
++


114
A123N + M181Y + Q291W + D369K + S434P + T540K

+++
++++


115
Q119E + T357L + D369M + S489L

+++
+


116
Q119E + D369F + Y491H

+++
+


117
Q119L + D369L

+++
+


118
Q119E + N220Y + V286I + S489L
c1749t + g2280t
+++
+


119
Q291A + D369L + Q381I

+++
++


120
A475F

+++
**


121
D369L + N536K

+++
++


122
Q119E + Y135I + D369H

+++
+


123
Q258N + S489L

+++
+


124
Q119E + Y135I + N437Y

+++
**


125
Q119E + Y135I + N437F + Y491F

+++
+


126
Q119L + D369L + A394V

+++
+


127
E183G + E360A + D369L + I428V

+++
+


128
D369L + E449Q + N536K

+++
+


129
Q119L + G202M + E360A + A475F

+++
**


130
I106V + D369L

+++
++


131
Y135I + D369M

+++
+


132
M234I + D369C + S434P

+++
+


133
D369L + A475Y

+++
++


134
Q119E + Y135I + S489L

+++
+


135
D369Y + N536K

+++
+


136
E360R + D369L

+++
+


137
G202V + A475H

+++
+


138
D369L + Q381V + N536K

+++
++


139
N220Y + Q258N + S489L + Y491F

+++
+


140
Q258N + T357L + D369M

+++
++


141
I179M + Q291A + D369L + Q381L + S388W + N398G
a69g
+++
+


142
D369Y + A394G + N536K

+++
++


143
Q291W + E360D + D369V + P374Y

++
+++


144
T120M + L149Q + T354Q + D369L + E385L

++
++


145
S489L + Y491H

++
+


146
N220S + Q291F + D369L

++
++


147
D369C + S434P + T540K

++
+


148
V318E + D369L + I428V

++
+


149
E183M + G202M + E360A + D369L + A378K + A394V

++
**


150
A394G + N536K

++
+


151
Q291W + T540K

++
+


152
N220L + Q291A + D369L + Q381L + S388W + K530M

++
+


153
T120V + R132W + E385L + N437D

++
+


154
Q119L + E360A + D369L + A378K

++
+


155
D369A + N536K
t726c
++
+


156
G202V + D369L + A475H

++
+


157
Q381V + A475Y + N536K

++
+


158
S434P

++
+


159
I106V + G180E + D369Y + A394G

++
++


160
G180E + Q381V + A475H

++
+


161
Q119E + N220Y + Q474I

++
+


162
I106V + D369Q

++
+


163
A475Y + N536K

++
+


164
K142R + Y219V + Q291W + S434P + V674I
c1398t
++
++


165
L149Q + S182L + Q313M + D369L + N437L

++
+


166
N112V + I179M + M234E + D369L + N398G
c1188t
++
++


167
E360A + D369L + A378K

++
+


168
E360R + D369Y + N536K

++
+


169
E360R + D369A + Q381V + N536K

++
+


170
D369L + Q381D
c858t
++
+


171
N437F + S489L

++
+


172
E183M + G202M + V318E + D369I + A394L + I428V

++
**


173
E360D + D369L + E402N + S434P

++
+


174
Q119L + A141F + G202M + A394L + I428V + A475F
c2488t
++
*


175
T357L + D369R + S489L + Y491H

++
+


176
Y135I + Q258N + T357L

++
+


177
K142R + Q291W + E360D + D369C + E402N

++
++


178
E183M + A243V + D369L + A378K + A475F

++
+


179
R132K + L149M + E385L

++
+


180
D369Y
a1266t
++
+


181
M234I + E402N + S434P

++
**


182
N437Y
c1641t
++
**


183
Q119E + N437F

++
+


184
N536K

++
+


185
Q119L + E183Q + G202M + D369P

++
+


186
N112V + I179M + Q291A + D358K + K406D
g1122a
++
+


187
A123N + Q291W + T540K

++
+


188
D369I + A394L + I428V
t2364n
++
*


189
A88S + N536K

++
+


190
Q119E + Y135I + N437F
c1473t
++
*


191
A141F + G202M + E360A + D369P + A378K

++
*


192
A123N + T482A

++
**


193
Q313M + N437D

++
+


194
E360R + D369Y

++
+


195
E183M + G202M + A475F

++
**


196
Q119L + E360A + A394V + A475F
t2364g
++
**


197
D369L + A378K

++
+


198
E360R + D369L + A394G
c90a
++
+


199
M181Y + D369E + S434P

++
+


200
D369I
t2364n
++
+


201
N112V + Y135Q + I375V + K406D + K530M + P870S

++
+


202
Q119E + Y135I + N220Y + Q258N

++
+


203
D369P + A394V + I428V

++
+


204
V318E + D369L

++
+


205
K186R + N536K

++
+


206
Q119L + D369L + A378K

++
+


207
M234I + D369K + S434P

+
+


208
N112V + I179M + N220L + Q291A + Q381I + S388W + N398G

+
+


209
E402N + S434P

+
+


210
Q119L + A141F + G202M + A394Q
c993t
+
*


211
Q313M + T354Q + N437D

+
+


212
N112V + M234E + D369L + I375V + K406D
g1437a
+
++


213
Q119E + A136E + N220Y

+
**


214
Q381D + A394G + N536K

+
**


215
Q119L + E183G + D369Q + A378T + V390I

+
**


216
Y135I + T357L + Q474I + S489L + Y491F

+
+


217
D369E + A394P + I428V

+
+


218
N112V + Q291A + Q381I + N398G

+
+


219
E360R + N536K

+
+


220
E360D + D369C

+
+


221
R132W + S182L + E385L

+
**


222
I179M + Q291A + D358K + N398G + K530G

+
+


223
N220Y + Q258N + T357L

+
+


224
T224N + D274Y + T357L + N437F

+
+


225
N437D

+
+


226
M234I + E360D + T482A

+
+


227
A141F + G202M + D274N + V318E + E360A + I428V

+
*


228
D369Q

+
+


229
N112V + N220L + D358K + D369L + I375V + Q381I + N398G

+
+


230
Y135I + D369F

+
+


231
A243V + V318E + E360A + A475W

+
**


232
N220L + D369L + Q381L + S388W + N398G + K530G

+
+


233
S489L

+
+


234
K142R + Y219V

+
*


235
Y135I + N220Y + Y491F

+
*


236
S182W + T354Q + E385L

+
+


237
I179M + D358K + S388W

+
**


238
L149M + Q313M + T565P

+
**


239
R132G + E385L

+
**


240
V318E

+
+


241
Q119L + E183K

+
**


242
R132W + E385L

+
**


243
E183G + V318E + E360A + A394Q + A475C

+
**


244
E183M + G202M + E360A

+
**


245
A394G

+
+


246
D369P

+
+


247
Y135M + Q291A + S388W + N398G

+
*


248
Y219V + D369C

+
++


249
A394V + I428V
g1428a
+
**


250
Q119L

+
+


251
Y135I

+
**


252
E360R + D369Q + Q381D + N536K

+
**


253
N220L + M234E + Q291A + I375V + K530M

+
+


254
Q119L + G202M + E360A

+
**


255
N220Y + Q258N + D369R

+
+++


256
M181Y + M234I + Q291W + E402N

+
+


257
A394V
g2382a
+
**


258
T120M + L149Q + Q313M
c1686t
+
+


259
Q119L + V318E + I428V

+
+


260
E360R + Q381V

+
+


261
K57R + G202M + E360A + A394V

+
**


262
I179M + D358K + I375V + Q381L

+
+


263
E360A

+
+


264
I179M + R682W

+
+


265
E360A + I428V

+
+


266
D369K + P374Y

+
+





Table 2, Thermoactivity conditions: pH 5, 65° C. for 21 hrs. Thermostability conditions: enzyme residual activity was determined after incubated at pH 5, 65° C. for 6 hrs.


1Amino acid changes are indicated with respect to SEQ ID NO: 2.


2Nucleotide changes are indicated with respect to SEQ ID NO: 1.


3Fold improvement is represented as follows: * = 0.3 to 0.5 fold improvement over the native C1 Bgl1 (SEQ ID NO: 2) ** = 0.6 to 1 fold improvement over the native C1 Bgl1 (SEQ ID NO: 2) + = 1.1 to 1.9 fold improvement over the native C1 Bgl1 (SEQ ID NO: 2) ++ = 2.0 to 2.9 fold improvement over the native C1 Bgl1 (SEQ ID NO: 2) +++ = 3.0 to 3.9 fold improvement over the native C1 Bgl1 (SEQ ID NO: 2) ++++ = 4.0 to 4.9 fold improvement over the native C1 Bgl1 (SEQ ID NO: 2) +++++ = 5.0 to 5.9 fold improvement over the native C1 Bgl1 (SEQ ID NO: 2) ++++++ = 6.0 to 6.9 fold improvement over the native C1 Bgl1 (SEQ ID NO: 2)

















TABLE 3









Stability





Activity Fold
Fold


Variant

Silent Nucleotide
improvement
improvement


Number
Amino Acid Changes1
Changes2
over Var. 33
over Var. 33



















3
Q291W + D369L + E402N





267
I106V + Q258N + Q291W + D369L + E402N + S434P

++++++
++


268
I106V + Y135Q + Q258N + Q291W + Q313M + D369H + E402N + K495N + G628W
t1620a
++++++
+++


269
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N + G628W

+++++
++


270
I106V + Q258N + Q291W + Q313M + D369H + E402N + S434P + A475C + K495I +

+++++
+++



T540K + G628W


271
I106V + Q258N + Q291W + D369R + E402N + S434P + K495H + G628L

+++++
++


272
D274Y + Q291W + D358K + D369L + E385L + E402N + N437I + S489N
g357a
++++
+


273
Q258N + Q291W + Q313M + D369H + E402N + S434P + T540K
c405t + t1884g
++++
++


274
I106V + Q258N + Q291W + D369L + E402N + S434P + A475F + K495H + G628V
t1620a
++++
++


275
Q258N + Q291W + Q313M + D369L + E402N + S434P + A475C + K495I + G628W
t60c + c246t + c318t +
++++
+++




c405t + t1620a


276
I106V + Y135Q + M181Y + Q258N + Q291W + Q313M + D369L + E402N + S434P +
t1620a + t1884g
++++
+++



K495N


277
Y135Q + Q258N + Q291W + D369L + E402N + K495N
t1620a + t1884g
++++
++


278
Q119E + D274Y + Q291W + D358K + D369L + E385L + E402N + N437V + S489N +
t846c
++++
+



S614A


279
Y135Q + Q258N + Q291W + Q313M + D369L + E402N + S434P + A475F + K495N +
c318t + c984a +
++++
++



G628V
t1620a


280
R132H + Q258N + Q291W + D369Y + E402N + K495Q
c318t + c405t +
++++
+




t1620a


281
Y135Q + Q258N + Q291W + D369L + E402N + S434P + A475F + K495H

++++
++


282
Y135Q + Q258N + Q291W + D369R + E402N + S434P + K495I + G628V
t1620a
++++
+


283
I106V + M181Y + Q258N + Q291W + D369L + E402N

++++
+


284
I106V + Q258N + Q291W + D369H + E402N + S434P + A475F + K495F + G628V
c405t + t1620a
++++
+


285
Q258N + Q291W + Q313M + D369L + E402N + S434P + G628V

++++
++


286
Q258N + Q291W + D369L + E402N + A475F + K495I
t1620a + t1884g +
+++
+




c2463t


287
Q119E + D274Y + Q291W + D369L + E385L + E402N + N437I

+++
+


288
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L
c318t + c405t
+++
++


289
Q258N + Q291W + D369Y + E402N + S434P + K495H + G628W
t1620a
+++
+


290
D274Y + Q291W + D369L + E385L + E402N + N437I + S489N + K530C
g357a + a1840t
+++
+


291
I106V + Y135Q + Q258N + Q291W + Q313M + D369Y + E402N + S434P + A475F +
t1884g
+++
+++



K495H + T540K


292
Q119E + Q291W + D358K + D369L + E385L + E402N
c822t
+++
+


293
I106V + M181Y + Q258N + Q291W + Q313M + D369H + E402N + S434P + A475L +
c405t + c1581t +
+++
++



K495Q + G628V
t1620a


294
Q119E + D274Y + Q291W + D358N + D369L + E385L + E402N + N437D

+++
+


295
Q258N + Q291W + D369R + E402N + S434P + G628V

+++
+


296
Q258N + Q291W + Q313M + D369H + E402N + S434P + G628L
t1620a
+++
++


297
Q258N + Q291W + D369L + E402N
c318t
+++
+


298
D274Y + Q291W + D358K + D369L + E385L + E402N + N437F + S614D
g357a + t1465a
+++
+


299
Q119E + D274Y + Q291W + D358N + D369L + E385L + E402N + N437L + K530V
a1840t
+++
+


300
Q119E + D274Y + Q291W + D358N + D369L + E385L + E402N + N437V + S489L +
a1840t
+++
++



K530N


301
Q258N + Q291W + D369L + E402N + S434P + A475F + K495F + T540K
c318t + c405t +
+++
+




t1884g


302
M181Y + Q291W + Q313M + D369Y + E402N
c318t + c405t
+++
+


303
A243V + D274Y + Q291W + D358K + D369L + E402N + N437I + K530C
g357a + t1465a +
+++
+




a1840t


304
Q119E + D274Y + Q291W + D358E + D369L + E385L + E402N + N437V + K530V +
t1465a
++
**



S614A


305
Q291W + D358K + D369L + E385L + E402N + N437W + K530C + S614A
g357a + c822t
++
+


306
Q119E + D274Y + Q291W + D369L + E385L + E402N + N437W

++
++


307
D274Y + Q291W + D358N + D369L + E385L + E402N + N437I + K530N + S614V
g357a + t1465a
++
+


308
M181Y + Q258N + Q291W + Q313M + D369L + E402N + S434P
c318t + c405t
++
+++


309
A109T + Q291W + D369L + E402N

++
+


310
Q119E + Q291W + D369L + E385L + E402N + N437Y + S489I + K530N
c822t + a1840t
++
++


311
Q119E + D274Y + Q291W + D358N + D369L + E402N + N437D
a1840t
++
+


312
D274Y + Q291W + D358K + D369L + E385L + E402N + N437W + K530V + S614D
g357a + t1465a
++
+


313
Q258N + Q291W + D369R + E402N + A475W + K495H + G628V
t1620a
++
+


314
A109S + Q291W + D369L + E402N
c1119t
++
+


315
Q119E + D274Y + Q291W + D358E + D369L + E385L + E402N + N437L + S614A
c1329t + t1465a
++
+


316
Q291W + D369L + E402N + E493Y + N504Y + T611A
c1563t + g1590a
++
+


317
Q119E + D274Y + Q291W + D358K + D369L + E402N + S614V

++
+


318
D274Y + Q291W + D358E + D369L + E385L + E402N + N437L + K530N + S614A
g357a + t1465a
++
+


319
Q258H + Q291W + Q313M + D369R + E402N + A475F + K495N + G628L
t1620a
++
+


320
Q258N + Q291W + Q313M + D369Y + E402N + A475W + K495V + T540K +

++
++



G628W


321
D274Y + Q291W + D358N + D369L + E385L + E402N + N437W + S614V
g357a + c936a
++
+


322
I106V + Y135Q + Q291W + Q313M + D369L + E402N + A475L + K495Q
t1620a + t1884g
++
++


323
Q258N + Q291W + D369R + E402N + S434P + A475F + G628V

++
+


324
Q119E + D274Y + Q291W + D369L + E385L + E402N + N437V
t1465a
++
+


325
D274Y + Q291W + D358K + D369L + E385L + E402N + N437Y + S489L + K530V
g357a
++
+


326
Q119E + D274Y + Q291W + D358N + D369L + E402N
a1840t
++
+


327
Q291W + D369L + E402N + K495V + S501R + A503E + K530N + T611H
t1465a
++
+


328
Q119E + D274Y + Q291W + D358N + D369L + E385L + E402N + N437V + S489I +

++
+



K530C


329
Q119E + Q291W + D358K + D369L + E402N + N437L + S489N
c822t
++
+


330
D274Y + Q291W + D369L + E385L + E402N + N437W + K530C + S614D
g357a
++
+


331
Q258N + Q291W + D369R + E402N + A475F + K495N + T540K + G628V
c318t + c405t
++
+


332
Q291W + D369L + E402N + E493V + N504Y
c1563t + t1698c +
++
+




c1773t + t1872c +




c1905t


333
Q291W + D358E + D369L + E385* + E402N + S489T
g357a + c822t
++
+


334
Q258N + Q291W + Q313M + D369R + E402N + A475L + K495V + A601T + G628W

++
++


335
D274Y + Q291W + D358N + D369L + E385L + E402N + S489N + S614C
g357a
++
+


336
D274Y + Q291W + D358K + D369L + E402N + N437V + S489L + K530C + S614A
g357a + g1140a
++
+


337
Q119E + D274Y + Q291W + D358E + D369L + E402N + N437D
a1840t
++
+


338
Q119E + D274Y + Q291W + D358N + D369L + E402N + N437L + S614R
t1465a
++
+


339
D274Y + Q291W + D358N + D369L + E402N + N437Y + K530V
g357a + t1465a +
++
**




a1840t


340
D274Y + Q291W + D358E + D369L + E402N + N437Y + S489N + K530N + S614V +
g357a
++
+



D781N


341
Q258H + Q291W + D369* + E402N + S434P + T540K + G628L
c318t + c405t
++
+


342
Q119E + Q291W + D358E + D369L + E385L + E402N + N437L + S489N + K530V
c822t
++
+


343
Q258N + Q291W + D369L + E402N + G628V
c405t + c1179t
++
+


344
Q291W + D358K + D369L + E385L + E402N + S489I + K530N
g357a + c822t +
++
+




a1840t


345
Q119E + Q291W + D369L + E385L + E402N + S489N + K530V + S614A
g6a + c822t
++
+


346
I106V + Y135Q + Q291W + D369L + E402N + S434P + A475F + K495H + G628L
t1620a
++
+


347
Q119E + Q291W + D358K + D369L + E402N + N437V + K497R + S614A
c822t + t1465a
++
+


348
Q119E + D274Y + Q291W + D358E + D369L + E385L + E402N + N437L
t1465a
++
+


349
D274Y + Q291W + D358K + D369L + E402N + N437L + S489I + K530V + S614A
g357a
++
+


350
I106V + Q291W + Q313M + D369L + E402N + S434P + A475W + K495N + G628W
c405t + t1620a
++
++


351
Q291W + D369L + E402N + A505C + L620M + T635I
g1479a + t1512c +
++
+




c1563t + t1698c +




c1773t + t1872c


352
Q291W + D369L + E402N + E493A + N504Y + A505C + L620M + T635A
c1563t + t1698c +
++
+




c1773t + t1872c


353
D274Y + Q291W + D358N + D369L + E402N + S489N + K530C + S614A
g357a + c981t
++
+


354
D274Y + Q291W + D358K + D369L + E385L + E402N + N437W + K530D
g357a + t1465a +
++
+




a1840t


355
Q291W + D369L + E402N + S489N + K495H + S501R + K530N
c1509g + c1833g
++
+


356
D274Y + Q291W + D358E + D369L + E385L + E402N + N437D + K530C + S614H
g357a
++
+


357
Q119E + Q291W + D358E + D369L + E385L + E402N + N437W + S489N + K530E +
c822t
++
+



S614A


358
Q291W + D369L + E402N + E493Y + N504Y + N521C + T591A + R612P + L620M +
t1698c + t1872c
++
+



T635I


359
Q291W + D358K + D369L + E385L + E402N + N437I + K530M + S614L
g357a + c822t +
+
**




t1465a


360
Q291W + D369L + E402N + R672I

+
**


361
D274Y + Q291W + D358E + D369L + E385L + E402N + N437L + S489L
g357a + a1840t
+
+


362
A265S + Q291W + D369L + E402N

+
**


363
Q215M + Q291W + D369L + E402N

+
+


364
Q291W + D369L + E402N + E493V + N504Y + N521C + T591A + L620M + T635I
t1872c
+
+


365
Q119E + D274Y + Q291W + D369L + E385L + E402N + N437L + S489N + K530M +

+
+



S614D


366
Q119E + Q291W + D369L + E385L + E402N
c822t
+
+


367
Q291W + D369L + E402N + E493A + N504Y + D566G + R612P + L620M + T635A
c1563t + c1773t +
+
+




c1863t + t1872c +




c2403a


368
Q119E + D274Y + Q291W + D369L + E385L + E402N + S614Y

+
+


369
I106V + Y135Q + M181Y + Q258N + Q291W + D369L + E402N

+
++


370
Q119E + Q291W + D358E + D369L + E385L + E402N + N437W + K530V
c822t
+
+


371
Q215E + Q291W + D369L + E402N

+
**


372
Q291W + D369L + E402N + N504Y + N521C + T591A + R612H + L620M + T635I
g1479a + t1698c +
+
+




t1872c


373
Q119E + D274Y + Q291W + D358K + D369L + E402N + N437Y + K530I
t1465a + a1840t
+
**


374
Q258H + Q291W + D369L + E402N + K495N
c318t + t1620a +
+
+




t1884g


375
Q291W + D358N + D369L + E385L + E402N + N437D + S489N + K530I
g357a + c822t +
+
+




a1840t


376
Q291W + D369L + P374Y + E402N + Y491L + S501R
c360t + c1773g
+
+


377
Q258N + Q291W + Q313M + G332D + D369H + E402N + S434P

+
++


378
Q291W + D358N + D369L + E385L + E402N + N437F + S489L + K530V
c822t + a1840t
+
+


379
Q291W + D369L + E402N + R672S

+
**


380
Q291W + D369L + E402N + T687M

+
+


381
Q291W + F314V + D369L + E402N

+
+


382
Q258N + Q291W + T357A + D369H + E402N + S434P + K495F
t1620a + t1884g
+
+


383
Q291W + D369L + P374Y + E402N + Y491F + S501R + N521C
c360t + c540a
+
+


384
Q291W + D369L + E402N + Q690K

+
+


385
Q291W + D358E + D369L + E385L + E402N + N437F + K530V + S614A
g357a + c822t +
+
+




t1465a


386
Q291W + D369L + E402N + R672A

+
**


387
Q291W + D369L + E402N + R672T

+
**


388
Q291W + D369L + E402N + D703K
c1947t
+
**


389
D274Y + Q291W + D369L + E402N
c1305t + t1465a +
+
+




a1840t


390
Q291W + D369L + E402N + R672F

+
**


391
Q291W + D369L + E402N + R672D

+
**


392
Q291W + D369L + E402N + Y491F + S501R + N536K + D566G
c1773g
+
+


393
Q291W + D369L + E402N + A732G

+
+


394
Q291W + D369L + E402N + E493Y + N504Y + N521C + T591C + R612P + L620M
t1698c + t1872c +
+
+




c1905t


395
I106V + Y135Q + Q291W + D369L + E402N + A475F + G628W

+
+


396
Q291W + D369L + E402N + R672G

+
**


397
Q291W + D369L + E402N + T777N
c2328t
+
+


398
Q291W + Q313M + D369L + E402N + T540K
c318t + c405t
+
+


399
Q291W + D369L + E402N + K708F

+
+


400
Q291W + D369L + P374Y + E402N + S501H

+
**


401
Q291W + D369L + E402N + Y715P
c2241t
+
+


402
Q291W + D369L + E402N + A732M

+
+


403
Q291W + D369L + E402N + E493A + N504Y + N521C + D566G + R612P + L620M
c1773t + t1872c +
+
+




c1905t


404
Q291W + D369L + E402N + Q690R

+
+


405
D274Y + Q291W + D369L + E385L + E402N + N437V + K530I + S614D
t1465a
+
**


406
Q291W + D369L + E402N + L757K
c2454t
+
+


407
Q291W + D369L + E402N + T687Y

+
+


408
Q291W + D369L + E402N + Y491H + S501R + N521C + T591A

+
+


409
Q291W + D369L + E402N + V775C

+
+


410
Q291W + D369L + E402N + R672V

+
**


411
Q291W + D369L + E402N + N670D

+
**


412
Q291W + D369L + E402N + T779S

+
**


413
Q291W + D369L + E402N + V638R

+
**


414
Q291W + D369L + E402N + T687F

+
+


415
Q291W + D369L + E402N + T687L

+
**


416
Q291W + D369L + E402N + K610S

+
**


417
Q291W + D369L + E402N + Y491L + S501R + N521C
c1773g
+
+


418
D274Y + Q291W + D358E + D369L + E402N + K530V + S614V
g357a
+
**


419
I106V + M181Y + Q258N + Q291W + D369R + E402N + S434P + A475W + K495V +
c405t
+
++



T540K + G628V


420
Q291W + D369L + E402N + N536K
t1501a + c1773g
+
+


421
Q291W + D369L + E402N + E493V + N504Y + R612P + L620M
c1563t + t1698c +
+
+




c1773t + t1872c +




c1905t


422
Q291W + D369L + E402N + S676C

+
**


423
Q291W + D369L + E402N + T540K + G628W
c318t
+
**


424
Q291W + D369L + E385L + E402N + N437D + S489L + K530C + S614D
g357a + c822t
+
+


425
Q119E + D274Y + Q291W + D358N + D369L + E402N + N437F + S489N + S614L

+
+


426
Q291W + D369L + E402N + S434P + A475W + K495V
t1620a
+
+


427
Q291W + D369L + E402N + A689I

+
+


428
Q291W + D369L + E402N + E493A + N504Y + N521C + D566G + R612H + L620M
c1773t + t1872c +
+
+




c1905t


429
Q291W + D369L + E402N + V638S

+
**


430
Q291W + D369L + E402N + V648W

+
**


431
Q291W + D369L + E402N + D650V

+
**


432
Q291W + D369L + E402N + V674M

+
**


433
Q291W + D369L + E402N + V638E

+
**


434
I106V + Q291W + D369L + E402N
t1884g
+
+


435
Y135Q + Q291W + D369L + E402N
t1884g
+
**


436
Q291W + D369L + E402N + S764F

+
+


437
Q291W + D369L + E402N + E493A + N504Y + A505C + N521C + T591A + R612P
t1698c + t1872c +
+
+




c1905t


438
Q291W + D369L + E402N + S489N + K495Q + S501R + K530N + T611Q
c1509g
+
+


439
Q291W + D369L + E402N + T685V

+
+


440
I106V + Q291W + D369L + E402N + S434P + A475C + K495N + T540K

+
+


441
Q119E + Q291W + D358N + D369L + E402N + N437D + K530N + S614V
c822t
+
+


442
Q291W + D369L + E402N + T687K

+
+


443
Q291W + D369L + E402N + S652D

+
+


444
Q291W + D369R + E402N + A475F + K495Q + T540K + G628L

+
+


445
Y135Q + Q291W + D369L + E402N + G628V
c318t
+
**


446
Q291W + D369L + E402N + E493Y + A505C + N521C + T591A + L620M
t1512c + t1698c +
+
+




t1872c + c1905t


447
Q291W + D369L + E402N + T687W

+
+


448
Q291W + D369L + E402N + D650F

+
**


449
Q291W + D369L + E402N + T687C

+
+


450
Q291W + D369L + E402N + S434P + K495N + G628V

+
**


451
Q291W + D369L + E402N + S501N
c540a + c1773g
+
*


452
D274Y + Q291W + D369L + E402N + N437K + S489I + K530V + S614L
g357a
+
+


453
Q291W + D369L + E402N + T699L

+
+


454
Q119E + V246I + Q291W + D358E + D369L + E402N + S614L
c267t
+
**


455
Q291W + D369L + E402N + N504Y + N521C + D566G + L620M + T635A
g1479a + c1773t +
**
+




t1872c


456
Q291W + D369L + E402N + E493Y + N504Y + D566G + T591A + R612P + L620M
c1563t + t1872c +
**
+




c1905t


457
Q291W + D369L + E402N + N536K + T591A

**
+


458
Q291W + D369L + E402N + Q690A

**
+


459
Q291W + D358K + D369L + E402N + N437L + S489I + K530D
g357a + c822t +
**
**




c1458t + a1840t


460
Q119E + D274Y + Q291W + D358E + D369L + E385L + E402N + N437V + S489N +

**
+



K530M + S614H


461
Q291W + D369L + P374Y + E402N + Y491L

**
**


462
Q291W + D369L + E402N + S501R
c1773g+
**
+


463
Q291W + D369L + E402N + Y491L + N521C + N536K
c360t + c540a +
**
+




t1501a + c1773g


464
Q291W + D369L + E402N + E493Y + N504Y + N521C + T591C + R612P + L620M +
t1698c + t1872c
**
+



T635V


465
Q119E + Q291W + D358N + D369L + E385L + E402N + N437I + S489I + S614L
c822t
**
++


466
T120Y + Q291W + D369L + E402N + S501R + N521C + D566G
c1773g
**
+


467
Q291W + D369L + E402N + Y491L + N521C
t1501a
**
**


468
Q291W + D369L + E402N + Y491L + S501N + D566G + T591A

**
**


469
D274Y + N278Y + Q291W + D358E + D369L + E402N + N437Y + S489L + K530E
g357a
**
+


470
I106V + Q291W + D369L + E402N + S434P + A475W + K495F + T540K + G628V
c405t + a636g
**
+


471
Q119E + Q291W + D358N + D369L + E402N + N437Y + E742G
c822t
**
**


472
Y135Q + Q291W + D369Y + E402N + A475F + K495F
t1620a + t1884g
**
**


473
T120H + Q291W + D369L + E402N + Y491H + S501H + T591C

*
+


474
Q119E + Q291W + D358N + D369L + E402N + N437F + S489N + K530E + S614L
c822t
*
+


475
Q119E + D274Y + Q291W + D358N + D369L + E402N + N437I + S489N + K530V +

*
+



S614Y


476
T120Y + Q291W + D369L + E402N + N521C + N536K
c540a + c1773g
*
++


477
Q291W + D369L + E402N + Y491F + S501N + N521C + N536K + T591R
c360t
*
+


478
Q291W + D369L + E402N + S501N + N521C + D566G + T591R
c1473t
*
**


479
Q291W + D369L + E402N + Y491L + S501R + N536K + D566G + T591R

*
+


480
D274Y + Q291W + D358E + D369L + E402N + N437Y + S489N + K530I + S614L
g357a
*
**





Table 3: Thermoactivity conditions: pH 5, 70° C. for 21 hrs. Thermostability conditions: enzyme residual activity was determined after incubated at pH 5, 65° C. for 16-48 hrs.


