Polypeptides Having Cellulase Activity

Abstract

The present disclosure relates to CBH II chimera fusion polypeptides, nucleic acids encoding the polypeptides, and host cells for producing the polypeptides.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The application is a continuation application of U.S. application Ser. No. 12/755,328, filed Apr. 6, 2010 (now U.S. Pat. No. 9,249,401), which claims priority under 35 U.S.C. .sctn.119 to U.S. Provisional Application Ser. No. 61/166,993, filed, Apr. 6, 2009, and 61/177,882, filed May 13, 2009, the disclosures of which are incorporated herein by reference.

TECHNICAL FIELD

[0003] The disclosure relates to biomolecular engineering and design, and engineered proteins and nucleic acids.

BACKGROUND

[0004] The performance of cellulase mixtures in biomass conversion processes depends on many enzyme properties including stability, product inhibition, synergy among different cellulase components, productive binding versus nonproductive adsorption and pH dependence, in addition to the cellulose substrate physical state and composition. Given the multivariate nature of cellulose hydrolysis, it is desirable to have diverse cellulases to choose from in order to optimize enzyme formulations for different applications and feedstocks.

SUMMARY

[0005] The disclosure provides recombinant polypeptides having cellulase activity and increased thermostability and activity compared to a wild-type protein. The disclosure provides and demonstrates that CBHII chimeras and the native enzymes having a Cys to Ser mutation at the C-terminal end (e.g., at about amino acid 310-315 depending upon the native protein sequence, see, e.g., SEQ ID NO:2 and 4) hydrolyze more solid cellulose than the native enzyme in long time hydrolysis assays.

[0006] The disclosure provides a recombinant polypeptide comprising a C.fwdarw.S substitution in the C-terminal region in a motif comprising the sequence GECDG (SEQ ID NO:2 from 312-316), wherein the variant comprises increased thermostability and cellulase activity compared to a wild-type cellobiohydrolase. For example, the disclosure provide polypeptides having increased thermostability and cellulase activity comprising a sequence that is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:2 comprising a C314S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:4 comprising a C311S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:12 comprising a C310S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:13 comprising a C312S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:14 comprising a C314S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:15 comprising a C315S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:16 comprising a C313S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:17 comprising a C311S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:19 comprising a C313S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:21 comprising a C312S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:22 comprising a C311S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:64 comprising a C400S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:65 comprising a C407S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:66 comprising a C394S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:67 comprising a C400S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:68 comprising a C400S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:69 comprising a C400S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:70 comprising a C400S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:71 comprising a C400S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:72 comprising a C400S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:73 comprising a C400S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:74 comprising a C400S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:75 comprising a C400S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:76 comprising a C407S; is at least 850, 900, 950, 980, 99% identical SEQ ID NO:77 comprising a C394S; or is at least 850, 900, 950, 980, 99% identical SEQ ID NO:78 comprising a C412S, wherein the foregoing polypeptides have cellulase activity and improved thermostability compared to their corresponding parental (wild-type) protein lacking a Cys.fwdarw.Ser mutation.

[0007] The disclosure also provides substantially purified polypeptides that are either recombinantly produced, synthetic made, or otherwise non-naturally generated wherein the polypeptide comprises a sequence as set forth below having from 1-10, 10-20 or 20-30 conservative amino acid substitutions except at the position identified below wherein a C.fwdarw.S substitution is present: SEQ ID NO:2 comprising a C314S; SEQ ID NO:4 comprising a C311S; SEQ ID NO:12 comprising a C310S; SEQ ID NO:13 comprising a C312S; SEQ ID NO:14 comprising a C314S; SEQ ID NO:15 comprising a C315S; SEQ ID NO:16 comprising a C313S; SEQ ID NO:17 comprising a C311S; SEQ ID NO:19 comprising a C313S; SEQ ID NO:21 comprising a C312S; SEQ ID NO:22 comprising a C311S; SEQ ID NO:64 comprising a C400S; SEQ ID NO:65 comprising a C407S; SEQ ID NO:66 comprising a C394S; SEQ ID NO:67 comprising a C400S; SEQ ID NO:68 comprising a C400S; SEQ ID NO:69 comprising a C400S; SEQ ID NO:70 comprising a C400S; SEQ ID NO:71 comprising a C400S; SEQ ID NO:72 comprising a C400S; SEQ ID NO:73 comprising a C400S; SEQ ID NO:74 comprising a C400S; SEQ ID NO:75 comprising a C400S; SEQ ID NO:76 comprising a C407S; SEQ ID NO:77 comprising a C394S; or SEQ ID NO:78 comprising a C412S.

[0008] The disclosure provides a recombinant polypeptide comprising a sequence selected from the group consisting of: (a) a polypeptide having at least 85% or greater identity to SEQ ID NO:2, having a Ser at position 314, and wherein the polypeptide has cellulase activity; (b) a polypeptide having at least 70% or greater identity to SEQ ID NO:4, having a Ser at position 311, and wherein the polypeptide has cellulase activity; (c) a polypeptide having 70% or greater identity to a sequence selected from the group consisting of: (i) SEQ ID NO:12 and having a Ser at position 310, (ii) SEQ ID NO:13 and having a Ser at position 312, (iii) SEQ ID NO:14 and having a Ser at position 314, (iv) SEQ ID NO:15 and having a Ser at position 315, (v) SEQ ID NO:16 and having a Ser at position 313, (vi) SEQ ID NO:17 and having a Ser at position 311, (vii) SEQ ID NO:19 and having a Ser at position 313, (viii) SEQ ID NO:21 and having a Ser at position 312, and (ix) SEQ ID NO:22 and having a Ser at position 311, and wherein each of the foregoing polypeptides has cellulase activity; and (d) a chimeric polypeptide comprising at least two domains from two different parental cellobiohydrolase polypeptides, wherein the domains comprise from N- to C-terminus: (segment 1)-(segment 2)-(segment 3)-(segment 4)-(segment 5)-(segment 6)-(segment 7)-(segment 8); wherein: segment 1 comprises a sequence that is at least 50-100% identity to amino acid residue from about 1 to about x.sub.1 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3"); segment 2 comprises a sequence that is at least 50-100% identity to amino acid residue x.sub.1 to about x.sub.2 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3"); segment 3 comprises a sequence that is at least 50-100% identity to amino acid residue x.sub.2 to about x.sub.3 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3"); segment 4 comprises a sequence that is at least 50-100% identity to amino acid residue x.sub.3 to about x.sub.4 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3"); segment 5 comprises a sequence that is at least 50-100% identity to about amino acid residue x.sub.4 to about x.sub.5 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3"); segment 6 comprises a sequence that is at least 50-100% identity to amino acid residue x.sub.5 to about x.sub.6 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3"); segment 7 comprises a sequence that is at least 50-100% identity to amino acid residue x.sub.6 to about x.sub.7 of SEQ ID NO:2 ("1") or SEQ ID NO:4 ("2"); and segment 8 comprises a sequence that is at least 50-100% identity to amino acid residue x.sub.7 to about x.sub.8 of SEQ ID NO:2 ("1") or SEQ ID NO:4 ("2"); wherein x.sub.1 is residue 43, 44, 45, 46, or 47 of SEQ ID NO:2, or residue 42, 43, 44, 45, or 46 of SEQ ID NO:4 or SEQ ID NO:6; x.sub.2 is residue 70, 71, 72, 73, or 74 of SEQ ID NO:2, or residue 68, 69, 70, 71, 72, 73, or 74 of SEQ ID NO:4 or SEQ ID NO:6; x.sub.3 is residue 113, 114, 115, 116, 117 or 118 of SEQ ID NO:2, or residue 110, 111, 112, 113, 114, 115, or 116 of SEQ ID NO:4 or SEQ ID NO:6; x.sub.4 is residue 153, 154, 155, 156, or 157 of SEQ ID NO:2, or residue 149, 150, 151, 152, 153, 154, 155 or 156 of SEQ ID NO:4 or SEQ ID NO:6; x.sub.5 is residue 220, 221, 222, 223, or 224 of SEQ ID NO:2, or residue 216, 217, 218, 219, 220, 221, 222 or 223 of SEQ ID NO:4 or SEQ ID NO:6; x.sub.6 is residue 256, 257, 258, 259, 260 or 261 of SEQ ID NO:2, or residue 253, 254, 255, 256, 257, 258, 259 or 260 of SEQ ID NO:4 or SEQ ID NO:6; x.sub.7 is residue 312, 313, 314, 315 or 316 of SEQ ID NO:2, or residue 309, 310, 311, 312, 313, 314, 315 or 318 of SEQ ID NO:4; and x.sub.8 is an amino acid residue corresponding to the C-terminus of the polypeptide have the sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, wherein the chimeric polypeptide comprises a Ser at position 314 of SEQ ID NO:2 or position 311 of SEQ ID NO:4 and wherein the chimeric polypeptide has cellulase activity and improved thermostability and/or pH stability compared to a CBH II polypeptide comprising SEQ ID NO:2, 4, or 6. In one embodiment of the recombinant polypeptide segment 1 comprises amino acid residue from about 1 to about x.sub.1 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3") and having 1-10 conservative amino acid substitutions; segment 2 is from about amino acid residue x.sub.1 to about x.sub.2 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3") and having about 1-10 conservative amino acid substitutions; segment 3 is from about amino acid residue x.sub.2 to about x.sub.3 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3") and having about 1-10 conservative amino acid substitutions; segment 4 is from about amino acid residue x.sub.3 to about x.sub.4 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3") and having about 1-10 conservative amino acid substitutions; segment 5 is from about amino acid residue x.sub.4 to about x.sub.5 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3") and having about 1-10 conservative amino acid substitutions; segment 6 is from about amino acid residue x.sub.5 to about x.sub.6 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3") and having about 1-10 conservative amino acid substitutions; segment 7 is from about amino acid residue x.sub.6 to about x.sub.7 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3") and having about 1-10 conservative amino acid substitutions; and segment 8 is from about amino acid residue x.sub.7 to about x.sub.8 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3") and having about 1-10 conservative amino acid substitutions except at position 314 of SEQ ID NO:2, position 311 or SEQ ID NO:4 or 313 of SEQ ID NO:6. In yet another embodiment, the chimeric polypeptide comprises a sequence that is at least 80%, 90%, 95%, 98% or 99% identical to a sequence selected from the group consisting of SEQ ID NO:12-62 and 63.

[0009] The disclosure also provides a recombinant polypeptide consisting of a sequence as set forth in SEQ ID NO:12-62 or 63.

[0010] The disclosure also provides a polynucleotide encoding any of the polypeptides as described above, vectors containing the polynucleotide and host cells containing the polynucleotide or vector.

[0011] The disclosure also provides an enzymatic preparation comprising a polypeptide of the disclosure in substantially purified form or as part of a cell lysate.

[0012] The disclosure also provides a method of treating a biomass comprising cellulose, the method comprising contacting the biomass with a polypeptide or enzymatic preparation of the disclosure.

BRIEF DESCRIPTION OF THE FIGURES

[0013] FIG. 1A-B SDS-PAGE gel of candidate CBH II parent gene yeast expression culture supernatants. (A) Gel Lanes (Left-to-Right): 1--H. jecorina, 2--Empty vector, 3--H. insolens, 4--C. thermophilum, 5--H. jecorina (duplicate), 6--P. chrysosporium, 7--T. emersonii, 8--Empty vector (duplicate), 9--H. jecorina (triplicate). Numbers at bottom of gel represent concentration of reducing sugar (ug/mL) present in reaction after 2-hr, 50.degree. C. PASC hydrolysis assay. Subsequent SDS-PAGE comparison with BSA standard allowed estimation of H. insolens expression level of 5-10 mg/L. (B) Shows SDS-PAGE gel analysis of S. cerevisiae CBH II expression culture supernatants. CBH II bands appear just below 60 kDa molecular weight standard. Lanes, left-to-right, 1--wild type H. jeco, 2--H. jeco B7P3, 3--H. jeco C311S, 4--wild type C. ther, 5--wild type H. inso, 6--H. inso B7P3, 7--H. inso C314S. Numbers denote .mu.g glucose equivalent/mL reaction volume per mL SDCAA expression culture supernatant equivalent produced during 100-minute incubation with PASC (1 mg/mL) at 50.degree. C. in 50 mM sodium acetate, pH 4.8. Values for lanes 1-4 have been divided by 2 to correct for twice the volume of concentrated culture supernatant being loaded where omitting this correction would make the specific activity values for the H. insolens enzymes appear artificially low.

[0014] FIG. 2A-C shows illustrations of CBH II chimera library block boundaries. (A) H. insolens CBH II catalytic domain ribbon diagram with blocks distinguished by color. CBH II enzyme is complexed with cellobio-derived isofagomine glycosidase inhibitor. (B) Linear representation of H. insolens catalytic domain showing secondary structure elements, disulfide bonds and block divisions denoted by black arrows. (C) Sidechain contact map denoting contacts (side chain heavy atoms within 4.5 .ANG.) that can be broken upon recombination. The majority of broken contacts occur between consecutive blocks.

[0015] FIG. 3 shows a number of broken contacts (E) and number of mutations from closest parent (m) for 23 secreted/active and 15 not secreted/not active sample set chimeras.

[0016] FIG. 4 shows specific activity, normalized to pH 5.0, as a function of pH for parent CBH II enzymes and three thermostable chimeras. Data presented are averages for two replicates, where error bars for HJPlus and H. jeco denote values for two independent trials. 16-hr reaction, 300 ug enzyme/g PASC, 50.degree. C., 12.5 mM sodium citrate/12.5 mM sodium phosphate buffer at pH as shown.

[0017] FIG. 5 shows long-time cellulose hydrolysis assay results (ug glucose reducing sugar equivalent/ug CBH II enzyme) for parents and thermostable chimeras across a range of temperatures. Error bars indicate standard errors for three replicates of HJPlus and H. insolens CBH II enzymes. 40-hr reaction, 100 ug enzyme/g PASC, 50 mM sodium acetate, pH 4.8.

[0018] FIG. 6 shows normalized residual activities for validation set chimeras after a 12-h incubation at 63.degree. C. Residual activities for CBH II enzymes in concentrated culture supernatants determined in 2-hr assay with PASC as substrate, 50.degree. C., 25 mM sodium acetate buffer, pH 4.8.

[0019] FIG. 7 Map for parent and chimera CBH II enzyme expression vector Yep352/PGK91-1-ss. Vector pictured contains wild type H. jecorina cel6a (CBH II enzyme) gene. For both chimeric and parent CBH II enzymes, the CBD/linker amino acid sequence following the ss Lys-Arg Kex.sub.2 site is:

TABLE-US-00001 (SEQ ID NO: 8) ASCSSVWGQCGGQNWSGPTCCASGSTCVYSNDYYSQCLPGAASSSSSTRA ASTTSRVSPTTSRSSSATPPPGSTTTRVPPVGSGTATYS.

[0020] FIG. 8 shows Observed and predicted T.sub.50 values for CBH II parents and 51 CBH II chimeras. Line denotes linear regression model equation (parameters in Table 7). Parent CBH II T.sub.50 values are denoted as squares.

[0021] FIG. 9A-C shows CBH II specific activities toward Avicel as a function of temperature. (a) CBH II parent and chimera specific activities. (b) CBH II parent, C311S mutant and B7P3 single block substitution chimera specific activities. Reactions were run for 16 hours in 50 mM sodium acetate, pH 4.8 with an Avicel concentration of 15 mg/mL. (c) CBH II parent, single point mutant and single block substitution chimera activities (.mu.g/glucose/mL reaction) toward avicel as a function of temperature. Reactions were run for 150 minutes in 50 mM sodium acetate, pH 4.8 with an avicel concentration of 15 mg/mL. CBH II yeast culture supernatants were dosed to achieve roughly equivalent reducing sugar product concentrations at 55.degree. C. Data presented are averages of two independent replicates with error bars indicating the duplicate activity values for each temperature point.

[0022] FIG. 10 shows ClustalW multiple sequence alignment for block 7 from parent 1, H. insolens and parent 3, C. thermophilum. Arrows denote residues changed in reversion mutants.

[0023] FIG. 11 shows T.sub.50 values for 21111331 chimera point mutants. Values shown as average of two independent duplicates, error bars indicate duplicate T.sub.50 values for each point mutant. Inactivation was carried out for 10 minutes at the temperature being tested in 50 mM sodium acetate buffer, pH 4.8. Residual activity was determined by incubation with 1 g/L phosphoric acid swollen cellulose (PASC) in above buffer for 100 minutes at 50.degree. C.

[0024] FIG. 12 shows T.sub.50 values for H. insolens and H. jecorina parent CBH IIs, Ser single point mutants and B7P3 block substitution chimeras. Values shown as average of three independent replicates, error bars indicate one standard deviation for each CBH II. Inactivation was carried out for 10 minutes at the temperature being tested in 50 mM sodium acetate buffer, pH 4.8. Residual activity was determined by incubation with 1 g/L phosphoric acid swollen cellulose (PASC) in above buffer for 100 minutes at 50.degree. C.

[0025] FIG. 13 shows T.sub.50 values for CBH II chimeras 31311112, 13231111 and the wild type CBH II catalytic domain from P. chrysosporium (fused to the H. jecorina CBM) and heterologously secreted from S. cerevisiae. Values shown as two independent replicates with error bars indicating values for each trial. Inactivation was carried out for 10 minutes at the temperature tested, in 50 mM sodium acetate buffer, pH 4.8. Residual activity was determined by incubation with 1 g/L phosphoric acid swollen cellulose (PASC) in above buffer for 100 minutes at 50.degree. C.

[0026] FIG. 14A-D shows CBH II recombination block interfaces. (a) Inter-block sites where novel non-parental residue pairs are possible (connected spheres) are usually surface-exposed, potentially allowing solvent to screen the interactions. (b) An example interface (B5-B6) illustrates conservation of the backbone (cartoons for aligned H. jecorina and H. insolens), variable residues on the surface, and the comparatively rare possibility of a novel buried hydrophobic pair at residues 173 and 253 (arrow). (c) Blocks 1-4 from H. jecorina (black cartoon) match cognate H. insolens blocks (color-coded cartoon) without large deviations, though movement associated with substrate binding is observed (arrow) in part of B3 (yellow). (d) Cognate blocks 5-8 are also similar, though the indel at the B6,B7 junction (arrow) will require conformational change.

[0027] FIG. 15 shows a structural analysis of C314S mutation and its stabilizing effect. (a) Hydrogen positions for high-resolution H. insolens structure (1ocn) were added with REDUCE.1 (b) The reconfigured geometry of the analogous serine structure was modeled in PyMOL (http:(//)www.pymol.org). Sidechain optimization in the SHARPEN2 modeling platform (with an all-atom Rosetta energy function) also suggested that both the Cys314 and Ser314 would donate hydrogen bonds to the carbonyl of Pro339, and accept hydrogen bonds from the amide of Gly316. The superior hydrogen bonding capacity of serine may play a role in the greater stability of the serine containing variants. Another possible explanation is geometric complementarity. Specifically, the Cys position from 1ocn shows evidence of conformational strain in that the sidechain is noticeably bent (i.e. the improper dihedral angle from N--C--C.alpha.--C.beta. is 6.degree. from the standard position), increasing the distance from the Pro carbonyl. Numbers in figure not preceded by letters denote hydrogen bond distances (.ANG.).

DETAILED DESCRIPTION

[0028] As used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a domain" includes a plurality of such domains and reference to "the protein" includes reference to one or more proteins, and so forth.

[0029] Also, the use of "or" means "and/or" unless stated otherwise. Similarly, "comprise," "comprises," "comprising" "include," "includes," and "including" are interchangeable and not intended to be limiting.

[0030] It is to be further understood that where descriptions of various embodiments use the term "comprising," those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language "consisting essentially of" or "consisting of."

[0031] Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein.

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Thus, as used throughout the instant application, the following terms shall have the following meanings.

[0033] Recent studies have documented the superior performance of cellulases from thermophilic fungi relative to their mesophilic counterparts in laboratory scale biomass conversion processes, where enhanced stability leads to retention of activity over longer periods of time at both moderate and elevated temperatures. Fungal cellulases are attractive because they are highly active and can be expressed in fungal hosts such as Hypocrea jecorina (anamorph Trichoderma reesei) at levels up to 40 g/L in the supernatant. Unfortunately, the set of documented thermostable fungal cellulases is small. In the case of the processive cellobiohydrolase class II (CBH II) enzymes, fewer than 10 natural thermostable gene sequences are annotated in the CAZy database.

[0034] As described more fully herein, using recursive chimeric polypeptide generation and analysis particular stabilizing domains and ultimately specific amino acid were identified the imparted thermostability and improved activity.

[0035] As will be described in more detail below, the invention is based, at least in part, on the generation and expression of novel enzymes that catalyze the hydrolysis of cellulose. In one embodiment, novel polypeptides that have been engineered to hydrolyze cellose at increased temperatures are provided. Such polypeptides include cellobiohydrolase variants that have been altered to include amino acid substitutions at specified residues. While these variants will be described in more detail below, it is understood that polypeptides of the disclosure may contain one or more modified amino acids. The presence of modified amino acids may be advantageous in, for example, (a) increasing a polypeptide's half-life, (b) thermostability, and (c) increased substrate turnover. Amino acid(s) are modified, for example, co-translationally or post-translationally during recombinant production (e.g., N-linked glycosylation at N--X--S/T motifs during expression in mammalian cells) or modified by synthetic means. Accordingly, a "mutant", "variant" or "modified" protein, enzyme, polynucleotide, gene, or cell, means a protein, enzyme, polynucleotide, gene, or cell, that has been altered or derived, or is in some way different or changed, from a parent protein, enzyme, polynucleotide, gene, or cell. A mutant or modified protein or enzyme is usually, although not necessarily, expressed from a mutant polynucleotide or gene.

[0036] A "mutation" means any process or mechanism resulting in a mutant protein, enzyme, polynucleotide, gene, or cell. This includes any mutation in which a protein, enzyme, polynucleotide, or gene sequence is altered, and any detectable change in a cell arising from such a mutation. Typically, a mutation occurs in a polynucleotide or gene sequence, by point mutations, deletions, or insertions of single or multiple nucleotide residues. A mutation includes polynucleotide alterations arising within a protein-encoding region of a gene as well as alterations in regions outside of a protein-encoding sequence, such as, but not limited to, regulatory or promoter sequences. A mutation in a gene can be "silent", i.e., not reflected in an amino acid alteration upon expression, leading to a "sequence-conservative" variant of the gene. This generally arises when one amino acid corresponds to more than one codon.

[0037] Non-limiting examples of a modified amino acid include a glycosylated amino acid, a sulfated amino acid, a prenlyated (e.g., farnesylated, geranylgeranylated) amino acid, an acetylated amino acid, an acylated amino acid, a pegylated amino acid, a biotinylated amino acid, a carboxylated amino acid, a phosphorylated amino acid, and the like. References adequate to guide one of skill in the modification of amino acids are replete throughout the literature. Example protocols are found in Walker (1998) Protein Protocols on CD-ROM (Humana Press, Towata, N.J.).

[0038] Recombinant methods for producing and isolating modified cellobiohydrolase polypeptides of the disclosure are described herein. In addition to recombinant production, the polypeptides may be produced by direct peptide synthesis using solid-phase techniques (e.g., Stewart et al. (1969) Solid-Phase Peptide Synthesis (WH Freeman Co, San Francisco); and Merrifield (1963) J. Am. Chem. Soc. 85: 2149-2154). Peptide synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City, Calif.) in accordance with the instructions provided by the manufacturer.

[0039] "Cellobiohydrolase II" or "CBH II enzyme" means an enzyme in the cellulase family 6 proteins, which are widely distributed in bacteria and fungi. The enzymes are involved in hydrolysis of cellulose.

[0040] By "cellulase activity" means an enzyme that is capable of hydrolyzing cellulose. Cellulase refers to a class of enzymes produced by fungi, bacteria, and protozoans that catalyze the hydrolysis of cellulose. However, there are also cellulases produced by other types of organisms such as plants and animals. The EC number for this group of enzymes is EC 3.2.1.4. There are five general types of cellulases based on the type of reaction catalyzed: endo-cellulase; exo-cellulase, within this category there are two main types of exo-cellulases (or cellobiohydrolases, abbreviate CBH)--one type working processively from the reducing end, and one type working processively from the non-reducing end of cellulose; cellobiase or beta-glucosidase hydrolyses; oxidative cellulases; and cellulose phosphorylases that depolymerize cellulose using phosphates instead of water. Most fungal cellulases have two-domains: a catalytic domain and a cellulose binding domain, that are connected by a flexible linker. In specific embodiments of the disclosure the cellulase activity is a CBH activity. The sequences described herein include, in some instances, both the cellulose binding domain and the catalytic domain or just the catalytic domain. In such instances where only the catalytic domain sequence is provided it will be recognized that a cellulose binding domain (CBD) such as that provided in SEQ ID NO:8, may be functional linked (either as part of the coding sequence or fused later) to the catalytic domain either directly or through a linker.

[0041] A "protein" or "polypeptide", which terms are used interchangeably herein, comprises one or more chains of chemical building blocks called amino acids that are linked together by chemical bonds called peptide bonds. An "enzyme" means any substance, preferably composed wholly or largely of protein, that catalyzes or promotes, more or less specifically, one or more chemical or biochemical reactions. A "native" or "wild-type" protein, enzyme, polynucleotide, gene, or cell, means a protein, enzyme, polynucleotide, gene, or cell that occurs in nature.

[0042] An "amino acid sequence" is a polymer of amino acids (a protein, polypeptide, etc.) or a character string representing an amino acid polymer, depending on context. The terms "protein," "polypeptide," and "peptide" are used interchangeably herein. "Amino acid" is a molecule having the structure wherein a central carbon atom is linked to a hydrogen atom, a carboxylic acid group (the carbon atom of which is referred to herein as a "carboxyl carbon atom"), an amino group (the nitrogen atom of which is referred to herein as an "amino nitrogen atom"), and a side chain group, R. When incorporated into a peptide, polypeptide, or protein, an amino acid loses one or more atoms of its amino acid carboxylic groups in the dehydration reaction that links one amino acid to another. As a result, when incorporated into a protein, an amino acid is referred to as an "amino acid residue."

[0043] A particular amino acid sequence of a given protein (i.e., the polypeptide's "primary structure," when written from the amino-terminus to carboxy-terminus) is determined by the nucleotide sequence of the coding portion of a mRNA, which is in turn specified by genetic information, typically genomic DNA (including organelle DNA, e.g., mitochondrial or chloroplast DNA). Thus, determining the sequence of a gene assists in predicting the primary sequence of a corresponding polypeptide and more particular the role or activity of the polypeptide or proteins encoded by that gene or polynucleotide sequence.

[0044] "Conservative amino acid substitution" or, simply, "conservative variations" of a particular sequence refers to the replacement of one amino acid, or series of amino acids, with essentially identical amino acid sequences. One of skill will recognize that individual substitutions, deletions or additions which alter, add or delete a single amino acid or a percentage of amino acids in an encoded sequence result in "conservative variations" where the alterations result in the deletion of an amino acid, addition of an amino acid, or substitution of an amino acid with a chemically similar amino acid.

[0045] Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, one conservative substitution group includes Alanine (A), Serine (S), and Threonine (T). Another conservative substitution group includes Aspartic acid (D) and Glutamic acid (E). Another conservative substitution group includes Asparagine (N) and Glutamine (Q). Yet another conservative substitution group includes Arginine (R) and Lysine (K). Another conservative substitution group includes Isoleucine, (I) Leucine (L), Methionine (M), and Valine (V). Another conservative substitution group includes Phenylalanine (F), Tyrosine (Y), and Tryptophan (W).

[0046] Thus, "conservative amino acid substitutions" of a listed polypeptide sequence (e.g., SEQ ID NOs: 2, 4, 6, and 12-78) include substitutions of a percentage, typically less than 10%, of the amino acids of the polypeptide sequence, with a conservatively selected amino acid of the same conservative substitution group. Accordingly, a conservatively substituted variation of a polypeptide of the disclosure can contain 100, 75, 50, 25, or 10 substitutions with a conservatively substituted variation of the same conservative substitution group.

[0047] It is understood that the addition of sequences which do not alter the encoded activity of a nucleic acid molecule, such as the addition of a non-functional or non-coding sequence, is a conservative variation of the basic nucleic acid. The "activity" of an enzyme is a measure of its ability to catalyze a reaction, i.e., to "function", and may be expressed as the rate at which the product of the reaction is produced. For example, enzyme activity can be represented as the amount of product produced per unit of time or per unit of enzyme (e.g., concentration or weight), or in terms of affinity or dissociation constants. As used interchangeably herein a "cellobiohydrolase activity or cellulase activity", "biological activity of cellobiohydrolase or cellulase" or "functional activity of cellobiohydrolase or cellulase", refers to an activity exerted by a protein, polypeptide having cellulase activity and in specific embodiments cellobiohydrolase activity on a cellulose substrate, as determined in vivo, or in vitro, according to standard techniques.

[0048] One of skill in the art will appreciate that many conservative variations of the nucleic acid constructs which are disclosed yield a functionally identical construct. For example, as discussed above, owing to the degeneracy of the genetic code, "silent substitutions" (i.e., substitutions in a nucleic acid sequence which do not result in an alteration in an encoded polypeptide) are an implied feature of every nucleic acid sequence which encodes an amino acid. Similarly, "conservative amino acid substitutions," in one or a few amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties, are also readily identified as being highly similar to a disclosed construct. Such conservative variations of each disclosed sequence are a feature of the polypeptides provided herein.

[0049] "Conservative variants" are proteins or enzymes in which a given amino acid residue has been changed without altering overall conformation and function of the protein or enzyme, including, but not limited to, replacement of an amino acid with one having similar properties, including polar or non-polar character, size, shape and charge. Amino acids other than those indicated as conserved may differ in a protein or enzyme so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and can be, for example, at least 30%, at least 50%, at least 70%, at least 80%, or at least 90%, as determined according to an alignment scheme. As referred to herein, "sequence similarity" means the extent to which nucleotide or protein sequences are related. The extent of similarity between two sequences can be based on percent sequence identity and/or conservation. "Sequence identity" herein means the extent to which two nucleotide or amino acid sequences are invariant. "Sequence alignment" means the process of lining up two or more sequences to achieve maximal levels of identity (and, in the case of amino acid sequences, conservation) for the purpose of assessing the degree of similarity. Numerous methods for aligning sequences and assessing similarity/identity are known in the art such as, for example, the Cluster Method, wherein similarity is based on the MEGALIGN algorithm, as well as BLASTN, BLASTP, and FASTA (Lipman and Pearson, 1985; Pearson and Lipman, 1988). When using all of these programs, the preferred settings are those that results in the highest sequence similarity.

[0050] Non-conservative modifications of a particular polypeptide are those which substitute any amino acid not characterized as a conservative substitution. For example, any substitution which crosses the bounds of the six groups set forth above. These include substitutions of basic or acidic amino acids for neutral amino acids, (e.g., Asp, Glu, Asn, or Gln for Val, Ile, Leu or Met), aromatic amino acid for basic or acidic amino acids (e.g., Phe, Tyr or Trp for Asp, Asn, Glu or Gln) or any other substitution not replacing an amino acid with a like amino acid. Basic side chains include lysine (K), arginine (R), histidine (H); acidic side chains include aspartic acid (D), glutamic acid (E); uncharged polar side chains include glycine (G), asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), cysteine (C); nonpolar side chains include alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W); beta-branched side chains include threonine (T), valine (V), isoleucine (I); aromatic side chains include tyrosine (Y), phenylalanine (F), tryptophan (W), histidine (H).

[0051] A "parent" protein, enzyme, polynucleotide, gene, or cell, is any protein, enzyme, polynucleotide, gene, or cell, from which any other protein, enzyme, polynucleotide, gene, or cell, is derived or made, using any methods, tools or techniques, and whether or not the parent is itself native or mutant. A parent polynucleotide or gene encodes for a parent protein or enzyme.

[0052] In addition to providing variants of CBH II polypeptides, chimeric polypeptides that comprise: 1) a variant domain isolated from a first parental strand and modified to include an amino acid substitution; and 2) a domain isolated from a second parental strand either unmodified or modified to include a new activity or an activity that a complements the domain, are provided. Methods for engineering a chimeric polypeptide of the disclosure are disclosed herein.

[0053] The disclosure provides cellulase and cellobiohydrolase (CBH) II variants, mutants and chimeras having increased thermostability compared to a wild-type or parental protein, wherein the wild-type protein consisting of SEQ ID NO:2, 4 or 6. The variant comprises a Serine in the C-terminal region in a motif comprising the sequence GEXDG, wherein X is C, A or G (SEQ ID NO:107), wherein X is substituted with Serine, the variant comprising cellulase activity and wherein the polypeptide has increased thermostability compared to a wild-type cellulase lacking a serine in the sequence GEXDG (SEQ ID NO:107). In one embodiment, the variants comprise at least a mutation of a Cys-Ser in the motif GECDG (see, e.g., SEQ ID NO:2 from amino acid 312-316) found in most cellulase and cellobiohydrolase II proteins (as described more fully below) and may comprise additional mutations that improve thermostability or activity. The identity between cellulases can be quite low. The serine substitution as described above is applicable to any cellulase having the motif of SEQ ID NO:107 (e.g., wherein the polypeptide has at least 60% or greater identity to SEQ ID NO:2 or 4).

[0054] For example, the disclosure provide polypeptides having increased thermostability and cellulase activity comprising a sequence that is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:2 comprising a C314S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:4 comprising a C311S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:12 comprising a C310S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:13 comprising a C312S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:14 comprising a C314S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:15 comprising a C315S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:16 comprising a C313S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:17 comprising a C311S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:19 comprising a C313S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:21 comprising a C312S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:22 comprising a C311S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:64 comprising a C400S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:65 comprising a C407S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:66 comprising a C394S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:67 comprising a C400S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:68 comprising a C400S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:69 comprising a C400S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:70 comprising a C400S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:71 comprising a C400S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:72 comprising a C400S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:73 comprising a C400S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:74 comprising a C400S; is at least 85%, 90%, 95%, 98%, 99% identical SEQ ID NO:75 comprising a C400S; is at least 85%, 90%, 950, 980, 99% identical SEQ ID NO:76 comprising a C407S; is at least 850, 900, 950, 980, 99% identical SEQ ID NO:77 comprising a C394S; or is at least 850, 900, 950, 980, 99% identical SEQ ID NO:78 comprising a C412S, wherein the foregoing polypeptides have cellulase activity and improved thermostability compared to their corresponding parental (wild-type) protein lacking a Cys.fwdarw.Ser mutation.

[0055] In yet another embodiment, the disclosure provides polypeptide as described above, however, they further comprise at least one addition mutation that can be determined by alignment to SEQ ID NO:64, wherein SEQ ID NO:64 comprises a Pro at position 413, or a Ser or Thr at position 231, or a Ser or Thr at position 305, or a Gln or Asn at position 410, or a Glu at position 82, or any combination of the foregoing. Similar substitutions can be identified by sequence alignment of the amino acid sequence of SEQ ID NO:64 with those of SEQ ID NOs:2, 4, 6, 12-63, and 65-78.

[0056] The disclosure also provides substantially purified polypeptides that are either recombinantly produced, synthetic made, or otherwise non-naturally generated wherein the polypeptide comprise a sequence as set forth below having from 1-10, 10-20 or 20-30 conservative amino acid substitutions except at the position identified below wherein a C.fwdarw.S substitution is present:

SEQ ID NO:2 comprising a C314S; SEQ ID NO:4 comprising a C311S; SEQ ID NO:12 comprising a C310S; SEQ ID NO:13 comprising a C312S; SEQ ID NO:14 comprising a C314S; SEQ ID NO:15 comprising a C315S; SEQ ID NO:16 comprising a C313S; SEQ ID NO:17 comprising a C311S; SEQ ID NO:19 comprising a C313S; SEQ ID NO:21 comprising a C312S; SEQ ID NO:22 comprising a C311S; SEQ ID NO:64 comprising a C400S; SEQ ID NO:65 comprising a C407S; SEQ ID NO:66 comprising a C394S; SEQ ID NO:67 comprising a C400S; SEQ ID NO:68 comprising a C400S; SEQ ID NO:69 comprising a C400S; SEQ ID NO:70 comprising a C400S; SEQ ID NO:71 comprising a C400S; SEQ ID NO:72 comprising a C400S; SEQ ID NO:73 comprising a C400S; SEQ ID NO:74 comprising a C400S; SEQ ID NO:75 comprising a C400S; SEQ ID NO:76 comprising a C407S; SEQ ID NO:77 comprising a C394S; or SEQ ID NO:78 comprising a C412S.

[0057] "Isolated polypeptide" refers to a polypeptide which is separated from other contaminants that naturally accompany it, e.g., protein, lipids, and polynucleotides. The term embraces polypeptides which have been removed or purified from their naturally-occurring environment or expression system (e.g., host cell or in vitro synthesis).

[0058] "Substantially pure polypeptide" refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. Generally, a substantially pure polypeptide composition will comprise about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition. In some embodiments, the object species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species.

[0059] "Reference sequence" refers to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence can be at least 20 nucleotide or amino acid residues in length, at least 25 nucleotide or residues in length, at least 50 nucleotides or residues in length, or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing sequences of the two polynucleotides or polypeptides over a "comparison window" to identify and compare local regions of sequence similarity.

[0060] "Sequence identity" means that two amino acid sequences are substantially identical (i.e., on an amino acid-by-amino acid basis) over a window of comparison. The term "sequence similarity" refers to similar amino acids that share the same biophysical characteristics. The term "percentage of sequence identity" or "percentage of sequence similarity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical residues (or similar residues) occur in both polypeptide sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity (or percentage of sequence similarity). With regard to polynucleotide sequences, the terms sequence identity and sequence similarity have comparable meaning as described for protein sequences, with the term "percentage of sequence identity" indicating that two polynucleotide sequences are identical (on a nucleotide-by-nucleotide basis) over a window of comparison. As such, a percentage of polynucleotide sequence identity (or percentage of polynucleotide sequence similarity, e.g., for silent substitutions or other substitutions, based upon the analysis algorithm) also can be calculated. Maximum correspondence can be determined by using one of the sequence algorithms described herein (or other algorithms available to those of ordinary skill in the art) or by visual inspection.

[0061] As applied to polypeptides, the term substantial identity or substantial similarity means that two peptide sequences, when optimally aligned, such as by the programs BLAST, GAP or BESTFIT using default gap weights or by visual inspection, share sequence identity or sequence similarity. Similarly, as applied in the context of two nucleic acids, the term substantial identity or substantial similarity means that the two nucleic acid sequences, when optimally aligned, such as by the programs BLAST, GAP or BESTFIT using default gap weights (described elsewhere herein) or by visual inspection, share sequence identity or sequence similarity.

[0062] One example of an algorithm that is suitable for determining percent sequence identity or sequence similarity is the FASTA algorithm, which is described in Pearson, W. R. & Lipman, D. J., (1988) Proc. Natl. Acad. Sci. USA 85:2444. See also, W. R. Pearson, (1996) Methods Enzymology 266:227-258. Preferred parameters used in a FASTA alignment of DNA sequences to calculate percent identity or percent similarity are optimized, BL50 Matrix 15: -5, k-tuple=2; joining penalty=40, optimization=28; gap penalty -12, gap length penalty=-2; and width=16.

[0063] Another example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity or percent sequence similarity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, (1987) J. Mol. Evol. 35:351-360. The method used is similar to the method described by Higgins & Sharp, CABIOS 5:151-153, 1989. The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. Using PILEUP, a reference sequence is compared to other test sequences to determine the percent sequence identity (or percent sequence similarity) relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al., (1984) Nuc. Acids Res. 12:387-395).

[0064] Another example of an algorithm that is suitable for multiple DNA and amino acid sequence alignments is the CLUSTALW program (Thompson, J. D. et al., (1994) Nuc. Acids Res. 22:4673-4680). CLUSTALW performs multiple pairwise comparisons between groups of sequences and assembles them into a multiple alignment based on sequence identity. Gap open and Gap extension penalties were 10 and 0.05 respectively. For amino acid alignments, the BLOSUM algorithm can be used as a protein weight matrix (Henikoff and Henikoff, (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919).

[0065] As mentioned above, cellobiohydrolase and cellulase family members can be identified by sequence alignment and a substitution in the motif GECDG (see, e.g., SEQ ID NO:2 from amino acid 312-316) made. The modified polypeptide may then be assayed for activity as described below at various temperatures and conditions to identify those modifications that introduce a favorable activity. Exemplary sequences can be found in the following GenBank accession numbers, the sequences of which are incorporated herein by reference.