1Amino acid changes are indicated with respect to SEQ ID NO: 2; the backbone sequence contains substitutions Q291W + D369L + E402N.


2Nucleotide changes are indicated relative to SEQ ID NO: 1.


3Fold improvement is represented as follows: * = 0.3 to 0.5 fold improvement over Variant 3 (SEQ ID NO: 5) ** = 0.6 to 1 fold improvement over Variant 3 (SEQ ID NO: 5) + = 1.1 to 1.9 fold improvement over Variant 3 (SEQ ID NO: 5) ++ = 2.0 to 2.9 fold improvement over Variant 3 (SEQ ID NO: 5) +++ = 3.0 to 3.9 fold improvement over Variant 3 (SEQ ID NO: 5) ++++ = 4.0 to 4.9 fold improvement over Variant 3 (SEQ ID NO: 5) +++++ = 5.0 to 5.9 fold improvement over Variant 3 (SEQ ID NO: 5) ++++++ = 6.0 to 6.9 fold improvement over Variant 3 (SEQ ID NO: 5)

















TABLE 4









Stability Fold





Activity Fold
improvement


Variant

Silent Nucleotide
improvement
over Var.


Number
Amino Acid Changes1
Changes2
over Var. 2693
2693



















269
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N + G628W





481
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

++++
++



K495N + G628W + A689I + Y715P


482
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +
g1290a + g2160a
+++
+



K495N + G628W + Y715P + T823K


483
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

++
+



K495N + G628W + T685V + Y715P +


484
I106V + Q258N + Q291W + Q313M + D369R + E402N + S434P +

++
+



A475L + K495N + G628W + S764F


485
A109T + Q258N + Q291W + Q313M + D369R + E402N + I428V +

++
+



S434P + A475L + K495N + G628W


486
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

++
+



Y491F + K495N + G628W + Y715P


487
Q258N + Q291W + Q313M + D369R + Q381V + E402N + S434P +

++
+



A475L + K495N + S501R + G628W + Y715P


488
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

++
+



K495N + G628W + S764F


489
Q258N + Q291W + Q313M + D369R + E385L + E402N + S434P +

++
+



A475L + K495N + G628W


490
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

++
+



K495N + N627H + G628W + A732G


491
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +
c852t
++
+



K495N + G628W + A689I


492
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

++
+



K495N + G628W + T685V + Y715P + T777N


493
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

++
+



K495N + G628W + D650Y + Q716R + L757K


494
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

++
+



K495N + G628W + Y715P


495
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +
c729t
++
**



K495N + V562L + G628W


496
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

++
+



K495N + A505C + G628W + S764F


497
Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P +

++
++



A475L + K495N + G628W


498
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

++
+



K495N + G628W + Y715P + E819V


499
A109T + Q258N + Q291W + Q313M + D369R + E402N + S434P +

++
+



A475L + K495N + G628W


500
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +
c27t
++
+



K495N + G628W + S652D + V846F


501
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
+



K495N + G628W + Q690K


502
D47I + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
++



A475L + K495N + G628W


503
Q258N + Q291W + Q313M + A343C + D369R + E402N + S434P +

+
+



A475L + K495N + G628W


504
E21Q + V175A + Q258N + Q291W + Q313M + D369R + E402N +
c249t
+
**



S434P + A475L + K495N + G628W


505
Q258N + Q291W + Q313M + E360D + D369R + E402N + S434P +

+
**



A475L + K495N + G628W + L757K


506
Q258N + Q291W + Q313M + F314L + D369R + E402N + S434P +

+
+



A475L + K495N + S604I + N627H + G628W + A732G


507
A109T + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
+



A475L + K495N + G628W + V775C


508
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
+



K495N + G628W + S764F


509
P29Q + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
**



A475L + K495N + G628W


510
A136L + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
+



A475L + K495N + G628W + S764F + P870S


511
Q258N + Q291W + Q313M + D369R + E385L + E402N + S434P +
c1425g
+
+



K495N + G628W


512
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +
g2160a
+
+



K495N + G628W + T687M


513
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
**



K495N + N588F + G628W


514
Q258N + Q291W + Q313M + D358K + D369R + E402N + S434P +
c951a
+
+



A475L + K495N + G628W


515
Y135M + Q258N + Q291W + Q313M + D369R + E402N + S434P +
t1341c
+
+



A475L + K495N + G628W + Y715P


516
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
**



K495N + G628W + T785L


517
A79G + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
+



A475L + K495N + G628W


518
A109T + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
+



A475L + K495N + G628W + S848N


519
I106V + Q258N + Q291W + Q313M + F314V + D369R + E402N +

+
+



S434P + A475L + K495N + G628W


520
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
+



K495N + G628W + A732G


521
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
+



K495N + G628W + L757K


522
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
+



K495N + N627H + G628W + T687M + E822K


523
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
+



K495N + G616D + G628W


524
A79M + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
+



A475L + K495N + G628W


525
I106V + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
+



A475L + K495N + G628W


526
Q258N + Q291W + Q313M + F314V + D369R + E402N + S434P +

+
**



A475L + K495N + A617V + G628W


527
Q258N + Q291W + Q313M + F314V + D369R + E402N + S434P +

+
+



A475L + K495N + G628W


528
Q258N + Q291W + Q313M + D369R + E402N + S434P + P436Q +

+
+



A475L + K495N + G626D + G628W


529
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
+



K495N + G628W + T687C


530
Q85N + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
+



A475L + K495N + G628W


531
A109S + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
+



A475L + K495N + N536K + G628W


532
S58G + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
+



A475L + K495N + G628W


533
E21Q + Q258N + Q291W + Q313M + D369R + Q381V + E402N +

+
**



S434P + A475L + K495N + G628W


534
Q258N + L275Y + Q291W + Q313M + D369R + E402N + S434P +

+
+



A475L + K495N + G628W


535
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
**



K495N + A505C + G628W


536
E21R + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
**



A475L + K495N + G628W


537
V25A + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
+



A475L + K495N + G628W


538
H26R + Q258N + Q291W + Q313M + D369R + E402N + S434P +
t282c
+
**



A475L + K495N + G628W


539
L30K + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
*



A475L + K495N + G628W


540
N45H + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
**



A475L + K495N + G628W


541
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
**



E493V + K495N + G628W


542
P29R + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
*



A475L + K495N + G628W


543
P29M + Q258N + Q291W + Q313M + D369R + E402N + S434P +
c21t + c1911t
+
**



A475L + K495N + G628W + A732S + A748T + V840I


544
Q55R + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
**



A475L + K495N + G628W


545
K24G + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
+



A475L + K495N + G628W


546
G180E + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
**



A475L + K495N + G628W


547
Q258N + Q291W + Q313M + D369R + E402N + S434P + K495N +
c1425g
+
**



G628W


548
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
+



Q479R + K495N + S501R + G628W + Y715P + E819L


549
A79E + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
+



A475L + K495N + G628W


550
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
**



K495N + G628W + R672G


551
Q258N + Q291W + Q313M + F314V + D369R + E402N + S434P +
g2493a
+
+



A475L + K495N + A505C + G628W


552
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
+



K495N + G628W + T687K


553
Q27R + Q258N + Q291W + Q313M + D369R + E402N + S434P +
c1689g
+
**



A475L + K495N + G628W


554
V253F + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
+



A475L + K495N + G628W


555
S22L + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
**



A475L + K495N + G628W


556
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +
a321t
+
+



K495N + G628W + Q690H


557
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
**



K495N + G628W + S799N


558
Q258N + Q291W + Q313M + F314V + E360D + D369R + E402N +

+
+



S434P + A475L + K495N + G628W


559
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
+



K495N + G628W + E822M


560
K24T + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
+



A475L + K495N + G628W


563
S22L + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
+



A475L + Q479R + K495N + G628W


561
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
+



K495N + G628W + S787G


562
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
*



K495N + R612H + G628W + L757K + S787G


564
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
**



K495N + G628W + T687K + E822M


565
Q258N + Q291W + Q313M + D369R + E402N + S434P + M454E +

+
**



A475L + K495N + G628W


566
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
**



K495N + V562C + G628W


567
S22R + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
**



A475L + K495N + G628W


568
V25G + Q258N + Q291W + Q313M + D369R + E402N + S434P +
c2478t
+
**



A475L + K495N + G628W


569
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +
c1842t
+
**



K495N + G628W + T685V


570
Q258N + Q291W + Q313M + D369R + E402N + A404S + S434P +

+
**



A475L + K495N + G628W


571
G216L + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
**



A475L + K495N + G628W


572
V25R + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
**



A475L + K495N + G628W


573
V25G + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
**



A475L + K495N + G628W


574
Q27H + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
**



A475L + K495N + G628W


575
K24L + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
**



A475L + K495N + G628W


576
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
**



K495N + G628W + D650F + L757K


577
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
**



T482A + K495N + G628W


578
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
**



K495N + A617V + G628W


579
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
**



K495N + S501C + G628W


580
Q258N + Q291W + Q313M + D369R + E402N + S434P + K495N +
c1425g
+
+



N536K + G628W + R817P


582
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
**



K495N + G628W + T777N


581
G180E + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
**



A475L + R476Q + K495N + G628W


583
Q119E + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
+



A475L + K495N + G628W


584
A109S + Q258N + Q291W + Q313M + D369R + E402N + S434P +
g558a + t1296c +
+
+



K495N + G628W + S848N
c1425g


585
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
**



K495N + G628W + Y850H


586
Q258N + Q291W + Q313M + D369R + E402N + S434P + P436E +

+
**



A475L + K495N + G628W


587
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
**



K495N + G628W + R769H + E819A


588
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
**



K495N + T496A + G628W


589
Q258N + V260L + Q291W + Q313M + D369R + E402N + S434P +

+
**



A475L + K495N + G628W


590
D244H + Q258N + Q291W + Q313M + D369R + E402N + S434P +
c814t
+
**



A475L + K495N + G628W


591
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +
c1197a
+
**



K495N + G628W + E819V


592
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
**



K495N + G628W + S652D


593
Q258N + Q291W + D311N + Q313M + D369R + E402N + S434P +
g1479a
+
*



A475L + K495N + R612H + G628W + A689I + K866I


594
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +
c1917t
+
+



K495N + G628W + T783H


595
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
**



K495N + G628W + T823K


596
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
**



K495N + G628W + D650N + S787G


597
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +
g1869a
+
+



K495N + G628W + K708F


598
Q258N + Q291W + Q313M + E360D + D369R + E402N + S434P +

+
**



A475L + K495N + G628W + S652D


599
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +
a321g
+
**



K495N + G628W + E822G


600
L237Y + Q258N + Q291W + Q313M + D369R + E402N + S434P +

+
+



A475L + K495N + G628W


601
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
**



E494K + K495N + G628W


602
Q258N + Q291W + Q313M + E360D + D369R + E402N + S434P +
c312t
+
*



D470N + A475L + K495N + G628W + L757K


603
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

**
+



K495N + G628W + S764F


604
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

**
**



K495N + G628W + D650F


605
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

**
**



K495N + G628W + S848N


606
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

**
**



K495N + T496A + N536K + G628W


607
Q258N + Q291W + Q313M + D369R + E402N + K421R + S434P +

**
**



A475L + K495N + G628W + T777N


608
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

**
**



K495N + N627H + G628W + E822M


609
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +
t1944g + c1962t
**
**



K495N + G628W + F634A


610
A4V + Q258N + Q291W + Q313M + D369R + E402N + S434P +
c1635t
**
**



A475L + K495N + G628W


611
Q258N + Q291W + Q313M + D369R + E402N + S434P + N437W +

**
**



A475L + K495N + G628W


612
Y135Q + Q258N + Q291W + Q313M + D369R + E402N + S434P +

**
+



A475L + K495N + G628W


613
Y135Q + Q258N + Q291W + Q313M + D369R + E402N + S434P +
t99c
**
**



A475L + K495N + G628W + V673A + T685V


614
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

**
**



K495N + G628W + L757K + P806L


615
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +
c1476t
**
**



K495N + V559T + G628W


616
Y135M + Q258N + Q291W + Q313M + D369R + E402N + S434P +

**
**



A475L + K495N + G628W


617
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

**
+



K495N + G628W + V775C


618
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

**
**



K495N + N627H + G628W


619
Q258N + Q291W + Q313M + D369R + E402N + A405T + S434P +

**
**



A475L + K495N + G628W


620
I221V + Q258N + Q291W + Q313M + D369R + E402N + S434P +

**
**



A475L + K495N + G628W


621
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

**
**



K495N + S501R + G628W + T685V


622
Q258N + Q291W + Q313M + D369R + A394G + E402N + S434P +

**
+



A475L + K495N + G628W + L757K


624
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

**
+



K495N + G616D + G628W


623
P161S + Q258N + Q291W + Q313M + D358K + D369R + E402N +

**
*



S434P + A475L + K495N + G628W


625
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

**
+



K495N + G628W + K807R


626
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

**
+



K495N + G628W + A732M


627
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

**
**



K495N + A505C + G628W + E822G


628
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

**
+



E493V + K495N + G628W + S652D


629
A15V + Q258N + Q291W + Q313M + D369R + E402N + S434P +

**
+



A475L + K495N + G628W


630
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

**
**



K495N + G628W + K866Q


631
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

**
+



E493A + K495N + G628W + S652D + Q690A


632
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

**
**



K495N + G628W + E819L


633
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

**
+



K495N + G628W + E819V


634
G180E + Q258N + Q291W + Q313M + D369R + E385L + E402N +

**
**



S434P + A475L + K495N + G628W


635
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +
g2430a
**
**



K495N + G628W + D751N


636
G202M + Q258N + Q291W + Q313M + D369R + E402N + S434P +

**
**



A475L + K495N + N627H + G628W + T687K + E822A


637
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

**
**



K495N + G628W + K866I


638
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

**
**



K495N + G628W + T783Q


639
Q258N + Q291W + Q313M + D369L + E402N + S434P + A475L +

**
**



K495N + G628W


640
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

**
**



E493Y + K495N + A505C + G628W


641
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

**
+



T482A + K495N + G628W + D646N


642
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

**
+



A477G + K495N + G628W + S764F + S848N


643
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +
c1434t
**
+



K495N + G628W + I847T


644
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +
g2472a
**
+



K495N + G628W + T685V + Y850Q


645
Y135M + Q258N + Q291W + Q313M + D369R + E402N + S434P +

**
+



A475L + K495N + S501R + G628W + T685V + T777N


646
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

**
+



K495N + G628W + Y850Q





Table 4: Thermoactivity conditions: pH 4.5, 70° C. for 21 hrs. Thermostability conditions: enzyme residual activity was determined after incubated at pH 4.5, 70° C. for 2 hrs.


1Amino acid changes are indicated with respect to SEQ ID NO: 2; the backbone sequence contains substitutions Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N + G628W


2Nucleotide changes are indicated relative to SEQ ID NO: 1.


3Fold improvement is with respect to Variant 269 (SEQ ID NO: 7) * = 0.2 to 0.5 fold improvement ** = 0.6 to 1.0 fold improvement + = 1.1 to 1.9 fold improvement ++ = 2.0 to 2.9 fold improvement +++ = 3.0 to 3.9 fold improvement ++++ = 4.0 to 4.9 fold improvement













TABLE 5







Table 5: Thermoactivity conditions: pH 4.2, 70° C. for 21 hrs. Thermostability conditions: enzyme residual activity was determined


after incubated at pH 4.5, 70° C. for 3 hrs.













Silent
Activity Fold
Stability Fold


Variant

Nucleotide
improvement
improvement


Number
Amino Acid Changes1
Changes2
over Var. 4813
over Var. 4813





481
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N + G628W +






A689I + Y715P


647
D47I + Q258N + Q291W + Q313M + A343C + D369R + E402N + S434P + A475L +

++++
++++



K495N + G628W + T687K + A689I + Y715P


648
A109S + Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N + S434P +

++++
++



A475L + K495N + G628W + A689I + Y715P


649
A109T + Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N + S434P +

++++
++



A475L + K495N + G628W + T687W + A689I + Y715P + S764F


650
D47I + Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N + S434P +

+++
+



A475L + K495N + G628W + T687C + A689I + Y715P + S764F


651
Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N + S434P + A475L +
c2292t
+++
+



K495N + G628W + A689I + Y715P


652
D47I + Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L +

+++
+



K495N + G628W + T687C + A689I + Y715P


653
D47I + A109T + Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N +

+++
++



S434P + A475L + K495N + G628W + A689I + Y715P


654
Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

+++
++



G628W + T687W + A689I + Y715P


655
Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N + S434P + A475L +

+++
+



K495N + G628W + A689I + Y715P + S764F


656
Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

+++
+



G628W + A689I + Y715P + A732G


657
D47I + A109T + Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N +

+++
+



S434P + A475L + K495N + G628W + A689I + Y715P + S764F


658
Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N + S434P + A475L +

+++
+



K495N + G628W + A689I + Y715P + S764F


659
D47I + A109T + Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N +

+++
+



S434P + A475L + K495N + G628W + A689I + Y715P + S764F


660
D47I + A109T + Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N +

++
+



S434P + A475L + K495N + G628W + A689I + Y715P


661
A109T + Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N + S434P +

++
++



A475L + K495N + G628W + T687C + A689I + Y715P


662
D47I + I106V + Q258N + V260G + Q291W + Q313M + F314L + D369R + E402N +

++
++



S434P + A475L + K495N + G628W + A689I + Y715P + A732G


663
Q258N + Q291W + Q313M + A343C + D369R + E402N + S434P + A475L + K495N +

++
+



G628W + T687W + A689I + Y715P


664
I106V + Q258N + V260G + Q291W + Q313M + F314L + D369R + E402N + S434P +
c1425g
++
+



K495N + G628W + A689I + Y715P + A732G


665
Q258N + V260G + Q291W + Q313M + F314L + D369R + E402N + S434P + A475L +

++
+



K495N + G628W + A689I + Y715P


666
D47I + A109S + Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N +

++
+



S434P + A475L + K495N + G628W + T687W + A689I + Y715P


667
Q258N + V260G + Q291W + Q313M + F314V + D369R + E402N + S434P + A475L +
c747t
++
+



K495N + G628W + S652D + A689I + Y715P + A732G


668
Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N + S434P + A475L +

++
+



K495N + G628W + A689I + Y715P


669
I106V + Q258N + V260G + Q291W + Q313M + F314V + D369R + E402N + S434P +

++
++



A475L + K495N + G628W + S652D + A689I + Y715P + A732G


670
Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N + S434P + A475L +

++
+



K495N + G628W + A689I + Y715P + S764F


671
D47I + Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N + S434P +

++
+



A475L + K495N + G628W + A689I + Y715P


672
I106V + Q258N + V260G + Q291W + Q313M + F314L + D369R + E402N + S434P +

++
+



A475L + K495N + G628W + S652D + A689I + Y715P + A732M


673
A109T + Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L +

++
+



K495N + G628W + A689I + Y715P + S764F


674
Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N + S434P + A475L +

++
+



K495N + G628W + A689I + Y715P


675
Q258N + V260G + Q291W + Q313M + A343G + D369R + E402N + S434P + Q474L +

++
+



A475L + K495N + G628W + A689I + Y715P


676
D47I + Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N + S434P +

++
+



A475L + K495N + G628W + A689I + Y715P + S764F


677
D47I + A109T + Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P +
t300c
++
+



A475L + K495N + G628W + A689I + Y715P + S764F


678
Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N + S434P + A475L +

++
+



K495N + G628W + A689I + Y715P + S764F


679
D47I + A109T + Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N +

++
+



S434P + A475L + K495N + G628W + A689I + Y715P


680
Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N + S434P + A475L +

++
+



K495N + G628W + A689I + Y715P + S764F


681
Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

++
+



G628W + A689I + Y715P


682
D47I + Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N + S434P +

++
+



A475L + K495N + G628W + A689I + Y715P


683
Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

++
+



G628W + A689I + Y715P


684
A109T + Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N + S434P +

++
+



A475L + K495N + G628W + A689I + Y715P


685
I106V + Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L +

++
++



K495N + G628W + A689I + Y715P + A732G + P870S


686
Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

++
+



G628W + A689I + Y715P


687
I106V + Q258N + V260G + Q291W + Q313M + F314V + D369R + E402N + S434P +

++
++



A475L + K495N + G628W + A689I + Y715P + A732M


688
Q258N + Q291W + Q313M + F314V + D369R + E402N + S434P + A475L + K495N +

++
+



G628W + A689I + Y715P + A732G


689
I106V + Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L +

++
+



K495N + G628W + A689I + Y715P + A732V


690
Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N + S434P + A475L +

++
+



K495N + G628W + A689I + Y715P


691
Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N + S434P + A475L +

++
+



K495N + G628W + A689I + Y715P


692
D47I + I106V + Q258N + Q291W + Q313M + F314L + D369R + E402N + S434P +

++
+



A475L + K495N + G628W + A689I + Y715P + A732G


693
Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +
c306t
++
+



G628W + A689I + Y715P


694
Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

++
+



G628W + A689I + Y715P


695
Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + K495N + G628W +
c1425g
++
+