TABLE-US-00002 P07987 Q6E5B1 GUX2_TRIRE Q6E5B1_9AGAR Q9NEY8 B7X9Z0 Q9HEY8_TRIRE B7X9Z0_COPCI Q7LSP2 A8NEJ3 Q7LSP2_TRIKO A8NEJ3_COPC7 Q6UJX9 Q96V98 Q6UJX9_TRIVI Q96V98_ORPSP A3QVU7 Q7Z7X6 A3QVU7_TRIVI Q727X6_PIREQ 1HCL5 Q870B2 Q1HCL5_TRIKO Q870B2_9FUNG Q66PN1 Q874E1 Q66PN1_9HYPO Q874E1_ORPSP B5TWC7 A9FHT2 B5TWC7_9HYPO A9FHT2_SORC5 Q9C1S9 BOFEV9 GUX6_HUMIN BOFEV9_9FUNG Q2GMP2 Q6EY63 Q2GMP2_CHAGB Q6EY63_9FUNG A7E6G7 Q6EH22 A7E6G7_SCLS1 Q6EH22_NEOFR Q0UPA5 B6EA50 Q0UPA5_PHANO B6EA50_NEOPA A6S7A6 B0FEV4 A6S7A6_BOTFB B0FEV4_NEOPA P49075 6EIY8 GUX3_AGABI Q6EIY8_NEOFR Q02321 Q9UW10 Q02321_PHACH Q9UW10_9FUNG Q9C1R4 Q12646 Q9C1R4_LENED Q12646_NEOPA Q96VU2 Q6A4K7 Q96VU2_LENED Q6A4K7_9FUNG B2ABX7 Q9UW11 B2ABX7_PODAN Q9UW11_9FUNG A4RPH6 Q9P8Q8 A4RPH6_MAGGR Q9P8Q8_9FUNG B0FEV8 B0FEV8_9FUNG

[0066] In yet other embodiments, the family of variant cellulase polypeptide having improved thermostability include those set forth in the following table having a C.fwdarw.S, G.fwdarw.S or A.fwdarw.S substitution. In addition, polypeptides having 85%, 90%, 95%, 98%, or 99% sequence identity to any of the following sequences having the identified substitutions in the following table, having cellulase activity and thermostability are also encompassed by the disclosure.

[0067] Alignment of amino acid frame bracketing H. jecorina CBH II Cys311 for protein sequences having highest identity to H. jecorina CBH II. Residues at 311 equivalent position denoted by bold, underline are shown. Sequences for recombinant H. insolens and P. chrys CBH IIs studied in this work are denoted as H inso and P. chrys. Fifty-four of the 250 most identical sequences were excluded due to redundancy (i.e. point mutants for structural studies or >95% identical isoforms). The accession number for the cellulase is identified and the corresponding sequence is incorporated herein by reference as if copied directly from the accession number. The sequences associated with the accession numbers are referred to as SEQ ID NO:79-106. A replacement of the bold-underlined residue (e.g., C, A or G) with S. The number in parenthesis following the sequence identified the SEQ ID NO:)

TABLE-US-00003 H.jeco ----T---G---D----S---L--LDSFVWVKPGGECDG--T----S-------------(4) XP_001903170 ----T---G---L----D---I--EDAFVWIKPGGECDG--T----S-------------(79) XP_001226029 ----T---G---H----D---L--LDAFVWIKPGGECDG--T----S-------------(80) XP_360146 ----T---G---S----E---L--ADAFVWIKPGGECDG--V----S-------------(81) H.inso ----T---G---H----Q---Y--VDAFVWVKPGGECDG--T----S-------------(2) XP_001598803 ----T---G---D----A---L--EDAFVWVKPGGEADG--T----S-------------(82) XP_001796781 ----T---D---D----P---L--LDAYVWVKPGGEGDG--T----S-------------(83) AAA50608 ----T---G---S----S---L--IDAIVWVKPGGECDG--T----S-------------(84) AAK28357 ----T---G---S----S---L--IDSIVWVKPGGECDG--T----S-------------(85) BAH59082 ----T---G---S----P---L--IDSIVWVKPGGECDG--T----S-------------(86) AAT64008 ----T---G---S----S---L--IDAIVWIKPGGECDG--T----T-------------(87) P.crys ----T---G---S----Q---F--IDSIVWVKPGGECDG--T----S-------------(12) BAH59083 ----T---P---S----S---L--IDSIVWVKPGGEADG--T----S-------------(88) XP_001833045 ----T---P---S----S---A--IDAIVWIKPGGEADG--T----S-------------(89) XP_002391276 ----T---G---S----S---L--IDSIVWVKPGGE------------------------(90) AAD51055 ----P---D---S----SKP-L--LDAYMWIKTPGEADG--S----S-------------(91) ABY52798 ----S---G---Y----P---L--LDAFMWLKTPGEADG--S----A-------------(92) AAF34679 ----P---D---ASMP-L---L--LDAYMWLKTPGEADG--S----A-------------(93) ABY52797 ----P---S---K----P---L--LDAYMWIKTPGEADG--S----S-------------(94) AAR08200 ----PNP-G---M----P---L--LDAYMWLKTPGEADG--S----S-------------(95) AAB92678 ----P---N---P----GSMPL--LDAYMWIKTPGEADG--S----S-------------(96) ABY52799 ----S---P---DPEKFP---L--LDAYFWLKPPGEADG--S----D-------------(97) AA060491 ----T---G---D----A---N--IDAYLWVKPPGEADG---------------------(98) AA009068 ----V---K---M----P---L--LDAYMWLKTPGEADG--S----D-------------(99) ZP_04371095 ----T---G---D----A---A--VDAFLWIKPPGEADG--C----A-------------(100) ZP_03818362 ----T---G---D----S---Q--IDAFLWVKIVGEADG---------------------(101) ZP_03817628 ----T---G---D----P---Q--IDAFLWVKIPGEADG---------------------(102) ZP_04331392 ----T---G---N----P---L--IDAFIWTKLPGEADG---------------------(103) 2BOE-X ----T---G---D----P---M--IDAFLWIKLPGEADG---------------------(104) ZP_04608509 ----T---G---D----S---A--IAAYLWVKLPGEADG---------------------(105) P26414 ----T---G---D----P---A--IDAFLWIKPPGEADG---------------------(106)

[0068] For the purposes of the disclosure, a polypeptide of the disclosure exhibits improved thermostability with respect to a corresponding parent polypeptide if it has a T.sub.50 which is at least about 4.degree. C., or at least about 9.degree. C. higher than that of the parent cellulase, or for example a cellobiohydrolase having a T.sub.50 from about 4.degree. C. to about 30.degree. C. higher, or any amount therebetween, or a T.sub.50 from about 9.degree. C. to about 30.degree. C. higher, or any amount therebetween, when compared to that of the parent cellobiohydrolase. The T.sub.50 is the temperature at which the modified or the natural enzyme retains 50% of its residual activity after a pre-incubation for 15 minutes and is determined by the assay detailed in Examples below or as known in the art.

[0069] The modified cellobiohydrolases or cellulases of the disclosure may have T.sub.50 which is about 4.degree. C. to about 30.degree. C. higher than that of a corresponding parent cellobiohydrolase (e.g., SEQ ID NO:2, 4 or 6), or any range therebetween, about 5.degree. C. to about 20.degree. C. higher, or any range therebetween, about 8.degree. C. to about 15.degree. C. higher, or any range therebetween, or from about 9.degree. C. to about 15.degree. C. higher, or any range therebetween. For example, the modified cellulase may have a T.sub.50 that is at least about 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30.degree. C. higher than that of the corresponding parent cellobiohydrolase.

[0070] The variants identified herein can also be used to generate chimeric cellobiohydrolases. For example, SCHEMA has been used previously to create families of hundreds of active .beta.-lactamase and cytochrome P450 enzyme chimeras. SCHEMA uses protein structure data to define boundaries of contiguous amino acid "blocks" which minimize <E>, the library average number of amino acid sidechain contacts that are broken when the blocks are swapped among different parents. It has been shown that the probability that a .beta.-lactamase chimera was folded and active was inversely related to the value of E for that sequence. The RASPP (Recombination as Shortest Path Problem) algorithm was used to identify the block boundaries that minimized <E> relative to the library average number of mutations, <m>. More than 20% of the .about.500 unique chimeras characterized from a .beta.-lactamase collection comprised of 8 blocks from 3 parents (3.sup.8=6,561 possible sequences) were catalytically active. A similar approach produced a 3-parent, 8-block cytochrome P450 chimera family containing more than 2,300 novel, catalytically active enzymes. Chimeras from these two collections were characterized by high numbers of mutations, 66 and 72 amino acids on average from the closest parent, respectively. SCHEMA/RASPP thus enabled design of chimera families having significant sequence diversity and an appreciable fraction of functional members.

[0071] It has also been shown that the thermostabilities of SCHEMA chimeras can be predicted based on sequence-stability data from a small sample of the sequences. Linear regression modeling of thermal inactivation data for 184 cytochrome P450 chimeras showed that SCHEMA blocks made additive contributions to thermostability. More than 300 chimeras were predicted to be thermostable by this model, and all 44 that were tested were more stable than the most stable parent. It was estimated that as few as 35 thermostability measurements could be used to predict the most thermostable chimeras. Furthermore, the thermostable P450 chimeras displayed unique activity and specificity profiles, demonstrating that chimeragenesis can lead to additional useful enzyme properties. Here SCHEMA recombination of CBH II enzymes can generate chimeric cellulases that are active on phosphoric acid swollen cellulose (PASC) at high temperatures, over extended periods of time, and broad ranges of pH.

[0072] Using the methods described herein a number of chimeric polypeptides having cellobiohydrolases activity were generated having improved characteristics compared to the wild-type parental CBH II proteins.

[0073] A diverse family of novel CBH II enzymes was constructed by swapping blocks of sequence from three fungal CBH II enzymes. Twenty-three of 48 chimeric sequences sampled from this set were secreted in active form by S. cerevisiae, and five have half-lives at 63.degree. C. that were greater than the most stable parent. Given that this 48-member sample set represents less than 1% of the total possible 6,561 sequences, we predict that this one collection of chimeras already contains hundreds of active, thermostable CBH II enzymes, a number that dwarfs the approximately twenty fungal CBH II enzymes in the CAZy database.

[0074] The approach of using the sample set sequence-stability data to identify blocks that contribute positively to chimera thermostability was validated by finding that all 10 catalytically active chimeras in the second CBH II validation set were more thermostable than the most stable parent, a naturally-thermostable CBH II from the thermophilic fungus, H. insolens. This disclosure has thus far generated a total of 33 new CBH II enzymes that are expressed in catalytically active form in S. cerevisiae, 15 of which are more thermostable than the most stable parent from which they were constructed. These 15 thermostable enzymes are diverse in sequence, differing from each other and their closest natural homologs at as many as 94 and 58 amino acid positions, respectively.

[0075] Analysis of the thermostabilities of CBH II chimeras in the combined sample and validation sets indicates that the four thermostabilizing blocks identified, B1P1, B6P3, B7P3 and B8P2, make cumulative contributions to thermal stability when present in the same chimera. Four of the five sample set chimeras that are more thermostable than the H. insolens CBH II contain either two or three of these stabilizing blocks (Table 1). The ten active members of the validation set, all of which are more stable than the H. insolens enzyme, contain at least two stabilizing blocks, with five of the six most thermostable chimeras in this group containing either three or four stabilizing blocks.

[0076] The disclosure demonstrates that stabilizing blocks can be recombined to create novel highly stable, active cellulases. The stability regression model predicts that the CBH II SCHEMA library contains 2,026 chimeras that are more stable than the most stable parent enzyme. These chimeras are diverse and distinct from the native cellulases: they differ from the parents by between 8 and 72 mutations (an average of 50) and from each other by an average of 63 mutations. A total of 33 genes from this set were synthesized and expressed in S. cerevisiae: every one of these chimeric CBH IIs was found to be more stable than the most stable parent cellulase, from the thermophilic fungus H. insolens, as measured either by its half-life of inactivation at 63.degree. C. or T.sub.50. Reducing the sequence complexity by making chimeras of only eight blocks allowed the generation of a sequence-stability model and identification of a single highly stabilizing sequence block. By testing only ten amino acid substitutions in this block a single, highly stabilizing substitution was identified. The very large stabilizing effect of the C313S (with reference to SEQ ID NO:6; C314S, SEQ ID NO:2 and C311S, SEQ ID NO:4) substitution observed across the chimeras and in the native P. chrysosporium, H. insolens and H. jecorina CBH II enzymes suggests that mutation of any residue at this position to Ser may stabilize any family 6 cellulase into which it is introduced.

[0077] Minimizing the number of broken contacts upon recombination (FIG. 2C) allows the blocks to be approximated as decoupled units that make independent contributions to the stability of the entire protein, thus leading to cumulative or even additive contributions to chimera thermostability. For this CBH II enzyme recombination, SCHEMA was effective in minimizing such broken contacts: whereas there are 303 total interblock contacts defined in the H. insolens parent CBH II crystal structure, the CBH II SCHEMA library design results in only 33 potential broken contacts. Given that the CBH II enzyme parents do not feature obvious structural subdomains, and only four of the eight blocks (1, 5, 7 and 8) resemble compact structural units, or modules, the low number of broken contacts demonstrates that the SCHEMA/RASPP algorithm is effective for cases in which the number of blocks appears greater than the number of structural subdivisions. As previously observed for .beta.-lactamase and cytochrome P450 chimeras, low E values were predictive of chimera folding and activity. Although not used here, this relationship should be valuable for designing chimera sample sets that contain a high fraction of active members.

[0078] The disclosure also used chimera to determine if the pH stability could be improved in CBH II enzymes. Whereas the specific activity of H. jecorina CBH II declines sharply as pH increases above the optimum value of 5, HJPlus, created by substituting stabilizing blocks onto the most industrially relevant H. jecorina CBH II enzyme, retains significantly more activity at these higher pHs (FIG. 4). The thermostable 11113132 and 13311332 chimeras, and also the H. insolens and C. thermophilum CBH II cellulase parents, have even broader pH/activity profiles than HJPlus. The narrow pH/activity profile of H. jecorina CBH II has been attributed to the deprotonation of several carboxyl-carboxylate pairs, which destabilizes the protein above pH .about.6. The substitution of parent 3 in block 7 in HJPlus changes aspartate 277 to histidine, eliminating the carboxyl-carboxylate pair between D277 and D316 (of block 8). Replacing D277 with the positively charged histidine may prevent destabilizing charge repulsion at nonacidic pH, allowing HJPlus to retain activity at higher pH than H. jecorina CBH II. The even broader pH/activity profiles of the remaining two thermostable chimeras and the H. insolens and C. thermophilum parent CBH II enzymes may be due to the absence of acidic residues at positions corresponding to the E57-E119 carboxyl-carboxylate pair of HJPlus and H. jecorina CBH II.

[0079] HJPlus exhibits both relatively high specific activity and high thermostability. FIG. 5 shows that these properties lead to good performance in long-time hydrolysis experiments: HJPlus hydrolyzed cellulose at temperatures 7-15.degree. C. higher than the parent CBH II enzymes and also had a significantly increased long-time activity relative to all the parents at their temperature optima, bettering H. jecorina CBH II by a factor of 1.7. Given that the specific activity of the HJPlus chimera is less than that of the H. jecorina CBH II parent, this increased long-time activity can be attributed to the ability of the thermostable HJPlus to retain activity at optimal hydrolysis temperatures over longer reaction timer.

[0080] The other two thermostable chimeras shared HJPlus's broad temperature range. This observation supports a positive correlation between t.sub.1/2 at elevated temperature and maximum operating temperature, and suggests that many of the thermostable chimeras among the 6,561 CBH II chimera sequences will also be capable of degrading cellulose at elevated temperatures. While this ability to hydrolyze the amorphous PASC substrate at elevated temperatures bodes well for the potential utility of thermostable fungal CBH II chimeras, studies with more challenging crystalline substrates and substrates containing lignin will provide a more complete assessment of this novel CBH II enzyme family's relevance to biomass degradation applications.

[0081] The majority of biomass conversion processes use mixtures of fungal cellulases (primarily CBH II, cellobiohydrolase class I (CBH I), endoglucanases and .beta.-glucosidase) to achieve high levels of cellulose hydrolysis. Generating a diverse group of thermostable CBH II enzyme chimeras is the first step in building an inventory of stable, highly active cellulases from which enzyme mixtures can be formulated and optimized for specific applications and feedstocks.

[0082] "Peptide segment" refers to a portion or fragment of a larger polypeptide or protein. A peptide segment need not on its own have functional activity, although in some instances, a peptide segment may correspond to a domain of a polypeptide wherein the domain has its own biological activity. A stability-associated peptide segment is a peptide segment found in a polypeptide that promotes stability, function, or folding compared to a related polypeptide lacking the peptide segment. A destabilizing-associated peptide segment is a peptide segment that is identified as causing a loss of stability, function or folding when present in a polypeptide.

[0083] "Fused," "operably linked," and "operably associated" are used interchangeably herein to broadly refer to a chemical or physical coupling of two otherwise distinct domains or peptide segments, wherein each domain or peptide segment when operably linked can provide a functional polypeptide having a desired activity. Domains or peptide segments can be connected through peptide linkers such that they are functional or can be fused through other intermediates or chemical bonds. For example, two domains can be part of the same coding sequence, wherein the polynucleotides are in frame such that the polynucleotide when transcribed encodes a single mRNA that when translated comprises both domains as a single polypeptide. Alternatively, both domains can be separately expressed as individual polypeptides and fused to one another using chemical methods. Typically, the coding domains will be linked "in-frame" either directly of separated by a peptide linker and encoded by a single polynucleotide. Various coding sequences for peptide linkers and peptide are known in the art.

[0084] "Polynucleotide" or "nucleic acid sequence" refers to a polymeric form of nucleotides. In some instances a polynucleotide refers to a sequence that is not immediately contiguous with either of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA) independent of other sequences. The nucleotides of the disclosure can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. A polynucleotides as used herein refers to, among others, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. The term polynucleotide encompasses genomic DNA or RNA (depending upon the organism, i.e., RNA genome of viruses), as well as mRNA encoded by the genomic DNA, and cDNA.

[0085] "Nucleic acid segment," "oligonucleotide segment" or "polynucleotide segment" refers to a portion of a larger polynucleotide molecule. The polynucleotide segment need not correspond to an encoded functional domain of a protein; however, in some instances the segment will encode a functional domain of a protein. A polynucleotide segment can be about 6 nucleotides or more in length (e.g., 6-20, 20-50, 50-100, 100-200, 200-300, 300-400 or more nucleotides in length). A stability-associated peptide segment can be encoded by a stability-associated polynucleotide segment, wherein the peptide segment promotes stability, function, or folding compared to a polypeptide lacking the peptide segment.

[0086] "Chimera" refers to a combination of at least two segments of at least two different parent proteins. As appreciated by one of skill in the art, the segments need not actually come from each of the parents, as it is the particular sequence that is relevant, and not the physical nucleic acids themselves. For example, a chimeric fungal class II cellobiohydrolases (CBH II cellulases) will have at least two segments from two different parent CBH II polypeptides. The two segments are connected so as to result in a new polypeptide having cellulase activity. In other words, a protein will not be a chimera if it has the identical sequence of either one of the full length parents. A chimeric polypeptide can comprise more than two segments from two different parent proteins. For example, there may be 2, 3, 4, 5-10, 10-20, or more parents for each final chimera or library of chimeras. The segment of each parent polypeptide can be very short or very long, the segments can range in length of contiguous amino acids from 1 to 90%, 95%, 98%, or 99% of the entire length of the protein. In one embodiment, the minimum length is 10 amino acids. In one embodiment, a single crossover point is defined for two parents. The crossover location defines where one parent's amino acid segment will stop and where the next parent's amino acid segment will start. Thus, a simple chimera would only have one crossover location where the segment before that crossover location would belong to one parent and the segment after that crossover location would belong to the second parent. In one embodiment, the chimera has more than one crossover location. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11-30, or more crossover locations. How these crossover locations are named and defined are both discussed below. In an embodiment where there are two crossover locations and two parents, there will be a first contiguous segment from a first parent, followed by a second contiguous segment from a second parent, followed by a third contiguous segment from the first parent. Contiguous is meant to denote that there is nothing of significance interrupting the segments. These contiguous segments are connected to form a contiguous amino acid sequence. For example, a CBH II chimera from Humicola insolens (hereinafter "1") and H. jecorina (hereinafter "2"), with two crossovers at 100 and 150, could have the first 100 amino acids from 1, followed by the next 50 from 2, followed by the remainder of the amino acids from 1, all connected in one contiguous amino acid chain. Alternatively, the CBH II chimera could have the first 100 amino acids from 2, the next 50 from 1 and the remainder followed by 2. As appreciated by one of skill in the art, variants of chimeras exist as well as the exact sequences. Thus, not 100% of each segment need be present in the final chimera if it is a variant chimera. The amount that may be altered, either through additional residues or removal or alteration of residues will be defined as the term variant is defined. Of course, as understood by one of skill in the art, the above discussion applies not only to amino acids but also nucleic acids which encode for the amino acids.

[0087] The disclosure describes in addition to specific variants, variants that can be used to generate CBH II chimeras. A directed SCHEMA recombination library was used to generate cellobiohydrolase enzymes based on a particularly well-studied member of this diverse enzyme family, and more particularly cellobiohydrolase II enzymes: H. insolens is parent "1" (SEQ ID NO:2), H. jecorina is parent "2" (SEQ ID NO:4) and C. thermophilum is parent "3" (SEQ ID NO:6). SCHEMA is a computational based method for predicting which fragments of homologous proteins can be recombined without affecting the structural integrity of the protein (see, e.g., Meyer et al., (2003) Protein Sci., 12:1686-1693). This computational approached identified seven recombination points in the CBH II parental proteins, thereby allowing the formation of a library of CBH II chimera polypeptides, where each polypeptide comprise eight segments. Chimeras with higher stability are identifiable by determining the additive contribution of each segment to the overall stability, either by use of linear regression of sequence-stability data, or by reliance on consensus analysis of the MSAs of folded versus unfolded proteins. SCHEMA recombination ensures that the chimeras retain biological function and exhibit high sequence diversity by conserving important functional residues while exchanging tolerant ones.

[0088] Thus, as illustrated by various embodiments herein, the disclosure provides CBH II polypeptides comprising a chimera of parental domains of which a parental strand or the resulting chimeric coding sequence may be modified to comprise a C.fwdarw.S substitution as described above. In some embodiments, the polypeptide comprises a chimera having a plurality of domains from N- to C-terminus from different parental CBH II proteins: (segment 1)-(segment 2)-(segment 3)-(segment 4)-(segment 5)-(segment 6)-(segment 7)-(segment 8);

[0089] wherein segment 1 comprises amino acid residue from about 1 to about x.sub.1 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3"): segment 2 is from about amino acid residue x.sub.1 to about x.sub.2 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3"); segment 3 is from about amino acid residue x.sub.2 to about x.sub.3 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3"); segment 4 is from about amino acid residue x.sub.3 to about x.sub.4 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3"); segment 5 is from about amino acid residue x.sub.4 to about x.sub.5 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3"); segment 6 is from about amino acid residue x.sub.5 to about x.sub.6 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3"); segment 7 is from about amino acid residue x.sub.6 to about x.sub.7 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3"); and segment 8 is from about amino acid residue x.sub.7 to about x.sub.8 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3");

[0090] wherein: x.sub.1 is residue 43, 44, 45, 46, or 47 of SEQ ID NO:2, or residue 42, 43, 44, 45, or 46 of SEQ ID NO:4 or SEQ ID NO:6; x.sub.2 is residue 70, 71, 72, 73, or 74 of SEQ ID NO:2, or residue 68, 69, 70, 71, 72, 73, or 74 of SEQ ID NO:4 or SEQ ID NO:6; x.sub.3 is residue 113, 114, 115, 116, 117 or 118 of SEQ ID NO:2, or residue 110, 111, 112, 113, 114, 115, or 116 of SEQ ID NO:4 or SEQ ID NO:6; x.sub.4 is residue 153, 154, 155, 156, or 157 of SEQ ID NO:2, or residue 149, 150, 151, 152, 153, 154, 155 or 156 of SEQ ID NO:4 or SEQ ID NO:6; x.sub.5 is residue 220, 221, 222, 223, or 224 of SEQ ID NO:2, or residue 216, 217, 218, 219, 220, 221, 222 or 223 of SEQ ID NO:4 or SEQ ID NO:6; x.sub.6 is residue 256, 257, 258, 259, 260 or 261 of SEQ ID NO:2, or residue 253, 254, 255, 256, 257, 258, 259 or 260 of SEQ ID NO:4 or SEQ ID NO:6; x.sub.7 is residue 312, 313, 314, 315 or 316 of SEQ ID NO:2, or residue 309, 310, 311, 312, 313, 314, 315 or 318 of SEQ ID NO:4 or SEQ ID NO:6; and x.sub.8 is an amino acid residue corresponding to the C-terminus of the polypeptide have the sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.

[0091] Using the foregoing domain references a number of chimeric structure were generated as set forth in Table 1. 1,588 CBH II chimera sequences with T.sub.50 values predicted to be greater than the measured T.sub.50 value of 64.8 C for the H. insolens parent CBH II.

TABLE-US-00004 31313232 13132231 13212231 21113231 22112331 33211132 22223232 32123131 31323333 11221233 13331133 21133232 21222133 33211131 31213132 11221333 11212133 33123232 13232232 22221133 32333132 12311232 22223231 11211231 31313231 33123231 13232231 21133231 32333131 12321333 22322232 11231232 31333232 21311333 22121133 23211232 22132332 33231132 31213131 11231231 11232133 21331333 33223232 23221333 22132331 12311231 31312132 33123332 31333231 32213332 23111232 23211231 33313332 33231131 22322231 33123331 21323112 32213331 23121333 23231232 33313331 12331232 31312131 31321232 21323111 32312332 33223231 11311333 33333332 12331231 31233132 31321231 32113332 21211133 33322232 23231231 33333331 23122232 31233131 33122132 32113331 32312331 23111231 21122133 33213132 23122231 31332132 33122131 31223133 13321112 33322231 11331333 33213131 11113232 31332131 12123232 21111133 32233332 23131232 11211133 33312132 11123333 23321133 12123231 31322133 13321111 23131231 11231133 33312131 11113231 23222232 32121332 32133332 32233331 11323112 33113132 12313232 11133232 23222231 32121331 32133331 32332332 11323111 33113131 12323333 12221133 11213232 21323133 21131133 21231133 11111133 33133132 33233132 11133231 11223333 13122232 32112132 32332331 11131133 33133131 12313231 31111332 11213231 13122231 32112131 32121232 32221232 31321133 33233131 31111331 11312232 22113132 32132132 32121231 32221231 31222232 33332132 31131332 11322333 22113131 32132131 13313133 22313232 31222231 33332131 13211232 11312231 22133132 21321312 32212132 22323333 12123133 12333232 31131331 11233232 22133131 33112332 32212131 22313231 32111132 12333231 13221333 11233231 23113332 21321311 13333133 22333232 13113232 32311332 13211231 11332232 23113331 33112331 32232132 22333231 13123333 32311331 13231232 11332231 23133332 11223233 32232131 31122232 32111131 32331332 22212332 31211332 23133331 33132332 33212332 31122231 13113231 32331331 13231231 31211331 12212332 11322233 33212331 11321312 32131132 12223133 22212331 31231332 21311232 33132331 22123133 11321311 13133232 12322133 11122133 31231331 12212331 31211232 33232332 22223133 32131131 31113332 22232332 12112332 21321333 31221333 33232331 22322133 13133231 31113331 22232331 31323232 21311231 31211231 23113232 23213232 33111332 31133332 33321232 12112331 12232332 21313333 23123333 23223333 33111331 32211132 33321231 11222133 21331232 31231232 23113231 23213231 33131332 13213232 23323133 31323231 12232331 31231231 23133232 23312232 11321233 31133331 31213332 12132332 21331231 21221112 23133231 23322333 33131331 13223333 31213331 12132331 23112132 21333333 11121112 23312231 13122133 32211131 31312332 32123332 23112131 21221111 12311133 23233232 12111232 13213231 31312331 32123331 23132132 12112232 11121111 11313333 12121333 13312232 31233332 21121133 23132131 12122333 12212232 23233231 12111231 13322333 31233331 32122132 32223332 12112231 12222333 23332232 12131232 13312231 31332332 32122131 22111332 12132232 12212231 23332231 12131231 32231132 31332331 33122332 32223331 12132231 21321233 11221112 32313332 13233232 31121232 33122331 32322332 21213133 12331133 11333333 32313331 32231131 31121231 31221232 22111331 21312133 12232232 11221111 21311133 13233231 22321133 31221231 21221133 13323112 12232231 23222133 21212232 13332232 22222232 21313232 32322331 13323111 13311333 11213133 21222333 13332231 31212132 21323333 22131332 21233133 23122133 11312133 32333332 33211332 22222231 21313231 22131331 21332133 13331333 11233133 21212231 33211331 31212131 21333232 13323133 32123232 11113133 11332133 32333331 33231332 31232132 21333231 32222132 32123231 11133133 22211232 21331133 33231331 31232131 12122232 32222131 13111133 32223232 22221333 21232232 22122232 23311232 12122231 33311132 13131133 22111232 22211231 21232231 31112132 23321333 22113332 33311131 12321112 22121333 22231232 32213132 22122231 23311231 22113331 33222332 12321111 32223231 22231231 32213131 31112131 23331232 21223133 33222331 33122232 32322232 31323133 32312132 31132132 23331231 21322133 33331132 33122231 22111231 21112232 32312131 33323232 21123133 22133332 33331131 21211333 32322231 21122333 32233132 31132131 23221133 22133331 23123232 13321312 22131232 21112231 32233131 13222133 11311133 13121133 23123231 13321311 22131231 21132232 32332132 33323231 11212232 22112132 12321133 21231333 13211133 21132231 32332131 12211232 11222333 22112131 12222232 11123112 13231133 32113132 33213332 12221333 11212231 22132132 12222231 12313133 11323312 32113131 33213331 12211231 11331133 22132131 13311232 11123111 11323311 11211333 33312332 12231232 11232232 33313132 13321333 21323233 33321133 32133132 33312331 12231231 11232231 33313131 13311231 12333133 33222232 32133131 33233332 23121133 31223232 23112332 22213332 13313333 33222231 11231333 33233331 11112232 21111232 23112331 13331232 32212332 11111333 33113332 33332332 11122333 21121333 33333132 22213331 32212331 11131333 33113331 33332331 11112231 31223231 33333131 22312332 13221112 11223112 33133332 33121232 11132232 31322232 23132332 13331231 13333333 11223111 11323233 33121231 32321232 21111231 23132331 22312331 13221111 11322112 33133331 33212132 11132231 31322231 21211232 11123133 32232332 11322111 31311232 33212131 32321231 21131232 21221333 22233332 32232331 13321233 31321333 12213232 31123232 21131231 21211231 22233331 22113232 22213232 31311231 12223333 31123231 32122332 21231232 22332332 22123333 22223333 31331232 12213231 22323133 32122331 21231231 22332331 22113231 22213231 31331231 12312232 33221232 13123133 12323133 22121232 22133232 22312232 21321112 12322333 33221231 33111132 32311132 31111132 22133231 22322333 33112132 33232132 23313232 33111131 13313232 22121231 13213133 22312231 21321111 12312231 23323333 33131132 13323333 31111131 13312133 22233232 33112131 33232131 23313231 33131131 32311131 31131132 13233133 22233231 12113232 12233232 23333232 21213232 13313231 31131131 13332133 22332232 12123333 12233231 23333231 21223333 32222332 13221133 11121312 22332231 12113231 12332232 11321112 21213231 32222331 22212132 12311333 31121133 33132132 12332231 11321111 21312232 32331132 22212131 11121311 11221312 33132131 32211332 23223133 21322333 13333232 22232132 22122133 11221311 12133232 32211331 23322133 21312231 32331131 22232131 12331333 22222133 12133231 23123133 11313133 21233232 13333231 23212332 33323133 23311133 32111332 32231332 11333133 21233231 33311332 23212331 23112232 23212232 32111331 32231331 22311232 21332232 33311331 23232332 23122333 23222333 31221133 32323232 22321333 21332231 33331332 23232331 23112231 23212231 32131332 12222133 22311231 12121133 22123232 11111232 11113333 23331133 21313133 32323231 31212332 13111232 33331331 11121333 23132232 11213333 32131331 31112332 31212331 13121333 31113132 11111231 23132231 23232232 21333133 31112331 22331232 13111231 22123231 11131232 11133333 11312333 12122133 13311133 22331231 32313132 31113131 11131231 12211133 23232231 13112232 31132332 31232332 32313131 31133132 31313332 21221233 11233333 13122333 13212232 31232331 13131232 31133131 31313331 12231133 11332333 13112231 31132331 21113232 22112332 13223133 31333332 13211333 11121233 13132232 13222333 21123333 13131231 13322133 31333331 22131131 22331331 13111331 33323132 33321132 22121132 11311132 11321132 33223332 21113332 13131332 33323131 33321131 22121131 11311131 11321131 23111332 21113331 13131331 23122332 11111332 23121332 11222332 11323132 33223331 21133332 21212132 23122331 11111331 23121331 11222331 11323131 33322332 22211132 21212131 11113332 11131332 11111132 11331132 11321332 23111331 21133331 21232132 11113331 11131331 11111131 11331131 11321331 33322331 22211131 21232131 11133332 13321232 11131132 21121332 11221132 23131332 22231132 12313332 12211132 13321231 11131131 21121331 11221131 23131331 22231131 12313331 11133331 22223332 22323332 13123132 21321132 33222132 23211332 12333332 12211131 22223331 22323331 13123131 21321131 33222131 23211331 12333331 21221232 22322332 22223132 21223332 13321132 12223232 23231332 33121132 21221231 22322331 22223131 21223331 13321131 12223231 23231331 33121131 12231132 31121132 22322132 21322332 11121132 12322232 21112132 12213132 12231131 31121131 22322131 21322331 11121131 12322231 21112131 12213131 13211332 22222132 23223332 12121132 11323332 32221332 21132132 12312132 13211331 22222131 23223331 12121131 11323331 32221331 23323232 12312131 13231332 23311132 23322332 13121332 11223132 22313332 21132131 21223232 13231331 23311131 23322331 13121331 11223131 22313331 23323231 21223231 11112132 23222332 11313332 21222132 11322132 22333332 11323133 21322232 11112131 23222331 11313331 21222131 11322131 22333331 22321232 12233132 11132132 23331132 11333332 12323332 11221332 31122332 31311132 21322231 32321132 11213332 11333331 12323331 11221331 31122331 22321231 12233131 13323232 23331131 23222132 12223132 21323132 13321133 31311131 12332132 11132131 11213331 23222131 12223131 21323131 13222232 31222332 12332131 32321131 11312332 11213132 12322132 21321332 13222231 31222331 13213332 13323231 11312331 11213131 12322131 21321331 22213132 31331132 13213331 33321332 11233332 11312132 13223332 21221132 22213131 31331131 13312332 33321331 11233331 11312131 13223331 21221131 22312132 12113132 13312331 31123132 11332332 11233132 13322332 13323132 22312131 12113131 13233332 31123131 11332331 11233131 13322331 13323131 22233132 21123232 13233331 33221132 11121232 11332132 13222132 12321132 22233131 21123231 13332332 33221131 11121231 11332131 13222131 12321131 22332132 12133132 13332331 23313132 31323332 22221332 12221332 13321332 22332131 12133131 13121232 12321232 11212132 22221331 12221331 13321331 23213332 13113332 13121231 23313131 31323331 31323132 23121132 11123132 23213331 13113331 32323132 12321231 11212131 31323131 23121131 11123131 23312332 13133332 32323131 23333132 11232132 21122332 11122332 13221132 23312331 13133331 22122332 23333131 11232131 21122331 11122331 13221131 23233332 23221232 22122331 11123232 31223132 11211332 22323132 11121332 23233331 23221231 33323332 11123231 21111132 11211331 22323131 11121331 23332332 11311232 13212132 31121332 31223131 11231332 23323332 23321132 23332331 11321333 33323331 31121331 31322132 11231331 23323331 23321131 23121232 11311231 13212131 13221232 21111131 11323232 23223132 11223332 23121231 11331232 13232132 13221231 31322131 11323231 23223131 11223331 23212132 11331231 13232131 22311132 21131132 31321332 23322132 11322332 23212131 13112132 12211332 22311131 21131131 31321331 23322131 11322331 23232132 13112131 12211331 22222332 11223232 12123332 11313132 11222132 23232131 13132132 12231332 22222331 11223231 12123331 11313131 11222131 22211332 13132131 12231331 22331132 11322232 31221132 11333132 21121132 22211331 12111332 33223132 22331131 11322231 31221131 11333131 21121131 11121133 12111331 23111132 23311332 31221332 21313132 22321332 21323332 22231332 11221133 33223131 23311331 31221331 21313131 22321331 21323331 22231331 12131332 33322132 23331332 21313332 21333132 21123332 21223132 22323232 12131331 23111131 23331331 21313331 21333131 21123331 21223131 31313132 33123132 33322131 21113132 21333332 12122132 22221132 21322132 22323231 33123131 12323232 21113131 21333331 12122131 22221131 21322131 31313131 21212332 12323231 21133132 12122332 13122332 23221332 13121132 21112332 21212331 23131132 21133131 12122331 13122331 23221331 13121131 21112331 21232332 23131131 23211132 21213132 11221232 11311332 21221332 31333132 21232331 11112332 23211131 21213131 11221231 11311331 21221331 31333131 32121132 11112331 23231132 21312132 21311332 21122132 12323132 21132332 13123232 11132332 23231131 21312131 21311331 21122131 12323131 21132331 32121131 32321332 11212332 21233132 21331332 11331332 13323332 23223232 13123231 11132331 11212331 21233131 21331331 11331331 13323331 23223231 33121332 32321331 11232332 21332132 21211132 11211132 13223132 23322232 33121331 31123332 11232331 21332131 21211131 11211131 13223131 23322231 12213332 31123331 31223332 13111132 21231132 11231132 13322132 11313232 12213331 32221132 21111332 13111131 21231131 11231131 13322131 11323333 12312332 13223232 31223331 13131132 13313132 31321132 12321332 11313231 12312331 32221131 31322332 13131131 13313131 31321131 12321331 11333232 12233332 13223231 21111331 21211332 13333132 12123132 11123332 11333231 12233331 13322232 31322331 21211331 13333131 12123131 11123331 31311332 12332332 13322231 21131332 21231332 22123132 13123332 12221132 31311331 12332331 22313132 21131331 21231331 22123131 13123331 12221131 31331332 23113132 22313131 31222132 12313132 23123332 11321232 13221332 31331331 12121232 22333132 31222131 12313131 23123331 11321231 13221331 12113332 23113131 33221332 23321232 21323232 12311132 13122132 11122132 12113331 12121231 22333131 23321231 21323231 12311131 13122131 11122131 11223133 23133132 33221331 13113132 12333132 12222332 12121332 23323132 11322133 23133131 23313332 13113131 12333131 21321232 12121331 23323131 12133332 32323332 23313331 13133132 13313332 12222331 21311132 22321132 12133331 12212132 31122132 13133131 13313331 21321231 21311131 22321131 22221232 32323331 23333332 11321133 13333332 12331132 21222332 23321332 31211132 12212131 31122131 11222232 13333331 12331131 21222331 23321331 22221231 21321133 23333331 11222231 22123332 13311332 21331132 21123132 31211131 21222232 23213132 21213332 22123331 13311331 21331131 21123131 31231132 21222231 12221232 21213331 13213132 23122132 12223332 23221132 31231131 12232132 23213131 21312332 13213131 23122131 12223331 23221131 12112132 12232131 23312132 21312331 13312132 13331332 12322332 12112131 13212332 12221231 21233332 13312131 13331331 12322331 21122232 13212331 23312131 21233331 13233132 11113132 23123132 21122231 13232332 23233132 21332332 13233131 11113131 23123131 12132132 13232331 23233131 12111132 13332132 11133132 12222132 12132131 32223132 23332132 21332331 13332131 11133131 12222131 13112332 22111132 23332131 12111131 12311332 22121332 13311132 13112331 32223131 22311332 21121232 12311331 22121331 13311131 13132332 32322132 22311331 21121231 22122132 13211132 13222332 13132331 22111131 11122232 12131132 22122131 13211131 13222331 32123132 32322131 22331332 12131131 12331332 13231132 13331132 11211232 22131132 11122231 13111332 12331331 13231131 13331131

[0092] Referring to the table above, each digit refers to a domain of a chimeric CBH II polypeptide. The number denotes the parental strand the domain was derived from. For example, a chimeric CBH II chimeric polypeptide having the sequence 12111131, indicates that the polypeptide comprises a sequence from the N-terminus to the C-terminus of: amino acids from about 1 to x.sub.1 of SEQ ID NO:2 ("1") linked to amino acids from about x.sub.1 to x.sub.2 of SEQ ID NO:4 ("2") linked to amino acids from about x.sub.2 to about x.sub.3 of SEQ ID NO:2 linked to amino acids from about x.sub.3 to about x.sub.4 of SEQ ID NO:2 linked to amino acids from about x.sub.4 to about x.sub.5 of SEQ ID NO:2 linked to amino acids from about x.sub.5 to about x.sub.6 of SEQ ID NO:2 linked to amino acids from about x.sub.6 to x.sub.7 of SEQ ID NO:6 ("3") linked to amino acids from about x.sub.7 to x.sub.8 (e.g., the C-terminus) of SEQ ID NO:2.