S652D + A689I + Y715P + A732G


696
D47I + I106V + Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P +
c1425g
++
++



K495N + G628W + S652D + A689I + Y715P + A732G


697
D47I + I106V + Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P +
c1425g
++
+



K495N + G628W + S652D + A689I + Y715P


698
Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N + S434P + A475L +

++
+



K495N + G628W + A689I + Y715P


699
C8A + L9F + D47I + Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P +
c1425g
++
+



K495N + G628W + A689I + Y715P + A732G


700
D47I + Q258N + V260G + Q291W + Q313M + F314V + D369R + E402N + S434P +

++
+



A475L + K495N + G628W + A689I + Y715P + A732G + D844G


701
Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

++
+



G628W + A689I + Y715P


702
I106V + Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L +

++
++



K495N + G628W + A689I + Y715P + A732G


703
D47N + Q258N + V260G + Q291W + Q313M + F314V + D369R + E402N + S434P +

++
++



A475L + K495N + G628W + A689I + Y715P + A732G


704
Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

++
+



G628W + A689I + Y715P


705
D47I + I106V + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

++
+



K495N + G628W + S652D + A689I + Y715P + A732M


706
A109T + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

++
+



G628W + T687W + A689I + Y715P


707
Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N + S434P + A475L +

++
+



K495N + G628W + A689I + Y715P


708
V25A + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

++
+



G628W + A689I + Y715P


709
D47I + Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L +

++
+



K495N + G628W + A689I + Y715P + A732M


710
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N + G628W +

++
+



T687W + A689I + Y715P


711
D47I + I106V + Q258N + V260G + Q291W + Q313M + F314V + D369R + E402N +

++
++



S434P + A475L + K495N + G628W + S652D + A689I + Y715P


712
D47I + A109T + Q258N + Q291W + Q313M + A343C + D369R + E402N + S434P +

++
+



A475L + K495N + G628W + A689I + Y715P


713
D47I + Q258N + Q291W + Q313M + A343C + D369R + E402N + S434P + A475L +

++
+



K495N + G628W + A689I + Y715P + S764F


714
Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +
c1173t
+
+



G628W + T687K + A689I + Y715P


715
A109T + Q258N + Q291W + Q313M + A343C + D369R + E402N + S434P + A475L +

+
+



K495N + G628W + A689I + Y715P + S764F


716
Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

+
+



G628W + A689I + Y715P


717
D47I + I106V + Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P +

+
+



A475L + K495N + G628W + A689I + Y715P


718
Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

+
+



G628W + A689I + Y715P + S764F


719
Q258N + V260G + Q291W + Q313M + F314L + D369R + E402N + S434P + K495N +
c1425g
+
+



G628W + S652D + A689I + Y715P


720
Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

+
+



G628W + A689I + Y715P


721
Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N + S434P + A475L +

+
+



K495N + G628W + A689I + Y715P


722
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N + G628W +

+
+



A689I + Y715P + S764F


723
Q258N + V260G + Q291W + Q313M + F314V + D369R + E402N + S434P + K495N +
c1425g
+
+



G628W + A689I + Y715P


724
L237Y + Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N + S434P +

+
**



A475L + K495N + G628W + A689I + Y715P


725
Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

+
+



G628W + A689I + Y715P


726
Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

+
+



G628W + A689I + Y715P


727
Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

+
+



G628W + A689I + Y715P


728
Q85N + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

+
+



G628W + A689I + Y715P + L757K


729
Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

+
+



G628W + A689I + Y715P


730
Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +
g2235a
+
+



G628W + A689I + Y715P


731
Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

+
+



S604C + G628W + A689I + Y715P


732
D47I + Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
+



R476G + K495N + N588F + G628W + D651E + A689I + Y715P


733
Q85N + Q258N + Q291W + Q313M + D369R + E402N + S434P + P436Q + A475L +

+
+



K495N + G628W + A689I + Y715P + L757K


734
I106V + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

+
+



G628W + A689I + Y715P + A732M


735
Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

+
+



G628W + A689I + Y715P


736
Q258N + V260G + Q291W + Q313M + F314L + D369R + E402N + S434P + A475L +

+
+



K495N + G628W + A689I + Y715P


737
Q258N + Q291W + Q313M + D369R + E402N + S434P + K495N + G628W + S652D +
c1425g
+
+



A689I + Y715P + A732G


738
A109T + Q258N + Q291W + Q313M + A343C + D369R + E402N + S434P + A475L +

+
+



K495N + G628W + A689I + Y715P


739
D47I + Q258N + Q291W + Q313M + F314L + D369R + E402N + S434P + A475L +
c2277a
+
+



K495N + G628W + A689I + Y715P + Y736N


740
Q258N + Q291W + Q313M + F314L + D369R + E402N + S434P + A475L + K495N +

+
+



G628W + S652D + A689I + Y715P + A732G


741
V25A + Q85N + Q258N + Q291W + Q313M + D369R + E402N + S434P + P436Q +

+
+



A475L + K495N + G616D + G628W + D650Y + A689I + Y715P + L757K


742
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N + G628W +

+
+



A689I + Y715P + S764F


743
V253F + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

+
+



G628W + A689I + Q690K + D709E + E710G + Y715P


744
Q85N + V175A + Q258N + L275Y + Q291W + Q313M + D369R + E402N + S434P +

+
+



A475L + K495N + G628W + A689I + Y715P


745
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N + G628W +

+
**



A689I + Q690K + Y715P


746
V25A + Q85N + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
+



K495N + G616D + G628W + A689I + Y715P + L757K


747
Q85N + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

+
+



G628W + A689I + Y715P


748
V25A + Q85N + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +
c57a
+
+



K495N + G628W + A689I + Y715P


749
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N + G628W +

+
**



T687C + A689I + Y715P


750
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N + G628W +

+
+



A689I + Q690H + Y715P + M816L


751
V25A + Q85N + Q258N + L275Y + Q291W + Q313M + D369R + E402N + S434P +

+
+



A475L + K495N + G616D + G628W + A689I + Y715P + V846F


752
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N + G628W +

+
+



A689I + Y715P + V846Q


753
A79E + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

+
**



G626D + G628W + A689I + Y715P


754
Q258N + Q291W + Q313M + A343C + D369R + E402N + S434P + A475L + K495N +

+
**



G628W + A689I + Y715P


755
A79E + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

+
+



A505C + G628W + A689I + Y715P


756
Q258N + Q291W + Q313M + F314L + D369R + Q381V + E402N + S434P + A475L +

+
+



K495N + G628W + A689I + Y715P


757
D47I + I106V + Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P +
c1704t
+
+



A475L + K495N + G628W + A689I + Y715P


758
Q85N + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

+
+



G628W + A689I + Y715P


759
V25A + Q85N + Q258N + Q291W + Q313M + D369R + E402N + S434P + P436Q +

+
+



A475L + K495N + G628W + A689I + Y715P + L757K + V846Q


760
V25A + Q85N + Q258N + Q291W + Q313M + D369R + E402N + S434P + P436Q +
g2274t
+
+



A475L + K495N + G628W + D650N + A689I + Y715P


761
D47I + I106V + Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P +

+
+



A475L + K495N + S550C + G628W + S652D + A689I + Y715P + A732G


762
A109S + Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
+



K495N + S604A + G628W + A689I + Y715P


763
V25A + Q85N + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
+



K495N + G628W + A689I + Y715P + V846Q


764
D47I + Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
+



K495N + G628W + A689I + Y715P


765
A109T + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

+
+



G628W + A689I + Y715P


766
Q258N + V260G + Q291W + Q313M + F314L + D369R + E402N + S434P + K495N +
c1425g
+
+



N588F + G628W + A689I + Y715P + A732M


767
Q85N + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

+
+



G616D + G628W + A689I + Y715P


768
V25A + Q85N + Q258N + Q291W + Q313M + D369R + E402N + S434P + P436Q +

+
+



A475L + K495N + G616D + G628W + D650N + A689I + Y715P


769
A79E + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

+
+



A505C + G628W + V674I + A689I + Y715P


770
V25A + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +
c483t
+
+



G628W + A689I + Y715P + L757K


771
V25A + Q85H + Q258N + L275F + Q291W + Q313M + D369R + E402N + S434P +

+
+



A475L + K495N + G628W + D650N + A689I + Y715P + L757I


772
K24T + A79E + A136L + Q258N + D274Y + Q291W + Q313M + D369R + E402N +

+
+



S434P + A475L + K495N + A505C + G628W + A689I + Y715P


773
V25A + Q85N + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
+



K495N + G628W + A689I + Y715P


774
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N + G628W +

+
**



A689I + Y715P


775
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N + G628W +
g633a
+
**



A689I + Y715P


776
K24G + A79E + Q258N + D274Y + Q291W + Q313M + D369R + E402N + S434P +

+
**



A475L + K495N + A505C + G628W + A689I + Y715P


777
Q85N + V175A + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
+



K495N + G628W + A689I + Y715P


778
V25A + Q85N + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L +

+
+



K495N + G628W + D650N + A689I + Y715P


779
V25A + Q85N + Q258N + L275Y + Q291W + Q313M + D369R + E402N + S434P +

+
+



A475L + K495N + G628W + A689I + Y715P + L757K


780
A79M + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

+
+



A505C + G628W + A689I + Y715P


781
Q85N + Q258N + Q291W + Q313M + D369R + E402N + S434P + P436Q + A475L +

+
+



K495N + G628W + A689I + Y715P+


782
Q85N + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

+
+



G628W + A689I + Y715P


783
Q85N + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

+
+



G628W + A689I + Y715P


784
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N + G628W +

+
+



A689I + Y715P


785
Q258N + Q291W + Q313M + A343C + D369R + E402N + S434P + A475L + K495N +

+
**



G628W + A689I + Y715P


786
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N + G628W +

+
**



A689I + Y715P


787
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N + G628W +

+
**



A689I + Y715P


788
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N + G628W +

+
+



A689I + Y715P


789
Q85N + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +
g927a
+
+



G628W + A689I + Y715P


790
I106V + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

**
+



G628W + A689I + Y715P


791
D47I + Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L +

**
+



K495N + S604A + G628W + A689I + Y715P


792
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N + G628W +

**
+



A689I + Y715P


793
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N + G628W +

**
**



A689I + Y715P


794
V25A + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

**
**



G628W + A689I + Y715P


795
Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N + S434P + Q474I +

**
+



A475L + K495N + G628W + A689I + Y715P + S764F


796
Q258N + Q291W + Q313M + A343C + D369R + E402N + S434P + A475L + K495N +

**
+



G628W + A689I + Y715P


797
D47I + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

**
+



N588F + G628W + A689I + Y715P


798
S58G + Q258N + Q291W + Q313M + D369R + Q381V + E402N + S434P + A475L +

**
+



K495N + G628W + A689I + Y715P


799
V253F + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

**
+



G628W + A689I + Y715P + T785L + M816L


800
A243G + Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L +

**
+



K495N + G628W + A689I + Y715P + S764F


801
I106V + Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L +
c597t
**
++



K495N + N588F + G628W + S652D + A689I + Y715P + D733G


802
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N + G628W +

**
**



A689I + Y715P + V846L


803
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N + S604A +

**
+



G628W + A689I + Y715P


804
A79G + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

**
+



G628W + A689I + Y715P + T777N


805
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N + G628W +

**
**



A689I + Y715P


806
V25A + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

**
**



G628W + A689I + Y715P


807
K24T + A79E + Q258N + D274Y + Q291W + Q313M + D369R + E402N + S434P +

**
+



A475L + K495N + A505C + G628W + A689I + Y715P + T777N


808
D47I + Q258N + Q291W + Q313M + A343C + D369R + E402N + S434P + A475L +

**
+



K495N + S604V + G628W + T687C + A689I + Y715P + S764F + L869R


809
Q258N + D274Y + Q291W + Q313M + D369R + E385L + E402N + S434P + A475L +

**
**



K495N + G626D + G628W + A689I + Y715P + T777N


810
S58G + Q258N + Q291W + Q313M + D369R + Q381V + E402N + S434P + N437D +
c489t
**
**



A475L + K495N + G628W + A689I + Q690H + D709E + E710G + Y715P


811
D47I + Q258N + V260G + Q291W + Q313M + A343C + D369R + E402N + S434P +

**
+



Q474I + A475L + K495N + G628W + A689I + Y715P


812
Q258N + Q291W + Q313M + D369R + E402N + S434P + N437K + A475L + K495N +

**
+



G628W + A689I + Y715P + T785L


813
A109S + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

**
+



S604I + G628W + A689I + Y715P


814
Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +
c624t
**
+



S604V + G628W + A689I + Y715P + K807R


815
Q258N + Q291W + Q313M + A343C + D369R + E402N + S434P + A475L + K495N +

**
+



S604V + G628W + A689I + Y715P


816
Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N + G628W +

**
**



A689I + Y715P + L757K


817
D47I + Q258N + Q291W + Q313M + D369R + E402N + S434P + K495N + N588F +
c1425g
**
+



G628W + S652D + A689I + Y715P


818
A79M + A136L + Q258N + D274Y + Q291W + Q313M + D369R + E402N + S434P +

**
+



A475L + K495N + A505C + G628W + A689I + Y715P + T783A


819
D47I + A109T + Q258N + V260G + Q291W + Q313M + D369R + E402N + S434P +

**
+



A475L + K495N + S604V + G628W + A689I + Y715P + S764F


820
S58G + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

*
**



G628W + A689I + Y715P + T785L


821
K24G + A136L + Q258N + D274Y + Q291W + Q313M + D369R + E402N + S434P +
g240a
*
**



A475L + K495N + A505C + G628W + A689I + Y715P + T777N


822
S58G + Q258N + Q291W + Q313M + D369R + Q381V + E402N + S434P + A475L +
c2007t
*
+



K495N + G628W + A689I + Y715P + T785L


823
S58G + Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N +

*
**



G628W + A689I + Y715P + T785L + M816L





1Amino acid changes are indicated with respect to SEQ ID NO: 2; the backbone sequence contains substitutions Q258N + Q291W + Q313M + D369R + E402N + S434P + A475L + K495N + G628W + A689I + Y715P.


2Nucleotide changes are indicated relative to SEQ ID NO: 1.


3Fold improvement is with respect to Variant 481 (SEQ ID NO: 9): * = 0.2 to 0.5 fold improvement ** = 0.6 to 1.0 fold improvement + = 1.1 to 1.9 fold improvement ++ = 2.0 to 2.9 fold improvement +++ = 3.0 to 3.9 fold improvement ++++ = 4.0 to 4.9 fold improvement













TABLE 6







Table 6: Thermoactivity conditions: pH 4, 70° C. for 21 hrs. Thermostability conditions: enzyme residual activity was determined


after incubated at pH 4.5, 70° C. for 24 hrs. Amino acid and silent nucleotide changes are indicated with respect to


wild type sequence.













Silent
Activity Fold
Stability Fold


Variant

Nucleotide
improvement over
improvement


Number
Amino Acid Changes1
Changes2
Var. 6473
over Var. 6473





647
D47I + Q258N + Q291W + Q313M + A343C + D369R + E402N +






S434P + A475L + K495N + G628W + T687K + A689I + Y715P


824
D47I + A79G + Q85N + Q258N + V260G + Q291W + Q313M + A343C +

++++
+



D369R + E402N + S434P + A475L + K495N + G628W +



T687K + A689I + Y715P + A732M


825
D47I + Q258N + V260G + Q291W + Q313M + F314L + A343C +

++++
+



D369R + E402N + S434P + A475L + K495N + G628W + T687C +



A689I + Y715P + A732G


826
D47I + A79E + Q85N + Q258N + Q291W + Q313M + A343C + D369R +
c1947t
+++
++



E402N + S434P + A475L + K495N + A505C + G628W +



T687W + A689I + Y715P


827
D47I + A79E + Q85N + Q258N + V260G + Q291W + Q313M + F314L +

+++
**



A343C + D369R + E402N + S434P + A475L + K495N +



G628W + T687K + A689I + Y715P


828
D47I + A79G + Q85N + Q258N + V260G + L275Y + Q291W + Q313M +
c1677t
+++
++



F314V + A343C + D369R + E402N + S434P + A475L +



K495N + A505C + G628W + T687C + A689I + Y715P + S764Y +



R769H


829
D47I + A79G + Q85N + Q258N + V260G + Q291W + Q313M + F314V +

+++
++



A343C + D369R + E402N + S434P + A475L + K495N +



G628W + T687K + A689I + Y715P


830
D47I + A79M + Q85N + Q258N + V260G + L275Y + Q291W + Q313M +

+++
++



F314L + A343C + D369R + E402N + S434P + A475L +



K495N + A505C + G628W + T687K + A689I + Y715P


831
D47I + A79M + Q85N + Q258N + Q291W + Q313M + A343C + D369R +
t756c
+++
++



E402N + S434P + A475L + K495N + A505C + G628W +



T687C + A689I + Y715P + A732G


832
D47I + Q258N + V260G + Q291W + Q313M + F314V + A343C +

+++
+



D369R + E402N + S434P + K495N + A505C + G628W + T687C +



A689I + Y715P + A732G


833
D47I + A79M + Q258N + V260G + Q291W + Q313M + F314V +

+++
**



A343C + D369R + E402N + S434P + K495N + G628W + T687K +



A689I + Y715P + A732G


834
D47I + Q258N + L275Y + Q291W + Q313M + F314V + A343C +

+++
+



D369R + E402N + S434P + K495N + G628W + T687K + A689I +



Y715P + A732G


835
D47I + A79E + Q85N + Q258N + V260G + Q291W + Q313M + F314V +

+++
++



A343C + D369R + E402N + S434P + K495N + A505C +



G628W + T687W + A689I + Y715P + A732V


836
D47I + A79M + Q258N + V260G + Q291W + Q313M + A343C +

+++
*



D369R + E402N + S434P + K495N + A505C + G628W + T687K +



A689I + Y715P + A732M


837
D47I + Q85N + Q258N + V260G + Q291W + Q313M + F314V +

+++
**



A343C + D369R + E402N + S434P + A475L + K495N + G628W +



T687C + A689I + Y715P


838
D47I + A79G + Q258N + V260G + L275Y + Q291W + Q313M +

+++
+



A343C + D369R + E402N + S434P + A475L + K495N + G628W +



T687K + A689I + Y715P + A732G


839
D47I + Q258N + V260G + Q291W + Q313M + F314V + A343C +

+++
++



D369R + E402N + S434P + A475L + K495N + G628W + T687W +



A689I + Y715P + A732G


840
D47I + Q258N + V260G + Q291W + Q313M + F314V + A343C +

+++
**



D369R + E402N + S434P + A475L + K495N + G628W + T687K +



A689I + Y715P + A732M


841
D47I + A79G + Q85N + Q258N + V260G + Q291W + Q313M + A343C +

+++
**



D369R + E402N + S434P + A475L + K495N + G628W +



T687K + A689I + Y715P


842
D47I + A79G + Q85N + Q258N + V260G + L275Y + Q291W + Q313M +

+++
**



F314V + A343C + D369R + E402N + S434P + A475L +



K495N + G628W + T687C + A689I + Y715P + A732G


843
D47I + A79G + Q258N + V260G + Q291W + Q313M + A343C +

+++
**



D369R + E402N + S434P + A475L + K495N + G628W + T687K +



A689I + Y715P + A732M


844
D47I + Q85N + Q258N + V260G + Q291W + Q313M + A343C +

+++
+



D369R + E402N + S434P + K495N + G628W + T687K + A689I +



Y715P


845
D47I + Q85N + Q258N + V260G + L275Y + Q291W + Q313M +

+++
+



A343C + D369R + E402N + S434P + K495N + G628W + T687K +



A689I + Y715P + A732G


846
D47I + A79M + Q85N + Q258N + V260G + L275Y + Q291W + Q313M +

+++
*



A343C + D369R + E402N + S434P + K495N + G628W +



T687W + A689I + Y715P


847
D47I + A79G + Q85N + Q258N + V260G + Q291W + Q313M + A343C +

+++
**



D369R + E402N + S434P + A475L + K495N + A505C +



G628W + T687K + A689I + Y715P


848
D47I + Q258N + V260G + Q291W + Q313M + F314V + A343C +
c498t
+++
+



D369R + E402N + S434P + A475L + K495N + G628W + T687C +



A689I + Y715P + A732G


849
D47I + A79G + Q85N + Q258N + V260G + L275Y + Q291W + Q313M +

+++
++



F314V + A343C + D369R + E402N + S434P + A475L +



K495N + G628W + T687K + A689I + Y715P


850
D47I + Q85N + Q258N + Q291W + Q313M + A343C + D369R +

+++
++



E402N + S434P + A475L + K495N + G628W + T687W + A689I +



Y715P + A732G


851
D47I + A79G + Q85N + Q258N + V260G + L275Y + Q291W + Q313M +
c1335t
+++
**



F314V + A343C + D369R + E402N + S434P + A475L +



K495N + A505C + G628W + T687C + A689I + Y715P


852
D47I + Q258N + V260G + Q291W + Q313M + A343C + D369R +

+++
+



E402N + S434P + A475L + K495N + A505C + G628W + T687K +



A689I + Y715P + A732G


853
D47I + Q258N + Q291W + Q313M + F314V + A343C + D369R +

+++
++



E402N + S434P + A475L + K495N + G628W + T687W + A689I +



Y715P + A732G


854
D47I + A79G + Q85N + Q258N + Q291W + Q313M + F314V + A343C +

+++
**



D369R + E402N + S434P + K495N + G628W + T687K +



A689I + Y715P


855
D47I + A79M + Q85N + Q258N + Q291W + Q313M + A343C + D369R +

+++
*



E402N + S434P + A475L + K495N + G628W + T687K +



A689I + Y715P + A732G


856
D47I + Q85N + Q258N + V260G + Q291W + Q313M + F314V +

+++
*



A343C + D369R + E402N + S434P + A475L + K495N + G628W +



T687K + A689I + Y715P


857
D47I + Q85N + Q258N + L275Y + Q291W + Q313M + F314V +

+++
++



A343C + D369R + E402N + S434P + K495N + G628W + T687C +



A689I + Y715P


858
D47I + A79G + Q85N + Q258N + V260G + L275Y + Q291W + Q313M +

+++
**



F314V + A343C + D369R + E402N + S434P + A475L +



K495N + G628W + T687K + A689I + Y715P + A732G


859
D47I + Q258N + V260G + Q291W + Q313M + A343C + D369R +
c1377t
++
+



E402N + S434P + A475L + K495N + G628W + T687K + A689I +



Y715P


860
D47I + Q85N + Q258N + V260G + L275Y + Q291W + Q313M +

++
+



A343C + D369R + E402N + S434P + A475L + K495N + G628W +



T687K + A689I + Y715P


861
D47I + A79G + Q258N + Q291W + Q313M + F314L + A343C +

+
++



D369R + E402N + S434P + A475L + K495N + G628W + T687K +



A689I + Y715P


862
D47I + Q258N + Q291W + Q313M + F314V + A343C + D369R +

+
+



E402N + S434P + K495N + G628W + T687K + A689I + Y715P


863
D47I + Q258N + V260G + L275Y + Q291W + Q313M + A343C +

+
+



D369R + E402N + S434P + A475L + K495N + A505C + G628W +



D646N + T687K + A689I + Y715P + A732G


864
D47I + Q258N + V260G + Q291W + Q313M + A343C + D369R +

+
+



E402N + S434P + A475L + K495N + G628W + T687K + A689I +



Y715P


865
D47I + Q258N + V260G + Q291W + Q313M + F314V + A343C +

+
+



D369R + E402N + S434P + K495N + G628W + T687K + A689I +



Y715P + A732V


866
D47I + A79G + Q85N + Q258N + Q291W + Q313M + A343C + D369R +

+
*



E402N + S434P + P439S + A475L + K495N + G628W +



T687K + A689I + Y715P


867
D47I + Q85N + Q258N + Q291W + Q313M + F314V + A343C +

+
+



D369R + D395N + E402N + S434P + A475L + K495N + G628W +



T687K + A689I + Y715P + A732V


868
D47I + A79G + Q258N + Q291W + Q313M + A343C + D369R +

**
+



E402N + S434P + A475L + K495N + A505C + G628W + T687C +



A689I + T693A + Y715P + T827I


869
D47I + Q85N + Q258N + V260G + Q291W + Q313M + A343C +
t237g
**
+



D369R + E402N + S434P + A475L + K495N + A505C + G628W +



T687K + A689I + Y715P + A732V


870
D47I + A79G + Q258N + L275Y + Q291W + Q313M + A343C +
a2185c
**
+



D369R + E402N + S434P + A475L + K495N + A505C + G628W +



T687K + A689I + T693E + N723G + A730S + Y855*





1Amino acid changes are indicated with respect to SEQ ID NO: 2; the backbone sequence contains substitutions D47I + Q258N + Q291W + Q313M + A343C + D369R + E402N + S434P + A475L + K495N + G628W + T687K + A689I + Y715P.


2Nucleotide changes are indicated relative to SEQ ID NO: 1.


3Fold improvement is with respect to Variant 647 (SEQ ID NO: 15): * = 0.2 to 0.5 fold improvement ** = 0.6 to 1.0 fold improvement + = 1.1 to 1.9 fold improvement ++ = 2.0 to 2.9 fold improvement +++ = 3.0 to 3.9 fold improvement ++++ = 4.0 to 4.9 fold improvement













TABLE 7







Table 7: Thermoactivity conditions: pH 4, 70° C. for 21 hrs. Thermostability conditions: enzyme residual activity was determined after incubated at


pH 4.5, 70° C. for 24 hrs.













Silent
Activity Fold
Stability Fold


Variant

Nucleotide
improvement over Var.
improvement over


Number
Amino Acid Changes1
Changes2
6643
Var. 6643





664
I106V + Q258N + V260G + Q291W + Q313M + F314L + D369R +
c1425g





E402N + S434P + K495N + G628W + A689I + Y715P + A732G


871
D47I + A79E + Q85N + I106V + A109T + Q258N + V260G + Q291W +
c1425g
++
+



Q313M + F314V + A343C + D369R + E402N + S434P + K495N +



G628W + A689I + Y715P + A732G


872
D47I + A79E + Q85N + I106V + Q258N + V260G + Q291W + Q313M +
c1425g
+++
+



F314V + A343C + D369R + E402N + S434P + K495N + A505C +



G628W + A689I + Y715P + A732G


873
A79M + Q85N + I106V + A109T + Q258N + V260G + Q291W +
c246t + c1425g +
+++
+



Q313M + F314V + A343C + D369R + E402N + S434P + K495N +
c2346t



G628W + A689I + Y715P + A732G


874
D47I + A79E + I106V + A109T + Q258N + V260G + Q291W + Q313M +
c1425g
+++
+



F314L + A343C + D369R + E402N + S434P + K495N +



G628W + A689I + Y715P + A732G


875
D47I + A79G + Q85N + I106V + A109T + Q258N + V260G + L275Y +
c1425g
+++
+



Q291W + Q313M + F314L + A343C + D369R + E402N + S434P +



K495N + G628W + A689I + Y715P + A732G


876
A79E + Q85N + I106V + A109T + Q258N + V260G + Q291W +
c1425g
+++
+



Q313M + F314L + A343C + D369R + E402N + S434P + K495N +



G628W + A689I + Y715P + A732G


877
A79G + Q85N + I106V + A109T + Q258N + V260G + L275F + Q291W +
c1425g
+++
+



Q313M + F314V + A343C + D369R + E402N + S434P +



K495N + A505C + G628W + T687C + A689I + Y715P + A732G


878
D47I + I106V + Q258N + V260G + Q291W + Q313M + F314L +
c1425g
++
+



A343C + D369R + E402N + S434P + K495N + G628W + A689I +



Y715P + A732G


879
D47I + A79E + Q85N + I106V + A109S + Q258N + V260G + Q291W +
c1425g
++
+



Q313M + F314V + A343C + D369R + E402N + S434P + K495N +



G628W + A689I + Y715P + A732G


880
D47I + A79E + Q85N + I106V + Q258N + V260G + L275Y + Q291W +
c1425g
++
**



Q313M + F314L + N315D + D369R + E402N + S434P + K495N +



A505C + G628W + T687W + A689I + Y715P + A732G


881
D47I + Q85N + I106V + Q258N + V260G + L275Y + Q291W + Q313M +
c1425g
++
+



F314L + D369R + E402N + S434P + K495N + G628W +



A689I + Y715P + A732G


882
A79E + Q85N + I106V + A109S + Q258N + V260G + Q291W +
c1425g
++
+



Q313M + F314V + D369R + E402N + S434P + K495N + G628W +



A689I + Y715P + A732G


883
A79G + I106V + Q258N + V260G + Q291W + Q313M + F314V +
c1425g
++
+



A343C + D369R + E402N + S434P + K495N + G628W + T687W +



A689I + Y715P + A732G


884
D47I + A79G + Q85N + I106V + Q258N + V260G + L275Y + Q291W +
c1425g + g2235a
++
+



Q313M + F314L + A343C + D369R + E402N + S434P + K495N +



A505C + G628W + A689I + Y715P + A732G


885
D47I + Q85N + I106V + A109S + Q258N + V260G + Q291W + Q313M +
c1425g+
++
+



F314V + A343C + D369R + E402N + S434P + K495N +



G628W + A689I + Y715P + A732G


886
D47I + A79E + Q85N + I106V + Q258N + V260G + Q291W + Q313M +
c811t + c1425g
++
+



F314L + A343C + D369R + E402N + S434P + K495N + G628W +



A689I + Y715P + A732G


887
D47I + A79M + Q85N + I106V + Q258N + V260G + Q291W + Q313M +
c1425g
++
+



F314V + A343C + D369R + E402N + S434P + K495N + G628W +



A689I + Y715P + A732G


888
D47I + I106V + A109T + Q258N + V260G + Q291W + Q313M +
c1425g + c1806t
++
+



F314V + A343C + D369R + E402N + S434P + K495N + G628W +



A689I + Y715P + A732G


889
D47I + A79G + Q85N + I106V + Q258N + V260G + Q291W + Q313M +
c1425g
++
+



F314V + D369R + E402N + S434P + K495N + A505C + G628W +



A689I + Y715P + A732G


890
A79M + I106V + Q258N + V260G + Q291W + Q313M + F314L +
c1425g
++
+



D369R + E402N + S434P + K495N + G628W + A689I + Y715P +



A732G


891
A79M + Q85N + I106V + Q258N + V260G + Q291W + Q313M +
c1425g
++
+



F314L + A343C + D369R + E402N + S434P + K495N + A505C +



G628W + A689I + Y715P + A732G


892
A79M + Q85N + I106V + Q258N + V260G + Q291W + Q313M +
c1425g
++
+



F314V + A343C + D369R + E402N + S434P + K495N + G628W +



A689I + Y715P + A732G


893
D47I + A79G + Q85N + I106V + A109S + Q258N + V260G + Q291W +
c1425g + c2541t
++
+



Q313M + F314L + D369R + E402N + S434P + K495N +



G628W + A689I + Y715P + A732G


894
A79E + Q85H + I106V + Q258N + V260G + Q291W + Q313M +
c540t + c1425g
++
+



F314L + D369R + E402N + S434P + K495N + G628W + A689I +



Y715P + A732G


895
D47I + A79G + Q85N + I106V + Q258N + V260G + Q291W + Q313M +
c1425g
++
+



F314L + D369R + E402N + S434P + K495N + G628W +



A689I + Y715P + A732G


896
A79G + Q85N + I106V + Q258N + V260G + Q291W + Q313M +
c1425
++
+



F314L + A343C + D369R + E402N + S434P + K495N + G628W +



A689I + Y715P + A732G


897
D47I + A79E + Q85N + I106V + Q258N + V260G + L275Y + Q291W +
c1425g
++
+



Q313M + F314L + A343C + D369R + E402N + S434P + K495N +



G628W + A689I + Y715P + A732G


898
D47I + I106V + A109T + Q258N + V260G + Q291W + Q313M +
c468t + c1425g
++
+



F314V + D369R + E402N + S434P + K495N + G628W + A689I +



Y715P + A732G


899
C8G + D47I + Q85N + I106V + Q258N + V260G + Q291W + Q313M +
c1425g
++
+



F314L + D369R + E402N + S434P + K495N + G628W + A689I +



Y715P + A732G


900
A79E + Q85N + I106V + Q258N + V260G + Q291W + Q313M +
c1425g
++
+



F314L + D369R + E402N + S434P + K495N + G628W + A689I +



Y715P + A732G


901
A79G + Q85N + I106V + Q258N + V260G + Q291W + Q313M +
c1425g
++
+



F314L + A343C + D369R + E402N + S434P + K495N + G628W +



A689I + Y715P + A732G


902
D47I + A79G + Q85N + I106V + Q258N + V260G + Q291W + Q313M +
c1425g
++
+



F314L + A343C + D369R + E402N + S434P + K495N + T591I +



G628W + A689I + Y715P + A732G


903
Q85N + I106V + A109T + Q258N + V260G + Q291W + Q313M +
g150a + c1425g
++
+



F314V + A343C + D369R + E402N + S434P + K495N + G628W +



A689I + Y715P + A732G


904
A79M + I106V + Q258N + V260G + Q291W + Q313M + F314V +
c1425g
++
+



A343C + D369R + E402N + S434P + K495N + G628W + A689I +



Y715P + A732G


905
Q85N + I106V + Q258N + V260G + Q291W + Q313M + F314V +
c1425g
++
+



A343C + D369R + E402N + S434P + K495N + G628W + A689I +



Y715P + A732G


906
Q85N + I106V + A109S + Q258N + V260G + Q291W + Q313M +
c1425g
++
+



F314L + D369R + E402N + S434P + K495N + A505C + G628W +



A689I + Y715P + A732G


907
I106V + Q258N + V260G + Q291W + Q313M + F314L + D369R +
c1425g
++
**



E402N + S434P + K495N + A505C + G628W + T687K + A689I +



Y715P + A732G


908
Q85N + I106V + A109T + Q258N + V260G + Q291W + Q313M +
c1425g
++
+



F314V + D369R + E402N + S434P + K495N + G628W + A689I +



Y715P + A732G


909
D47I + Q85N + I106V + A109T + Q258N + V260G + Q291W + Q313M +
t237g + c1425g
++
+



F314V + A343G + D369R + E402N + S434P + K495N +



G628W + A689I + Y715P + A732G


910
D47I + Q85N + I106V + A109T + Q258N + V260G + Q291W + Q313M +
c1425g
++
+



F314L + A343C + D369R + E402N + S434P + K495N +



G628W + A689I + Y715P + A732G


911
D47I + Q85N + I106V + A109S + Q258N + V260G + L275Y + Q291W +
c1425g
++
+



Q313M + F314L + A343C + D369R + E402N + S434P +



K495N + G628W + A689I + Y715P + A732G


912
D47I + I106V + Q258N + V260G + Q291W + Q313M + F314V +
c933t + c1425g
++
+



A343C + D369R + E402N + S434P + K495N + G628W + A689I +



Y715P + A732G


913
I106V + Q258N + V260G + Q291W + Q313M + F314V + D369R +
c1425g
++
**



E402N + S434P + K495N + G628W + A689I + Y715P + A732G


914
D47I + I106V + Q258N + V260G + Q291W + Q313M + F314L +
c1425g
++
**



A343C + D369R + E402N + S434P + K495N + G628W + A689I +



Y715P + A732G


915
D47I + Q85N + I106V + A109S + Q258N + V260G + L275Y + Q291W +

++
+



Q313M + F314V + A343C + D369R + E402N + S434P +



A475L + K495N + G628W + T687C + A689I + Y715P + A732G


916
D47I + A79M + I106V + Q258N + V260G + Q291W + Q313M + F314L +
c1425g
++
+



D369R + E402N + S434P + K495N + G628W + A689I +



Y715P + A732G


917
A79G + Q85N + I106V + A109S + Q258N + V260G + Q291W +
c1425g
++
+



Q313M + F314V + D369R + E402N + S434P + K495N + A505C +



G628W + A689I + Y715P + A732G


918
D47I + A79E + Q85N + I106V + Q258N + V260G + Q291W + Q313M +
c1425g
++
+



F314L + D369R + E402N + S434P + K495N + G628W +



A689I + Y715P + A732G


919
Q85N + I106V + Q258N + V260G + L275Y + Q291W + Q313M +
c1425g
++
**



F314V + A343C + D369R + E402N + S434P + K495N + G628W +



T687W + A689I + Y715P + A732G


920
I106V + Q258N + V260G + L275Y + Q291W + Q313M + F314L +
c1425g
++
**



A343C + D369R + E402N + S434P + K495N + G628W + A689I +



Y715P + A732G


921
D47I + Q85N + I106V + A109T + Q258N + V260G + Q291W + Q313M +
c1425g
++
+



F314V + D369R + E402N + S434P + K495N + G628W +



A689I + Y715P + A732G


922
Q85N + I106V + A109S + Q258N + V260G + L275Y + Q291W +
c1425g
++
+



Q313M + F314V + A343C + D369R + E402N + S434P + K495N +



G628W + A689I + Y715P + A732G


923
D47I + A79M + I106V + A109S + Q258N + V260G + Q291W + Q313M +
c1425g
++
+



F314L + A343C + D369R + E402N + S434P + K495N +



G628W + A689I + Y715P + A732G


924
A79M + Q85N + I106V + Q258N + V260G + Q291W + Q313M +
c1425g
++
+



F314L + A343C + D369R + E402N + S434P + K495N + G628W +



A689I + Y715P + A732G


925
A79M + Q85N + I106V + Q258N + V260G + L275Y + Q291W +
c699t + c1425g
++
**



Q313M + F314L + A343C + D369R + E402N + S434P + K495N +



A505C + G628W + A689I + Y715P + A732G





1Amino acid changes are indicated with respect to SEQ ID NO: 2; the backbone sequence contains substitutions I106V + Q258N + V260G + Q291W + Q313M + F314L + D369R + E402N + S434P + K495N + G628W + A689I + Y715P + A732G.


2Nucleotide changes are indicated relative to SEQ ID NO: 1.


3Fold improvement is with respect to Variant 664 (SEQ ID NO: 13)


* = 0.2 to 0.5 fold improvement


** = 0.6 to 1.0 fold improvement


+ = 1.1 to 1.9 fold improvement


++ = 2.0 to 2.9 fold improvement


+++ = 3.0 to 3.9 fold improvement






Table 8 illustrates exemplary properties of C1 Bgl variants with increased thermoactivity and/or thermostability compared to the wild-type enzyme.