[0093] In some embodiments, the CBH II polypeptide has a chimeric segment structure selected from the group consisting of 11113132, 21333331, 21311131, 22232132, 33133132, 33213332, 13333232, 12133333, 13231111, 11313121, 11332333, 12213111, 23311333, 13111313, 31311112, 23231222, 33123313, 22212231, 21223122, 21131311, 23233133, 31212111 and 32333113.

[0094] In some embodiments, the polypeptide has improved thermostability compared to a wild-type polypeptide of SEQ ID NO:2, 4, or 6. The activity of the polypeptide can be measured with any one or combination of substrates as described in the examples. As will be apparent to the skilled artisan, other compounds within the class of compounds exemplified by those discussed in the examples can be tested and used.

[0095] In some embodiments, the polypeptide can have various changes to the amino acid sequence with respect to a reference sequence. The changes can be a substitution, deletion, or insertion of one or more amino acids. Where the change is a substitution, the change can be a conservative, a non-conservative substitution, or a combination of conservative and non-conservative substitutions. For example, the chimera can comprises a C.fwdarw.S substitution at C314 of SEQ ID NO:2 or C311 of SEQ ID NO:4.

[0096] Thus, in some embodiments, the polypeptides can comprise a general structure from N-terminus to C-terminus: (segment 1)-(segment 2)-(segment 3)-(segment 4)-(segment 5)-(segment 6)-(segment 7)-(segment 8),

[0097] wherein segment 1 comprises amino acid residue from about 1 to about x.sub.1 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3") and having 1-10 conservative amino acid substitutions; segment 2 is from about amino acid residue x.sub.1 to about x.sub.2 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3") and having about 1-10 conservative amino acid substitutions; segment 3 is from about amino acid residue x.sub.2 to about x.sub.3 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3") and having about 1-10 conservative amino acid substitutions; segment 4 is from about amino acid residue x.sub.3 to about x.sub.4 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3") and having about 1-10 conservative amino acid substitutions; segment 5 is from about amino acid residue x.sub.4 to about x.sub.5 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3") and having about 1-10 conservative amino acid substitutions; segment 6 is from about amino acid residue x.sub.5 to about x.sub.6 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3") and having about 1-10 conservative amino acid substitutions; segment 7 is from about amino acid residue x.sub.6 to about x.sub.7 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3") and having about 1-10 conservative amino acid substitutions; and segment 8 is from about amino acid residue x.sub.7 to about x.sub.8 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3") and having about 1-10 conservative amino acid substitutions;

[0098] wherein x.sub.1 is residue 43, 44, 45, 46, or 47 of SEQ ID NO:2, or residue 42, 43, 44, 45, or 46 of SEQ ID NO:4 or SEQ ID NO:6; x.sub.2 is residue 70, 71, 72, 73, or 74 of SEQ ID NO:2, or residue 68, 69, 70, 71, 72, 73, or 74 of SEQ ID NO:4 or SEQ ID NO:6; x.sub.3 is residue 113, 114, 115, 116, 117 or 118 of SEQ ID NO:2, or residue 110, 111, 112, 113, 114, 115, or 116 of SEQ ID NO:4 or SEQ ID NO:6; x.sub.4 is residue 153, 154, 155, 156, or 157 of SEQ ID NO:2, or residue 149, 150, 151, 152, 153, 154, 155 or 156 of SEQ ID NO:4 or SEQ ID NO:6; x.sub.5 is residue 220, 221, 222, 223, or 224 of SEQ ID NO:2, or residue 216, 217, 218, 219, 220, 221, 222 or 223 of SEQ ID NO:4 or SEQ ID NO:6; x.sub.6 is residue 256, 257, 258, 259, 260 or 261 of SEQ ID NO:2, or residue 253, 254, 255, 256, 257, 258, 259 or 260 of SEQ ID NO:4 or SEQ ID NO:6; x.sub.7 is residue 312, 313, 314, 315 or 316 of SEQ ID NO:2, or residue 309, 310, 311, 312, 313, 314, 315 or 318 of SEQ ID NO:4 or SEQ ID NO:6; and x.sub.8 is an amino acid residue corresponding to the C-terminus of the polypeptide have the sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 and wherein the chimera has an algorithm as set forth in Table 1 and wherein the chimera comprises a C.fwdarw.S substitution corresponding to C314 of SEQ ID NO:2 or C311 of SEQ ID NO:4.

[0099] In some embodiments, the number of substitutions can be 2, 3, 4, 5, 6, 8, 9, or 10, or more amino acid substitutions (e.g., 10-20, 21-30, 31-40 and the like amino acid substitutions).

[0100] In some embodiments, the functional CBH II polypeptides can have cellulase activity along with increased thermostability, such as for a defined substrate discussed in the Examples, and also have a level of amino acid sequence identity to a reference cellobiohydrolase, or segments thereof. The reference enzyme or segment, can be that of a wild-type (e.g., naturally occurring) or an engineered enzyme. Thus, in some embodiments, the polypeptides of the disclosure can comprise a general structure from N-terminus to C-terminus:

[0101] wherein segment 1 comprises a sequence that is at least 50-100% identity to amino acid residue from about 1 to about x.sub.1 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3"); segment 2 comprises a sequence that is at least 50-100% identity to amino acid residue x.sub.1 to about x.sub.2 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3"); segment 3 comprises a sequence that is at least 50-100% identity to amino acid residue x.sub.2 to about x.sub.3 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3"); segment 4 comprises a sequence that is at least 50-100% identity to amino acid residue x.sub.3 to about x.sub.4 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3"); segment 5 comprises a sequence that is at least 50-100% identity to about amino acid residue x.sub.4 to about x.sub.5 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3"); segment 6 comprises a sequence that is at least 50-100% identity to amino acid residue x.sub.5 to about x.sub.6 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3"); segment 7 comprises a sequence that is at least 50-100% identity to amino acid residue x.sub.6 to about x.sub.7 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3"); and segment 8 comprises a sequence that is at least 50-100% identity to amino acid residue x.sub.7 to about x.sub.8 of SEQ ID NO:2 ("1"), SEQ ID NO:4 ("2") or SEQ ID NO:6 ("3");

[0102] wherein x.sub.1 is residue 43, 44, 45, 46, or 47 of SEQ ID NO:2, or residue 42, 43, 44, 45, or 46 of SEQ ID NO:4 or SEQ ID NO:6; x.sub.2 is residue 70, 71, 72, 73, or 74 of SEQ ID NO:2, or residue 68, 69, 70, 71, 72, 73, or 74 of SEQ ID NO:4 or SEQ ID NO:6; x.sub.3 is residue 113, 114, 115, 116, 117 or 118 of SEQ ID NO:2, or residue 110, 111, 112, 113, 114, 115, or 116 of SEQ ID NO:4 or SEQ ID NO:6; x.sub.4 is residue 153, 154, 155, 156, or 157 of SEQ ID NO:2, or residue 149, 150, 151, 152, 153, 154, 155 or 156 of SEQ ID NO:4 or SEQ ID NO:6; x.sub.5 is residue 220, 221, 222, 223, or 224 of SEQ ID NO:2, or residue 216, 217, 218, 219, 220, 221, 222 or 223 of SEQ ID NO:4 or SEQ ID NO:6; x.sub.6 is residue 256, 257, 258, 259, 260 or 261 of SEQ ID NO:2, or residue 253, 254, 255, 256, 257, 258, 259 or 260 of SEQ ID NO:4 or SEQ ID NO:6; x.sub.7 is residue 312, 313, 314, 315 or 316 of SEQ ID NO:2, or residue 309, 310, 311, 312, 313, 314, 315 or 318 of SEQ ID NO:4 or SEQ ID NO:6; and x.sub.8 is an amino acid residue corresponding to the C-terminus of the polypeptide have the sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 and wherein the chimera has an algorithm as set forth in Table 1 and wherein the chimera comprises a C.fwdarw.S substitution corresponding to C314 of SEQ ID NO:2 or C311 of SEQ ID NO:4.

[0103] In some embodiments, each segment of the chimeric polypeptide can have at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity as compared to the reference segment indicated for each of the (segment 1), (segment 2), (segment 3), (segment 4)-(segment 5), (segment 6), (segment 7), and (segment 8) of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

[0104] In some embodiments, the polypeptide variants can have improved thermostability compared to the enzyme activity of the wild-type polypeptide of SEQ ID NO:2, 4, or 6 and wherein the chimera comprises a C.fwdarw.S substitution corresponding to C314 of SEQ ID NO:2 or C311 of SEQ ID NO:4.

[0105] The chimeric enzymes described herein may be prepared in various forms, such as lysates, crude extracts, or isolated preparations. The polypeptides can be dissolved in suitable solutions; formulated as powders, such as an acetone powder (with or without stabilizers); or be prepared as lyophilizates. In some embodiments, the polypeptide can be an isolated polypeptide.

[0106] In some embodiments, the polypeptides can be in the form of arrays. The enzymes may be in a soluble form, for example, as solutions in the wells of mircotitre plates, or immobilized onto a substrate. The substrate can be a solid substrate or a porous substrate (e.g, membrane), which can be composed of organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as co-polymers and grafts thereof. A solid support can also be inorganic, such as glass, silica, controlled pore glass (CPG), reverse phase silica or metal, such as gold or platinum. The configuration of a substrate can be in the form of beads, spheres, particles, granules, a gel, a membrane or a surface. Surfaces can be planar, substantially planar, or non-planar. Solid supports can be porous or non-porous, and can have swelling or non-swelling characteristics. A solid support can be configured in the form of a well, depression, or other container, vessel, feature, or location. A plurality of supports can be configured on an array at various locations, addressable for robotic delivery of reagents, or by detection methods and/or instruments.

[0107] The disclosure also provides polynucleotides encoding the engineered CBH II polypeptides disclosed herein. The polynucleotides may be operatively linked to one or more heterologous regulatory or control sequences that control gene expression to create a recombinant polynucleotide capable of expressing the polypeptide. Expression constructs containing a heterologous polynucleotide encoding the CBH II chimera can be introduced into appropriate host cells to express the polypeptide.

[0108] Given the knowledge of specific sequences of the CBH II chimera enzymes (e.g., the segment structure of the chimeric CBH II), the polynucleotide sequences will be apparent form the amino acid sequence of the engineered CBH II chimera enzymes to one of skill in the art. The knowledge of the codons corresponding to various amino acids coupled with the knowledge of the amino acid sequence of the polypeptides allows those skilled in the art to make different polynucleotides encoding the polypeptides of the disclosure. Thus, the disclosure contemplates each and every possible variation of the polynucleotides that could be made by selecting combinations based on possible codon choices, and all such variations are to be considered specifically disclosed for any of the polypeptides described herein.

[0109] In some embodiments, the polynucleotides encode the polypeptides described herein but have about 80% or more sequence identity, about 85% or more sequence identity, about 90% or more sequence identity, about 91% or more sequence identity, about 92% or more sequence identity, about 93% or more sequence identity, about 94% or more sequence identity, about 95% or more sequence identity, about 96% or more sequence identity, about 97% or more sequence identity, about 98% or more sequence identity, or about 99% or more sequence identity at the nucleotide level to a reference polynucleotide encoding the CBH II variant of chimera polypeptides and having a C.fwdarw.S substitution as described above (e.g., wherein the polypeptide or chimera comprises a C.fwdarw.S substitution corresponding to C314 of SEQ ID NO:2 or C311 of SEQ ID NO:4).

[0110] In some embodiments, the isolated polynucleotides encoding the polypeptides may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the isolated polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are well known in the art. Guidance is provided in Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press; and Current Protocols in Molecular Biology, Ausubel. F. ed., Greene Pub. Associates, 1998, updates to 2007.

[0111] In some embodiments, the polynucleotides are operatively linked to control sequences for the expression of the polynucleotides and/or polypeptides. In some embodiments, the control sequence may be an appropriate promoter sequence, which can be obtained from genes encoding extracellular or intracellular polypeptides, either homologous or heterologous to the host cell. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure, include the promoters obtained from the E. coli lac operon, Bacillus subtilis xylA and xylB genes, Bacillus megatarium xylose utilization genes (e.g., Rygus et al., (1991) Appl. Microbiol. Biotechnol. 35:594-599; Meinhardt et al., (1989) Appl. Microbiol. Biotechnol. 30:343-350), prokaryotic beta-lactamase gene (Villa-Kamaroff et al., (1978) Proc. Natl Acad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., (1983) Proc. Natl Acad. Sci. USA 80: 21-25). Various suitable promoters are described in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; and in Sambrook et al., supra.

[0112] In some embodiments, the control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used.

[0113] In some embodiments, the control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used.

[0114] In some embodiments, the control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway. The 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. The foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region. Effective signal peptide coding regions for bacterial host cells can be the signal peptide coding regions obtained from the genes for Bacillus NClB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-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.

[0115] The disclosure is further directed to a recombinant expression vector comprising a polynucleotide encoding the engineered CBH II variant or chimera polypeptide, and one or more expression regulating regions such as a promoter and a terminator, a replication origin, etc., depending on the type of hosts into which they are to be introduced. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

[0116] The recombinant expression vector may be any vector (e.g., a plasmid or virus), which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the polynucleotide sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.

[0117] The expression vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

[0118] In some embodiments, the expression vector of the disclosure contains one or more selectable markers, which permit easy selection of transformed cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers, which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol (Example 1) or tetracycline resistance. Other useful markers will be apparent to the skilled artisan.

[0119] In another embodiment, the disclosure provides a host cell comprising a polynucleotide encoding the CBH II variant or chimera polypeptide, the polynucleotide being operatively linked to one or more control sequences for expression of the polypeptide in the host cell. Host cells for use in expressing the polypeptides encoded by the expression vectors of the disclosure are well known in the art and include, but are not limited to, bacterial cells, such as E. coli and Bacillus megaterium; eukaryotic cells, such as yeast cells, CHO cells and the like, insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. Other suitable host cells will be apparent to the skilled artisan. Appropriate culture mediums and growth conditions for the above-described host cells are well known in the art.

[0120] The CBH II variant or chimera polypeptides of the disclosure can be made by using methods well known in the art. Polynucleotides can be synthesized by recombinant techniques, such as that provided in Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press; and Current Protocols in Molecular Biology, Ausubel. F. ed., Greene Pub. Associates, 1998, updates to 2007. Polynucleotides encoding the enzymes, or the primers for amplification can also be prepared by standard solid-phase methods, according to known synthetic methods, for example using phosphoramidite method described by Beaucage et al., (1981) Tet Lett 22:1859-69, or the method described by Matthes et al., (1984) EMBO J. 3:801-05, e.g., as it is typically practiced in automated synthetic methods. In addition, essentially any nucleic acid can be obtained 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.

[0121] Engineered enzymes expressed in a host cell can be recovered from the cells and or the culture medium using any one or more of the well known techniques for protein purification, including, among others, lysozyme treatment, sonication, filtration, salting-out, ultra-centrifugation, chromatography, and affinity separation (e.g., substrate bound antibodies). Suitable solutions for lysing and the high efficiency extraction of proteins from bacteria, such as E. coli, are commercially available under the trade name CelLytic BTM from Sigma-Aldrich of St. Louis Mo.

[0122] Chromatographic techniques for isolation of the polypeptides include, among others, reverse phase chromatography high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying a particular enzyme will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those having skill in the art.

[0123] Descriptions of SCHEMA directed recombination and synthesis of chimeric polypeptides are described in the examples herein, as well as in Otey et al., (2006), PLoS Biol. 4(5):e112; Meyer et al., (2003) Protein Sci., 12:1686-1693; U.S. patent application Ser. No. 12/024,515, filed Feb. 1, 2008; and U.S. patent application Ser. No. 12/027,885, filed Feb. 7, 2008; such references incorporated herein by reference in their entirety.

[0124] As discussed above, the polypeptide can be used in a variety of applications, such as, among others, biofuel generation, cellulose breakdown and the like.

[0125] The following examples are meant to further explain, but not limited the foregoing disclosure or the appended claims.

Examples

CBH II Expression Plasmid Construction

[0126] Parent and chimeric genes encoding CBH II enzymes were cloned into yeast expression vector YEp352/PGK91-1-ass (FIG. 6). DNA sequences encoding parent and chimeric CBH II catalytic domains were designed with S. cerevisiae codon bias using GeneDesigner software (DNA2.0) and synthesized by DNA2.0. The CBH II catalytic domain genes were digested with XhoI and KpnI, ligated into the vector between the XhoI and KpnI sites and transformed into E. coli XL-1 Blue (Stratagene). CBH II genes were sequenced using primers: CBH2L (5'-GCTGAACGTGTCATCGGTTAC-3' (SEQ ID NO:9) and RSQ3080 (5'-GCAACACCTGGCAATTCCTTACC-3' (SEQ ID NO:10)). C-terminal His.sub.6 parent and chimera CBH II constructs were made by amplifying the CBH II gene with forward primer CBH2LPCR (5'-GCTGAACGTGTCATCGTTACTTAG-3' (SEQ ID NO:11)) and reverse primers complementary to the appropriate CBH II gene with His.sub.6 overhangs and stop codons. PCR products were ligated, transformed and sequenced as above.

[0127] CBH II Enzyme Expression in S. cerevisiae.

[0128] S. cerevisiae strain YDR483W BY4742 (Mat.alpha. his3.DELTA.1 leu2.DELTA.0 lys2.DELTA.0 ura3.DELTA.0 .DELTA.KRE2, ATCC No. 4014317) was made competent using the EZ Yeast II Transformation Kit (Zymo Research), transformed with plasmid DNA and plated on synthetic dropout-uracil agar. Colonies were picked into 5 mL overnight cultures of synthetic dextrose casamino acids (SDCAA) media (20 g/L dextrose, 6.7 g/L Difco yeast nitrogen base, 5 g/L Bacto casamino acids, 5.4 g/L Na.sub.2HPO.sub.4, 8.56 g/L NaH.sub.2PO.sub.4.H.sub.2O) supplemented with 20 ug/mL tryptophan and grown overnight at 30.degree. C., 250 rpm. 5 mL cultures were expanded into 40 mL SDCAA in 250 mL Tunair flasks (Shelton Scientific) and shaken at 30.degree. C., 250 rpm for 48 hours. Cultures were centrifuged, and supernatants were concentrated to 500 uL, using an Amicon ultrafiltration cell fitted with 30-kDa PES membrane, for use in t.sub.1/2 assays. Concentrated supernatants were brought to1 mM phenylmethylsulfonylfluoride and 0.02% NaN.sub.3. His.sub.6-tagged CBH II proteins were purified using Ni-NTA spin columns (Qiagen) per the manufacturer's protocol and the proteins exchanged into 50 mM sodium acetate, pH 4.8, using Zeba-Spin desalting columns (Pierce). Purified protein concentration was determined using Pierce Coomassie Plus protein reagent with BSA as standard. SDS-PAGE analysis was performed by loading either 20 uL of concentrated culture supernatant or approximately 5 ug of purified CBH II enzyme onto a 7.5% Tris-HCl gel (Biorad) and staining with SimplyBlue safe stain (Invitrogen). CBH II supernatants or purified proteins were treated with EndoH (New England Biolabs) for 1 hr at 37.degree. C. per the manufacturer's instructions. CBH II enzyme activity in concentrated yeast culture supernatants was measured by adding 37.5 uL concentrated culture supernatant to 37.5 uL PASC and incubating for 2 hr at 50.degree. C. Reducing sugar equivalents formed were determined via Nelson-Somogyi assay as described below.

[0129] Half-Life, Specific Activity, pH-Activity and Long-Time Cellulose Hydrolysis Measurements.

[0130] Phosphoric acid swollen cellulose (PASC) was prepared. To enhance CBH II enzyme activity on the substrate, PASC was pre-incubated at a concentration of 10 g/L with 10 mg/mL A. niger endoglucanase (Sigma) in 50 mM sodium acetate, pH 4.8 for 1 hr at 37.degree. C. Endoglucanase was inactivated by heating to 95.degree. C. for 15 minutes, PASC was washed twice with 50 mM acetate buffer and resuspended at 10 g/L in deionzed water.

[0131] CBH II enzyme t.sub.1/2s were measured by adding concentrated CBH II expression culture supernatant to 50 mM sodium acetate, pH 4.8 at a concentration giving A.sub.520 of 0.5 as measured in the Nelson-Somogyi reducing sugar assay after incubation with treated PASC as described below. 37.5 uL CBH II enzyme/buffer mixtures were inactivated in a water bath at 63.degree. C. After inactivation, 37.5 uL endoglucanase-treated PASC was added and hydrolysis was carried out for 2 hr at 50.degree. C. Reaction supernatants were filtered through Multiscreen HTS plates (Millipore). Nelson-Somogyi assay log(A.sub.520) values, obtained using a SpectraMax microplate reader (Molecular Devices) corrected for background absorbance, were plotted versus time and CBH II enzyme half-lives obtained from linear regression using Microsoft Excel.

[0132] For specific activity measurements, purified CBH II enzyme was added to PASC to give a final reaction volume of 75 uL 25 mM sodium acetate, pH 4.8, with 5 g/L PASC and CBH II enzyme concentration of 3 mg enzyme/g PASC. Incubation proceeded for 2 hr in a 50.degree. C. water bath and the reducing sugar concentration determined. For pH/activity profile measurements, purified CBH II enzyme was added at a concentration of 300 ug/g PASC in a 75 uL reaction volume. Reactions were buffered with 12.5 mM sodium citrate/12.5 mM sodium phosphate, run for 16 hr at 50.degree. C. and reducing sugar determined. Long-time cellulose hydrolysis measurements were performed with 300 uL volumes of 1 g/L treated PASC in 100 mM sodium acetate, pH 4.8, 20 mM NaCl. Purified CBH II enzyme was added at 100 ug/g PASC and reactions carried out in water baths for 40 hr prior to reducing sugar determination.

[0133] Five candidate parent genes encoding CBH II enzymes were synthesized with S. cerevisiae codon bias. All five contained identical N-terminal coding sequences, where residues 1-89 correspond to the cellulose binding module (CBM), flexible linker region and the five N-terminal residues of the H. jecorina catalytic domain. Two of the candidate CBH II enzymes, from Humicola insolens and Chaetomium thermophilum, were secreted from S. cerevisiae at much higher levels than the other three, from Hypocrea jecorina, Phanerochaete chrysosporium and Talaromyces emersonii (FIG. 1). Because bands in the SDS-PAGE gel for the three weakly expressed candidate parents were difficult to discern, activity assays in which concentrated culture supernatants were incubated with phosphoric acid swollen cellulose (PASC) were performed to confirm the presence of active cellulase. The values for the reducing sugar formed, presented in FIG. 1, confirmed the presence of active CBH II in concentrated S. cerevisiae culture supernatants for all enzymes except T. emersonii CBH II. H. insolens and C. thermophilum sequences were chose to recombine with the most industrially relevant fungal CBH II enzyme, from H. jecorina. The respective sequence identities of the catalytic domains are 64% (1:2), 66% (2:3) and 82% (1:3), where H. insolens is parent 1, H. jecorina is parent 2 and C. thermophilum is parent 3. These respective catalytic domains contain 360, 358 and 359 amino acid residues.

[0134] Heterologous protein expression in the filamentous fungus H. jecorina, the organism most frequently used to produce cellulases for industrial applications, is much more arduous than in Saccharomyces cerevisiae. The observed secretion of H. jecorina CBH II from S. cerevisiae motivated the choice of this heterologous host. To minimize hyperglycosylation, which has been reported to reduce the activity of recombinant cellulases, the recombinant CBH II genes were expressed in a glycosylation-deficient dKRE2 S. cerevisiae strain. This strain is expected to attach smaller mannose oligomers to both N-linked and O-linked glycosylation sites than wild type strains, which more closely resembles the glycosylation of natively produced H. jecorina CBH II enzyme. SDS-PAGE gel analysis of the CBH II proteins, both with and without EndoH treatment to remove high-mannose structures, showed that EndoH treatment did not increase the electrophoretic mobility of the enzymes secreted from this strain, confirming the absence of the branched mannose moieties that wild type S. cerevisiae strains attach to glycosylation sites in the recombinant proteins.

[0135] The high resolution structure of H. insolens (pdb entry 1ocn) was used as a template for SCHEMA to identify contacts that could be broken upon recombination. RASPP returned four candidate libraries, each with <E> below 15. The candidate libraries all have lower <E> than previously constructed chimera libraries, suggesting that an acceptable fraction of folded, active chimeras could be obtained for a relatively high <m>. Chimera sequence diversity was maximized by selecting the block boundaries leading to the greatest <m>=50. The blocks for this design are illustrated in FIG. 2B and detailed in Table 2.

TABLE-US-00005 TABLE 2 ClustalW multiple sequence alignment for parent CBH II enzyme catalytic domains. Blocks 2, 4, 6 and 8 are denoted by boxes and grey shading. Blocks 1, 3, 5 and 7 are not shaded. (H, inso: SEQ ID NO: 2; H. Jeco: SEQ ID NO: 4 and C. Ther: SEQ ID NO: 6). ##STR00001## ##STR00002## ##STR00003## ##STR00004## ##STR00005## ##STR00006## ##STR00007##

[0136] The H. insolens CBH II catalytic domain has an .alpha./.beta. barrel structure in which the eight helices define the barrel perimeter and seven parallel .beta.-sheets form the active site (FIG. 2A). Two extended loops form a roof over the active site, creating a tunnel through which the substrate cellulose chains pass during hydrolysis. Five of the seven block boundaries fall between elements of secondary structure, while block 4 begins and ends in the middle of consecutive .alpha.-helices (FIGS. 2A, 2B). The majority of interblock sidechain contacts occur between blocks that are adjacent in the primary structure (FIG. 2C).

[0137] A sample set of 48 chimera genes was designed as three sets of 16 chimeras having five blocks from one parent and three blocks from either one or both of the remaining two parents (Table 3); the sequences were selected to equalize the representation of each parent at each block position. The corresponding genes were synthesized and expressed.

TABLE-US-00006 TABLE 3 Sequences of sample set CBH II enzyme chimeras. Inactive Active 13121211 11332333 12122221 21131311 33332321 31212111 33321331 22232132 21322232 33213332 21112113 23233133 31121121 13231111 32312222 12213111 23223223 31311112 31313323 11113132 32121222 13111313 12121113 21311131 22133222 11313121 33222333 21223122 11131231 22212231 11112321 23231222 12111212 32333113 31222212 12133333 22322312 13333232 12222213 33123313 12221122 21333331 22212323 23311333 23222321 33133132 32333223 33331213

[0138] Twenty-three of the 48 sample set S. cerevisiae concentrated culture supernatants exhibited hydrolytic activity toward PASC. These results suggest that thousands of the 6,561 possible CBH II chimera sequences (see e.g., Table 1) encode active enzymes. The 23 active CBH II sample set chimeras show considerable sequence diversity, differing from the closest parental sequence and each other by at least 23 and 36 amino acid substitutions and as many as 54 and 123, respectively. Their average mutation level <m> is 36.

[0139] As Meyer et al. found correlations between E, m and the probability that a chimera is folded and active, analysis of whether similar correlations existed for the sample set CBH II chimeras was analyzed. The amount of CBH II enzyme activity in concentrated expression culture supernatants, as measured by assaying for activity on PASC, was correlated to the intensity of CBH II bands in SDS-PAGE gels (FIG. 1). As with the H. jecorina CBH II parent, activity could be detected for some CBH II chimeras with undetectable gel bands. There were no observations of CBH II chimeras presenting gel bands but lacking activity. The probability of a CBH II chimera being secreted in active form was inversely related to both E and m (FIG. 3).

[0140] Half-lives of thermal inactivation (t.sub.1/2) were measured at 63.degree. C. for concentrated culture supernatants of the parent and active chimeric CBH II enzymes. The H. insolens, H. jecorina and C. thermophilum CBH II parent half-lives were 95, 2 and 25 minutes, respectively (Table 1). The active sample set chimeras exhibited a broad range of half-lives, from less than 1 minute to greater than 3,000. Five of the 23 active chimeras had half-lives greater than that of the most thermostable parent, H. insolens CBH II.

[0141] In attempting to construct a predictive quantitative model for CBH II chimera half-life, five different linear regression data modeling algorithms were used (Table 4). Each algorithm was used to construct a model relating the block compositions of each sample set CBH II chimera and the parents to the log(t.sub.1/2). These models produced thermostability weight values that quantified a block's contribution to log(t.sub.1/2). For all five modeling algorithms, this process was repeated 1,000 times, with two randomly selected sequences omitted from each calculation, so that each algorithm produced 1,000 weight values for each of the 24 blocks. The mean and standard deviation (SD) were calculated for each block's thermostability weight. The predictive accuracy of each model algorithm was assessed by measuring how well each model predicted the t.sub.1/2s of the two omitted sequences. The correlation between measured and predicted values for the 1,000 algorithm iterations is the model algorithm's cross-validation score. For all five models, the cross-validation scores (X-val) were less than or equal to 0.57 (Table 4), indicating that linear regression modeling could not be applied to this small, 23 chimera t.sub.1/2 data set for quantitative CBH II chimera half-life prediction.

TABLE-US-00007 TABLE 4 Cross validation values for application of 5 linear regression algorithms to CBH II enzyme chimera block stability scores. Algorithm abbreviations: ridge regression (RR), partial least square regression (PLSR), support vector machine regression (SVMR), linear programming support vector machine regression (LPSVMR) and linear programming boosting regression (LPBoostR). Method Ridge PLS SVMR LSVM LPBoost X-val 0.56 0.55 0.50 0.42 0.43

[0142] Linear regression modeling was used to qualitatively classify blocks as stabilizing, destabilizing or neutral. Each block's impact on chimera thermostability was characterized using a scoring system that accounts for the thermostability contribution determined by each of the regression algorithms. For each algorithm, blocks with a thermostability weight value more than 1 SD above neutral were scored "+1", blocks within 1 SD of neutral were assigned zero and blocks 1 or more SD below neutral were scored "-1". A "stability score" for each block was obtained by summing the 1, 0, -1 stability scores from each of the five models. Table 5 summarizes the scores for each block. Block 1/parent 1 (B1P1), B6P3, B7P3 and B8P2 were identified as having the greatest stabilizing effects, while B1P3, B2P1, B3P2, B6P2, B7P1, B7P2 and B8P3 were found to be the most strongly destabilizing blocks.

TABLE-US-00008 TABLE 5 Qualitative block classification results generated by five linear regression algorithms.sup.1 for sample set CBH II enzyme chimeras. Score of +1 denotes a block with thermostability weight (dimensionless metric for contribution of a block to chimera thermostability) greater than one standard deviation above neutral (stabilizing), score of 0 denotes block with weight within one standard deviation of neutral and -1 denotes block with weight more than one standard deviation below neutral (destabilizing). Block Ridge PLS SVMR LSVM LPBoost Sum B1P1 1 0 1 1 0 3 B1P2 0 0 0 -1 0 -1 B1P3 -1 0 -1 -1 -1 -4 B2P1 -1 0 0 -1 -1 -3 B2P2 1 0 0 0 0 1 B2P3 1 0 0 0 0 1 B3P1 1 0 1 0 0 2 B3P2 -1 0 -1 -1 -1 -4 B3P3 1 0 1 0 0 2 B4P1 0 0 0 0 0 0 B4P2 0 0 0 0 0 0 B4P3 0 0 0 -1 0 -1 B5P1 0 0 0 0 0 0 B5P2 0 0 0 0 -1 -1 B5P3 -1 0 0 -1 0 -2 B6P1 1 0 0 -1 -1 -1 B6P2 -1 0 -1 -1 -1 -4 B6P3 1 1 1 1 1 5 B7P1 -1 0 -1 -1 -1 -4 B7P2 -1 0 -1 -1 -1 -4 B7P3 1 0 1 1 1 4 B8P1 1 0 1 -1 0 1 B8P2 1 0 1 1 0 3 B8P3 -1 0 -1 -1 -1 -4

[0143] A second set of genes encoding CBH II enzyme chimeras was synthesized in order to validate the predicted stabilizing blocks and identify cellulases more thermostable than the most stable parent. The 24 chimeras included in this validation set (Table 6) were devoid of the seven blocks predicted to be most destabilizing and enriched in the four most stabilizing blocks, where representation was biased toward higher stability scores. Additionally, the "HJPlus" 12222332 chimera was constructed by substituting the predicted most stabilizing blocks into the H. jecorina CBH II enzyme (parent 2).

TABLE-US-00009 TABLE 6 Sequences of 24 validation set CBH II enzyme chimeras, nine of which were expressed in active form. Inactive Active 12122132 12111131 12132332 12132331 12122331 12131331 12112132 12332331 13122332 13332331 13111132 13331332 13111332 13311331 13322332 13311332 22122132 22311331 22322132 22311332 23111332 23321131 23321332 23321331

[0144] Concentrated supernatants of S. cerevisiae expression cultures for nine of the 24 validation set chimeras, as well as the HJPlus chimera, showed activity toward PASC (Table 6). Of the 15 chimeras for which activity was not detected, nine contained block B4P2. Of the 16 chimeras containing B4P2 in the initial sample set, only one showed activity toward PASC. Summed over both chimera sets and HJPlus, just two of 26 chimeras featuring B4P2 were active, indicating that this particular block is highly detrimental to expression of active cellulase in S. cerevisiae.

[0145] The stabilities of the 10 functional chimeric CBH II enzymes from the validation set were evaluated. Because the stable enzymes already had half-lives of more than 50 hours, residual hydrolytic activity toward PASC after a 12-hour thermal inactivation at 63.degree. C. was used as the metric for preliminary evaluation. This 12-hour incubation produced a measurable decrease in the activity of the sample set's most thermostable chimera, 11113132, and completely inactivated the thermostable H. insolens parent CBH II. All ten of the functional validation set chimeras retained a greater fraction of their activities than the most stable parent, H. insolens CBH II.

[0146] The activities of selected thermostable chimeras using purified enzymes was analyzed. The parent CBH II enzymes and three thermostable chimeras, the most thermostable sample set chimera 11113132, the most thermostable validation set chimera 13311332 and the HJPlus chimera 12222332, were expressed with C-terminal His.sub.6 purification tags and purified. To minimize thermal inactivation of CBH II enzymes during the activity test, we used a shorter, two-hour incubation with the PASC substrate at 50.degree. C., pH 4.8. As shown in Table 3, the parent and chimera CBH II specific activities were within a factor of four of the most active parent CBH II enzyme, from H. jecorina. The specific activity of HJPlus was greater than all other CBH II enzymes tested, except for H. jecorina CBH II.

[0147] The pH dependence of cellulase activity is also important, as a broad pH/activity profile would allow the use of a CBH II chimera under a wider range of potential cellulose hydrolysis conditions. H. jecorina CBH II has been observed to have optimal activity in the pH range 4 to 6, with activity markedly reduced outside these values..sup.16 FIG. 4 shows that the H. insolens and C. thermophilum CBH II enzymes and all three purified thermostable CBH II chimeras have pH/activity profiles that are considerably broader than that of H. jecorina CBH II. Although Liu et al. report an optimal pH of 4 for C. thermophilum CBH II, the optimal pH of the recombinant enzyme here was near 7. Native H. insolens CBH II has a broad pH/activity profile, with maximum activity around pH 9 and approximately 60% of this maximal activity at pH 4. A similarly broad profile was observed for the recombinant enzyme. The HJPlus chimera has a much broader pH/activity profile than H. jecorina CBH II, showing a pH dependence similar to the other two parent CBH II enzymes.

[0148] Achieving activity at elevated temperature and retention of activity over extended time intervals are two primary motivations for engineering highly stable CBH II enzymes. The performance of thermostable CBH II chimeras in cellulose hydrolysis was tested across a range of temperatures over a 40-hour time interval. As shown in FIG. 5, all three thermostable chimeras were active on PASC at higher temperatures than the parent CBH II enzymes. The chimeras retained activity at 70.degree. C., whereas the H. jecorina CBH II did not hydrolyze PASC above 57.degree. C. and the stable H. insolens enzyme showed no hydrolysis above 63.degree. C. The activity of HJPlus in long-time cellulose hydrolysis assays exceeded that of all the parents at their respective optimal temperatures.

[0149] The CBH II library has fewer potential disruptions for several reasons. In addition to the higher identity of the CBH II parent sequences, the barrel topology of the CBH II fold limits the number of long-range contacts that can be broken by recombination. Between-block contacts (heavy atoms within 4.5 .ANG.) comprise only 27% (503/1831) of the total in a contact map derived from H. insolens structure 1ocn. When only counting contacts for which novel residue pairs are possible in chimeras, the inter-block total is reduced to 23% (68/294). Furthermore, most of these interactions are between residues on the protein surface, and the possibility of solvent screening further decreases the chances of dramatic disruptive residue-residue interactions (FIG. 14a). One exception, a buried interaction between positions 176 and 256, is illustrated in FIG. 14b. At this site, chimeras with B6P2 and either B5P1 or B5P3 pair Met173:Trp253 (larger amino acid than parental pairs Met176:Phe256 or Leu173:Trp253). Nevertheless, upon inspection of the parental crystallographic models, a steric clash at this position was deemed unlikely due to movement in the portion of the protein backbone which positions Trp253 and the intrinsic flexibility of Met side chains. Notably, one characterized chimera fits this pattern (13333232) and is more stable than the parents (67.degree. C.), in accord with the regression model fit (68.degree. C.)

[0150] Another mechanism by which coupling could arise, block structural divergence, does not depend on the presence of novel residue pairs at block interfaces. Instead, as parental sequences diverge, intrinsic block structures may diverge, hindering modular block transplants. In the case of the CBH II library, the high parent pair sequence identity values (82%, 66%, and 64%) suggest that only minor structure deviations are likely (<1 .ANG. RMSD). This possibility can be evaluated by comparing crystallographic structures for H. insolens and H. jecorina CBH II (C. thermophilum CBH II lacks a crystal structure but is 82% identical to H. insolens). Aligning blocks from structures for each parent (1ocn and 1cb2), generates low alpha carbon RMSD values (0.5, 0.5, 0.6, 0.5, 0.3, 0.7, 0.3, and 0.4 .ANG. RMSD). H. jecorina blocks superimposed onto H. insolens are illustrated in Supplemental FIG. 5c. To check for context-dependent effects an in silico structural recombination was performed, splicing each aligned block onto the opposing host structure. It is possible to construct non-clashing structural models (alpha carbons>3 .ANG. apart) for all single-block substitution chimeras (e.g., 11112111 or 22122222), with the exception of a minor clash (2.65 .ANG.) when using B7P2 (11111121) due to the Asn insertion between blocks 6 and 7 (FIG. 14D).

[0151] Further experiments were performed to determine the contributions of various blocks/segments to the chimera's stability and improved thermostability and/or pH stability. Parent and chimeric genes encoding CBH II enzymes were cloned into yeast expression vector YEp352/PGK91-1-.alpha.ss and expression in synthetic dextrose casamino acids (SDCAA) media. For Avicel activity assays, yeast peptone dextrose (YPD) culture supernatants were brought to 1 mM phenylmethylsulfonylfluoride and 0.02% NaN.sub.3 and used without concentration. CBH II enzyme activity in concentrated SDCAA yeast culture supernatants was measured by adding dilutions of concentrated culture supernatant to 37.5 .mu.L PASC and 225 .mu.L 50 mM sodium acetate, pH 4.8 and incubating for 2 hr at 50.degree. C. Reducing sugar equivalents formed were determined via Nelson-Somogyi assay.