TABLE 8







An exemplary embodiment has












at least x

than





fold greater

variant

Exemplary
See


x=
activity
No.:
under conditions:
Variant(s)
Table















5
Thermoactivity
wildtype
pH 5, 65° C., 21 h
3
2


2
Thermostability*
wildtype
pH 5, 65° C., 6 h
3
2


2, 3, 4, 5 or 6
Thermoactivity
3
pH 5, 70° C., 21 h
269
3


1.1, 2, or 3
Thermostability
3
pH 5, 65° C., 16 h
269
3


2, 3, or 4
Thermoactivity
269
pH 4.5, 70° C., 21 h
481
4


1.1 or 2
Thermostability
269
pH 4.5, 70° C., 2 h
481
4


2, 3, or 4
Thermoactivity
481
pH 4.2, 70° C., 21 h
647
5


1.1, 2, or 4
Thermostability
481
pH 4.5, 70° C., 3 h
647
5


3 or 4
Thermoactivity
647
pH 4, 70° C., 21 h
824, 825
6


1.1 or 2
Thermostability
647
pH 4.5, 70° C., 24 h
824, 825
6


2 or 3
Thermoactivity
664
pH 4, 70° C., 21 h
871, 885, 916
7


1.1
Thermostability
664
pH 4.5, 70° C., 24 h
871, 885, 916
7









For example, in some embodiments, a variant has at least 5-fold greater thermoactivity and/or at least 2-fold greater thermostability than wildtype Bgl1. For example and not limitation, Variant 3 has these properties. For example, in another embodiment, a variant has at least 4-fold greater thermoactivity and/or at least 4-fold greater thermostability than Variant 481 Bgl1. For example and not limitation, Variant 647 has these properties.
The amino acid sequences of β-glucosidase variants not specifically described herein can be readily generated and identified using methods that are well known to those having ordinary skill in the art. Some β-glucosidase variants of the invention having at least 70% sequence identity to residues 20-870 of SEQ ID NO:2 and one or more substitutions disclosed herein, also have one or more substitutions, deletions or insertions in addition to those specifically disclosed herein. The effect, if any, of such substitutions, deletions or insertions on β-glucosidase activity and thermostability can be determined using assays known in the art and described herein (see e.g., Examples 3 and 5, infra). For illustration, variant number 1 in Table 2 has the following substitutions relative to residues 20-870 of SEQ ID NO:2: M181Y+Q291W+E402N+S434P. To determine the effect of a further substitution (e.g., replacement of isoleucine at position 20 with leucine) the variant (in this case, 120L+M181Y+Q291W+E402N+S434P) is expressed and its properties compared to the parent (in this case, M181Y+Q291W+E402N+S434P).
Further, libraries of β-glucosidase polypeptide variants (and polynucleotides encoding the variants) may be generated from a parental sequence (e.g., such as one or more variants exemplified herein) and screened using the high throughput screen for presence of β-glucosidase activity described in, for example, Example 5. Mutagenesis and directed evolution methods known in the art can be readily applied to polynucleotides encoding β-glucosidase variants exemplified herein 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., 1999, “Approaches to DNA mutagenesis: an overview,” Anal. Biochem., 254(2):157-78; Dale, et al., 1996, “Oligonucleotide-directed random mutagenesis using the phosphorothioate method,” Methods Mol. Biol., 57:369-74; Smith, 1985, “In vitro mutagenesis,” Ann. Rev. Genet., 19:423-462; Botstein, et al., 1985, “Strategies and applications of in vitro mutagenesis,” Science, 229:1193-1201; Carter, 1986, “Site-directed mutagenesis,” Biochem. J., 237:1-7; Kramer, et al., 1984, “Point Mismatch Repair,” Cell, 38:879-887; Wells, et al., 1985, “Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites,” Gene, 34:315-323; Minshull, et al., 1999, “Protein evolution by molecular breeding,” Current Opinion in Chemical Biology, 3:284-290; Christians, et al., 1999, “Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling,” Nature Biotechnology, 17:259-264; Crameri, et al., 1998, “DNA shuffling of a family of genes from diverse species accelerates directed evolution,” Nature, 391:288-291; Crameri, et al., 1997, “Molecular evolution of an arsenate detoxification pathway by DNA shuffling,” Nature Biotechnology, 15:436-438; Zhang, et al., 1997 “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., 1996, “Improved green fluorescent protein by molecular evolution using DNA shuffling,” Nature Biotechnology, 14:315-319; Stemmer, 1994, “Rapid evolution of a protein in vitro by DNA shuffling,” Nature, 370:389-391; Stemmer, 1994, “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; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; and WO 01/75767, all of which are incorporated herein by reference.
In generating variants that comprise substitutions, insertions or deletions at positions in addition to those described supra, the ordinarily skilled practitioner will be aware that certain regions of the β-glucosidase protein are less tolerant than others to substitutions (especially non-conservative substitutions). Thus, in some embodiments, variant Bgl1 proteins retain conserved residues and functional domains from the parent.
β-Glucosidase Activity and Thermostability Assays
β-glucosidase activity can be determined by methods described in Examples 3 and 5, as well as any other methods known in the art. β-glucosidase activity may be determined, for example, using a para-nitrophenyl-β-D-glucopyranoside (pNPG) assay or using a cellobiose assay.
For example, a colorimetric pNPG (p-nitrophenyl-β-D-glucopyranosideybased assay may be used to measure β-glucosidase activity. One such assay is described in Example 3, infra. In another exemplary pNPG assay, in a total volume of 100 μL, 20 μL clear media supernatant containing β-glucosidase enzyme is added to 4 mM pNPG (Sigma-Aldrich, Inc. St. Louis, Mo.) solution in 50 mM sodium phosphate buffer at pH 5. The reactions are incubated at pH 5, 50° C. for 1.5 hours. The reaction mixture is quenched with 100 μL of 1M sodium carbonate pH 11 solution. The absorbance of the solution is 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) is measured at 405 nm to calculate β-glucosidase activity. Detectable β-glucosidase activity is observed under high throughput screening conditions (pH 7, 50° C.). See Breves et al., 1997, Appl. Environmental Microbiol. 63:3902, incorporated herein by reference.
Alternatively, β-glucosidase activity may be determined using an assay in which cellobiose is the substrate. One suitable assay is described in Example 3, infra. Another suitable assay is carried out as follows: In a total volume of 100 μL, 25 μL clear media supernatant containing β-glucosidase enzyme is 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 is incubated at 45-70° C. for an appropriate time (25 minutes to overnight depending on the enzyme concentration) while shaking. Glucose production may be determined using any number of art-known methods for measuring glucose. In one approach, glucose production is determined using an enzymatic glucose assay (K-GLUC, Megazyme, Ireland). 10 μl of each reaction is added to 190 μl GOPOD reagent (supplied as part of the K-GLUC assay kit). The reaction is 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.
Signal Peptide
In general, the β-glucosidase polypeptides are secreted from the host cell in which they are expressed (e.g., a yeast or fungal cell) and are expressed as a pre-protein including a signal peptide, i.e., an amino acid sequence linked to the amino terminus of a polypeptide and which directs the encoded polypeptide into the cell secretory pathway. In one embodiment, the signal peptide is the endogenous C1 β-glucosidase signal peptide having the sequence set forth as residues 1-19 of SEQ ID NO:2. In other embodiments, signal peptides from other C1 secreted proteins are used.
Still other signal peptides may be used, depending on the host cell and other factors. 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, Humicola lanuginosa lipase, and T. reesei cellobiohydrolase II (TrCBH2).
Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiol Rev 57: 109-137 (incorporated herein by reference).
Useful signal peptides for yeast host cells also include those from the genes for Saccharomyces cerevisiae alpha-factor, Saccharomyces cerevisiae SUC2 invertase (see Taussig and Carlson, 1983, Nucleic Acids Res 11:1943-54; SwissProt Accession No. P00724), and others. See, e.g., Romanos et al., 1992, Yeast 8:423-488. Variants of these signal peptides and other signal peptides are suitable.
Fusion Polypeptides and Additional Sequence Elements
In some embodiments, a β-glucosidase polypeptide variant of the invention includes additional sequences which do not alter the encoded activity of a β-glucosidase. For example, the β-glucosidase may be linked to an epitope tag or to other sequence useful in β-glucosidase purification.
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, where 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 T. aurentiacus β-glucosidase variant polypeptide), or a polypeptide exhibiting CBH or EG 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 species. 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), Lys and Arg (the trypsin protease recognition sites). See, for example, WO 2007/075899, which is incorporated herein by reference.
IV. β-Glucosidase Polynucleotides and Expression Systems
The present invention provides polynucleotide sequences that encode C1 β-glucosidase variants of the invention. The C1 genomic and cDNA sequences are described in Section II, supra.
In one embodiment, for expression of a β-glucosidase variant described herein, the wild-type C1 β-glucosidase cDNA sequence (SEQ ID NO:1), or the portion thereof comprising the open reading frame, can be used (with changes as required at codons corresponding to substitutions (residues mutated relative to the wild-type sequence). In addition, one or more of the “silent” nucleotide changes shown in Table 2, Table 3, Table, 4, Table 5, Table 6, or Table 7 can be incorporated.
Those having ordinary skill in the art will understand that due to the degeneracy of the genetic code, a multitude of nucleotide sequences encoding β-glucosidase polypeptides of the present invention exist. Table 9 provides the standard triplet genetic code 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. 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.
A DNA sequence may also be designed for high codon usage bias codons (codons that are used at higher frequency in the protein coding regions than other codons that code for the same amino acid). The preferred codons may be determined in relation to codon usage in a single gene, a set of genes of common function or origin, highly expressed genes, the codon frequency in the aggregate protein coding regions of the whole organism, codon frequency in the aggregate protein coding regions of related organisms, or combinations thereof. Codons whose frequency increases with the level of gene expression are typically optimal codons for expression. In particular, a DNA sequence can be optimized for expression in a particular host organism. References providing preference information for a wide range of organisms are readily available See e.g., Henaut and Danchin in “Escherichia Salmonella,” Neidhardt, et al. Eds., ASM Pres, Washington D.C. (1996), pp. 2047-2066, which is incorporated herein by reference. For illustration, and not for limitation, SEQ ID NO:3 shows a C1 Bgl1-encoding polynucleotide sequence designed with codon biasing for expression in Saccharomyces cerevisiae.