[0152] CBH II enzyme T.sub.50 values were measured by adding concentrated CBH II SDCAA expression culture supernatant to 50 mM sodium acetate, pH 4.8 at a concentration giving A.sub.520 of 0.5 as measured in the Nelson-Somogyi reducing sugar assay after incubation with endoglucanase-treated PASC. 200 .mu.L CBH II enzyme/buffer mixtures were incubated in a water bath at the temperature of interest for 10 minutes. After incubation, 37.5 .mu.L endoglucanase-treated PASC and 62.5 .mu.L of 50 mM sodium acetate were added, and hydrolysis was carried out for 2 hr at 50.degree. C. The incubation temperature at which the enzyme lost one-half of its activity was determined by linear interpolation of the Nelson-Somogyi assay A.sub.520 values plotted versus temperature.

[0153] For long-time Avicel PH101 (Fluka) hydrolysis measurements, 0.3 .mu.g of purified CBH II was incubated with 3 mg of Avicel in 270 .mu.L of 50 mM sodium acetate, pH 4.8, in PCR tubes placed in a water bath for 16 hours. Tubes were cooled in a room temperature water bath for 10 minutes, centrifuged at 1000 g for 10 minutes and supernatants withdrawn for reducing sugar analysis.

[0154] For estimation of CBH II activity in YPD expression culture supernatants, supernatant volumes ranging from 2 mL to 40 mL were added to 800 .mu.L of 33 mg/mL Avicel suspended in 50 mM sodium acetate, pH 4.8 in conical tubes. CBH IIs were allowed to bind Avicel at 4.degree. C. for one hour, centrifuged at 2000 g for 2 minutes and washed twice with 50 mM sodium acetate, pH 4.8. After the second wash, CBH II-bound Avicel was resuspended in 2.75 mL of sodium acetate buffer, split into 270 .mu.L aliquots and incubated at 50.degree. C. for 2.5 hours. Centrifugation and supernatant reducing sugar analysis were carried out as above.

[0155] The Linear Regression package in Mathematica was used to fit CBH II chimera T.sub.50 data to a 17-parameter, block additive model and was also used for cross validation analysis. Block effects are reported relative to a parent 1 (H. insolens CBH II) reference state with 16 parameters representing substitution of each of the 8 blocks from parents 2 and 3.

[0156] Values of T.sub.50, defined here as the temperature at which an enzyme loses 50% of its activity during a ten-minute incubation, were determined for the three parent cellobiohydrolases, 33 active CBH II chimeras from prior experiments and 18 additional chimeras that qualitative stability modeling predicted to be among the most thermostable, i.e. containing none of the 7 predicted destabilizing blocks and either 3 or 4 of the 4 predicted stabilizing blocks. All 51 chimera sequences are listed in Table 8. Re-culturing and re-concentrating all of the predicted thermostable chimeras previously classified as not secreted allowed for the obtaining of sufficient amounts of 12112132, 13111132 and 13322332 CBH IIs for T.sub.50 determination. The complete set of T.sub.50 values for the chimeras and parent CBH IIs is provided in Table 8. The amino acid sequences for all these CBH IIs appear in Table 7. All 31 predicted thermostable chimeras tested have T.sub.50 values more than two degrees higher than that of the most thermostable parent enzyme (64.8.degree. C.). The table also identifies the Cys residue in block/domain 7 that can be mutated to a Ser to provide increased thermostability. Accordingly, the disclosure provides polypeptide of any of the following sequences wherein the underlined/italicized/bold Cys is substituted with a Ser residue and wherein the resulting polypeptide has improved thermostability compared to a wild-type enzyme.

TABLE-US-00010 TABLE 7 Amino acid sequences for CBH II parent and chimera catalytic domains shown in Table 8. Table also includes catalytic domain for P. chrysosporium CBH II. All recombinant CBH IIs share the N-terminal CBM and linker from the native H. jecorina CBH II, CSSVWGQCGGQNWSGPTCCASGSTCVYSNDYYSQCLPGAASSSSSTRAASTTSRVSPTTSRSSSATPP PGSTTTRVPPVGSGTATYS (SEQ ID NO: 8). Parent 1 (H. insolens) (SEQ ID NO: 2) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFQWLDRNVTVDTLLVQTLSEIREA NQAGANPQYAAQIVVYDLPDRDCAAAASNGEWAIANNGVNNYKAYINRIREILISFSDVRTILVIEPD SLANMVTNMNVPKCSGAASTYRELTIYALKQLDLPHVAMYMDAGHAGWLGWPANIQPAAELFAKIYED AGKPRAVRGLATNVANYNAWSVSSPPPYTSPNPNYDEKHYIEAFRPLLEARGFPAQFIVDQGRSGKQP TGQKEWGHWCNAIGTGFGMRPTANTGHQYVDAFVWVKPGGE DGTSDTTAARYDYHCGLEDALKPAPE AGQWFNEYFIQLLRNANPPF Parent 2 (H. jecorina) (SEQ ID NO: 4) GNPFVGVTPWANAYYASEVSSLAIPSLTGAMATAAAAVAKVPSFMWLDTLDKTPLMEQTLADIRTANK NGGNYAGQFVVYDLPDRDCAALASNGEYSIADGGVAKYKNYIDTIRQIVVEYSDIRTLLVIEPDSLAN LVTNLGTPKCANAQSAYLECINYAVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANVYKNASSP RALRGLATNVANYNGWNITSPPSYTQGNAVYNEKLYIHAIGPLLANHGWSNAFFITDQGRSGKQPTGQ QQWGDWCNVIGTGFGIRPSANTGDSLLDSFVWVKPGGE DGTSDSSAPRFDSHCALPDALQPAPQAGA WFQAYFVQLLTNANPSFL Parent 3 (C. thermophilum) (SEQ ID NO: 6) GNPFSGVQLWANTYYSSEVHTLAIPSLSPELAAKAAKVAEVPSFQWLDRNVTVDTLFSGTLAEIRAAN QRGANPPYAGIFVVYDLPDRDCAAAASNGEWSIANNGANNYKRYIDRIRELLIQYSDIRTILVIEPDS LANMVTNMNVQKCSNAASTYKELTVYALKQLNLPHVAMYMDAGHAGWLGWPANIQPAAELFAQIYRDA GRPAAVRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAKFIVDTGRNGKQPT GQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDTSAARYDYHCGLSDALTPAPEA GQWFQAYFEQLLINANPPF P. chrysosporium CBH II (SEQ ID NO: 12) NNPWTGFQIFLSPYYANEVAAAAKQITDPTLSSKAASVANIPTFTWLDSVAKIPDLGTYLASASALGK STGTKQLVQIVIYDLPDRDCAAKASNGEFSIANNGQANYENYIDQIVAQIQQFPDVRVVAVIEPDSLA NLVTNLNVQKCANAKTTYLACVNYALTNLAKVGVYMYMDAGHAGWLGWPANLSPAAQLFTQVWQNAGK SPFIKGLATNVANYNALQAASPDPITQGNPNYDEIHYINALAPLLQQAGWDATFIVDQGRSGVQNIRQ QWGDWCNIKGAGFGTRPTTNTGSQFIDSIVWVKPGGE DGTSNSSSPRYDSTCSLPDAAQPAPEAGTW FQAYFQTLVSAANPPL 32333113 (SEQ ID NO: 13) GNPFSGVQLWANTYYSSEVHTLAIPSLSPELAAKAAKVAEVPSFMWLDTLDKTPLMEQTLADIRTANK NGANPPYAGIFVVYDLPDRDCAAAASNGEWSIANNGANNYKRYIDRIRELLIQYSDIRTILVIEPDSL ANMVTNMNVQKCSNAASTYKELTVYALKQLNLPHVAMYMDAGHAGWLGWPANIQPAAELFAQIYRDAG RPAAVRGLATNVANYNAWSVSSPPPYTSPNPNYDEKHYIEAFRPLLEARGFPAQFIVDQGRSGKQPTG QKEWGHWCNAIGTGFGMRPTANTGHQYVDAFVWVKPGGE DGTSDTSAARYDYHCGLSDALTPAPEAG QWFQAYFEQLLINANPPF 13111313 (SEQ ID NO: 14) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFQWLDRNVTVDTLFSGTLAEIRAA NQRGANPQYAAQIVVYDLPDRDCAAAASNGEWAIANNGVNNYKAYINRIREILISFSDVRTILVIEPD SLANMVTNMNVPKCSGAASTYRELTIYALKQLDLPHVAMYMDAGHAGWLGWPANIQPAAELFAKIYED AGKPRAVRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAQFIVDQGRSGKQP TGQKEWGHWCNAIGTGFGMRPTANTGHQYVDAFVWVKPGGE DGTSDTSAARYDYHCGLSDALTPAPE AGQWFQAYFEQLLINANPPF 11313121 (SEQ ID NO: 15) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFQWLDRNVTVDTLLVQTLSEIREA NQAGANPPYAGIFVVYDLPDRDCAAAASNGEWSIANNGANNYKRYIDRIREILISFSDVRTILVIEPD SLANMVTNMNVPKCSGAASTYKELTVYALKQLNLPHVAMYMDAGHAGWLGWPANIQPAAELFAQIYRD AGRPAAVRGLATNVANYNAWSVSSPPPYTSPNPNYDEKHYIEAFRPLLEARGFPNAFFITDQGRSGKQ PTGQQQWGDWCNVIGTGFGIRPSANTGDSLLDSFVWVKPGGE DGTSDTTAARYDYHCGLEDALKPAP EAGQWFNEYFIQLLRNANPPF 21131311 (SEQ ID NO: 16) GNPFVGVTPWANAYYASEVSSLAIPSLTGAMATAAAAVAKVPSFQWLDRNVTVDTLLVQTLSEIREAN QAGANPQYAAQIVVYDLPDRDCAAAASNGEWAIANNGVNNYKAYINRIRELLIQYSDIRTILVIEPDS LANMVTNMNVQKCSNAASTYRELTIYALKQLDLPHVAMYMDAGHAGWLGWPANIQPAAELFAKIYEDA GKPRAVRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAQFIVDQGRSGKQPT GQKEWGHWCNAIGTGFGMRPTANTGHQYVDAFVWVKPGGE DGTSDTTAARYDYHCGLEDALKPAPEA GQWFNEYFIQLLRNANPPF 31212111 (SEQ ID NO: 17) GNPFSGVQLWANTYYSSEVHTLAIPSLSPELAAKAAKVAEVPSFQWLDRNVTVDTLLVQTLSEIREAN QAGGNYAGQFVVYDLPDRDCAALASNGEYSIADGGVAKYKNYIDRIREILISFSDVRTILVIEPDSLA NMVTNMNVPKCSGAASAYLECINYAVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANVYKNASS PRALRGLATNVANYNAWSVSSPPPYTSPNPNYDEKHYIEAFRPLLEARGFPAQFIVDQGRSGKQPTGQ KEWGHWCNAIGTGFGMRPTANTGHQYVDAFVWVKPGGE DGTSDTTAARYDYHCGLEDALKPAPEAGQ WFNEYFIQLLRNANPPF 23233133 (SEQ ID NO: 18) GNPFVGVTPWANAYYASEVSSLAIPSLTGAMATAAAAVAKVPSFQWLDRNVTVDTLFSGTLAEIRAAN QRGGNYAGQFVVYDLPDRDCAALASNGEYSIADGGVAKYKNYIDRIRELLIQYSDIRTILVIEPDSLA NMVTNMNVQKCSNAASTYKELTVYALKQLNLPHVAMYMDAGHAGWLGWPANIQPAAELFAQIYRDAGR PAAVRGLATNVANYNAWSVSSPPPYTSPNPNYDEKHYIEAFRPLLEARGFPAKFIVDTGRNGKQPTGQ LEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDTSAARYDYHCGLSDALTPAPEAGQ WFQAYFEQLLINANPPF 31311112 (SEQ ID NO: 19) GNPFSGVQLWANTYYSSEVHTLAIPSLSPELAAKAAKVAEVPSFQWLDRNVTVDTLLVQTLSEIREA NQAGANPPYAGIFVVYDLPDRDCAAAASNGEWSIANNGANNYKRYIDRIREILISFSDVRTILVIEPD SLANMVTNMNVPKCSGAASTYRELTIYALKQLDLPHVAMYMDAGHAGWLGWPANIQPAAELFAKIYED AGKPRAVRGLATNVANYNAWSVSSPPPYTSPNPNYDEKHYIEAFRPLLEARGFPAQFIVDQGRSGKQ PTGQKEWGHWCNAIGTGFGMRPTANTGHQYVDAFVWVKPGGE DGTSDSSAPRFDSHCALPDALQPAP QAGAWFQAYFVQLLTNANPSFL 22212231 (SEQ ID NO: 20) GNPFVGVTPWANAYYASEVSSLAIPSLTGAMATAAAAVAKVPSFMWLDTLDKTPLMEQTLADIRTANK NGGNYAGQFVVYDLPDRDCAALASNGEYSIADGGVAKYKNYIDRIREILISFSDVRTILVIEPDSLAN MVTNMNVPKCSGAASAYLECINYAVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANVYKNASSP RALRGLATNVANYNGWNITSPPSYTQGNAVYNEKLYIHAIGPLLANHGWSAKFIVDTGRNGKQPTGQ LEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDTTAARYDYHCGLEDALKPAPEAGQ WFNEYFIQLLRNANPPF 13231111 (SEQ ID NO: 21) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFQWLDRNVTVDTLFSGTLAEIRAA NQRGGNYAGQFVVYDLPDRDCAALASNGEYSIADGGVAKYKNYIDRIRELLIQYSDIRTILVIEPDSL ANMVTNMNVQKCSNAASTYRELTIYALKQLDLPHVAMYMDAGHAGWLGWPANIQPAAELFAKIYEDAG KPRAVRGLATNVANYNAWSVSSPPPYTSPNPNYDEKHYIEAFRPLLEARGFPAQFIVDQGRSGKQPTG QKEWGHWCNAIGTGFGMRPTANTGHQYVDAFVWVKPGGE DGTSDTTAARYDYHCGLEDALKPAPEAG QWFNEYFIQLLRNANPPF 12213111 (SEQ ID NO: 22) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFMWLDTLDKTPLMEQTLADIRTAN KNGGNYAGQFVVYDLPDRDCAALASNGEYSIADGGVAKYKNYIDRIREILISFSDVRTILVIEPDSLA NMVTNMNVPKCSGAASTYKELTVYALKQLNLPHVAMYMDAGHAGWLGWPANIQPAAELFAQIYRDAGR PAAVRGLATNVANYNAWSVSSPPPYTSPNPNYDEKHYIEAFRPLLEARGFPAQFIVDQGRSGKQPTGQ KEWGHWCNAIGTGFGMRPTANTGHQYVDAFVWVKPGGE DGTSDTTAARYDYHCGLEDALKPAPEAGQ WFNEYFIQLLRNANPPF 12133333 (SEQ ID NO: 23) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFMWLDTLDKTPLMEQTLADIRTAN KNGANPQYAAQIVVYDLPDRDCAAAASNGEWAIANNGVNNYKAYINRIRELLIQYSDIRTILVIEPDS LANMVTNMNVQKCSNAASTYKELTVYALKQLNLPHVAMYMDAGHAGWLGWPANIQPAAELFAQIYRDA GRPAAVRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAKFIVDTGRNGKQPT GQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDTSAARYDYHCGLSDALTPAPEA GQWFQAYFEQLLINANPPF 33133132 (SEQ ID NO: 24) GNPFSGVQLWANTYYSSEVHTLAIPSLSPELAAKAAKVAEVPSFQWLDRNVTVDTLFSGTLAEIRAAN QRGANPQYAAQIVVYDLPDRDCAAAASNGEWAIANNGVNNYKAYINRIRELLIQYSDIRTILVIEPDS LANMVTNMNVQKCSNAASTYKELTVYALKQLNLPHVAMYMDAGHAGWLGWPANIQPAAELFAQIYRDA GRPAAVRGLATNVANYNAWSVSSPPPYTSPNPNYDEKHYIEAFRPLLEARGFPAKFIVDTGRNGKQPT GQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAPQA GAWFQAYFVQLLTNANPSFL 11332333 (SEQ ID NO: 25) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFQWLDRNVTVDTLLVQTLSEIREA NQAGANPPYAGIFVVYDLPDRDCAAAASNGEWSIANNGANNYKRYIDRIRELLIQYSDIRTILVIEPD SLANMVTNMNVQKCSNAASAYLECINYAVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANVYKN ASSPRALRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAKFIVDTGRNGKQP TGQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDTSAARYDYHCGLSDALTPAPE AGQWFQAYFEQLLINANPPF 23311333 (SEQ ID NO: 26) GNPFVGVTPWANAYYASEVSSLAIPSLTGAMATAAAAVAKVPSFQWLDRNVTVDTLFSGTLAEIRAAN QRGANPPYAGIFVVYDLPDRDCAAAASNGEWSIANNGANNYKRYIDRIREILISFSDVRTILVIEPDS LANMVTNMNVPKCSGAASTYRELTIYALKQLDLPHVAMYMDAGHAGWLGWPANIQPAAELFAKIYEDA GKPRAVRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAKFIVDTGRNGKQPT GQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDTSAARYDYHCGLSDALTPAPEA GQWFQAYFEQLLINANPPF 33213332 (SEQ ID NO: 27) GNPFSGVQLWANTYYSSEVHTLAIPSLSPELAAKAAKVAEVPSFQWLDRNVTVDTLFSGTLAEIRAAN QRGGNYAGQFVVYDLPDRDCAALASNGEYSIADGGVAKYKNYIDRIREILISFSDVRTILVIEPDSLA NMVTNMNVPKCSGAASTYKELTVYALKQLNLPHVAMYMDAGHAGWLGWPANIQPAAELFAQIYRDAGR PAAVRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAKFIVDTGRNGKQPTGQ LEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAPQAGA WFQAYFVQLLTNANPSFL 13333232 (SEQ ID NO: 28) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFQWLDRNVTVDTLFSGTLAEIRAA NQRGANPPYAGIFVVYDLPDRDCAAAASNGEWSIANNGANNYKRYIDRIRELLIQYSDIRTILVIEPD SLANMVTNMNVQKCSNAASTYKELTVYALKQLNLPHVAMYMDAGHAGWLGWPANIQPAAELFAQIYRD AGRPAAVRGLATNVANYNGWNITSPPSYTQGNAVYNEKLYIHAIGPLLANHGWS_AKFIVDTGRNGKQ PTGQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAP QAGAWFQAYFVQLLTNANPSFL 22232132 (SEQ ID NO: 29) GNPFVGVTPWANAYYASEVSSLAIPSLTGAMATAAAAVAKVPSFMWLDTLDKTPLMEQTLADIRTANK NGGNYAGQFVVYDLPDRDCAALASNGEYSIADGGVAKYKNYIDRIRELLIQYSDIRTILVIEPDSLAN MVTNMNVQKCSNAASAYLECINYAVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANVYKNASSP RALRGLATNVANYNAWSVSSPPPYTSPNPNYDEKHYIEAFRPLLEARGFPAKFIVDTGRNGKQPTGQL EWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAPQAGAW FQAYFVQLLTNANPSFL 11113132 (SEQ ID NO: 30) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFQWLDRNVTVDTLLVQTLSEIREA NQAGANPQYAAQIVVYDLPDRDCAAAASNGEWAIANNGVNNYKAYINRIREILISFSDVRTILVIEPD SLANMVTNMNVPKCSGAASTYKELTVYALKQLNLPHVAMYMDAGHAGWLGWPANIQPAAELFAQIYRD AGRPAAVRGLATNVANYNAWSVSSPPPYTSPNPNYDEKHYIEAFRPLLEARGFPAKFIVDTGRNGKQP TGQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAPQ AGAWFQAYFVQLLTNANPSFL 21333331 (SEQ ID NO: 31) GNPFVGVTPWANAYYASEVSSLAIPSLTGAMATAAAAVAKVPSFQWLDRNVTVDTLLVQTLSEIREAN QAGANPPYAGIFVVYDLPDRDCAAAASNGEWSIANNGANNYKRYIDRIRELLIQYSDIRTILVIEPDS LANMVTNMNVQKCSNAASTYKELTVYALKQLNLPHVAMYMDAGHAGWLGWPANIQPAAELFAQIYRDA GRPAAVRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAKFIVDTGRNGKQPT GQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDTTAARYDYHCGLEDALKPAPEA GQWFNEYFIQLLRNANPPF 21311131 (SEQ ID NO: 32) GNPFVGVTPWANAYYASEVSSLAIPSLTGAMATAAAAVAKVPSFQWLDRNVTVDTLLVQTLSEIREAN QAGANPPYAGIFVVYDLPDRDCAAAASNGEWSIANNGANNYKRYIDRIREILISFSDVRTILVIEPDS LANMVTNMNVPKCSGAASTYRELTIYALKQLDLPHVAMYMDAGHAGWLGWPANIQPAAELFAKIYEDA GKPRAVRGLATNVANYNAWSVSSPPPYTSPNPNYDEKHYIEAFRPLLEARGFPAKFIVDTGRNGKQPT GQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDTTAARYDYHCGLEDALKPAPEA GQWFNEYFIQLLRNANPPF 12332331 (SEQ ID NO: 33) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFMWLDTLDKTPLMEQTLADIRTAN KNGANPPYAGIFVVYDLPDRDCAAAASNGEWSIANNGANNYKRYIDRIRELLIQYSDIRTILVIEPDS LANMVTNMNVQKCSNAASAYLECINYAVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANVYKNA SSPRALRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAKFIVDTGRNGKQPT GQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDTTAARYDYHCGLEDALKPAPEA GQWFNEYFIQLLRNANPPF 13112332 (SEQ ID NO: 34) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFQWLDRNVTVDTLFSGTLAEIRAA NQRGANPQYAAQIVVYDLPDRDCAAAASNGEWAIANNGVNNYKAYINRIREILISFSDVRTILVIEPD SLANMVTNMNVPKCSGAASAYLECINYAVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANVYKN ASSPRALRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAKFIVDTGRNGKQP TGQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAPQ AGAWFQAYFVQLLTNANPSFL 22311331 (SEQ ID NO: 35) GNPFVGVTPWANAYYASEVSSLAIPSLTGAMATAAAAVAKVPSFMWLDTLDKTPLMEQTLADIRTANK NGANPPYAGIFVVYDLPDRDCAAAASNGEWSIANNGANNYKRYIDRIREILISFSDVRTILVIEPDSL ANMVTNMNVPKCSGAASTYRELTIYALKQLDLPHVAMYMDAGHAGWLGWPANIQPAAELFAKIYEDAG KPRAVRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAKFIVDTGRNGKQPTG QLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDTTAARYDYHCGLEDALKPAPEAG QWFNEYFIQLLRNANPPF 12111332 (SEQ ID NO: 36) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFMWLDT_LDKTPLMEQTLADIRTA NKNGANPQYAAQIVVYDLPDRDCAAAASNGEWAIANNGVNNYKAYINRIREILISFSDVRTILVIEPD SLANMVTNMNVPKCSGAASTYRELTIYALKQLDLPHVAMYMDAGHAGWLGWPANIQPAAELFAKIYED AGKPRAVRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFD_AKFIVDTGRNGKQ PTGQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAP QAGAWFQAYFVQLLTNANPSFL 12112332 (SEQ ID NO: 37) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFMWLDT_LDKTPLMEQTLADIRTA NKNGANPQYAAQIVVYDLPDRDCAAAASNGEWAIANNGVNNYKAYINRIREILISFSDVRTILVIEPD SLANMVTNMNVPKCSGAASAYLECINYAVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANVYKN ASSPRALRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDA_KFIVDTGRNGKQ PTGQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAP QAGAWFQAYFVQLLTNANPSFL 12131331 (SEQ ID NO: 38) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFMWLDTLDKTPLMEQTLADIRTAN KNGANPQYAAQIVVYDLPDRDCAAAASNGEWAIANNGVNNYKAYINRIRELLIQYSDIRTILVIEPDS LANMVTNMNVQKCSNAASTYRELTIYALKQLDLPHVAMYMDAGHAGWLGWPANIQPAAELFAKIYEDA GKPRAVRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAKFIVDTGRNGKQPT GQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDTTAARYDYHCGLEDALKPAPEA GQWFNEYFIQLLRNANPPF