TABLE 9







GENETIC CODE








Amino acid
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









A variety of methods are known for determining the codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in specific organisms, including multivariate analysis, for example, using cluster analysis or correspondence analysis, and the effective number of codons used in a gene (see GCG CodonPreference, Genetics Computer Group Wisconsin Package; Codon W, John Peden, University of Nottingham; McInerney, J. O, 1998, Bioinformatics 14:372-73; Stenico et al., 1994, Nucleic Acids Res. 222437-46; Wright, F., 1990, Gene 87:23-29; Wada et al., 1992, Nucleic Acids Res. 20:2111-2118; Nakamura et al., 2000, Nucl. Acids Res. 28:292; Henaut and Danchin, “Escherichia coli and Salmonella,” 1996, Neidhardt, et al. Eds., ASM Press, Washington D.C., p. 2047-2066, all of which are incorporated herein be reference). The data source for obtaining codon usage may rely on any available nucleotide sequence capable of coding for a protein. These data sets include nucleic acid sequences actually known to encode expressed proteins (e.g., complete protein coding sequences-CDS), expressed sequence tags (ESTs), or predicted coding regions of genomic sequences (see for example, Mount, D., Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Uberbacher, E. C., 1996, Methods Enzymol. 266:259-281; Tiwari et al., 1997, Comput. Appl. Biosci. 13:263-270, all of which are incorporated herein by reference).
For expression of a β-glucosidase variant in a C1 or M. thermophilia host, the wild-type C1 β-glucosidase cDNA sequence (SEQ ID NO:1), or the portion thereof comprising the open reading frame, can be used (with changes as required at codons corresponding to substitutions (residues mutated relative to the wild-type sequence). In addition, one or more of the “silent” nucleotide changes shown in Table 2 can be incorporated. These changes may affect β-glucosidase activity in a variety of ways. For example, without intending to be bound by a particular mechanism, silent mutations may increase the stability of mRNAs encoding the variant protein.
Expression Vectors
The present invention makes use of recombinant constructs comprising a sequence encoding a β-glucosidase as described above. In a particular aspect the present invention provides an expression vector comprising a β-glucosidase polynucleotide operably linked to a heterologous promoter. Expression vectors of the present invention may be used to transform an appropriate host cell to permit the host to express β-glucosidase protein. Methods for recombinant expression of proteins in fungi and other organisms are well known in the art, and a number expression vectors are available or can be constructed using routine methods. See, e.g., Tkacz and Lange, 2004, ADVANCES IN FUNGAL BIOTECHNOLOGY FOR INDUSTRY, AGRICULTURE, AND MEDICINE, KLUWER ACADEMIC/PLENUM PUBLISHERS. New York; Zhu et al., 2009, Construction of two Gateway vectors for gene expression in fungi Plasmid 6:128-33; Kavanagh, K. 2005, FUNGI: BIOLOGY AND APPLICATIONS Wiley, all of which are incorporated herein by reference.
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. 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.
In one aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the protein encoding sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art.
Promoter/Gene Constructs
As discussed above, to obtain high levels of expression in a particular host it is often useful to express C1 β-glucosidase under control of a heterologous promoter. Typically a promoter sequence may be operably linked to the 5′ region of the C1 β-glucosidase coding sequence using routine methods.
Examples of useful promoters for expression of β-glucosidase polynucleotides include promoters from fungi. For example, promoter sequences that drive expression of genes other than the β-glucosidase 1 gene in C1 may be used. For example, a fungal promoter from a gene encoding cellobiohydrolase may be used.
Examples of other suitable promoters useful for directing the transcription of the nucleotide constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (WO 96/00787, which is incorporated herein by reference), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), promoters such as cbh1, cbh2, egl1, egl2, pepA, hfb1, hfb2, xynl, amy, and g/aA (Nunberg et al., 1984, Mol. Cell Biol., 4:2306-2315, Boel et al., 1984, EMBO J. 3:1581-85 and EPA 137280, all of which are incorporated herein by reference), and mutant, truncated, and hybrid promoters thereof. In a yeast host, useful promoters can be from the genes for Saccharomyces cerevisiae enolase (eno-1), Saccharomyces cerevisiae galactokinase (gall), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), and S. cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488, incorporated herein by reference. Promoters associated with chitinase production in fungi may be used. See, e.g., Blaiseau and Lafay, 1992, Gene 120243-248 (filamentous fungus Aphanocladium album); Limon et al., 1995, Curr. Genet, 28:478-83 (Trichoderma harzianum), both of which are incorporated herein by reference.
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. In bacterial host cells, suitable promoters include the promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucranse gene (sacB), Bacillus licheniformis alpha-amylase gene (amyl), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus subtilis xylA and xylB genes and prokaryotic β-lactamase gene.
Any other promoter sequence that drives expression in a suitable host cell may be used. Suitable promoter sequences can be identified using well known methods. In one approach, a putative promoter sequence is linked 5′ to a sequence encoding a reporter protein, the construct is transfected into the host cell (e.g., a C1 cell) and the level of expression of the reporter is measured. Expression of the reporter can be determined by measuring, for example, mRNA levels of the reporter sequence, an enzymatic activity of the reporter protein, or the amount of reporter protein produced. For example, promoter activity may be determined by using the green fluorescent protein as coding sequence (Henriksen et al, 1999, Microbiology 145:729-34, incorporated herein by reference) or a lacZ reporter gene (Punt et al, 1997, Gene, 197:189-93, incorporated herein by reference). Functional promoters may be derived from naturally occurring promoter sequences by directed evolution methods. See, e.g. Wright et al., 2005, Human Gene Therapy, 16:881-892, incorporated herein by reference.
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.
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 antimicrobial resistance such as, ampicillin (ampR), kanamycin, chloramphenicol, or tetracycline resistance. Further examples include the antimicrobial streptomycin or spectinomycin (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. 
Synthesis and Manipulation of β-Glucosidase Polynucleotides
Polynucleotides encoding β-glucosidase 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., 1982, Cold Spring Harbor Symp. Quant. Biol., 47:411-18 and Adams et al., 1983, J. Am. Chem. Soc. 105:661, 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, protocols sufficient to direct persons of skill through in vitro amplification methods, including the polymerase chain reaction (PCR) and the ligase chain reaction (LCR), and many other relevant methods, 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 2009) (“Ausubel”), all of which are incorporated herein by reference. 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; Lomeli 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. 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.
Expression Hosts
The present invention also provides engineered (recombinant) host cells that are transformed with an expression vector or DNA construct encoding β-glucosidase. Optionally, β-glucosidase expression in the cell is under the control of a heterologous promoter. Host cells of the invention may be used to produce β-glucosidase polypeptides. Thus, the present invention is directed to a 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 recombinant polypeptide of the invention. Often, the genetically modified or recombinant host cell is a microorganism. In some embodiments, the genetically modified or recombinant host cell is a prokaryote. In some embodiments, the genetically modified or recombinant host cell is a eukaryotic cell. Generally the eukaryotic host cell is a non-human cell. Suitable eukaryotic host cells include, but are not limited to, fungal cells, algal cells, insect cells, and plant cells. In some cases host cells may be modified to increase protein expression, secretion or stability, or to confer other desired characteristics. Cells (e.g., fungi) that have been mutated or selected to have low protease activity are particularly useful for expression. For example, protease deficient strains of C. lucknowense (e.g., in which the alkaline protease locus has been deleted or disrupted) may be used. In some embodiments, the host cells, e.g., fungi, may modified to express a recombinant cellulase.
Suitable fungal host cells include, but are not limited to, Ascomycota, Basidiomycota, Deuteromycota, Zygomycota, and Fungi imperfecti. In some embodiments the fungal host cells are yeast cells and filamentous fungal cells. The filamentous fungal 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 fungal host cells of the present invention are morphologically distinct from yeast.
In some embodiments the 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, Endothia, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora, 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 Chrysosporium species, e.g., C. lucknowense, C. keratinophilum, C. tropicum, C. merdarium, C. inops, C. pannicola, and C. zonatum. 
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), or T. reesei, an anamorph of Hypocrea jecorina, (NRRL 15709, ATTC 13631, 56764, 56765, 56466, 56767 and RL-P37 and derivatives thereof—See Sheir-Neiss et al., 1984, Appl. Microbiol. Biotechnology, 20:46-53, which is incorporated herein by reference), 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. fumigatus, 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 et al., 1984, Proc. Natl. Acad. Sci. USA, 81, 1470-1474; Tilburn et al., 1982, Gene 26, 205-221; and Johnston 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 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 Myceliophthora species, e.g., M. thermophilia. 
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 Penicillium 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. 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, Saccharomyces 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 algae 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, Acidothermus, 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, Ilyobacter, Micrococcus, Microbacterium, Mesorhizobium, Methylobacteriu, Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus, Rhodobacter, Rhodopseudomonas, Roseburia, Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces, Streptococcus, Synecoccus, Saccharomonospora, Staphylococcus, Serratia, Salmonella, Shigella, Thermoanaerobactenurn, Tropheryma, Francisella, Temecula, Thermosynechococcus, Thermococcus, Ureapiasma, Xanthomonas, Xylella, Yersinia and Zymomanas. 
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 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, C. thermocellum, 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. fluorescens, P. aeruginosa, P. mevalonii, and P. sp. D-0I 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).
Host cells may be genetically modified to have characteristics that improve protein secretion, protein stability or other properties desirable for expression and/or secretion of a protein. For example, knock out of Alp1 function results in a cell that is protease deficient. Knock out of pyr5 function results in a cell with a pyrimidine deficient phenotype. In particular embodiments host cells are modified to delete endogenous cellulase protein-encoding sequences or otherwise eliminate expression of one or more endogenous cellulases. In one embodiment expression of one or more endogenous cellulases is inhibited to increase production of cellulases of interest. Genetic modification can be achieved by genetic engineering techniques or using classical microbiological techniques, such as chemical or UV mutagenesis and subsequent selection. A combination of recombinant modification and classical selection techniques may be used to produce the organism of interest. Using recombinant technology, nucleic acid molecules can be introduced, deleted, inhibited or modified, in a manner that results in increased yields of β-glucosidase within the organism or in the culture. For example, knock out of Alp1 function results in a cell that is protease deficient. Knock out of pyr5 function results in a cell with a pyrimidine deficient phenotype. In one genetic engineering approach, homologous recombination can be used to induce targeted gene modifications by specifically targeting a gene in vivo to suppress expression of the encoded protein. In an alternative approach, siRNA, antisense, or ribozyme technology can be used to inhibit gene expression
Transformation and Culture
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 et al., 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. 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 polypeptides 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 state 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.
Cell-free transcription/translation systems can also be employed to produce β-glucosidase olypeptides 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.
V. Production and Recovery of β-Glucosidase Polypeptides
The present invention is also 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 in which the host cell expresses the encoded β-glucosidase polypeptide; and optionally recovering or isolating the expressed β-glucosidase polypeptide, or recovering or isolating the culture medium containing the expressed β-glucosidase polypeptide. The method further provides optionally lysing the transformed host cells after expressing the encoded β-glucosidase polypeptide and optionally recovering or isolating the expressed β-glucosidase polypeptide from the cell lysate. The present invention further provides a method of making a β-glucosidase polypeptide, said method comprising cultivating a host cell transformed with a β-glucosidase polynucleotide under conditions suitable for the production of the β-glucosidase polypeptide and recovering the β-glucosidase polypeptide.
Typically, recovery or isolation of the β-glucosidase polypeptide 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. 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.
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 interaction, 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. Purification of BGL1 is described in Parry et al., 2001, Biochem. J. 353:117, and Hong et al., 2007, Appl. Microbiol. Biotechnol. 73:1331, both incorporated herein by reference. 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.
Immunological methods may be used to purify β-glucosidase polypeptides. In one approach, antibody raised against the β-glucosidase polypeptides (e.g., against a polypeptide comprising SEQ ID NO:2 or an immunogenic fragment thereof) using conventional methods is immobilized on beads, mixed with cell culture media under conditions in which the β-glucosidase is bound, and precipitated. In a related approach immunochromatography is used.
As noted, in some embodiments the β-glucosidase is expressed as a fusion protein including a non-enzyme portion. In some embodiments the β-glucosidase sequence is fused to a purification facilitating domain. As used herein, the term “purification facilitating domain” refers to a domain that mediates purification of the polypeptide to which it is fused. Suitable purification domains include metal chelating peptides, histidine-tryptophan modules that allow purification on immobilized metals, a sequence which binds glutathione (e.g., GST), a hemagglutinin (HA) tag (corresponding to an epitope derived from the influenza hemagglutinin protein; Wilson et al., 1984, Cell 37:767), maltose binding protein sequences, the FLAG epitope utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle, Wash.), and the like. The inclusion of a protease-cleavable polypeptide linker sequence between the purification domain and the HHDH polypeptide is useful to facilitate purification. One expression vector contemplated for use in the compositions and methods described herein provides for expression of a fusion protein comprising a polypeptide of the invention fused to a polyhistidine region separated by an enterokinase cleavage site. The histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography, as described in Porath et al., 1992, Protein Expression and Purification 3:263-281) while the enterokinase cleavage site provides a means for separating the HHDH polypeptide from the fusion protein. pGEX vectors (Promega; Madison, Wis.) may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to ligand-agarose beads (e.g., glutathione-agarose in the case of GST-fusions) followed by elution in the presence of free ligand.
VI. Methods of using β-Glucosidase Polypeptides and Cells Expressing β-Glucosidase Polypeptides
As described supra, β-glucosidase polypeptides 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, by (a) providing a cellobiose; and (b) contacting the cellobiose with a β-glucosidase polypeptide of the invention under conditions sufficient to form a reaction mixture for converting the cellobiose to glucose. The β-glucosidase polypeptide 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 may also be provided in cell culturing media or in a cell lysate. For example, after producing the β-glucosidase polypeptide 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. Any composition, cell culture medium, or cell lysate containing a β-glucosidase polypeptide 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, by: (a) providing a cellobiose; and (b) contacting the cellobiose with a culture medium or cell lysate or composition comprising a β-glucosidase polypeptide 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 polypeptides 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.
Enzyme Mixtures
In another aspect, the invention provides an enzyme mixture that comprises a C1 Bgl1 variant polypeptide as described herein. The enzyme mixture may be cell-free, or in alternative embodiments, may not be separated from host cells that secrete an enzyme mixture component. A cell-free enzyme mixture typically comprises enzymes that have been separated from any cells. Cell-free enzyme mixtures can be prepared by any of a variety of methodologies that are known in the art, such as filtration or centrifugation methodologies. In certain embodiments, the enzyme mixture can be, for example, partially cell-free, substantially cell-free, or entirely cell-free.
The C1 Bgl1 variant and any additional enzymes present in the enzyme mixture may be secreted from a single genetically modified fungal cell or by different microbes in combined or separate fermentations. Similarly, the C1 Bgl1 variant and any additional enzymes present in the enzyme mixture may be expressed individually or in sub-groups from different strains of different organisms and the enzymes combined in vitro to make the enzyme mixture. It is also contemplated that the C1 Bgl1 variant and any additional enzymes in the enzyme mixture may be expressed individually or in sub-groups from different strains of a single organism, and the enzymes combined to make the enzyme mixture. In some embodiments, all of the enzymes are expressed from a single host organism, such the genetically modified fungal cell.
In some embodiments, the enzyme mixture comprises other types of cellulases, selected from CBH, EG, and BG cellulase. In some embodiments, the cellobiohydrolase is T. reesei cellobiohydrolase II. In some embodiments, the endoglucanase comprises a catalytic domain derived from the catalytic domain of a Streptomyces avermitilis endoglucanase. See, copending application Ser. No. 12/751,985, incorporated herein by reference. In some embodiments, the at least one cellulase is Acidothermus cellulolyticus, Thermobifida fusca, Humicola grisea or a Chrysosporium sp. Cellulase 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).
Cellulase mixtures for efficient enzymatic hydrolysis of cellulose are known (see, e.g., Viikari et al., 2007, “Thermostable enzymes in lignocellulose hydrolysis” Adv Biochem Eng Biotechnol 108:121-45, and US Pat. publications US 2009/0061484; US 2008/0057541; and US 2009/0209009 to logen Energy Corp.), each of which is incorporated herein by reference for all purposes. In some embodiments, mixtures of purified naturally occurring or recombinant enzymes are combined with cellulosic feedstock or a product of cellulose hydrolysis. Alternatively or in addition, one or more cell populations, each producing one or more naturally occurring or recombinant cellulases, may be combined with cellulosic feedstock or a product of cellulose hydrolysis.
A variant β-glucosidase polypeptide of the invention may also be present in mixtures with enzymes other than cellulases that degrade cellulose, hemicellulose, pectin, and/or lignocellulose.
A “hemicellulase” as used herein, refers to a polypeptide that can catalyze hydrolysis of hemicellulose into small polysaccharides such as oligosaccharides, or monomeric saccharides. Hemicellulloses include xylan, glucuonoxylan, arabinoxylan, glucomannan and xyloglucan. Hemicellulases include, for example, the following: endoxylanases, β-xylosidases, α-L-arabinofuranosidases, a-D-glucuronidases, feruloyl esterases, coumarolyl esterases, α-galactosidases, β-galactosidases, β-mannanases, and β-mannosidases. An enzyme mixture may therefore comprise a β-glucosidase variant of the invention and one or more hemicellulases.
An endoxylanase (EC 3.2.1.8) catalyzes the endohydrolysis of 1,4-β-D-xylosidic linkages in xylans. This enzyme may also be referred to as endo-1,4-β-xylanase or 1,4-β-D-xylan xylanohydrolase. An alternative is EC 3.2.1.136, a glucuronoarabinoxylan endoxylanase, an enzyme that is able to hydrolyse 1,4 xylosidic linkages in glucuronoarabinoxylans.
A β-xylosidase (EC 3.2.1.37) catalyzes the hydrolysis of 1,4-β-D-xylans, to remove successive D-xylose residues from the non-reducing termini. This enzyme may also be referred to as xylan 1,4-β-xylosidase, 1,4-β-D-xylan xylohydrolase, exo-1,4-β-xylosidase or xylobiase.
An α-L-arabinofuranosidase (EC 3.2.1.55) catalyzes the hydrolysis of terminal non-reducing alpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzyme acts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)- and/or (1,5)-linkages, arabinoxylans, and arabinogalactans. Alpha-L-arabinofuranosidase is also known as arabinosidase, alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase, arabinofuranosidase, polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranoside hydrolase, L-arabinosidase and alpha-L-arabinanase.
An alpha-glucuronidase (EC 3.2.1.139) catalyzes the hydrolysis of an alpha-D-glucuronoside to D-glucuronate and an alcohol.
An acetylxylanesterase (EC 3.1.1.72) catalyzes the hydrolysis of acetyl groups from polymeric xylan, acetylated xylose, acetylated glucose, alpha-napthyl acetate, and p-nitrophenyl acetate.
A feruloyl esterase (EC 3.1.1.73) has 4-hydroxy-3-methoxycinnamoyl-sugar hydrolase activity (EC 3.1.1.73) that catalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl) group from an esterified sugar, which is usually arabinose in “natural” substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate). Feruloyl esterase is also known as ferulic acid esterase, hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, or FAE-II.
A coumaroyl esterase (EC 3.1.1.73) catalyzes a reaction of the form: coumaroyl-saccharide+H(2)O=coumarate+saccharide. The saccharide may be, for example, an oligosaccharide or a polysaccharide. This enzyme may also be referred to as trans-4-coumaroyl esterase, trans-p-coumaroyl esterase, p-coumaroyl esterase or p-coumaric acid esterase. The enzyme also falls within EC 3.1.1.73 so may also be referred to as a feruloyl esterase.
An α-galactosidase (EC 3.2.1.22) catalyzes the hydrolysis of terminal, non-reducing α-D-galactose residues in α-D-galactosides, including galactose oligosaccharides, galactomannans, galactans and arabinogalactans. This enzyme may also be referred to as melibiase.
A β-galactosidase (EC 3.2.1.23) catalyzes the hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides. Such a polypeptide may also be capable of hydrolyzing α-L-arabinosides. This enzyme may also be referred to as exo-(1->4)-β-D-galactanase or lactase.
A β-mannanase (EC 3.2.1.78) catalyzes the random hydrolysis of 1,4-β-D-mannosidic linkages in mannans, galactomannans and glucomannans. This enzyme may also be referred to as mannan endo-1,4-β-mannosidase or endo-1,4-mannanase.
A β-mannosidase (EC 3.2.1.25) catalyzes the hydrolysis of terminal, non-reducing β-D-mannose residues in β-D-mannosides. This enzyme may also be referred to as mannanase or mannase.
One or more enzymes that derade pectin may also be included in an enzyme mixture that comprises a β-glucosidase variant of the invention. A pectinase catalyzes the hydrolysis of pectin into smaller units such as oligosaccharide or monomeric saccharides. An ezyme mixture may comprise any pectinase, for example an endo-polygalacturonase, a pectin methyl esterase, an endo-galactanase, a pectin acetyl esterase, an endo-pectin lyase, pectate lyase, alpha rhamnosidase, an exo-galacturonase, an exo-polygalacturonate lyase, a rhamnogalacturonan hydrolase, a rhamnogalacturonan lyase, a rhamnogalacturonan acetyl esterase, a rhamnogalacturonan galacturonohydrolase or a xylogalacturonase.
An endo-polygalacturonase (EC 3.2.1.15) catalyzes the random hydrolysis of 1,4-α-D-galactosiduronic linkages in pectate and other galacturonans. This enzyme may also be referred to as polygalacturonase pectin depolymerase, pectinase, endopolygalacturonase, pectolase, pectin hydrolase, pectin polygalacturonase, poly-α-1,4-galacturonide glycanohydrolase, endogalacturonase; endo-D-galacturonase or poly(1,4-α-D-galacturonide) glycanohydrolase.
A pectin methyl esterase (EC 3.1.1.11) catalyzes the reaction: pectin+n H2O=n methanol+pectate. The enzyme may also been known as pectinesterase, pectin demethoxylase, pectin methoxylase, pectin methylesterase, pectase, pectinoesterase or pectin pectylhydrolase.
A endo-galactanase (EC 3.2.1.89) catalyzes the endohydrolysis of 1,4-β-D-galactosidic linkages in arabinogalactans. The enzyme may also be known as arabinogalactan endo-1,4-β-galactosidase, endo-1,4-β-galactanase, galactanase, arabinogalactanase or arabinogalactan 4-β-D-galactanohydrolase.
A pectin acetyl esterase catalyzes the deacetylation of the acetyl groups at the hydroxyl groups of GalUA residues of pectin.
An endo-pectin lyase (EC 4.2.2.10) catalyzes the eliminative cleavage of (1→4)-α-D-galacturonan methyl ester to give oligosaccharides with 4-deoxy-6-O-methyl-α-D-galact-4-enuronosyl groups at their non-reducing ends. The enzyme may also be known as pectin lyase, pectin trans-eliminase; endo-pectin lyase, polymethylgalacturonic transeliminase, pectin methyltranseliminase, pectolyase, PL, PNL or PMGL or (1→4)-6-O-methyl-α-D-galacturonan lyase.
A pectate lyase (EC 4.2.2.2) catalyzes the eliminative cleavage of (1→4)-α-D-galacturonan to give oligosaccharides with 4-deoxy-α-D-galact-4-enuronosyl groups at their non-reducing ends. The enzyme may also be known polygalacturonic transeliminase, pectic acid transeliminase, polygalacturonate lyase, endopectin methyltranseliminase, pectate transeliminase, endogalacturonate transeliminase, pectic acid lyase, pectic lyase, α-1,4-D-endopolygalacturonic acid lyase, PGA lyase, PPase-N, endo-α-1,4-polygalacturonic acid lyase, polygalacturonic acid lyase, pectin trans-eliminase, polygalacturonic acid trans-eliminase or (1→4)-α-D-galacturonan lyase.
An alpha rhamnosidase (EC 3.2.1.40) catalyzes the hydrolysis of terminal non-reducing α-L-rhamnose residues in α-L-rhamnosides or alternatively in rhamnogalacturonan. This enzyme may also be known as α-L-rhamnosidase T, α-L-rhamnosidase N or α-L-rhamnoside rhamnohydrolase.
An exo-galacturonase (EC 3.2.1.82) hydrolyzes pectic acid from the non-reducing end, releasing digalacturonate. The enzyme may also be known as exo-poly-α-galacturonosidase, exopolygalacturonosidase or exopolygalacturanosidase.
An exo-galacturonase (EC 3.2.1.67) catalyzes a reaction of the following type: (1,4-α-D-galacturonide)n+H2O=(1,4-α-D-galacturonide)n-i+D-galacturonate. The enzyme may also be known as galacturan 1,4-α-galacturonidase, exopolygalacturonase, poly(galacturonate) hydrolase, exo-D-galacturonase, exo-D-galacturonanase, exopoly-D-galacturonase or poly(1,4-α-D-galacturonide) galacturonohydrolase.
An exopolygalacturonate lyase (EC 4.2.2.9) catalyzes eliminative cleavage of 4-(4-deoxy-α-D-galact-4-enuronosyl)-D-galacturonate from the reducing end of pectate, i.e. de-esterified pectin. This enzyme may be known as pectate disaccharide-lyase, pectate exo-lyase, exopectic acid transeliminase, exopectate lyase, exopolygalacturonic acid-trans-eliminase, PATE, exo-PATE, exo-PGL or (1→4)-α-D-galacturonan reducing-end-disaccharide-lyase.
A rhamnogalacturonan hydrolyzes the linkage between galactosyluronic acid and rhamnopyranosyl in an endo-fashion in strictly alternating rhamnogalacturonan structures, consisting of the disaccharide [(1,2-alpha-L-rhamnoyl-(1,4)-alpha-galactosyluronic acid].
A rhamnogalacturonan lyase cleaves α-L-Rhap-(1→4)-α-D-GalpA linkages in an endo-fashion in rhamnogalacturonan by beta-elimination.
A rhamnogalacturonan acetyl esterase catalyzes the deacetylation of the backbone of alternating rhamnose and galacturonic acid residues in rhamnogalacturonan.
A rhamnogalacturonan galacturonohydrolase hydrolyzes galacturonic acid from the non-reducing end of strictly alternating rhamnogalacturonan structures in an exo-fashion. This enzyme may also be known as xylogalacturonan hydrolase.
An endo-arabinanase (EC 3.2.1.99) catalyzes endohydrolysis of 1,5-α-arabinofuranosidic linkages in 1,5-arabinans. The enzyme may also be know as endo-arabinase, arabinan endo-1,5-α-L-arabinosidase, endo-1,5-α-L-arabinanase, endo-α-1,5-arabanase; endo-arabanase or 1,5-α-L-arabinan 1,5-α-L-arabinanohydrolase.
One or more enzymes that participate in lignin degradation may also be included in an enzyme mixture that comprises a β-glucosidase variant of the invention. Enzymatic lignin depolymerization can be accomplished by lignin peroxidases, manganese peroxidases, laccases and cellobiose dehydrogenases (CDH), often working in synergy. These extracellular enzymes are often referred to as lignin-modifying enzymes or LMEs. Three of these enzymes comprise two glycosylated heme-containing peroxidases: lignin peroxidase (LIP); Mn-dependent peroxidase (MNP); and, a copper-containing phenoloxidase laccase (LCC).
Laccase. Laccases are copper containing oxidase enzymes that are found in many plants, fungi and microorganisms. Laccases are enzymatically active on phenols and similar molecules and perform a one electron oxidation. Laccases can be polymeric and the enzymatically active form can be a dimer or trimer.
Mn-dependent peroxidase. The enzymatic activity of Mn-dependent peroxidase (MnP) in is dependent on Mn2+. Without being bound by theory, it has been suggested that the main role of this enzyme is to oxidize Mn2+ to Mn3+ (Glenn et al. (1986) Arch. Biochem. Biophys. 251:688-696). Subsequently, phenolic substrates are oxidized by the Mn3+ generated.
Lignin peroxidase. Lignin peroxidase is an extracellular heme that catalyses the oxidative depolymerization of dilute solutions of polymeric lignin in vitro. Some of the substrates of LiP, most notably 3,4-dimethoxybenzyl alcohol (veratryl alcohol, VA), are active redox compounds that have been shown to act as redox mediators. VA is a secondary metabolite produced at the same time as LiP by ligninolytic cultures of P. chrysosporium and without being bound by theory, has been proposed to function as a physiological redox mediator in the LiP-catalysed oxidation of lignin in vivo (Harvey, et al. (1986) FEBS Lett. 195, 242-246).
An enzymatic mixture comprising a β-glucosidase variant of the invention may further comprise at least one of the following; a protease or a lipase that participates in cellulose degradation.
“Protease” includes enzymes that hydrolyze peptide bonds (peptidases), as well as enzymes that hydrolyze bonds between peptides and other moieties, such as sugars (glycopeptidases). Many proteases are characterized under EC 3.4, and are suitable for use in the invention. Some specific types of proteases include, cysteine proteases including pepsin, papain and serine proteases including chymotrypsins, carboxypeptidases and metalloendopeptidases.
“Lipase” includes enzymes that hydrolyze lipids, fatty acids, and acylglycerides, including phosphoglycerides, lipoproteins, diacylglycerols, and the like. In plants, lipids are used as structural components to limit water loss and pathogen infection. These lipids include waxes derived from fatty acids, as well as cutin and suberin.
An enzyme mixture that comprises a β-glucosidase variant of the invention may also comprise at least one expansin or expansin-like protein, such as a swollenin (see Salheimo et al., Eur. J. Biohem. 269, 4202-4211, 2002) or a swollenin-like protein.
Expansins are implicated in loosening of the cell wall structure during plant cell growth. Expansins have been proposed to disrupt hydrogen bonding between cellulose and other cell wall polysaccharides without having hydrolytic activity. In this way, they are thought to allow the sliding of cellulose fibers and enlargement of the cell wall. Swollenin, an expansin-like protein contains an N-terminal Carbohydrate Binding Module Family 1 domain (CBD) and a C-terminal expansin-like domain. For the purposes of this invention, an expansin-like protein or swollenin-like protein may comprise one or both of such domains and/or may disrupt the structure of cell walls (such as disrupting cellulose structure), optionally without producing detectable amounts of reducing sugars.
An enzyme mixture that comprises a β-glucosidase variant of the invention may also comprise at least one of the following: a polypeptide product of a cellulose integrating protein, scaffoldin or a scaffoldin-like protein, for example CipA or CipC from Clostridium thermocellum or Clostridium cellulolyticum respectively. Scaffoldins and cellulose integrating proteins are multi-functional integrating subunits which may organize cellulolytic subunits into a multi-enzyme complex. This is accomplished by the interaction of two complementary classes of domain, i.e. a cohesion domain on scaffoldin and a dockerin domain on each enzymatic unit. The scaffoldin subunit also bears a cellulose-binding module that mediates attachment of the cellulosome to its substrate. A scaffoldin or cellulose integrating protein for the purposes of this invention may comprise one or both of such domains.
An enzyme mixture that comprises a β-glucosidase variant of the invention may also comprise at least one cellulose induced protein or modulating protein, for example as encoded by cip1 or cip2 gene or similar genes from Trichoderma reesei (see Foreman et al., J. Biol. Chem. 278(34), 31988-31997, 2003).
An enzyme mixture that comprises a β-glucosidase variant of the invention may comprise a member of each of the classes of the polypeptides described above, several members of one polypeptide class, or any combination of these polypeptide classes.
Other Components of β-Glucosidase Compositions
β-glucosidase polypeptides of the present invention may be used in combination with other optional ingredients such as a buffer, a surfactant, and/or a scouring agent. A buffer may be used with a β-glucosidase polypeptide 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, optionally, with any other cellulases being used. 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.
Production of Soluble Sugars From Cellulosic Biomass
β-glucosidase polypeptides of the present invention, as well as any composition, culture medium, or cell lysate comprising such β-glucosidase polypeptides, 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 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 soluble (or “fermentable”) sugar by (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 of the present invention (and optionally other cellulases) under conditions suitable for the production of the fermentable sugar.
In some embodiments, the biomass includes cellulosic substrates that contain cellulose, hemicellulose, lignocellulose. Cellulosic substrates include, but are not limited to 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 biomass comprises transgenic plants that express ligninase and/or cellulase enzymes which degrade lignin and cellulose. See, e.g., US 20080104724, which is incorporated herein by reference.
In some embodiments, the β-glucosidase polypeptide and β-glucosidase polypeptide-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 polypeptides and β-glucosidase polypeptide-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 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 and about pH 4.0 to about 6.5. 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 to about 240 hours, from about 5 to about 180 hrs and from about 10 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 C1. In some embodiments, the amount of soluble sugar made available may be at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold greater than that made available by the wildtype C1 under the same conditions. 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 produced using a β-glucosidase polypeptide of the present invention in the methods described supra; (b) contacting the fermentable sugar with a fermenting microorganism to produce the alcohol or other metabolic product; and (c) recovering the alcohol or other metabolic product.
In some embodiments, the β-glucosidase polypeptide of the present invention, or composition, cell culture medium, or cell lysate containing the β-glucosidase polypeptide 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 (e.g., Zymomonas sp., E. coli), to produce an end-product such as ethanol. In one example, a simultaneous saccharification and fermentation (SSF) process is used in which 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 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 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.
In addition to use for conversion of cellulosic biomass, β-glucosidase polypeptides 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 polypeptides 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.
VII: Examples
Example 1
Wild-type C1 β-glucosidase 1 (C1 Bgl1) Gene Acquisition and Construction of Expression Vectors
The wild-type C1 bg11 cDNA was synthesized and the DNA sequence verified. The gene was cloned into a Saccharomyces cerevisiae/C1 shuttle vector pYTDX20 using Pml1 cloning sites. The signal peptide and gene were under the control of a chitinase promoter (Pchi). The vector contained the REP2, rep1 and protein D (partial) origin of replication for S. cerevisiae and a URA3 resistance marker. The resulting plasmid (pYTDX20-C1 bgl1) was transformed into S. cerevisiae INVSC1 strain, and the transformed host cells were grown in HTP for C1 Bgl1 protein production. The C1 bgl1 sequence from the transformants was verified. The protein-encoding portion of the wild-type C1 bgl1 cDNA sequence is provided as SEQ ID NO: 1 and the encoded polypeptide is provided as SEQ ID NO: 2. The first 19 residues of SEQ ID NO:2 are shown in bold and comprise the signal peptide.
Example 2
Shake Flask Procedure
A single colony of S. cerevisiae containing a plasmid with the C1 bgl1 cDNA gene was inoculated into 1 mL Synthetic Defined-uracil (SD-ura) Broth (2 g/L synthetic prop-out minus uracil w/o yeast nitrogen base (from United States Biological, Swampscott, Mass.), 5 g/L Ammonium Sulphate, 0.1 g/L Calcium Chloride, 2 mg/L Inositol, 0.5 g/L Magnesium Sulphate, 1 g/L Potassium Phosphate monobasic (KH2PO4), 0.1 g/L Sodium Chloride) containing 6% glucose. Cells were grown for 24 hrs in an incubator at 30° C. with shaking at 250 rpm. 500 μL of the overnight culture was then diluted into 50 mL SD-ura media containing 2% glucose in a 250 mL baffled sterile shake flask and incubated at 37° C. for 48 hrs. Cells were pelleted by centrifugation (4000 rpm, 15 min, 4° C.). The clear media supernatant containing the secreted C1 Bgl1 enzyme was collected and stored at −20° C. until used.
Example 3
Assays to Determine β-Glucosidase Activity
β-glucosidase activity may be determined either by a para-nitrophenyl-β-D-glucoside (pNPG) assay, or a cellobiose assay.
A colorimetric pNPG (p-nitrophenyl-β-D-glucoside)-based assay was used for measuring β-glucosidase activity. In a total volume of 150 μL, 75 μL of clear media supernatant containing C1 Bgl1 enzyme was added to 75 μL of 3 mM pNPG (Sigma-Aldrich, Inc., St. Louis, Mo.) solution in 300 mM sodium acetate buffer, pH 5. The reactions were incubated at pH 5, 50° C. for 1.5 hrs. After reaction, 75 μL of the reaction mixture was quenched with 75 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-nitrophenyl. The amount of p-nitrophenol product was measured spectrophotometrically at 405 nm to calculate β-glucosidase activity as described by Breves, et al (1997), Appl. Environmental Microbiol. 63:3902-3910. Detectable β-glucosidase activity was observed under high throughput screening conditions (pH 5, 65° C.).
β-glucosidase activity was also determined using a cellobiose assay, which used cellobiose (Sigma-Aldrich, Inc., St. Louis, Mo.) as substrate. In a total volume of 150 μL, 75 μL of clear media supernatant containing C1 Bgl1 enzyme was added to 75 μL of 6.6 g/L cellobiose in 300 mM sodium acetate buffer (pH 5). The reaction was incubated at 65° C. for 21 hours with shaking. Glucose production was determined using an enzymatic glucose assay kit (K-GLUC, Megazyme, Bray, Co. Wicklow, Ireland). In a total volume of 200 μL, 15 μL of C1 Bgl1 reaction mixture was added to 185 μL of Glucose Determination Reagent (GOPOD Reagent, supplied as part of the K-GLUC assay kit). The reaction was incubated at 50° C. for 30 minutes and the absorbance of the solution was measured at 510 nm. The glucose oxidase enzyme in the GOPOD reagent reacts with glucose and produces hydrogen peroxide which then reacts with the 4-aminoantipyrine in the reagent to produce a quinoneimine dye. The amount of quinoneimine dye was measured spectrophotometrically at 510 nm to calculate β-glucosidase activity. Conversion of cellobiose to glucose was also measured using an Agilent HPLC 1200 equipped with HPX-87H ion exclusion column (300 mm×7.8 mm) with water as eluent at a flow rate of 1.0 mL/min at 80° C. The retention times of the cellobiose and glucose were 4.7 and 5.8 minute respectively. Detectable β-glucosidase activity was observed under high throughput screening conditions (pH 5, 65° C.).
Example 4
Evaluation of Optimal C1 Bgl1 Activity
The native C1 Bgl1 activity profile was investigated at different temperatures (50° C., 60° C. and 65° C.) and pH (3.5-7.5) using cellobiose (3.3 g/L) as a substrate. The experimental and analytical procedures are described in Example 3. C1 Bgl1 exhibited optimum activity at pH 5 and 50° C., and detectable β-glucosidase activity (20% of optimal activity) was observed under high throughput screening conditions (pH 5 and 65° C.).
Example 5
High Throughput Assays to Identify Improved C1 Bgl1 Variants
Plasmid libraries containing variant C1 bgl1 genes were transformed into S. cerevisiae INVSC1 strain and plated on SD-ura agar plate containing 2% glucose. After incubation for at least 48 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 200 μL SD-ura media and 6% glucose. Cells were grown for 24 hours at 30° C. with shaking at 250 rpm and 85% humidity. 20 μL of this overnight culture was then transferred into 96-well microtiter plates (deep well) containing 380 μL SD-ura medium and 2% glucose as described in Example 2. The plates were incubated at 37° C. with shaking at 250 rpm and 85% humidity for 48 hours. The deep plates were centrifuged at 4000 rpm for 15 minutes and the clear media supernatant containing the secreted C1 Bgl1 enzyme was used for the high throughput pNPG or cellobiose assay.
The C1 Bgl1 libraries were screened in high throughput using both thermoactivity and thermostability assays. In the thermoactivity assay, C1 Bgl1 variants were screened with a cellobiose-based high throughput assay (Substrate: cellbiose; pH: 5.0; temperature: 65° C.; time: 21 hrs). Active C1 Bgl1 variants identified from the thermoactivity assay were subsequently subjected to the thermostability assay. In the thermostability assay, the HTP media supernatant samples containing C1 Bgl1 variant enzymes were pre-incubated at pH 5, 65° C. for 0 or 6 hours. The residual enzyme activity after the thermal challenge was measured using pNPG as substrate at pH 5, 50° C. for 1.5 hrs as described in Example 3.
Thermoactivity Assay
Thermoactivity screening was a cellobiose-based high throughput assay (HTA). In shallow, 96-well microtiter plates 75 μL of media supernatant containing C1 Bgl1 variant enzyme was added to 75 μL of 6.6 g/L cellobiose in 300 mM sodium acetate buffer pH 5.0. After sealing with aluminum/polypropylene laminate heat seal tape (Velocity 11 (Menlo Park, Calif.), Cat#06643-001), the plates were shaken at 65° C. for up to 21 hrs. The plates were centrifuged for 5 minutes at 4000 rpm. In shallow well clear microtiter plates, 15 μL of the reaction mixture was quenched with 185 μL of GOPOD Reagent solution per well. The solutions were gently mixed for 3 times and absorbance was measured at 510 nm for the identification of thermoactivity improved C1 Bgl1 variants.
Thermostability Assay
Thermostability screening was a pNPG-based high throughput assay. In shallow, 96-well microtiter plates, 180 μL of media supernatant containing active C1 Bgl1 variant enzyme was mixed with 30 μL of 1 M sodium acetate buffer pH 5.0. From a total 210 μL of the enzyme solution, 120 μL of enzyme solution was transferred into 96-well PCR plates for thermal challenge treatment, and 90 μL of enzyme solution left in the shallow 96-well plates was used as unchallenged C1 Bgl1 enzyme sample. After sealing with aluminum/polypropylene laminate heat seal tape (Velocity 11 (Menlo Park, Calif.), Cat#06643-001), the PCR plates were heated in the thermocycler (MJ Research, Waltham, Mass.) at 65° C. for 6 hrs. After thermal challenge, 90 μL of challenged enzyme solution was transferred into 96-well shallow plates. To initiate the pNPG reaction, in shallow 96-well plates, 90 μL of unchallenged or challenged enzyme solutions were mixed with 10 μL of 15 mM pNPG (Sigma-Aldrich, Inc., St. Louis, Mo.) solution in 150 mM sodium acetate buffer, pH 5. The reactions were incubated at pH 5, 50° C. for 1.5 hrs. After reaction, 100 μL of 1M sodium carbonate pH 11 solution was added to the reaction mixture to quench the reaction. The absorbance of the solution was measured at 405 nm to determine the conversion of pNPG to p-nitrophenyl. The residual activity was calculated using the formula:

% residual activity=100×(Absorbance of challenged samples/Absorbance of unchallenged samples).

Residual activities of C1 Bgl1 variants were compared with that of the native enzyme to identify the thermostability improved variants.
Example 6
Improved β-glucosidase Activities and Stabilities of Engineered C1 Bgl1 Variants
Improved C1 Bgl1 variants were identified from the high throughput screening of various C1 Bgl1 variant libraries as described in Example 5. A variant reference sequence exhibiting improved activity and stability compared to wildtype C1 Bgl1 was selected as a reference protein (Variant 3, as shown in Tables 2 and 3) and additional C1 Bgl1 variants were generated and evaluated as indicated in the legend to Table 3 to identify variants that had improved stability and activity relative to the Variant 3 reference sequence. One of the improved variants from this round was then selected as a reference protein (Variant 269, as shown in Tables 3 and 4) and additional C1 Bgl1 variants were generated and evaluated as described in the legend to Table 4 to identify variants that had improved stability and activity relative to Variant 269. A variant (Variant 481, as shown in Tables 4 and 5) was selected from this round as a reference protein and additional C1 Bgl1 variants were generated and evaluated as described in the legend to table 5 to identify variants that had improved stability and activity relative to Variant 481. Two variants (Variant 647, as shown in Tables 5 and 6; and Variant 664 as shown in Tables 5 and 7) were then selected. Each variant served as a reference protein for separate rounds of screening. Additional C1 Bgl1 variants were generated and evaluated as described in the legend for Table 6 to identify variants that had improved stability and activity relative to variant 647. C1 Bgl1 variants were also generated and evaluated as described in the legend to Table 7 to identify variants that had improved stability and activity relative to Variant 664.
Tables 2, 3, 4, 5, 6, and 7 summarize the improvement in thermoactivities and thermostabilities of certain C1 Bgl1 variants encompassed by the invention.
Example 7
Production of Improved β-glucosidase Variants in the C1 Host
A two-step fermentation process (inoculation and main fermentations starting from spores) was used to express C1 bgl1 variant genes in C1. Six plasmids containing C1 bgl1 variant genes were transformed into C1 strain and plated on agar plates containing M3-01 medium with 22.93% sucrose (ingredients of M3-01 Medium: 6.0 g/L Sodium Nitrate, 0.52 g/L Potassium Chloride, 1.52 g/L Potassium Phosphate monobasic (KH2PO4), 0.24 g/L Magnesium Sulfate, 1.6 mg/L Copper(II) Sulfate pentahydrate (CuSO45H2O), 5 mg/L Ferrous Sulfate heptahydrate (FeSO47H2O), 22 mg/L Zinc Sulfate heptahydrate (ZnSO47H2O), 5 mg/L Manganese(II) Chloride tetrahydrate (MnCl24H2O), 1.8 mg/L Cobalt(II) Sulfate heptahydrate (CoSO47H2O), 1.5 mg/L Sodium Molybdate dihydrate (Na2MoO42H2O), 11 mg/L Boric Acid, 50 mg/L EDTA, 10.0 g/L Glucose, 1.0 g/L CAS amino acids (Tritium Microbiologie B. V., The Netherlands), 16 g/L agar, 1 ml/L 1000×Pen/Strep after sterilization (1000×Pen/Strep: 2 g Penicillin G and 5 g Streptomycin dissolved in 100 ml H2O, sterilized by filtration). The pH of the medium was adjusted to 6.5 with 10 M KOH and autoclaved for 25 minutes at 121° C. The plates were incubated at 35° C. for 5 days. Spores harvested from the agar plates were used to inoculate a 100 mL F1-01 inoculum medium sterilized in a 500 mL Erlenmeyer flask to reach 5*104-105 spores/mL initial spore number. (Ingredients of F1-01 Inoculum Medium: 0.50 g/L Potassium Phosphate dibasic (K2HPO4), 0.05 g/L Potassium Chloride, 0.007 g/L Ferrous Sulfate heptahydrate (FeSO47H2O), 1.00 g/L Yeast Extract (only KAT), 10 g/L Pharmamedia (Traders Protein, Lubbock, Tex., USA), 10 g/L D(+)Lactose monohydrate, 10 g/L Glucose after sterilization, 1 ml/L 1000×Pen/Strep after sterilization (1000×Pen/Strep: 2 g Penicillin G and 5 g Streptomycin dissolved in 100 ml H2O, sterilized by filtration). The pH of the medium was adjusted to 7.0 with 10 M NaOH and autoclaved for 25 minutes at 121° C. (The pH of the medium after sterilization was 6.5). To prepare inoculum culture, the flask was incubated at 35° C., 85% humidity for 3 days with shaking at 250 rpm and 25 mm displacement. 15 mL F1-01 Main Fermentation Medium sterilized in a 100 mL Erlenmeyer flask was inoculated with 750 μL of the obtained inoculum culture (ingredients of F1-01 Main Fermentation Medium: 0.66 g/L Potassium Phosphate dibasic (K2HPO4), 0.24 g/L Potassium Phosphate monobasic (KH2PO4), 8.00 g/L Ammonium Sulphate, 12.00 g/L Sodium Citrate tribasic dehydrate, 0.15 g/L Yeast Extract (only KAT), 0.09 g/L Magnesium Sulfate heptahydrate, 0.80 g/L Calcium Chloride dihydrate, 24.80 g/L Pharmamedia (Traders Protein, Lubbock, Tex., USA), 26.40 g/L D(+)Lactose monohydrate, 64.80 g/L Cellulose (AlphaCel BH200A)). The medium was autoclaved for 25 minutes at 121° C. The main fermentation was carried out by incubation at 35° C., 85% humidity for 6 days with shaking at 300 rpm and 25 mm displacement. After finishing the main fermentation the cells were pelleted by centrifugation (4500 rpm, 15 min, 4° C.). The clear medium supernatant containing the secreted C1 Bgl1 enzyme was collected and stored at −20° C. until used.
Example 8
Improved Thermostabilities of 6-Glucosidase Variants Produced in the C1 Host
Eight C1 Bgl1 variants (3, 8, 70, 109, 143, 194, 269 and 270) and native enzyme, produced in the C1 host, were characterized to determine their stabilities at high temperature (65° C.). The samples containing various C1 Bgl1 variant enzymes were pre-incubated at pH 5, 65° C. for 0, 6 or 24 hrs. The residual enzyme activity after the thermal challenge was measured using pNPG as substrate at pH 5, 50° C. for 20 mins. The best variant of the eight exhibited up to 34-fold improvement in stability over the native enzyme (FIG. 1A and FIG. 1B). Comparison of stability profiles of the native enzyme and eight C1 Bgl1 variants, produced from yeast and from C1, showed good correlation between the two hosts. (FIG. 1C).
Example 9
Thermostabilities of C1 Bql1 Variants
The thermostabilities of improved C1 Bgl1 variants 3, 269, and 481 were compared to that of the native C1 Bgl1 enzyme. The residual enzyme activity after 24 hr incubation at pH 4.5, 65° C. was measured using pNPG as substrate at pH 5, 50° C. for 20 mins. FIG. 2A shows that Variant 481 had the greatest increase in activity following thermal challenge.
The thermostability of improved C1 Bgl1 Variant 664 was compared to that of Variant 481. The residual enzyme activity after 4 hr incubation at pH 4, 65° C. was measured using pNPG as substrate at pH 5, 50° C. for 20 mins. FIG. 2B shows that Variant 664 had improved enzyme activity after thermal challenge relative to Variant 481.
The thermostability of improved C1 Bgl1 variants 3, 481, 664, 916, 885, and 871 was compared to the native enzyme (FIG. 3). Residual enzyme activity was measured after 72 hr incubation at pH 4.5, 65° C. using a pNPG assay at pH 5, 50° C. for 20 mins. The results showed that variants 664, 916, 885, and 871 retained substantial activity.
The specific activity of variant 871 was compared to the native, i.e., wildtype enzyme using a cellobiose assay (pH 4.5 or pH 5, 55° C.-75° C.; 8 g/L cellobiose, 18 hr reaction). FIG. 4 shows that variant 871 produced more glucose in the assay than the native enzyme.
FIG. 5 shows an alignment of Variants 871, 916, 885, 664, 647, 481, 269, and 3 with the native C1 Bgl1 amino acid sequence.
While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes can be made and equivalents can be substituted without departing from the scope of the invention. In addition, many modifications can be made to adapt a particular situation, material, composition of matter, process, process step or steps, to achieve the benefits provided by the present invention without departing from the scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.
All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an indication that any such document is pertinent prior art, nor does it constitute any admission as to the contents or date of the same.








SEQ ID NO: 1 Wild-type C1 bgl1 cDNA Sequence










atgaaggctg ctgcgctttc ctgcctcttc ggcagtaccc ttgccgttgc aggcgccatt
60



 


gaatcgagaa aggttcacca gaagcccctc gcgagatctg aaccttttta cccgtcgcca
120


 


tggatgaatc ccaacgccga cggctgggcg gaggcctatg cccaggccaa gtcctttgtc
180


 


tcccaaatga ctctgctaga gaaggtcaac ttgaccacgg gagtcggctg gggggctgag
240


 


cagtgcgtcg gccaagtggg cgcgatccct cgccttggac ttcgcagtct gtgcatgcat
300


 


gactcccctc tcggcatccg aggagccgac tacaactcag cgttcccctc tggccagacc
360


 


gttgctgcta cctgggatcg cggtctgacg taccgtcgcg gctacgcaat gggccaggag
420


 


gccaaaggca agggcatcaa tgtccttctc ggaccagtcg ccggccccct tggccgcatg
480


 


cccgagggcg gtcgtaactg ggaaggcttc gctccggatc ccgtccttac cggcatcggc
540


 


atgtccgaga cgatcaaggg cattcaggat gctggcgtca tcgcttgtgc gaagcacttt
600


 


attggaaacg agcaggagca cttcagacag gtgccagaag cccagggata cggttacaac
660


 


atcagcgaaa ccctctcctc caacattgac gacaagacca tgcacgagct ctacctttgg
720


 


ccgtttgccg atgccgtccg ggccggcgtc ggctctgtca tgtgctcgta ccagcaggtc
780


 


aacaactcgt acgcctgcca gaactcgaag ctgctgaacg acctcctcaa gaacgagctt
840


 


gggtttcagg gcttcgtcat gagcgactgg caggcacagc acactggcgc agcaagcgcc
900


 


gtggctggtc tcgatatgtc catgccgggc gacacccagt tcaacactgg cgtcagtttc
960


 


tggggcgcca atctcaccct cgccgtcctc aacggcacag tccctgccta ccgtctcgac
1020


 


gacatggcca tgcgcatcat ggccgccctc ttcaaggtca ccaagaccac cgacctggaa
1080


 


ccgatcaact tctccttctg gaccgacgac acttatggcc cgatccactg ggccgccaag
1140


 


cagggctacc aggagattaa ttcccacgtt gacgtccgcg ccgaccacgg caacctcatc
1200


 


cgggagattg ccgccaaggg tacggtgctg ctgaagaata ccggctctct acccctgaac
1260


 


aagccaaagt tcgtggccgt catcggcgag gatgctgggt cgagccccaa cgggcccaac
1320


 


ggctgcagcg accgcggctg taacgaaggc acgctcgcca tgggctgggg atccggcaca
1380


 


gccaactatc cgtacctcgt ttcccccgac gccgcgctcc aggcccgggc catccaggac
1440


 


ggcacgaggt acgagagcgt cctgtccaac tacgccgagg aaaagacaaa ggctctggtc
1500


 


tcgcaggcca atgcaaccgc catcgtcttc gtcaatgccg actcaggcga gggctacatc
1560


 


aacgtggacg gtaacgaggg cgaccgtaag aacctgactc tctggaacaa cggtgatact
1620


 


ctggtcaaga acgtctcgag ctggtgcagc aacaccatcg tcgtcatcca ctcggtcggc
1680


 


ccggtcctcc tgaccgattg gtacgacaac cccaacatca cggccattct ctgggctggt
1740


 


cttccgggcc aggagtcggg caactccatc accgacgtgc tttacggcaa ggtcaacccc
1800


 


gccgcccgct cgcccttcac ttggggcaag acccgcgaaa gctatggcgc ggacgtcctg
1860


 


tacaagccga ataatggcaa tggtgcgccc caacaggact tcaccgaggg cgtcttcatc
1920


 


gactaccgct acttcgacaa ggttgacgat gactcggtca tctacgagtt cggccacggc
1980


 


ctgagctaca ccaccttcga gtacagcaac atccgcgtcg tcaagtccaa cgtcagcgag
2040


 


taccggccca cgacgggcac cacggcccag gccccgacgt ttggcaactt ctccaccgac
2100


 


ctcgaggact atctcttccc caaggacgag ttcccctaca tctaccagta catctacccg
2160


 


tacctcaaca cgaccgaccc ccggagggcc tcggccgatc cccactacgg ccagaccgcc
2220


 


gaggagttcc tcccgcccca cgccaccgat gacgaccccc agccgctcct ccggtcctcg
2280


 


ggcggaaact cccccggcgg caaccgccag ctgtacgaca ttgtctacac aatcacggcc
2340


 


gacatcacga atacgggctc cgttgtaggc gaggaggtac cgcagctcta cgtctcgctg
2400


 


ggcggtcccg aggatcccaa ggtgcagctg cgcgactttg acaggatgcg gatcgaaccc
2460


 


ggcgagacga ggcagttcac cggccgcctg acgcgcagag atctgagcaa ctgggacgtc
2520


 


acggtgcagg actgggtcat cagcaggtat cccaagacgg catatgttgg gaggagcagc
2580


 


cggaagttgg atctcaagat tgagcttcct tga
2613


 








SEQ ID NO: 2: Wild-type C1 Bgl1 Polypeptide Sequence; signal peptide indicated in bold










MKAAALSCLF GSTLAVAGAI ESRKVHQKPL ARSEPFYPSP WMNPNADGWA EAYAQAKSFV
60



 


SQMTLLEKVN LTTGVGWGAE QCVGQVGAIP RLGLRSLCMH DSPLGIRGAD YNSAFPSGQT
120


 


VAATWDRGLM YRRGYAMGQE AKGKGINVLL GPVAGPLGRM PEGGRNWEGF APDPVLTGIG
180


 


MSETIKGIQD AGVIACAKHF IGNEQEHFRQ VPEAQGYGYN ISETLSSNID DKTMHELYLW
240


 


PFADAVRAGV GSVMCSYQQV NNSYACQNSK LLNDLLKNEL GFQGFVMSDW QAQHTGAASA
300


 


VAGLDMSMPG DTQFNTGVSF WGANLTLAVL NGTVPAYRLD DMAMRIMAAL FKVTKTTDLE
360


 


PINFSFWTDD TYGPIHWAAK QGYQEINSHV DVRADHGNLI REIAAKGTVL LKNTGSLPLN
420


 


KPKFVAVIGE DAGSSPNGPN GCSDRGCNEG TLAMGWGSGT ANYPYLVSPD AALQARAIQD
480


 


GTRYESVLSN YAEEKTKALV SQANATAIVF VNADSGEGYI NVDGNEGDRK NLTLWNNGDT
540


 


LVKNVSSWCS NTIVVIHSVG PVLLTDWYDN PNITAILWAG LPGQESGNSI TDVLYGKVNP
600


 


AARSPFTWGK TRESYGADVL YKPNNGNGAP QQDFTEGVFI DYRYFDKVDD DSVIYEFGHG
660


 


LSYTTFEYSN IRVVKSNVSE YRPTTGTTAQ APTFGNFSTD LEDYLFPKDE FPYIYQYIYP
720


 


YLNTTDPRRA SADPHYGQTA EEFLPPHATD DDPQPLLRSS GGNSPGGNRQ LYDIVYTITA
780


 


DITNTGSVVG EEVPQLYVSL GGPEDPKVQL RDFDRMRIEP GETRQFTGRL TRRDLSNWDV
840


 


TVQDWVISRY PKTAYVGRSS RKLDLKIELP
870


 








SEQ ID NO: 3: Polynucleotide Sequence Encoding Wild-Type Chrysosporium lucknowense



(β-glucosidase 1 protein using Codons Biased For Expression in Saccharomyces


cerevisiae. The protein encoded by SEQ ID NO: 3 is SEQ ID NO: 22.


                                                                        ATAGAAAGTAGAAAGGTA


                                                                         I  E  S  R  K  V


 


CATCAAAAACCATTAGCTAGATCAGAACCATTCTACCCTTCTCCATGGATGAACCCTAATGCAGATGGATGGGCAGAAGCATATGCTCAG


 H  Q  K  P  L  A  R  S  E  P  F  Y  P  S  P  W  M  N  P  N  A  D  G  W  A  E  A  Y  A  Q


 


GCCAAGAGTTTTGTCTCCCAGATGACTCTGTTGGAAAAGGTTAATCTGACAACAGGAGTAGGATGGGGTGCAGAACAGTGTGTCGGCCAA


 A  K  S  F  V  S  Q  M  T  L  L  E  K  V  N  L  T  T  G  V  G  W  G  A  E  Q  C  V  G  Q


 


GTTGGTGCTATCCCTAGATTGGGTCTTAGAAGTTTGTGTATGCACGATTCTCCCTTAGGTATAAGAGGCGCTGACTATAACTCAGCATTC


 V  G  A  I  P  R  L  G  L  R  S  L  C  M  H  D  S  P  L  G  I  R  G  A  D  Y  N  S  A  F


 


CCATCCGGGCAAACTGTTGCTGCGACATGGGACAGGGGTTTGATGTATAGAAGGGGTTATGCGATGGGTCAAGAGGCAAAGGGTAAAGGA


 P  S  G  Q  T  V  A  A  T  W  D  R  G  L  M  Y  R  R  G  Y  A  M  G  Q  E  A  K  G  K  G


 


ATTAATGTATTGTTGGGGCCGGTGGCGGGGCCACTGGGAAGAATGCCAGAAGGTGGAAGGAACTGGGAAGGATTCGCCCCCGACCCAGTG


 I  N  V  L  L  G  P  V  A  G  P  L  G  R  M  P  E  G  G  R  N  W  E  G  F  A  P  D  P  V


 


CTAACAGGTATAGGTATGTCCGAAACGATCAAAGGCATACAAGATGCAGGTGTTATCGCCTGTGCGAAGCATTTTATTGGTAATGAACAA


 L  T  G  I  G  M  S  E  T  I  K  G  I  Q  D  A  G  V  I  A  C  A  K  H  F  I  G  N  E  Q


 


GAGCATTTTCGTCAAGTGCCAGAGGCTCAAGGTTATGGTTATAATATTTCTGAAACTTTAAGTTCCAACATCGATGACAAAACCATGCAC


 E  H  F  R  Q  V  P  E  A  Q  G  Y  G  Y  N  I  S  E  T  L  S  S  N  I  D  D  K  T  M  H


 


GAGTTATACTTATGGCCTTTTGCAGACGCTGTGAGAGCTGGCGTTGGCTCTGTTATGTGCTCTTATCAGCAAGTTAATAACTCTTACGCC


 E  L  Y  L  W  P  F  A  D  A  V  R  A  G  V  G  S  V  M  C  S  Y  Q  Q  V  N  N  S  Y  A


 


TGTCAAAATTCCAAGTTACTAAATGACTTATTGAAGAACGAACTAGGATTCCAAGGATTCGTCATGAGCGATTGGCAAGCACAGCATACT


 C  Q  N  S  K  L  L  N  D  L  L  K  N  E  L  G  F  Q  G  F  V  M  S  D  W  Q  A  Q  H  T


 


GGTGCTGCATCCGCTGTGGCAGGATTAGATATGTCAATGCCAGGAGATACACAATTTAATACTGGCGTTAGTTTTTGGGGTGCAAACCTA


 G  A  A  S  A  V  A  G  L  D  M  S  M  P  G  D  T  Q  F  N  T  G  V  S  F  W  G  A  N  L


 


ACTTTAGCTGTTCTAAACGGTACGGTACCTGCATATCGTTTAGACGACATGGCCATGCGTATAATGGCTGCTTTATTCAAAGTTACAAAA


 T  L  A  V  L  N  G  T  V  P  A  Y  R  L  D  D  M  A  M  R  I  M  A  A  L  F  K  V  T  K


 


ACCACCGATTTAGAACCAATTAATTTTAGTTTTTGGACAGATGACACATATGGTCCTATACACTGGGCTGCTAAGCAAGGGTACCAAGAA


 T  T  D  L  E  P  I  N  F  S  F  W  T  D  D  T  Y  G  P  I  H  W  A  A  K  Q  G  Y  Q  E


 


ATAAATAGTCACGTTGACGTAAGAGCGGATCACGGCAATCTTATCAGAGAGATAGCAGCAAAGGGAACTGTATTGTTGAAGAATACTGGT


 I  N  S  H  V  D  V  R  A  D  H  G  N  L  I  R  E  I  A  A  K  G  T  V  L  L  K  N  T  G


 


TCATTACCACTAAACAAACCAAAGTTTGTCGCAGTCATTGGTGAAGATGCTGGTTCATCCCCTAATGGACCAAATGGTTGTAGTGACAGA


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


 


GGCTGCAATGAAGGCACGTTGGCAATGGGCTGGGGCTCAGGGACTGCCAATTACCCCTATTTGGTCTCTCCGGATGCGGCTTTACAGGCT


 G  C  N  E  G  T  L  A  M  G  W  G  S  G  T  A  N  Y  P  Y  L  V  S  P  D  A  A  L  Q  A


 


AGAGCAATCCAGGATGGTACTAGATACGAGAGCGTCCTAAGTAACTATGCCGAAGAAAAGACTAAGGCCTTAGTCAGTCAAGCCAATGCC


 R  A  I  Q  D  G  T  R  Y  E  S  V  L  S  N  Y  A  E  E  K  T  K  A  L  V  S  Q  A  N  A


 


ACTGCTATCGTTTTCGTAAACGCGGATTCTGGCGAAGGTTATATCAATGTTGATGGTAATGAAGGTGACAGAAAGAATTTAACTTTATGG


 T  A  I  V  F  V  N  A  D  S  G  E  G  Y  I  N  V  D  G  N  E  G  D  R  K  N  L  T  L  W


 


AATAACGGCGACACATTAGTTAAAAATGTATCAAGTTGGTGTTCCAATACTATCGTCGTGATACATTCTGTTGGTCCAGTTTTACTGACA


 N  N  G  D  T  L  V  K  N  V  S  S  W  C  S  N  T  I  V  V  I  H  S  V  G  P  V  L  L  T


 


GACTGGTACGATAACCCAAACATTACCGCCATTTTATGGGCAGGTCTGCCAGGGCAGGAATCAGGAAATTCCATTACGGACGTACTATAC


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


 


GGAAAGGTTAACCCAGCCGCCAGGAGCCCTTTCACATGGGGTAAGACAAGAGAGAGCTACGGAGCTGATGTTCTTTATAAACCGAACAAC


 G  K  V  N  P  A  A  R  S  P  F  T  W  G  K  T  R  E  S  Y  G  A  D  V  L  Y  K  P  N  N


 


GGGAATGGAGCGCCACAGCAAGATTTTACTGAAGGTGTGTTCATTGACTATAGATACTTCGACAAAGTTGACGATGACTCAGTTATATAT


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


 


GAATTCGGTCACGGTCTATCTTATACTACTTTTGAATATTCAAATATAAGAGTAGTCAAAAGTAATGTTTCTGAATATAGGCCGACCACC


 E  F  G  H  G  L  S  Y  T  T  F  E  Y  S  N  I  R  V  V  K  S  N  V  S  E  Y  R  P  T  T


 


GGAACGACGGCTCAAGCGCCTACCTTCGGTAATTTTTCAACGGATTTAGAAGATTATTTATTTCCCAAAGACGAATTTCCATACATCTAC


 G  T  T  A  Q  A  P  T  F  G  N  F  S  T  D  L  E  D  Y  L  F  P  K  D  E  F  P  Y  I  Y


 


CAATACATATACCCCTATCTGAATACTACCGATCCAAGAAGAGCTTCTGCCGATCCACATTACGGGCAGACTGCCGAAGAGTTCTTGCCA


 Q  Y  I  Y  P  Y  L  N  T  T  D  P  R  R  A  S  A  D  P  H  Y  G  Q  T  A  E  E  F  L  P


 


CCACACGCTACTGACGACGATCCTCAACCTCTTCTGAGGTCCAGTGGCGGAAATTCACCTGGTGGTAATAGGCAGCTGTATGATATTGTG


 P  H  A  T  D  D  D  P  Q  P  L  L  R  S  S  G  G  N  S  P  G  G  N  R  Q  L  Y  D  I  V


 


TATACTATAACGGCTGATATTACTAATACTGGTAGCGTTGTTGGTGAAGAAGTGCCGCAATTATATGTGTCTTTAGGTGGTCCGGAAGAT


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


 


CCTAAGGTTCAGTTAAGAGACTTTGATAGGATGAGAATAGAACCTGGAGAAACTAGGCAATTTACAGGTAGATTGACCCGTAGGGATCTG


 P  K  V  Q  L  R  D  F  D  R  M  R  I  E  P  G  E  T  R  Q  F  T  G  R  L  T  R  R  D  L


 


TCAAACTGGGATGTAACAGTGCAAGATTGGGTAATCAGCAGGTACCCGAAAACTGCATACGTGGGTAGATCTTCCCGTAAGTTAGATTTG


 S  N  W  D  V  T  V  Q  D  W  V  I  S  R  Y  P  K  T  A  Y  V  G  R  S  S  R  K  L  D  L


 


AAAATTGAATTGCCATAA


 K  I  E  L  P  *


 


SEQ ID NO: 4 C1 Variant 3 cDNA sequence









atgaaggctg ctgcgctttc ctgcctcttc ggcagtaccc ttgccgttgc aggcgccatt
60



 


gaatcgagaa aggttcacca gaagcccctc gcgagatctg aaccttttta cccgtcgcca
120


 


tggatgaatc ccaacgccga cggctgggcg gaggcctatg cccaggccaa gtcctttgtc
180


 


tcccaaatga ctctgctaga gaaggtcaac ttgaccacgg gagtcggctg gggggctgag
240


 


cagtgcgtcg gccaagtggg cgcgatccct cgccttggac ttcgcagtct gtgcatgcat
300


 


gactcccctc tcggcatccg aggagccgac tacaactcag cgttcccctc tggccagacc
360


 


gttgctgcta cctgggatcg cggtctgatg taccgtcgcg gctacgcaat gggccaggag
420


 


gccaaaggca agggcatcaa tgtccttctc ggaccagtcg ccggccccct tggccgcatg
480


 


cccgagggcg gtcgtaactg ggaaggcttc gctccggatc ccgtccttac cggcatcggc
540


 


atgtccgaga cgatcaaggg cattcaggat gctggcgtca tcgcttgtgc gaagcacttt
600


 


attggaaacg agcaggagca cttcagacag gtgccagaag cccagggata cggttacaac
660


 


atcagcgaaa ccctctcctc caacattgac gacaagacca tgcacgagct ctacctttgg
720


 


ccgtttgccg atgccgtccg ggccggcgtc ggctctgtca tgtgctcgta ccagcaggtc
780


 


aacaactcgt acgcctgcca gaactcgaag ctgctgaacg acctcctcaa gaacgagctt
840


 


gggtttcagg gcttcgtcat gagcgactgg tgggcacagc acactggcgc agcaagcgcc
900


 


gtggctggtc tcgatatgtc catgccgggc gacacccagt tcaacactgg cgtcagtttc
960


 


tggggcgcca atctcaccct cgccgtcctc aacggcacag tccctgccta ccgtctcgac
1020


 


gacatggcca tgcgcatcat ggccgccctc ttcaaggtca ccaagaccac cgacctggaa
1080


 


ccgatcaact tctccttctg gaccctggac acttatggcc cgatccactg ggccgccaag
1140


 


cagggctacc aggagattaa ttcccacgtt gacgtccgcg ccgaccacgg caacctcatc
1200


 


cggaacattg ccgccaaggg tacggtgctg ctgaagaata ccggctctct acccctgaac
1260


 


aagccaaagt tcgcggccgt catcggcgag gatgctgggt cgagccccaa cgggcccaac
1320


 


ggctgcagcg accgcggctg taacgaaggc acgctcgcca tgggctgggg atccggcaca
1380


 


gccaactatc cgtacctcgt ttcccccgac gccgcgctcc aggcccgggc catccaggac
1440


 


ggcacgaggt acgagagcgt cctgtccaac tacgccgagg aaaagacaaa ggctctggtc
1500


 


tcgcaggcca atgcaaccgc catcgtcttc gtcaatgccg actcaggcga gggctacatc
1560


 


aacgtggacg gtaacgaggg cgaccgtaag aacctgactc tctggaacaa cggtgatact
1620


 


ctggtcaaga acgtctcgag ctggtgcagc aacaccatcg tcgtcatcca ctcggtcggc
1680


 


ccggtcctcc tgaccgattg gtacgacaac cccaacatca cggccattct ctgggctggt
1740


 


cttccgggcc aggagtcggg caactccatc accgacgtgc tttacggcaa ggtcaacccc
1800


 


gccgcccgct cgcccttcac ttggggcaag acccgcgaaa gctatggcgc ggacgtcctg
1860


 


tacaagccga ataatggcaa tggtgcgccc caacaggact tcaccgaggg cgtcttcatc
1920


 


gactaccgct acttcgacaa ggttgacgat gactcggtca tctacgagtt cggccacggc
1980


 


ctgagctaca ccaccttcga gtacagcaac acccgcgtcg tcaagtccaa cgtcagcgag
2040


 


taccggccca cgacgggcac cacggcccag gccccgacgt ttggcaactt ctccaccgac
2100


 


ctcgaggact atctcttccc caaggacgag ttcccctaca tctaccagta catctacccg
2160


 


tacctcaaca cgaccgaccc ccggagggcc tcggccgatc cccactacgg ccagaccgcc
2220


 


gaggagttcc tcccgcccca cgccaccgat gacgaccccc agccgctcct ccggtcctcg
2280


 


ggcggaaact cccccggcgg caaccgccag ctgtacgaca ttgtctacac aatcacggcc
2340


 


gacatcacga atacgggctc cgttgtaggc gaggaggtac cgcagctcta cgtctcgctg
2400


 


ggcggtcccg aggatcccaa ggtgcagctg cgcgactttg acaggatgcg gatcgaaccc
2460


 


ggcgagacga ggcagttcac cggccgcctg acgcgcagag atctgagcaa ctgggacgtc
2520


 


acggtgcagg actgggtcat cagcaggtat cccaagacgg catatgttgg gaggagcagc
2580


 


cggaagttgg atctcaagat tgagcttcct tga
2613


 








SEQ ID NO: 5 C1 Variant 3 Polypeptide Sequence










MKAAALSCLF GSTLAVAGAI ESRKVHQKPL ARSEPFYPSP WMNPNADGWA EAYAQAKSFV
60



 


SQMTLLEKVN LTTGVGWGAE QCVGQVGAIP RLGLRSLCMH DSPLGIRGAD YNSAFPSGQT
120


 


VAATWDRGLM YRRGYAMGQE AKGKGINVLL GPVAGPLGRM PEGGRNWEGF APDPVLTGIG
180


 


MSETIKGIQD AGVIACAKHF IGNEQEHFRQ VPEAQGYGYN ISETLSSNID DKTMHELYLW
240


 


PFADAVRAGV GSVMCSYQQV NNSYACQNSK LLNDLLKNEL GFQGFVMSDW WAQHTGAASA
300


 


VAGLDMSMPG DTQFNTGVSF WGANLTLAVL NGTVPAYRLD DMAMRIMAAL FKVTKTTDLE
360


 


PINFSFWTLD TYGPIHWAAK QGYQEINSHV DVRADHGNLI RNIAAKGTVL LKNTGSLPLN
420


 


KPKFVAVIGE DAGSSPNGPN GCSDRGCNEG TLAMGWGSGT ANYPYLVSPD AALQARAIQD
480


 


GTRYESVLSN YAEEKTKALV SQANATAIVF VNADSGEGYI NVDGNEGDRK NLTLWNNGDT
540


 


LVKNVSSWCS NTIVVIHSVG PVLLTDWYDN PNITAILWAG LPGQESGNSI TDVLYGKVNP
600


 


AARSPFTWGK TRESYGADVL YKPNNGNGAP QQDFTEGVFI DYRYFDKVDD DSVIYEFGHG
660


 


LSYTTFEYSN IRVVKSNVSE YRPTTGTTAQ APTFGNFSTD LEDYLFPKDE FPYIYQYIYP
720


 


YLNTTDPRRA SADPHYGQTA EEFLPPHATD DDPQPLLRSS GGNSPGGNRQ LYDIVYTITA
780


 


DITNTGSVVG EEVPQLYVSL GGPEDPKVQL RDFDRMRIEP GETRQFTGRL TRRDLSNWDV
840


 


TVQDWVISRY PKTAYVGRSS RKLDLKIELP
870


 








SEQ ID NO: 6 C1 Variant 269 cDNA Sequence










atgaaggctg ctgcgctttc ctgcctcttc ggcagtaccc ttgccgttgc aggcgccatt
60



 


gaatcgagaa aggttcacca gaagcccctc gcgagatctg aaccttttta cccgtcgcca
120


 


tggatgaatc ccaacgccga cggctgggcg gaggcctatg cccaggccaa gtcctttgtc
180


 


tcccaaatga ctctgctaga gaaggtcaac ttgaccacgg gagtcggctg gggggctgag
240


 


cagtgcgtcg gccaagtggg cgcgatccct cgccttggac ttcgcagtct gtgcatgcat
300


 


gactcccctc tcggcatccg aggagccgac tacaactcag cgttcccctc tggccagacc
360


 


gttgctgcta cctgggatcg cggtctgatg taccgtcgcg gctacgcaat gggccaggag
420


 


gccaaaggca agggcatcaa tgtccttctc ggaccagtcg ccggccccct tggccgcatg
480


 


cccgagggcg gtcgtaactg ggaaggcttc gctccggatc ccgtccttac cggcatcggc
540


 


atgtccgaga cgatcaaggg cattcaggat gctggcgtca tcgcttgtgc gaagcacttt
600


 


attggaaacg agcaggagca cttcagacag gtgccagaag cccagggata cggttacaac
660


 


atcagcgaaa ccctctcctc caacattgac gacaagacca tgcacgagct ctacctttgg
720


 


ccgtttgccg atgccgtccg ggccggcgtc ggctctgtca tgtgctcgta caaccaggtc
780


 


aacaactcgt acgcctgcca gaactcgaag ctgctgaacg acctcctcaa gaacgagctt
840


 


gggtttcagg gcttcgtcat gagcgactgg tgggcacagc acactggcgc agcaagcgcc
900


 


gtggctggtc tcgatatgtc catgccgggc gacaccatgt tcaacactgg cgtcagtttc
960


 


tggggcgcca atctcaccct cgccgtcctc aacggcacag tccctgccta ccgtctcgac
1020


 


gacatggcca tgcgcatcat ggccgccctc ttcaaggtca ccaagaccac cgacctggaa
1080


 


ccgatcaact tctccttctg gacccgcgac acttatggcc cgatccactg ggccgccaag
1140


 


cagggctacc aggagattaa ttcccacgtt gacgtccgcg ccgaccacgg caacctcatc
1200


 


cggaacattg ccgccaaggg tacggtgctg ctgaagaata ccggctctct acccctgaac
1260


 


aagccaaagt tcgtggccgt catcggcgag gatgctgggc cgagccccaa cgggcccaac
1320


 


ggctgcagcg accgcggctg taacgaaggc acgctcgcca tgggctgggg atccggcaca
1380


 


gccaactatc cgtacctcgt ttcccccgac gccgcgctcc agttgcgggc catccaggac
1440


 


ggcacgaggt acgagagcgt cctgtccaac tacgccgagg aaaatacaaa ggctctggtc
1500


 


tcgcaggcca atgcaaccgc catcgtcttc gtcaatgccg actcaggcga gggctacatc
1560


 


aacgtggacg gtaacgaggg cgaccgtaag aacctgactc tctggaacaa cggtgatact
1620


 


ctggtcaaga acgtctcgag ctggtgcagc aacaccatcg tcgtcatcca ctcggtcggc
1680


 


ccggtcctcc tgaccgattg gtacgacaac cccaacatca cggccattct ctgggctggt
1740


 


cttccgggcc aggagtcggg caactccatc accgacgtgc tttacggcaa ggtcaacccc
1800


 


gccgcccgct cgcccttcac ttggggcaag acccgcgaaa gctatggcgc ggacgtcctg
1860


 


tacaagccga ataatggcaa ttgggcgccc caacaggact tcaccgaggg cgtcttcatc
1920


 


gactaccgct acttcgacaa ggttgacgat gactcggtca tctacgagtt cggccacggc
1980


 


ctgagctaca ccaccttcga gtacagcaac atccgcgtcg tcaagtccaa cgtcagcgag
2040


 


taccggccca cgacgggcac cacggcccag gccccgacgt ttggcaactt ctccaccgac
2100


 


ctcgaggact atctcttccc caaggacgag ttcccctaca tctaccagta catctacccg
2160


 


tacctcaaca cgaccgaccc ccggagggcc tcggccgatc cccactacgg ccagaccgcc
2220


 


gaggagttcc tcccgcccca cgccaccgat gacgaccccc agccgctcct ccggtcctcg
2280


 


ggcggaaact cccccggcgg caaccgccag ctgtacgaca ttgtctacac aatcacggcc
2340


 


gacatcacga atacgggctc cgttgtaggc gaggaggtac cgcagctcta cgtctcgctg
2400


 


ggcggtcccg aggatcccaa ggtgcagctg cgcgactttg acaggatgcg gatcgaaccc
2460


 


ggcgagacga ggcagttcac cggccgcctg acgcgcagag atctgagcaa ctgggacgtc
2520


 


acggtgcagg actgggtcat cagcaggtat cccaagacgg catatgttgg gaggagcagc
2580


 


cggaagttgg atctcaagat tgagcttcct tga
2613


 








SEQ ID NO: 7 C1 Variant 269 Polypeptide Sequence










MKAAALSCLF GSTLAVAGAI ESRKVHQKPL ARSEPFYPSP WMNPNADGWA EAYAQAKSFV
60



 


SQMTLLEKVN LTTGVGWGAE QCVGQVGAIP RLGLRSLCMH DSPLGIRGAD YNSAFPSGQT
120


 


VAATWDRGLM YRRGYAMGQE AKGKGINVLL GPVAGPLGRM PEGGRNWEGF APDPVLTGIG
180


 


MSETIKGIQD AGVIACAKHF IGNEQEHFRQ VPEAQGYGYN ISETLSSNID DKTMHELYLW
240


 


PFADAVRAGV GSVMCSYNQV NNSYACQNSK LLNDLLKNEL GFQGFVMSDW WAQHTGAASA
300


 


VAGLDMSMPG DTMFNTGVSF WGANLTLAVL NGTVPAYRLD DMAMRIMAAL FKVTKTTDLE
360


 


PINFSFWTRD TYGPIHWAAK QGYQEINSHV DVRADHGNLI RNIAAKGTVL LKNTGSLPLN
420


 


KPKFVAVIGE DAGPSPNGPN GCSDRGCNEG TLAMGWGSGT ANYPYLVSPD AALQLRAIQD
480


 


GTRYESVLSN YAEENTKALV SQANATAIVF VNADSGEGYI NVDGNEGDRK NLTLWNNGDT
540


 


LVKNVSSWCS NTIVVIHSVG PVLLTDWYDN PNITAILWAG LPGQESGNSI TDVLYGKVNP
600


 


AARSPFTWGK TRESYGADVL YKPNNGNWAP QQDFTEGVFI DYRYFDKVDD DSVIYEFGHG
660


 


LSYTTFEYSN IRVVKSNVSE YRPTTGTTAQ APTFGNFSTD LEDYLFPKDE FPYIYQYIYP
720


 


YLNTTDPRRA SADPHYGQTA EEFLPPHATD DDPQPLLRSS GGNSPGGNRQ LYDIVYTITA
780


 


DITNTGSVVG EEVPQLYVSL GGPEDPKVQL RDFDRMRIEP GETRQFTGRL TRRDLSNWDV
840


 


TVQDWVISRY PKTAYVGRSS RKLDLKIELP
870


 








SEQ ID NO: 8 C1 Variant 481 cDNA Sequence










atgaaggctg ctgcgctttc ctgcctcttc ggcagtaccc ttgccgttgc aggcgccatt
60



 


gaatcgagaa aggttcacca gaagcccctc gcgagatctg aaccttttta cccgtcgcca
120


 


tggatgaatc ccaacgccga cggctgggcg gaggcctatg cccaggccaa gtcctttgtc
180


 


tcccaaatga ctctgctaga gaaggtcaac ttgaccacgg gagtcggctg gggggctgag
240


 


cagtgcgtcg gccaagtggg cgcgatccct cgccttggac ttcgcagtct gtgcatgcat
300


 


gactcccctc tcggcatccg aggagccgac tacaactcag cgttcccctc tggccagacc
360


 


gttgctgcta cctgggatcg cggtctgatg taccgtcgcg gctacgcaat gggccaggag
420


 


gccaaaggca agggcatcaa tgcccttctc ggaccagtcg ccggccccct tggccgcatg
480


 


cccgagggcg gtcgtaactg ggaaggcttc gctccggatc ccgtccttac cggcatcggc
540


 


atgtccgaga cgatcaaggg cattcaggat gctggcgtca tcgcttgtgc gaagcacttt
600


 


attggaaacg agcaggagca cttcagacag gtgccagaag cccagggata cggttacaac
660


 


atcagcgaaa ccctctcctc caacattgac gacaagacca tgcacgagct ctacctttgg
720


 


ccgtttgccg atgccgtccg ggccggcgtc ggctctgtca tgtgctcgta caaccaggtc
780


 


aacaactcgt acgcctgcca gaactcgaag ctgctgaacg acctcctcaa gaacgagctt
840


 


gggtttcagg gcttcgtcat gagcgactgg tgggcacagc acactggcgc agcaagcgcc
900


 


gtggctggtc tcgatatgtc catgccgggc gacaccatgt tcaacactgg cgtcagtttc
960


 


tggggcgcca atctcaccct cgccgtcctc aacggcacag tccctgccta ccgtctcgac
1020


 


gacatggcca tgcgcatcat ggccgccctc ttcaaggtca ccaagaccac cgacctggaa
1080


 


ccgatcaact tctccttctg gacccgcgac acttatggcc cgatccactg ggccgccaag
1140


 


cagggctacc aggagattaa ttcccacgtt gacgtccgcg ccgaccacgg caacctcatc
1200


 


cggaacattg ccgccaaggg tacggtgctg ctgaagaata ccggctctct acccctgaac
1260


 


aagccaaagt tcgtggccgt catcggcgag gatgctgggc cgagccccaa cgggcccaac
1320


 


ggctgcagcg accgcggctg taacgaaggc acgctcgcca tgggctgggg atccggcaca
1380


 


gccaactatc cgtacctcgt ttcccccgac gccgcgctcc agttgcgggc catccaggac
1440


 


ggcacgaggt acgagagcgt cctgtccaac tacgccgagg aaaatacaaa ggctctggtc
1500


 


tcgcaggcca atgcaaccgc catcgtcttc gtcaatgccg actcaggcga gggctacatc
1560


 


aacgtggacg gtaacgaggg cgaccgtaag aacctgactc tctggaacaa cggtgatact
1620


 


ctggtcaaga acgtctcgag ctggcgcagc aacaccatcg tcgtcatcca ctcggtcggc
1680


 


ccggtcctcc tgaccgattg gtacgacaac cccaacatca cggccattct ctgggctggt
1740


 


cttccgggcc aggagtcggg caactccatc accgacgtgc tttacggcaa ggtcaacccc
1800


 


gccgcccgct cgcccttcac ttggggcaag acccgcgaaa gctatggcgc ggacgtcctg
1860


 


tacaagccga ataatggcaa ttgggcgccc caacaggact tcaccgaggg cgtcttcatc
1920


 


gactaccgct acttcgacaa ggttgacgat gactcggtca tctacgagtt cggccacggc
1980


 


ctgagctaca ccaccttcga gtacagcaac atccgcgtcg tcaagtccaa cgtcagcgag
2040


 


taccggccca cgacgggcac cacgattcag gccccgacgt ttggcaactt ctccaccgac
2100


 


ctcgaggact atctcttccc caaggacgag ttcccctaca tcccgcagta catctacccg
2160


 


tacctcaaca cgaccgaccc ccggagggcc tcggccgatc cccactacgg ccagaccgcc
2220


 


gaggagttcc tcccgcccca cgccaccgat gacgaccccc agccgctcct ccggtcctcg
2280


 


ggcggaaact cccccggcgg caaccgccag ctgtacgaca ttgtctacac aatcacggcc
2340


 


gacatcacga atacgggctc cgttgtaggc gaggaggtac cgcagctcta cgtctcgctg
2400


 


ggcggtcccg aggatcccaa ggtgcagctg cgcgactttg acaggatgcg gatcgaaccc
2460


 


ggcgagacga ggcagttcac cggccgcctg acgcgcagag atctgagcaa ctgggacgtc
2520


 


acggtgcagg actgggtcat cagcaggtat cccaagacgg catatgttgg gaggagcagc
2580


 


cggaagttgg atctcaagat tgagcttcct tga
2613


 








SEQ ID NO: 9 C1 Variant 481 Polypeptide Sequence










MKAAALSCLF GSTLAVAGAI ESRKVHQKPL ARSEPFYPSP WMNPNADGWA EAYAQAKSFV
60



 


SQMTLLEKVN LTTGVGWGAE QCVGQVGAIP RLGLRSLCMH DSPLGIRGAD YNSAFPSGQT
120


 


VAATWDRGLM YRRGYAMGQE AKGKGINVLL GPVAGPLGRM PEGGRNWEGF APDPVLTGIG
180


 


MSETIKGIQD AGVIACAKHF IGNEQEHFRQ VPEAQGYGYN ISETLSSNID DKTMHELYLW
240


 


PFADAVRAGV GSVMCSYNQV NNSYACQNSK LLNDLLKNEL GFQGFVMSDW WAQHTGAASA
300


 


VAGLDMSMPG DTMFNTGVSF WGANLTLAVL NGTVPAYRLD DMAMRIMAAL FKVTKTTDLE
360


 


PINFSFWTRD TYGPIHWAAK QGYQEINSHV DVRADHGNLI RNIAAKGTVL LKNTGSLPLN
420


 


KPKFVAVIGE DAGPSPNGPN GCSDRGCNEG TLAMGWGSGT ANYPYLVSPD AALQLRAIQD
480


 


GTRYESVLSN YAEENTKALV SQANATAIVF VNADSGEGYI NVDGNEGDRK NLTLWNNGDT
540


 


LVKNVSSWCS NTIVVIHSVG PVLLTDWYDN PNITAILWAG LPGQESGNSI TDVLYGKVNP
600


 


AARSPFTWGK TRESYGADVL YKPNNGNWAP QQDFTEGVFI DYRYFDKVDD DSVIYEFGHG
660


 


LSYTTFEYSN IRVVKSNVSE YRPTTGTTIQ APTFGNFSTD LEDYLFPKDE FPYIPQYIYP
720


 


YLNTTDPRRA SADPHYGQTA EEFLPPHATD DDPQPLLRSS GGNSPGGNRQ LYDIVYTITA
780


 


DITNTGSVVG EEVPQLYVSL GGPEDPKVQL RDFDRMRIEP GETRQFTGRL TRRDLSNWDV
840


 


TVQDWVISRY PKTAYVGRSS RKLDLKIELP
870


 








SEQ ID NO: 10 C1 Variant 482 cDNA Sequence










atgaaggctg ctgcgctttc ctgcctcttc ggcagtaccc ttgccgttgc aggcgccatt
60



 


gaatcgagaa aggttcacca gaagcccctc gcgagatctg aaccttttta cccgtcgcca
120


 


tggatgaatc ccaacgccga cggctgggcg gaggcctatg cccaggccaa gtcctttgtc
180


 


tcccaaatga ctctgccaga gaaggccaac ccgaccacgg gagtcggccg gggggctgag
240


 


cagtgcgtcg gccaagtggg cgcgatccct cgccttggac ttcgcagtct gtgcatgcat
300


 


gactcccctc tcggcatccg aggagccgac tacaactcag cgttcccctc tggccagacc
360


 


gttgctgcta cctgggatcg cggtctgatg taccgtcgcg gctacgcaat gggccaggag
420


 


gccaaaggca agggcatcaa tgtccttctc ggaccagtcg ccggccccct tggccgcatg
480


 


cccgagggcg gtcgtaactg ggaaggcttc gctccggatc ccgtccttac cggcatcggc
540


 


atgtccgaga cgatcaaggg cattcaggat gctggcgtca tcgcttgtgc gaagcacttt
600


 


attggaaacg agcaggagca cttcagacag gtgccagaag cccagggata cggttacaac
660


 


atcagcgaaa ccctctcctc caacattgac gacaagacca tgcacgagct ctacctttgg
720


 


ccgtttgccg atgccgtccg ggccggcgtc ggctctgtca tgtgctcgta caaccaggtc
780


 


aacaactcgt acgcctgcca gaactcgaag ctgctgaacg acctcctcaa gaacgagctt
840


 


gggtttcagg gcttcgtcat gagcgactgg tgggcacagc acactggcgc agcaagcgcc
900


 


gtggctggtc tcgatatgtc catgccgggc gacaccatgt tcaacactgg cgtcagtttc
960


 


tggggcgcca atctcaccct cgccgtcctc aacggcacag tccctgccta ccgtctcgac
1020


 


gacatggcca tgcgcatcat ggccgccctc ttcaaggtca ccaagaccac cgacctggaa
1080


 


ccgatcaact tctccttctg gacccgcgac acttatggcc cgatccactg ggccgccaag
1140


 


cagggctacc aggagattaa ttcccacgtt gacgtccgcg ccgaccacgg caacctcatc
1200


 


cggaacattg ccgccaaggg tacggtgctg ctgaagaata ccggctctct acccctgaac
1260


 


aagccaaagt tcgtggccgt catcggcgaa gatgctgggc cgagccccaa cgggcccaac
1320


 


ggctgcagcg accgcggctg taacgaaggc acgctcgcca tgggctgggg atccggcaca
1380


 


gccaactatc cgtacctcgt ttcccccgac gccgcgctcc agttgcgggc catccaggac
1440


 


ggcacgaggt acgagagcgt cctgtccaac tacgccgagg aaaatacaaa ggctctggtc
1500


 


tcgcaggcca atgcaaccgc catcgtcttc gtcaatgccg actcaggcga gggctacatc
1560


 


aacgtggacg gtaacgaggg cgaccgtaag aacctgactc tctggaacaa cggtgatact
1620


 


ctggtcaaga acgtctcgag ctggtgcagc aacaccatcg tcgtcatcca ctcggtcggc
1680


 


ccggtcctcc tgaccgattg gtacgacaac cccaacatca cggccattct ctgggctggt
1740


 


cttccgggcc aggagtcggg caactccatc accgacgtgc tttacggcaa ggtcaacccc
1800


 


gccgcccgct cgcccttcac ttggggcaag acccgcgaaa gctatggcgc ggacgtcctg
1860


 


tacaagccga ataatggcaa ttgggcgccc caacaggact tcaccgaggg cgtcttcatc
1920


 


gactaccgct acttcgacaa ggttgacgat gactcggtca tctacgagtt cggccacggc
1980


 


ctgagctaca ccaccttcga gtacagcaac atccgcgtcg tcaagtccaa cgtcagcgag
2040


 


taccggccca cgacgggcac cacggcccag gccccgacgt ttggcaactt ctccaccgac
2100


 


ctcgaggact atctcttccc caaggacgag ttcccctaca tcccgcagta catctaccca
2160


 


tacctcaaca cgaccgaccc ccggagggcc tcggccgatc cccactacgg ccagaccgcc
2220


 


gaggagttcc tcccgcccca cgccaccgat gacgaccccc agccgctcct ccggtcctcg
2280


 


ggcggaaact cccccggcgg caaccgccag ctgtacgaca ttgtctacac aatcacggcc
2340


 


gacatcacga atacgggctc cgttgtaggc gaggaggtac cgcagctcta cgtctcgctg
2400


 


ggcggtcccg aggatcccaa ggtgcagctg cgcgactttg acaggatgcg gatcgaaccc
2460


 


ggcgagaaaa ggcagttcac cggccgcctg acgcgcagag atctgagcaa ctgggacgtc
2520


 


acggtgcagg actgggtcat cagcaggtat cccaagacgg catatgttgg gaggagcagc
2580


 


cggaagttgg atctcaagat tgagcttcct tga
2613


 








SEQ ID NO: 11 C1 Variant 482 polypeptide sequence










MKAAALSCLF GSTLAVAGAI ESRKVHQKPL ARSEPFYPSP WMNPNADGWA EAYAQAKSFV
60



 


SQMTLLEKVN LTTGVGWGAE QCVGQVGAIP RLGLRSLCMH DSPLGIRGAD YNSAFPSGQT
120


 


VAATWDRGLM YRRGYAMGQE AKGKGINVLL GPVAGPLGRM PEGGRNWEGF APDPVLTGIG
180


 


MSETIKGIQD AGVIACAKHF IGNEQEHFRQ VPEAQGYGYN ISETLSSNID DKTMHELYLW
240


 


PFADAVRAGV GSVMCSYNQV NNSYACQNSK LLNDLLKNEL GFQGFVMSDW WAQHTGAASA
300


 


VAGLDMSMPG DTMFNTGVSF WGANLTLAVL NGTVPAYRLD DMAMRIMAAL FKVTKTTDLE
360


 


PINFSFWTRD TYGPIHWAAK QGYQEINSHV DVRADHGNLI RNIAAKGTVL LKNTGSLPLN
420


 


KPKFVAVIGE DAGPSPNGPN GCSDRGCNEG TLAMGWGSGT ANYPYLVSPD AALQLRAIQD
480


 


GTRYESVLSN YAEENTKALV SQANATAIVF VNADSGEGYI NVDGNEGDRK NLTLWNNGDT
540


 


LVKNVSSWCS NTIVVIHSVG PVLLTDWYDN PNITAILWAG LPGQESGNSI TDVLYGKVNP
600


 


AARSPFTWGK TRESYGADVL YKPNNGNWAP QQDFTEGVFI DYRYFDKVDD DSVIYEFGHG
660


 


LSYTTFEYSN IRVVKSNVSE YRPTTGTTAQ APTFGNFSTD LEDYLFPKDE FPYIPQYIYP
720


 


YLNTTDPRRA SADPHYGQTA EEFLPPHATD DDPQPLLRSS GGNSPGGNRQ LYDIVYTITA
780


 


DITNTGSVVG EEVPQLYVSL GGPEDPKVQL RDFDRMRIEP GEKRQFTGRL TRRDLSNWDV
840


 


TVQDWVISRY PKTAYVGRSS RKLDLKIELP
870


 








SEQ ID NO: 12 C1 Variant 664 cDNA Sequence










atgaaggctg ctgcgctttc ctgcctcttc ggcagtaccc ttgccgttgc aggcgccatt
60



 


gaatcgagaa aggttcacca gaagcccctc gcgagatctg aaccttttta cccgtcgcca
120


 


tggatgaatc ccaacgccga cggctgggcg gaggcctatg cccaggccaa gtcctttgtc
180


 


tcccaaatga ctctgctaga gaaggtcaac ttgaccacgg gagtcggctg gggggctgag
240


 


cagtgcgtcg gccaagtggg cgcgatccct cgccttggac ttcgcagtct gtgcatgcat
300


 


gactcccctc tcggcgtgcg aggagccgac tacaactcag cgttcccctc tggccagacc
360


 


gttgctgcta cctgggatcg cggtctgatg taccgtcgcg gctacgcaat gggccaggag
420


 


gccaaaggca agggcatcaa tgtccttctc ggaccagtcg ccggccccct tggccgcatg
480


 


cccgagggcg gtcgtaactg ggaaggcttc gctccggatc ccgtccttac cggcatcggc
540


 


atgtccgaga cgatcaaggg cattcaggat gctggcgtca tcgcttgtgc gaagcacttt
600


 


attggaaacg agcaggagca ctccagacag gtgccagaag cccagggata cggttacaac
660


 


atcagcgaaa ccctctcctc caacattgac gacaagacca tgcacgagct ctacctttgg
720


 


ccgtttgccg atgccgtccg ggccggcgtc ggctctgtca tgtgctcgta caaccagggc
780


 


aacaactcgt acgcctgcca gaactcgaag ctgctgaacg acctcctcaa gaacgagctt
840


 


gggtttcagg gcttcgtcat gagcgactgg tgggcacagc acactggcgc agcaagcgcc
900


 


gtggctggtc tcgatatgtc catgccgggc gacaccatgc tgaacactgg cgtcagtttc
960


 


tggggcgcca atctcaccct cgccgtcctc aacggcacag tccctgccta ccgtctcgac
1020


 


gacatggcca tgcgcatcat ggccgccctc ttcaaggtca ccaagaccac cgacctggaa
1080


 


ccgatcaact tctccttctg gacccgcgac acttatggcc cgatccactg ggccgccaag
1140


 


cagggctacc aggagattaa ttcccacgtt gacgtccgcg ccgaccacgg caacctcatc
1200


 


cggaacattg ccgccaaggg tacggtgctg ctgaagaata ccggctctct acccctgaac
1260


 


aagccaaagt tcgtggccgt catcggcgag gatgctgggc cgagccccaa cgggcccaac
1320


 


ggctgcagcg accgcggctg taacgaaggc acgctcgcca tgggctgggg atccggcaca
1380


 


gccaactatc cgtacctcgt ttcccccgac gccgcgctcc aggcgcgggc catccaggac
1440


 


ggcacgaggt acgagagcgt cctgtccaac tacgccgagg aaaatacaaa ggctctggtc
1500


 


tcgcaggcca atgcaaccgc catcgtcttc gtcaatgccg actcaggcga gggctacatc
1560


 


aacgtggacg gtaacgaggg cgaccgtaag aacctgactc tctggaacaa cggtgatact
1620


 


ctggtcaaga acgtctcgag ctggtgcagc aacaccatcg tcgtcatcca ctcggtcggc
1680


 


ccggtcctcc tgaccgattg gtacgacaac cccaacatca cggccattct ctgggctggt
1740


 


cttccgggcc aggagtcggg caactccatc accgacgtgc tttacggcaa ggtcaacccc
1800


 


gccgcccgct cgcccttcac ttggggcaag acccgcgaaa gctatggcgc ggacgtcctg
1860


 


tacaagccga ataatggcaa ttgggcgccc caacaggact tcaccgaggg cgtcttcatc
1920


 


gactaccgct acttcgacaa ggttgacgat gactcggtca tctacgagtt cggccacggc
1980


 


ctgagctaca ccaccttcga gtacagcaac atccgcgtcg tcaagtccaa cgtcagcgag
2040


 


taccggccca cgacgggcac cacgattcag gccccgacgt ttggcaactt ctccaccgac
2100


 


ctcgaggact atctcttccc caaggacgag ttcccctaca tcccgcagta catctacccg
2160


 


tacctcaaca cgaccgaccc ccggagggcc tcgggcgatc cccactacgg ccagaccgcc
2220


 


gaggagttcc tcccgcccca cgccaccgat gacgaccccc agccgctcct ccggtcctcg
2280


 


ggcggaaact cccccggcgg caaccgccag ctgtacgaca ttgtctacac aatcacggcc
2340


 


gacatcacga atacgggctc cgttgtaggc gaggaggtac cgcagctcta cgtctcgctg
2400


 


ggcggtcccg aggatcccaa ggtgcagctg cgcgactttg acaggatgcg gatcgaaccc
2460


 


ggcgagacga ggcagttcac cggccgcctg acgcgcagag atctgagcaa ctgggacgtc
2520


 


acggtgcagg actgggtcat cagcaggtat cccaagacgg catatgttgg gaggagcagc
2580


 


cggaagttgg atctcaagat tgagcttcct tga
2613


 








SEQ ID NO: 13 C1 Variant 664 polypeptide sequence










MKAAALSCLF GSTLAVAGAI ESRKVHQKPL ARSEPFYPSP WMNPNADGWA EAYAQAKSFV
60



 


SQMTLLEKVN LTTGVGWGAE QCVGQVGAIP RLGLRSLCMH DSPLGVRGAD YNSAFPSGQT
120


 


VAATWDRGLM YRRGYAMGQE AKGKGINVLL GPVAGPLGRM PEGGRNWEGF APDPVLTGIG
180


 


MSETIKGIQD AGVIACAKHF IGNEQEHFRQ VPEAQGYGYN ISETLSSNID DKTMHELYLW
240


 


PFADAVRAGV GSVMCSYNQG NNSYACQNSK LLNDLLKNEL GFQGFVMSDW WAQHTGAASA
300


 


VAGLDMSMPG DTMLNTGVSF WGANLTLAVL NGTVPAYRLD DMAMRIMAAL FKVTKTTDLE
360


 


PINFSFWTRD TYGPIHWAAK QGYQEINSHV DVRADHGNLI RNIAAKGTVL LKNTGSLPLN
420


 


KPKFVAVIGE DAGPSPNGPN GCSDRGCNEG TLAMGWGSGT ANYPYLVSPD AALQARAIQD
480


 


GTRYESVLSN YAEENTKALV SQANATAIVF VNADSGEGYI NVDGNEGDRK NLTLWNNGDT
540


 


LVKNVSSWCS NTIVVIHSVG PVLLTDWYDN PNITAILWAG LPGQESGNSI TDVLYGKVNP
600


 


AARSPFTWGK TRESYGADVL YKPNNGNWAP QQDFTEGVFI DYRYFDKVDD DSVIYEFGHG
660


 


LSYTTFEYSN IRVVKSNVSE YRPTTGTTIQ APTFGNFSTD LEDYLFPKDE FPYIPQYIYP
720


 


YLNTTDPRRA SGDPHYGQTA EEFLPPHATD DDPQPLLRSS GGNSPGGNRQ LYDIVYTITA
780


 


DITNTGSVVG EEVPQLYVSL GGPEDPKVQL RDFDRMRIEP GETRQFTGRL TRRDLSNWDV
840


 


TVQDWVISRY PKTAYVGRSS RKLDLKIELP
870


 








SEQ ID NO: 14 C1 Variant 647 cDNA Sequence










atgaaggctg ctgcgctttc ctgcctcttc ggcagtaccc ttgccgttgc aggcgccatt
60



 


gaatcgagaa aggttcacca gaagcccctc gcgagatctg aaccttttta cccgtcgcca
120


 


tggatgaatc ccaacgccat tggctgggcg gaggcctatg cccaggccaa gtcctttgtc
180


 


tcccaaatga ctctgctaga gaaggtcaac ttgaccacgg gagtcggctg gggggctgag
240


 


cagtgcgtcg gccaagtggg cgcgatccct cgccttggac ttcgcagtct gtgcatgcat
300


 


gactcccctc tcggcatccg aggagccgac tacaactcag cgttcccctc tggccagacc
360


 


gttgctgcta cctgggatcg cggtctgatg taccgtcgcg gctacgcaat gggccaggag
420


 


gccaaaggca agggcatcaa tgtccttctc ggaccagtcg ccggccccct tggccgcatg
480


 


cccgagggcg gtcgtaactg ggaaggcttc gctccggatc ccgtccttac cggcatcggc
540


 


atgtccgaga cgatcaaggg cattcaggat gctggcgtca tcgcttgtgc gaagcacttt
600


 


attggaaacg agcaggagca cttcagacag gtgccagaag cccagggata cggttacaac
660


 


atcagcgaaa ccctctcctc caacattgac gacaagacca tgcacgagct ctacctttgg
720


 


ccgtttgccg atgccgtccg ggccggcgtc ggctctgtca tgtgctcgta caaccaggtc
780


 


aacaactcgt acgcctgcca gaactcgaag ctgctgaacg acctcctcaa gaacgagctt
840


 


gggtttcagg gcttcgtcat gagcgactgg tgggcacagc acactggcgc agcaagcgcc
900


 


gtggctggtc tcgatatgtc catgccgggc gacaccatgt tcaacactgg cgtcagtttc
960


 


tggggcgcca atctcaccct cgccgtcctc aacggcacag tccctgccta ccgtctcgac
1020


 


gacatgtgca tgcgcatcat ggccgccctc ttcaaggtca ccaagaccac cgacctggaa
1080


 


ccgatcaact tctccttctg gacccgcgac acttatggcc cgatccactg ggccgccaag
1140


 


cagggctacc aggagattaa ttcccacgtt gacgtccgcg ccgaccacgg caacctcatc
1200


 


cggaacattg ccgccaaggg tacggtgctg ctgaagaata ccggctctct acccctgaac
1260


 


aagccaaagt tcgtggccgt catcggcgag gatgctgggc cgagccccaa cgggcccaac
1320


 


ggctgcagcg accgcggctg taacgaaggc acgctcgcca tgggctgggg atccggcaca
1380


 


gccaactatc cgtacctcgt ttcccccgac gccgcgctcc agttgcgggc catccaggac
1440


 


ggcacgaggt acgagagcgt cctgtccaac tacgccgagg aaaatacaaa ggctctggtc
1500


 


tcgcaggcca atgcaaccgc catcgtcttc gtcaatgccg actcaggcga gggctacatc
1560


 


aacgtggacg gtaacgaggg cgaccgtaag aacctgactc tctggaacaa cggtgatact
1620


 


ctggtcaaga acgtctcgag ctggtgcagc aacaccatcg tcgtcatcca ctcggtcggc
1680


 


ccggtcctcc tgaccgattg gtacgacaac cccaacatca cggccattct ctgggctggt
1740


 


cttccgggcc aggagtcggg caactccatc accgacgtgc tttacggcaa ggtcaacccc
1800


 


gccgcccgct cgcccttcac ttggggcaag acccgcgaaa gctatggcgc ggacgtcctg
1860


 


tacaagccga ataatggcaa ttgggcgccc caacaggact tcaccgaggg cgtcttcatc
1920


 


gactaccgct acttcgacaa ggttgacgat gactcggtca tctacgagtt cggccacggc
1980


 


ctgagctaca ccaccttcga gtacagcaac atccgcgtcg tcaagtccaa cgtcagcgag
2040


 


taccggccca cgacgggcaa aacgattcag gccccgacgt ttggcaactt ctccaccgac
2100


 


ctcgaggact atctcttccc caaggacgag ttcccctaca tcccgcagta catctacccg
2160


 


tacctcaaca cgaccgaccc ccggagggcc tcggccgatc cccactacgg ccagaccgcc
2220


 


gaggagttcc tcccgcccca cgccaccgat gacgaccccc agccgctcct ccggtcctcg
2280


 


ggcggaaact cccccggcgg caaccgccag ctgtacgaca ttgtctacac aatcacggcc
2340


 


gacatcacga atacgggctc cgttgtaggc gaggaggtac cgcagctcta cgtctcgctg
2400


 


ggcggtcccg aggatcccaa ggtgcagctg cgcgactttg acaggatgcg gatcgaaccc
2460


 


ggcgagacga ggcagttcac cggccgcctg acgcgcagag atctgagcaa ctgggacgtc
2520


 


acggtgcagg actgggtcat cagcaggtat cccaagacgg catatgttgg gaggagcagc
2580


 


cggaagttgg atctcaagat tgagcttcct tga
2613


 








SEQ ID NO: 15 C1 Variant 647 polypeptide sequence










MKAAALSCLF GSTLAVAGAI ESRKVHQKPL ARSEPFYPSP WMNPNAIGWA EAYAQAKSFV
60



 


SQMTLLEKVN LTTGVGWGAE QCVGQVGAIP RLGLRSLCMH DSPLGIRGAD YNSAFPSGQT
120


 


VAATWDRGLM YRRGYAMGQE AKGKGINVLL GPVAGPLGRM PEGGRNWEGF APDPVLTGIG
180


 


MSETIKGIQD AGVIACAKHF IGNEQEHFRQ VPEAQGYGYN ISETLSSNID DKTMHELYLW
240


 


PFADAVRAGV GSVMCSYNQV NNSYACQNSK LLNDLLKNEL GFQGFVMSDW WAQHTGAASA
300


 


VAGLDMSMPG DTMFNTGVSF WGANLTLAVL NGTVPAYRLD DMCMRIMAAL FKVTKTTDLE
360


 


PINFSFWTRD TYGPIHWAAK QGYQEINSHV DVRADHGNLI RNIAAKGTVL LKNTGSLPLN
420


 


KPKFVAVIGE DAGPSPNGPN GCSDRGCNEG TLAMGKGSGT ANYPYLVSPD AALQLRAIQD
480


 


GTRYESVLSN YAEENTKALV SQANATAIVF VNADSGEGYI NVDGNEGDRK NLTLWNNGDT
540


 


LVKNVSSWCS NTIVVIHSVG PVLLTDWYDN PNITAILWAG LPGQESGNSI TDVLYGKVNP
600


 


AARSPFTWGK TRESYGADVL YKPNNGNWAP QQDPTEGVFI DYRYFDKVDD DSVIYEPGHG
660


 


LSYTTFEYSN IRVVKSNVSE YRPTTGKTIQ APTFGNFSTD LEDYLFPKDE FPYIPQYIYP
720


 


YLNTTDPRRA SADPHYGQTA EEFLPPHATD DDPQPLLRSS GGNSPGGNRQ LYDIVYTITA
780


 


DITNTGSVVG EEVPQLYVSL GGPEDPKVQL RDPDRMRIEP GETRQFTGRL TRRDLSNWDV
840


 


TVQDWVISRY PKTAYVGRSS RKLDLKIELP
870


 








SEQ ID NO: 16 C1 Variant 871 cDNA Sequence










atgaaggctg ctgcgctttc ctgcctcttc ggcagtaccc ttgccgttgc aggcgccatt
60



 


gaatcgagaa aggttcacca gaagcccctc gcgagatctg aaccttttta cccgtcgcca
120


 


tggatgaatc ccaacgccat cggctgggcg gaggcctatg cccaggccaa gtcctttgtc
180


 


tcccaaatga ctctgctaga gaaggtcaac ttgaccacgg gagtcggctg gggggaggag
240


 


cagtgcgtcg gcaacgtggg cgcgatccct cgccttggac ttcgcagtct gtgcatgcat
300


 


gactcccctc tcggcgtgcg aggaaccgac tacaactcag cgttcccctc tggccagacc
360


 


gttgctgcta cctgggatcg cggtctgatg taccgtcgcg gctacgcaat gggccaggag
420


 


gccaaaggca agggcatcaa tgtccttctc ggaccagtcg ccggccccct tggccgcatg
480


 


cccgagggcg gtcgtaactg ggaaggcttc gctccggatc ccgtccttac cggcatcggc
540


 


atgtccgaga cgatcaaggg cattcaggat gctggcgtca tcgcttgtgc gaagcacttt
600


 


attggaaacg agcaggagca cttcagacag gtgccagaag cccagggata cggttacaac
660


 


atcagcgaaa ccctctcctc caacattgac gacaagacca tgcacgagct ctacctttgg
720


 


ccgtttgccg atgccgtccg ggccggcgtc ggctctgtca tgtgctcgta caaccagggc
780


 


aacaactcgt acgcctgcca gaactcgaag ctgctgaacg acctcctcaa gaacgagctt
840


 


gggtttcagg gcttcgtcat gagcgactgg tgggcacagc acactggcgc agcaagcgcc
900


 


gtggctggtc tcgatatgtc catgccgggc gacaccatgg tcaacactgg cgtcagtttc
960


 


tggggcgcca atctcaccct cgccgtcctc aacggcacag tccctgccta ccgtctcgac
1020


 


gacatgtgca tgcgcatcat ggccgccctc ttcaaggtca ccaagaccac cgacctggaa
1080


 


ccgatcaact tctccttctg gacccgcgac acttatggcc cgatccactg ggccgccaag
1140


 


cagggctacc aggagattaa ttcccacgtt gacgtccgcg ccgaccacgg caacctcatc
1200


 


cggaacattg ccgccaaggg tacggtgctg ctgaagaata ccggctctct acccctgaac
1260


 


aagccaaagt tcgtggccgt catcggcgag gatgctgggc cgagccccaa cgggcccaac
1320


 


ggctgcagcg accgcggctg taacgaaggc acgctcgcca tgggctgggg atccggcaca
1380


 


gccaactatc cgtacctcgt ttcccccgac gccgcgctcc aggcgcgggc catccaggac
1440


 


ggcacgaggt acgagagcgt cctgtccaac tacgccgagg aaaatacaaa ggctctggtc
1500


 


tcgcaggcca atgcaaccgc catcgtcttc gtcaatgccg actcaggcga gggctacatc
1560


 


aacgtggacg gtaacgaggg cgaccgtaag aacctgactc tctggaacaa cggtgatact
1620


 


ctggtcaaga acgtctcgag ctggtgcagc aacaccatcg tcgtcatcca ctcggtcggc
1680


 


ccggtcctcc tgaccgattg gtacgacaac cccaacatca cggccattct ctgggctggt
1740


 


cttccgggcc aggagtcggg caactccatc accgacgtgc tttacggcaa ggtcaacccc
1800


 


gccgcccgct cgcccttcac ttggggcaag acccgcgaaa gctatggcgc ggacgtcctg
1860


 


tacaagccga ataatggcaa ttgggcgccc caacaggact tcaccgaggg cgtcttcatc
1920


 


gactaccgct acttcgacaa ggttgacgat gactcggtca tctacgagtt cggccacggc
1980


 


ctgagctaca ccaccttcga gtacagcaac atccgcgtcg tcaagtccaa cgtcagcgag
2040


 


taccggccca cgacgggcac cacgattcag gccccgacgt ttggcaactt ctccaccgac
2100


 


ctcgaggact atctcttccc caaggacgag ttcccctaca tcccgcagta catctacccg
2160


 


tacctcaaca cgaccgaccc ccggagggcc tcgggcgatc cccactacgg ccagaccgcc
2220


 


gaggagttcc tcccgcccca cgccaccgat gacgaccccc agccgctcct ccggtcctcg
2280


 


ggcggaaact cccccggcgg caaccgccag ctgtacgaca ttgtctacac aatcacggcc
2340


 


gacatcacga atacgggctc cgttgtaggc gaggaggtac cgcagctcta cgtctcgctg
2400


 


ggcggtcccg aggatcccaa ggtgcagctg cgcgactttg acaggatgcg gatcgaaccc
2460


 


ggcgagacga ggcagttcac cggccgcctg acgcgcagag atctgagcaa ctgggacgtc
2520


 


acggtgcagg actgggtcat cagcaggtat cccaagacgg catatgttgg gaggagcagc
2580


 


cggaagttgg atctcaagat tgagcttcct tga
2613


 








SEQ ID NO: 17 C1 Variant 871 polypeptide sequence










MKAAALSCLF GSTLAVAGAI ESRKVHQKPL ARSEPFYPSP WMNPNAIGWA EAYAQAKSFV
60



 


SQMTLLEKVN LTTGVGWGEE QCVGNVGAIP RLGLRSLCMH DSPLGVRGTD YNSAFPSGQT
120


 


VAATWDRGLM YRRGYAMGQE AKGKGINVLL GPVAGPLGRM PEGGRNWEGF APDPVLTGIG
180


 


MSETIKGIQD AGVIACAKHF IGNEQEHFRQ VPEAQGYGYN ISETLSSNID DKTMHELYLW
240


 


PFADAVRAGV GSVMCSYNQG NNSYACQNSK LLNDLLKNEL GFQGFVMSDW WAQHTGAASA
300


 


VAGLDMSMPG DTMVNTGVSF WGANLTLAVL NGTVPAYRLD DMCMRIMAAL FKVTKTTDLE
360


 


PINFSFWTRD TYGPIHWAAK QGYQEINSHV DVRADHGNLI RNIAAKGTVL LKNTGSLPLN
420


 


KPKFVAVIGE DAGPSPNGPN GCSDRGCNEG TLAMGWGSGT ANYPYLVSPD AALQARAIQD
480


 


GTRYESVLSN YAEENTKALV SQANATAIVF VNADSGEGYI NVDGNEGDRK NLTLWNNGDT
540


 


LVKNVSSWCS NTIVVIHSVG PVLLTDWYDN PNITAILWAG LPGQESGNSI TDVLYGKVNP
600


 


AARSPFTWGK TRESYGADVL YKPNNGNWAP QQDFTEGVFI DYRYFDKVDD DSVIYEFGHG
660


 


LSYTTFEYSN IRVVKSNVSE YRPTTGTTIQ APTFGNFSTD LEDYLFPKDE FPYIPQYIYP
720


 


YLNTTDPRRA SGDPHYGQTA EEFLPPHATD DDPQPLLRSS GGNSPGGNRQ LYDIVYTITA
780


 


DITNTGSVVG EEVPQLYVSL GGPEDPKVQL RDFDRMRIEP GETRQFTGRL TRRDLSNWDV
840


 


TVQDWVISRY PKTAYVGRSS RKLDLKIELP
870


 








SEQ ID NO: 18 C1 Variant 885 cDNA sequence










atgaaggctg ctgcgctttc ctgcctcttc ggcagtaccc ttgccgttgc aggcgccatt
60



 


gaatcgagaa aggttcacca gaagcccctc gcgagatctg aaccttttta cccgtcgcca
120


 


tggatgaatc ccaacgccat cggctgggcg gaggcctatg cccaggccaa gtcctttgtc
180


 


tcccaaatga ctctgctaga gaaggtcaac ttgaccacgg gagtcggctg gggggctgag
240


 


cagtgcgtcg gcaacgtggg cgcgatccct cgccttggac ttcgcagtct gtgcatgcat
300


 


gactcccctc tcggcgtgcg aggaagcgac tacaactcag cgttcccctc tggccagacc
360


 


gttgctgcta cctgggatcg cggtctgatg taccgtcgcg gctacgcaat gggccaggag
420


 


gccaaaggca agggcatcaa tgtccttctc ggaccagtcg ccggccccct tggccgcatg
480


 


cccgagggcg gtcgtaactg ggaaggcttc gctccggatc ccgtccttac cggcatcggc
540


 


atgtccgaga cgatcaaggg cattcaggat gctggcgtca tcgcttgtgc gaagcacttt
600


 


attggaaacg agcaggagca cttcagacag gtgccagaag cccagggata cggttacaac
660


 


atcagcgaaa ccctctcctc caacattgac gacaagacca tgcacgagct ctacctttgg
720


 


ccgtttgccg atgccgtccg ggccggcgtc ggctctgtca tgtgctcgta caaccagggc
780


 


aacaactcgt acgcctgcca gaactcgaag ctgctgaacg acctcctcaa gaacgagctt
840


 


gggtttcagg gcttcgtcat gagcgactgg tgggcacagc acactggcgc agcaagcgcc
900


 


gtggctggtc tcgatatgtc catgccgggc gacaccatgg tcaacactgg cgtcagtttc
960


 


tggggcgcca atctcaccct cgccgtcctc aacggcacag tccctgccta ccgtctcgac
1020


 


gacatgtgca tgcgcatcat ggccgccctc ttcaaggtca ccaagaccac cgacctggaa
1080


 


ccgatcaact tctccttctg gacccgcgac acttatggcc cgatccactg ggccgccaag
1140


 


cagggctacc aggagattaa ttcccacgtt gacgtccgcg ccgaccacgg caacctcatc
1200


 


cggaacattg ccgccaaggg tacggtgctg ctgaagaata ccggctctct acccctgaac
1260


 


aagccaaagt tcgtggccgt catcggcgag gatgctgggc cgagccccaa cgggcccaac
1320


 


ggctgcagcg accgcggctg taacgaaggc acgctcgcca tgggctgggg atccggcaca
1380


 


gccaactatc cgtacctcgt ttcccccgac gccgcgctcc aggcgcgggc catccaggac
1440


 


ggcacgaggt acgagagcgt cctgtccaac tacgccgagg aaaatacaaa ggctctggtc
1500


 


tcgcaggcca atgcaaccgc catcgtcttc gtcaatgccg actcaggcga gggctacatc
1560


 


aacgtggacg gtaacgaggg cgaccgtaag aacctgactc tctggaacaa cggtgatact
1620


 


ctggtcaaga acgtctcgag ctggtgcagc aacaccatcg tcgtcatcca ctcggtcggc
1680


 


ccggtcctcc tgaccgattg gtacgacaac cccaacatca cggccattct ctgggctggt
1740


 


cttccgggcc aggagtcggg caactccatc accgacgtgc tttacggcaa ggtcaacccc
1800


 


gccgcccgct cgcccttcac ttggggcaag acccgcgaaa gctatggcgc ggacgtcctg
1860


 


tacaagccga ataatggcaa ttgggcgccc caacaggact tcaccgaggg cgtcttcatc
1920


 


gactaccgct acttcgacaa ggttgacgat gactcggtca tctacgagtt cggccacggc
1980


 


ctgagctaca ccaccttcga gtacagcaac atccgcgtcg tcaagtccaa cgtcagcgag
2040


 


taccggccca cgacgggcac cacgattcag gccccgacgt ttggcaactt ctccaccgac
2100


 


ctcgaggact atctcttccc caaggacgag ttcccctaca tcccgcagta catctacccg
2160


 


tacctcaaca cgaccgaccc ccggagggcc tcgggcgatc cccactacgg ccagaccgcc
2220


 


gaggagttcc tcccgcccca cgccaccgat gacgaccccc agccgctcct ccggtcctcg
2280


 


ggcggaaact cccccggcgg caaccgccag ctgtacgaca ttgtctacac aatcacggcc
2340


 


gacatcacga atacgggctc cgttgtaggc gaggaggtac cgcagctcta cgtctcgctg
2400


 


ggcggtcccg aggatcccaa ggtgcagctg cgcgactttg acaggatgcg gatcgaaccc
2460


 


ggcgagacga ggcagttcac cggccgcctg acgcgcagag atctgagcaa ctgggacgtc
2520


 


acggtgcagg actgggtcat cagcaggtat cccaagacgg catatgttgg gaggagcagc
2580


 


cggaagttgg atctcaagat tgagcttcct tga
2613


 








SEQ ID NO: 19 C1 Variant 885 polypeptide sequence










MKAAALSCLF GSTLAVAGAI ESRKVHQKPL ARSEPFYPSP WMNPNAIGWA EAYAQAKSFV
60



 


SQMTLLEKVN LTTGVGWGAE QCVGNVGAIP RLGLRSLCMH DSPLGVRGSD YNSAFPSGQT
120


 


VAATWDRGLM YRRGYAMGQE AKGKGINVLL GPVAGPLGRM PEGGRNWEGF APDPVLTGIG
180


 


MSETIKGIQD AGVIACAKHF IGNEQEHFRQ VPEAQGYGYN ISETLSSNID DKTMHELYLW
240


 


PFADAVRAGV GSVMCSYNQG NNSYACQNSK LLNDLLKNEL GFQGFVMSDW WAQHTGAASA
300


 


VAGLDMSMPG DTMVNTGVSF WGANLTLAVL NGTVPAYRLD DMCMRIMAAL FKVTKTTDLE
360


 


PINFSFWTRD TYGPIHWAAK QGYQEINSHV DVRADHGNLI RNIAAKGTVL LKNTGSLPLN
420


 


KPKFVAVIGE DAGPSPNGPN GCSDRGCNEG TLAMGWGSGT ANYPYLVSPD AALQARAIQD
480


 


GTRYESVLSN YAEENTKALV SQANATAIVF VNADSGEGYI NVDGNEGDRK NLTLWNNGDT
540


 


LVKNVSSWCS NTIVVIHSVG PVLLTDWYDN PNITAILWAG LPGQESGNSI TDVLYGKVNP
600


 


AARSPFTWGK TRESYGADVL YKPNNGNWAP QQDFTEGVFI DYRYFDKVDD DSVIYEFGHG
660


 


LSYTTFEYSN IRVVKSNVSE YRPTTGTTIQ APTFGNFSTD LEDYLFPKDE FPYIPQYIYP
720


 


YLNTTDPRRA SGDPHYGQTA EEFLPPHATD DDPQPLLRSS GGNSPGGNRQ LYDIVYTITA
780


 


DITNTGSVVG EEVPQLYVSL GGPEDPKVQL RDFDRMRIEP GETRQFTGRL TRRDLSNWDV
840


 


TVQDWVISRY PKTAYVGRSS RKLDLKIELP
870


 








SEQ ID NO: 20 C1 Variant 916 cDNA sequence










atgaaggctg ctgcgctttc ctgcctcttc ggcagtaccc ttgccgttgc aggcgccatt
60



 


gaatcgagaa aggttcacca gaagcccctc gcgagatctg aaccttttta cccgtcgcca
120


 


tggatgaatc ccaacgccat cggctgggcg gaggcctatg cccaggccaa gtcctttgtc
180


 


tcccaaatga ctctgctaga gaaggtcaac ttgaccacgg gagtcggctg ggggatggag
240


 


cagtgcgtcg gccaagtggg cgcgatccct cgccttggac ttcgcagtct gtgcatgcat
300


 


gactcccctc tcggcgtgcg aggagccgac tacaactcag cgttcccctc tggccagacc
360


 


gttgctgcta cctgggatcg cggtctgatg taccgtcgcg gctacgcaat gggccaggag
420


 


gccaaaggca agggcatcaa tgtccttctc ggaccagtcg ccggccccct tggccgcatg
480


 


cccgagggcg gtcgtaactg ggaaggcttc gctccggatc ccgtccttac cggcatcggc
540


 


atgtccgaga cgatcaaggg cattcaggat gctggcgtca tcgcttgtgc gaagcacttt
600


 


attggaaacg agcaggagca cttcagacag gtgccagaag cccagggata cggttacaac
660


 


atcagcgaaa ccctctcctc caacattgac gacaagacca tgcacgagct ctacctttgg
720


 


ccgtttgccg atgccgtccg ggccggcgtc ggctctgtca tgtgctcgta caaccagggc
780


 


aacaactcgt acgcctgcca gaactcgaag ctgctgaacg acctcctcaa gaacgagctt
840


 


gggtttcagg gcttcgtcat gagcgactgg tgggcacagc acactggcgc agcaagcgcc
900


 


gtggctggtc tcgatatgtc catgccgggc gacaccatgc tgaacactgg cgtcagtttc
960


 


tggggcgcca atctcaccct cgccgtcctc aacggcacag tccctgccta ccgtctcgac
1020


 


gacatggcca tgcgcatcat ggccgccctc ttcaaggtca ccaagaccac cgacctggaa
1080


 


ccgatcaact tctccttctg gacccgcgac acttatggcc cgatccactg ggccgccaag
1140


 


cagggctacc aggagattaa ttcccacgtt gacgtccgcg ccgaccacgg caacctcatc
1200


 


cggaacattg ccgccaaggg tacggtgctg ctgaagaata ccggctctct acccctgaac
1260


 


aagccaaagt tcgtggccgt catcggcgag gatgctgggc cgagccccaa cgggcccaac
1320


 


ggctgcagcg accgcggctg taacgaaggc acgctcgcca tgggctgggg atccggcaca
1380


 


gccaactatc cgtacctcgt ttcccccgac gccgcgctcc aggcgcgggc catccaggac
1440


 


ggcacgaggt acgagagcgt cctgtccaac tacgccgagg aaaatacaaa ggctctggtc
1500


 


tcgcaggcca atgcaaccgc catcgtcttc gtcaatgccg actcaggcga gggctacatc
1560


 


aacgtggacg gtaacgaggg cgaccgtaag aacctgactc tctggaacaa cggtgatact
1620


 


ctggtcaaga acgtctcgag ctggtgcagc aacaccatcg tcgtcatcca ctcggtcggc
1680


 


ccggtcctcc tgaccgattg gtacgacaac cccaacatca cggccattct ctgggctggt
1740


 


cttccgggcc aggagtcggg caactccatc accgacgtgc tttacggcaa ggtcaacccc
1800


 


gccgcccgct cgcccttcac ttggggcaag acccgcgaaa gctatggcgc ggacgtcctg
1860


 


tacaagccga ataatggcaa ttgggcgccc caacaggact tcaccgaggg cgtcttcatc
1920


 


gactaccgct acttcgacaa ggttgacgat gactcggtca tctacgagtt cggccacggc
1980


 


ctgagctaca ccaccttcga gtacagcaac atccgcgtcg tcaagtccaa cgtcagcgag
2040


 


taccggccca cgacgggcac cacgattcag gccccgacgt ttggcaactt ctccaccgac
2100


 


ctcgaggact atctcttccc caaggacgag ttcccctaca tcccgcagta catctacccg
2160


 


tacctcaaca cgaccgaccc ccggagggcc tcgggcgatc cccactacgg ccagaccgcc
2220


 


gaggagttcc tcccgcccca cgccaccgat gacgaccccc agccgctcct ccggtcctcg
2280


 


ggcggaaact cccccggcgg caaccgccag ctgtacgaca ttgtctacac aatcacggcc
2340


 


gacatcacga atacgggctc cgttgtaggc gaggaggtac cgcagctcta cgtctcgctg
2400


 


ggcggtcccg aggatcccaa ggtgcagctg cgcgactttg acaggatgcg gatcgaaccc
2460


 


ggcgagacga ggcagttcac cggccgcctg acgcgcagag atctgagcaa ctgggacgtc
2520


 


acggtgcagg actgggtcat cagcaggtat cccaagacgg catatgttgg gaggagcagc
2580


 


cggaagttgg atctcaagat tgagcttcct tga
2613


 








SEQ ID NO: 21 C1 Variant 916 polypeptide sequence










MKAAALSCLF GSTLAVAGAI ESRKVHQKPL ARSEPFYPSP WMNPNAIGWA EAYAQAKSFV
60



 


SQMTLLEKVN LTTGVGWGME QCVGQVGAIP RLGLRSLCMH DSPLGVRGAD YNSAFPSGQT
120


 


VAATWDRGLM YRRGYAMGQE AKGKGINVLL GPVAGPLGRM PEGGRNWEGF APDPVLTGIG
180


 


MSETIKGIQD AGVIACAKHF IGNEQEHFRQ VPEAQGYGYN ISETLSSNID DKTMHELYLW
240


 


PFADAVRAGV GSVMCSYNQG NNSYACQNSK LLNDLLKNEL GFQGFVMSDW WAQHTGAASA
300


 


VAGLDMSMPG DTMLNTGVSF WGANLTLAVL NGTVPAYRLD DMAMRIMAAL FKVTKTTDLE
360


 


PINFSFWTRD TYGPIHWAAK QGYQEINSHV DVRADHGNLI RNIAAKGTVL LKNTGSLPLN
420


 


KPKFVAVIGE DAGPSPNGPN GCSDRGCNEG TLAMGWGSGT ANYPYLVSPD AALQARAIQD
480


 


GTRYESVLSN YAEENTKALV SQANATAIVF VNADSGEGYI NVDGNEGDRK NLTLWNNGDT
540


 


LVKNVSSWCS NTIVVIHSVG PVLLTDWYDN PNITAILWAG LPGQESGNSI TDVLYGKVNP
600


 


AARSPFTWGK TRESYGADVL YKPNNGNWAP QQDFTEGVFI DYRYFDKVDD DSVIYEFGHG
660


 


LSYTTFEYSN IRVVKSNVSE YRPTTGTTIQ APTFGNFSTD LEDYLFPKDE FPYIPQYIYP
720


 


YLNTTDPRRA SGDPHYGQTA EEFLPPHATD DDPQPLLRSS GGNSPGGNRQ LYDIVYTITA
780


 


DITNTGSVVG EEVPQLYVSL GGPEDPKVQL RDFDRMRIEP GETRQFTGRL TRRDLSNWDV
840


 


TVQDWVISRY PKTAYVGRSS RKLDLKIELP
870

## Claims

1. A recombinant nucleic acid encoding a β-glucosidase (Bgl1) variant having β-glucosidase activity, said variant comprising an amino acid sequence that has at least 90% identity to amino acid residues 20-870 of SEQ ID NO:2 and comprises amino acid substitutions at positions Q291, D369, and E402, wherein the positions are numbered with reference to SEQ ID NO:2.

2. The recombinant nucleic acid of claim 1, wherein the Bgl1 variant comprises an amino acid sequence that has at least 95% identity to amino acid residue 20-870 of SEQ ID NO:2.

3. The recombinant nucleic acid of claim 1, wherein the Bgl1 variant comprises amino acid substitutions Q291W, D369R, and E402N.

4. The recombinant nucleic acid of claim 1, wherein the Bgl1 variant comprises at least one amino acid substitution at a position selected from the group consisting of Q258, Q313, S434, A475, K495, and G628.

5. The recombinant nucleic acid of claim 4, wherein the at least one amino acid substitution is Q258N/H, Q313M, S434P, A475L, K495N, or G628W.

6. The recombinant nucleic acid of claim 3, wherein the Bgl1 variant further comprises amino acid substitutions Q258N, Q313M, S434P, K495N, and G628W.

7. The recombinant nucleic acid of claim 1, wherein the Bgl1 variant further comprises at least one amino acid substitution at a position A689 or Y715.

8. The recombinant nucleic acid of claim 7, wherein the amino acid substitution is A689I or Y715P.

9. The recombinant nucleic acid of claim 6, wherein the Bgl1 variant further comprises amino acid substitutions A6891 and Y715P.

10. The recombinant nucleic acid of claim 9, wherein the Bgl1 variant further comprises the amino acid substitution A475L.

11. The recombinant nucleic acid of claim 1, wherein the Bgl1 variant further comprises at least one amino acid substitution at a position D47, A343 or T687.

12. The recombinant nucleic acid of claim 11, where the at least one amino acid substitution is D47I, A343C, or T687W/K/C.

13. The recombinant nucleic acid of claim 9, further comprising amino acid substitutions D47I, A343C, and T687K.

14. The recombinant nucleic acid of claim 1, wherein the Bgl1 variant further comprises at least one amino acid substitution at a position I106, V260, F314, or A732.

15. The recombinant nucleic acid of claim 14, wherein the at least one amino acid substitution is I106V, V260G, F314L/V, or A732G/M/V.

16. The recombinant nucleic acid of claim 9, wherein the Bgl1 variant further comprises amino acid substitutions I106V, V260G, F314V/L and A732G.

17. The recombinant nucleic acid of claim 1, wherein the Bgl1 variant further comprises at least one amino acid substitution at a position D47, A79, Q85, A109, or A343.

18. The recombinant nucleic acid of claim 17, wherein the at least one amino acid substitution is D47I, A79E/G/M, Q85N, A109T/S, or A343C.

19. The recombinant nucleic acid of claim 15, wherein the Bgl1 variant further comprises amino acid substitutions D47I, A79E, Q85N, A109T, and A343C.

20. The recombinant nucleic acid of claim 15, wherein the Bgl1 variant further comprises amino acid substitutions D47I, Q85N, A109S, and A343C.

21. The recombinant nucleic acid of claim 15, wherein the Bgl1 variant further comprises amino acid substitutions D47I and A79M.

22. The recombinant nucleic acid of claim 1, wherein the Bgl1 variant has: (i) increased thermostability after incubation at pH 5 at 65° C. for six hours relative to wild-type Bgl1 having amino acid residues 20-870 of SEQ ID NO:2; or (ii) increased thermoactivity after incubation at pH 5 at 65° C. for twenty one hours relative to wild-type Bgl1 having amino acid residues 20-870 of SEQ ID NO:2; or (iii) both (i) and (ii).

23. The recombinant nucleic acid of claim 1 that encodes a preprotein comprising the Bgl1 variant fused to a signal peptide.

24. The recombinant nucleic acid of claim 1, wherein the nucleic acid is codon-optimized for expression in a host cell selected to express the nucleic acid.

25. An expression vector comprising the recombinant nucleic acid of claim 1.

26. An isolated host cell that comprises the recombinant nucleic acid of claim 1.

27. The host cell of claim 26, wherein the nucleic acid is operably linked to a promoter other than the C1 Bgl1 promoter.

28. The host cell of claim 26 that is a species of filamentous fungus.

29. The host cell of claim 28 that is an Aspergillus, Trichoderma, Humicola, Chrysosporium, Myceliophthora or Thielavia species.

30. The host cell of claim 26 that also expresses at least one additional nonnaturally occurring recombinant cellulase polypeptide, selected from a recombinant endoglucanase (EG) polypeptide and a recombinant cellobiohydrolase polypeptide (CBH).

31. A method of producing a Bgl1 polypeptide comprising culturing a host cell of claim 26 under conditions in which the Bgl1 variant is expressed.

32. The method of claim 31 wherein the Bgl1 variant is secreted by the host cell.

33. The method of claim 31 further comprising a step of recovering the Bgl1 variant from the medium in which the cell is cultured.

34. A composition for use in a saccharification reaction, wherein the composition comprises a host cell of claim 26 and enzymes for the saccharification reaction.