12131332 (SEQ ID NO: 39) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFMWLDTLDKTPLMEQTLADIRTAN KNGANPQYAAQIVVYDLPDRDCAAAASNGEWAIANNGVNNYKAYINRIRELLIQYSDIRTILVIEPDS LANMVTNMNVQKCSNAASTYRELTIYALKQLDLPHVAMYMDAGHAGWLGWPANIQPAAELFAKIYEDA GKPRAVRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAKFIVDTGRNGKQPT GQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAPQA GAWFQAYFVQLLTNANPSFL 12332332 (SEQ ID NO: 40) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFMWLDTLDKTPLMEQTLADIRTAN KNGANPPYAGIFVVYDLPDRDCAAAASNGEWSIANNGANNYKRYIDRIRELLIQYSDIRTILVIEPDS LANMVTNMNVQKCSNAASAYLECINYAVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANVYKNA SSPRALRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAKFIVDTGRNGKQPT GQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAPQA GAWFQAYFVQLLTNANPSFL 12111131 (SEQ ID NO: 41) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFMWLDTLDKTPLMEQTLADIRTAN KNGANPQYAAQIVVYDLPDRDCAAAASNGEWAIANNGVNNYKAYINRIREILISFSDVRTILVIEPDS LANMVTNMNVPKCSGAASTYRELTIYALKQLDLPHVAMYMDAGHAGWLGWPANIQPAAELFAKIYEDA GKPRAVRGLATNVANYNAWSVSSPPPYTSPNPNYDEKHYIEAFRPLLEARGFPAKFIVDTGRNGKQPT GQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDTTAARYDYHCGLEDALKPAPEA GQWFNEYFIQLLRNANPPF 12311332 (SEQ ID NO: 42) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFMWLDTLDKTPLMEQTLADIRTAN KNGANPPYAGIFVVYDLPDRDCAAAASNGEWSIANNGANNYKRYIDRIREILISFSDVRTILVIEPDS LANMVTNMNVPKCSGAASTYRELTIYALKQLDLPHVAMYMDAGHAGWLGWPANIQPAAELFAKIYEDA GKPRAVRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAKFIVDTGRNGKQPT GQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAPQA GAWFQAYFVQLLTNANPSFL 13332331 (SEQ ID NO: 43) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFQWLDRNVTVDTLFSGTLAEIRAA NQRGANPPYAGIFVVYDLPDRDCAAAASNGEWSIANNGANNYKRYIDRIRELLIQYSDIRTILVIEPD SLANMVTNMNVQKCSNAASAYLECINYAVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANVYKN ASSPRALRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAKFIVDTGRNGKQP TGQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDTTAARYDYHCGLEDALKPAPE AGQWFNEYFIQLLRNANPPF 12132331 (SEQ ID NO: 44) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFMWLDTLDKTPLMEQTLADIRTAN KNGANPQYAAQIVVYDLPDRDCAAAASNGEWAIANNGVNNYKAYINRIRELLIQYSDIRTILVIEPDS LANMVTNMNVQKCSNAASAYLECINYAVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANVYKNA SSPRALRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAKFIVDTGRNGKQPT GQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDTTAARYDYHCGLEDALKPAPEA GQWFNEYFIQLLRNANPPF 12132332 (SEQ ID NO: 45) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFMWLDTLDKTPLMEQTLADIRTAN KNGANPQYAAQIVVYDLPDRDCAAAASNGEWAIANNGVNNYKAYINRIRELLIQYSDIRTILVIEPDS LANMVTNMNVQKCSNAASAYLECINYAVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANVYKNA SSPRALRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAKFIVDTGRNGKQPT GQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAPQA GAWFQAYFVQLLTNANPSFL 13332332 (SEQ ID NO: 46) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFQWLDRNVTVDTLFSGTLAEIRAA NQRGANPPYAGIFVVYDLPDRDCAAAASNGEWSIANNGANNYKRYIDRIRELLIQYSDIRTILVIEPD SLANMVTNMNVQKCSNAASAYLECINYAVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANVYKN ASSPRALRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAKFIVDTGRNGKQP TGQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAPQ AGAWFQAYFVQLLTNANPSFL 12112132 (SEQ ID NO: 47) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFMWLDTLDKTPLMEQTLADIRTAN KNGANPQYAAQIVVYDLPDRDCAAAASNGEWAIANNGVNNYKAYINRIREILISFSDVRTILVIEPDS LANMVTNMNVPKCSGAASAYLECINYAVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANVYKNA SSPRALRGLATNVANYNAWSVSSPPPYTSPNPNYDEKHYIEAFRPLLEARGFPAKFIVDTGRNGKQPT GQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAPQA GAWFQAYFVQLLTNANPSFL 13322332 (SEQ ID NO:48) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFQWLDRNVTVDTLFSGTLAEIRAA NQRGANPPYAGIFVVYDLPDRDCAAAASNGEWSIANNGANNYKRYIDTIRQIVVEYSDIRTLLVIEPD SLANLVTNLGTPKCANAQSAYLECINYAVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANVYKN ASSPRALRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAKFIVDTGRNGKQP TGQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAPQ AGAWFQAYFVQLLTNANPSFL 13131332 (SEQ ID NO: 49) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFQWLDRNVTVDTLFSGTLAEIRAA NQRGANPQYAAQIVVYDLPDRDCAAAASNGEWAIANNGVNNYKAYINRIRELLIQYSDIRTILVIEPD SLANMVTNMNVQKCSNAASTYRELTIYALKQLDLPHVAMYMDAGHAGWLGWPANIQPAAELFAKIYED AGKPRAVRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAKFIVDTGRNGKQP TGQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAPQ AGAWFQAYFVQLLTNANPSFL 12331332 (SEQ ID NO: 50) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFMWLDTLDKTPLMEQTLADIRTAN KNGANPPYAGIFVVYDLPDRDCAAAASNGEWSIANNGANNYKRYIDRIRELLIQYSDIRTILVIEPDS LANMVTNMNVQKCSNAASTYRELTIYALKQLDLPHVAMYMDAGHAGWLGWPANIQPAAELFAKIYEDA GKPRAVRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAKFIVDTGRNGKQPT GQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAPQA GAWFQAYFVQLLTNANPSFL 13312332 (SEQ ID NO: 51) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFQWLDRNVTVDTLFSGTLAEIRAA NQRGANPPYAGIFVVYDLPDRDCAAAASNGEWSIANNGANNYKRYIDRIREILISFSDVRTILVIEPD SLANMVTNMNVPKCSGAASAYLECINYAVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANVYKN ASSPRALRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAKFIVDTGRNGKQP TGQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAPQ AGAWFQAYFVQLLTNANPSFL 11113332 (SEQ ID NO: 52) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFQWLDRNVTVDTLLVQTLSEIREA NQAGANPQYAAQIVVYDLPDRDCAAAASNGEWAIANNGVNNYKAYINRIREILISFSDVRTILVIEPD SLANMVTNMNVPKCSGAASTYKELTVYALKQLNLPHVAMYMDAGHAGWLGWPANIQPAAELFAQIYRD AGRPAAVRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAKFIVDTGRNGKQP TGQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAPQ AGAWFQAYFVQLLTNANPSFL 13113132 (SEQ ID NO: 53) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFQWLDRNVTVDTLFSGTLAEIRAA NQRGANPQYAAQIVVYDLPDRDCAAAASNGEWAIANNGVNNYKAYINRIREILISFSDVRTILVIEPD SLANMVTNMNVPKCSGAASTYKELTVYALKQLNLPHVAMYMDAGHAGWLGWPANIQPAAELFAQIYRD AGRPAAVRGLATNVANYNAWSVSSPPPYTSPNPNYDEKHYIEAFRPLLEARGFPAKFIVDTGRNGKQP TGQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAPQ AGAWFQAYFVQLLTNANPSFL 11112132 (SEQ ID NO: 54) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFQWLDRNVTVDTLLVQTLSEIREA NQAGANPQYAAQIVVYDLPDRDCAAAASNGEWAIANNGVNNYKAYINRIREILISFSDVRTILVIEPD SLANMVTNMNVPKCSGAASAYLECINYAVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANVYKN ASSPRALRGLATNVANYNAWSVSSPPPYTSPNPNYDEKHYIEAFRPLLEARGFPAKFIVDTGRNGKQ PTGQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAP QAGAWFQAYFVQLLTNANPSFL 12113132 (SEQ ID NO: 55) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFMWLDTLDKTPLMEQTLADIRTAN KNGANPQYAAQIVVYDLPDRDCAAAASNGEWAIANNGVNNYKAYINRIREILISFSDVRTILVIEPDS LANMVTNMNVPKCSGAASTYKELTVYALKQLNLPHVAMYMDAGHAGWLGWPANIQPAAELFAQIYRDA GRPAAVRGLATNVANYNAWSVSSPPPYTSPNPNYDEKHYIEAFRPLLEARGFPAKFIVDTGRNGKQPT GQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAPQA GAWFQAYFVQLLTNANPSFL 13132332 (SEQ ID NO: 56) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFQWLDRNVTVDTLFSGTLAEIRAA NQRGANPQYAAQIVVYDLPDRDCAAAASNGEWAIANNGVNNYKAYINRIRELLIQYSDIRTILVIEPD SLANMVTNMNVQKCSNAASAYLECINYAVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANVYKN ASSPRALRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAKFIVDTGRNGKQP TGQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAPQ AGAWFQAYFVQLLTNANPSFL 11111132 (SEQ ID NO: 57) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFQWLDRNVTVDTLLVQTLSEIREA NQAGANPQYAAQIVVYDLPDRDCAAAASNGEWAIANNGVNNYKAYINRIREILISFSDVRTILVIEPD SLANMVTNMNVPKCSGAASTYRELTIYALKQLDLPHVAMYMDAGHAGWLGWPANIQPAAELFAKIYED AGKPRAVRGLATNVANYNAWSVSSPPPYTSPNPNYDEKHYIEAFRPLLEARGFPAKFIVDTGRNGKQP TGQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAPQ AGAWFQAYFVQLLTNANPSFL 13331332 (SEQ ID NO: 58) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFQWLDRNVTVDTLFSGTLAEIRAA NQRGANPPYAGIFVVYDLPDRDCAAAASNGEWSIANNGANNYKRYIDRIRELLIQYSDIRTILVIEPD SLANMVTNMNVQKCSNAASTYRELTIYALKQLDLPHVAMYMDAGHAGWLGWPANIQPAAELFAKIYED AGKPRAVRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAKFIVDTGRNGKQP TGQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAPQ AGAWFQAYFVQLLTNANPSFL 13111132 (SEQ ID NO: 59) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFQWLDRNVTVDTLFSGTLAEIRAA NQRGANPQYAAQIVVYDLPDRDCAAAASNGEWAIANNGVNNYKAYINRIREILISFSDVRTILVIEPD SLANMVTNMNVPKCSGAASTYRELTIYALKQLDLPHVAMYMDAGHAGWLGWPANIQPAAELFAKIYED AGKPRAVRGLATNVANYNAWSVSSPPPYTSPNPNYDEKHYIEAFRPLLEARGFPAKFIVDTGRNGKQ PTGQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAP QAGAWFQAYFVQLLTNANPSFL 12222132 (SEQ ID NO: 60) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFMWLDTLDKTPLMEQTLADIRTAN KNGGNYAGQFVVYDLPDRDCAALASNGEYSIADGGVAKYKNYIDTIRQIVVEYSDIRTLLVIEPDSLA NLVTNLGTPKCANAQSAYLECINYAVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANVYKNASS PRALRGLATNVANYNAWSVSSPPPYTSPNPNYDEKHYIEAFRPLLEARGFPAKFIVDTGRNGKQPTGQ LEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAPQAGA WFQAYFVQLLTNANPSFL 12222332 (SEQ ID NO: 61) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFMWLDTLDKTPLMEQTLADIRTAN KNGGNYAGQFVVYDLPDRDCAALASNGEYSIADGGVAKYKNYIDTIRQIVVEYSDIRTLLVIEPDSLA NLVTNLGTPKCANAQSAYLECINYAVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANVYKNASS PRALRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAKFIVDTGRNGKQPTGQ LEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAPQAGA WFQAYFVQLLTNANPSFL 13311332 (SEQ ID NO: 62) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFQWLDRNVTVDTLFSGTLAEIRAA NQRGANPPYAGIFVVYDLPDRDCAAAASNGEWSIANNGANNYKRYIDRIREILISFSDVRTILVIEPD SLANMVTNMNVPKCSGAASTYRELTIYALKQLDLPHVAMYMDAGHAGWLGWPANIQPAAELFAKIYED AGKPRAVRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDAKFIVDTGRNGKQP TGQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDSSAPRFDSHCALPDALQPAPQ AGAWFQAYFVQLLTNANPSFL 13311331 (SEQ ID NO: 63) GNPFEGVQLWANNYYRSEVHTLAIPQITDPALRAAASAVAEVPSFQWLDRNVTVDTLFSGTLAEIRAA NQRGANPPYAGIFVVYDLPDRDCAAAASNGEWSIANNGANNYKRYIDRIREILISFSDVRTILVIEPD SLANMVTNMNVPKCSGAASTYRELTIYALKQLDLPHVAMYMDAGHAGWLGWPANIQPAAELFAKIYED AGKPRAVRGLATNVANYNAWSIASPPSYTSPNPNYDEKHYIEAFAPLLRNQGFDA_KFIVDTGRNGKQ PTGQLEWGHWCNVKGTGFGVRPTANTGHELVDAFVWVKPGGESDGTSDTTAARYDYHCGLEDALKPAP EAGQWFNEYFIQLLRNANPPF

TABLE-US-00011 TABLE 8 Two independent duplicate T.sub.50 values (.degree. C.) for parent CBH IIs, 23 original sample set CBH II chimeras and predicted thermostable CBH II chimeras. The 18 chimeras synthesized for this work are preceded by an asterisk. Sample Set Chimeras & Parents Predicted Thermostable Chimeras Sequence T.sub.50 (1) T.sub.50 (2) MeanT.sub.50 Sequence T.sub.50 (1) T.sub.50 (2) MeanT.sub.50 32333113 52 51 51.5 12332331 66.5 67 66.8 13111313 56 53.5 54.8 *13112332 67 67 67 11313121 55 55.5 55.3 22311331 68 68 68 21131311 57.5 57 57.3 *12111332 68 68 68 31212111 59 58 58.5 *12112332 68.5 67.5 68 Parent 2 60 58 59 12131331 68.5 69 68.8 23233133 61 61 61 *12131332 70 67.5 68.8 31311112 60 62 61 *12332332 69 69 69 22212231 63 61 62 12111131 70 68.5 69.3 13231111 63 6.5 63.3 12311332 70 69 69.5 12213111 63 63.5 63.3 13332331 70 69 69.5 Parent 3 63.5 64.5 64 12132331 70.5 69 69.8 12133333 64 64 64 *12132332 70.5 69 69.8 Parent 1 64 65.5 64.8 *13332332 69.5 70 69.8 33133132 65 66 65 12112132 71 68.5 69.8 11332333 64.5 66 65.3 13322332 71 68.5 69.8 23311333 65 66 65.5 *13131332 70 70 70 33213332 66 66 66 *12331332 71 69 70 13333232 67.5 67 67.3 *13312332 70 70 70 22232132 68 68 68 *11113332 69.5 70.5 70 11113132 71.5 71 71.3 *13113132 70.5 69.5 70 21333331 73.5 75.5 74.5 *11112132 70.5 70 70.3 21311131 75.5 75.5 75 *12113132 70.5 70.5 70.5 *13132332 69.5 71.5 70.5 *11111132 71 70.5 70.8 13331332 72 70 71 *13111132 72 69.5 71.3 *12222132 72.5 70 71.3 12222332 72 69.5 71.3 13311332 71 71.5 71.7 13311331 73.5 72.5 73

[0157] Applying linear regression to the sequence-stability data resulted in a ten-parameter model that fit the observed T.sub.50 values with R.sup.2=0.88 (FIG. 8). To better estimate the predictive capacity of the regression model outside the training set, an eleven-fold cross-validation was performed resulting in a R.sup.2 of 0.57, where removal of two outliers, (11313121 and 22222222) increases the cross validation R.sup.2 to 0.76. The regression model model uses the most stable parent 1 (H. insolens) as the reference state T.sub.50 and includes nine additional terms having p values 0.1. The model parameters (Table 9) show that a single block, block 7 from parent 3 (B7P3), is by far the strongest contributor to chimera thermostability relative to H. insolens CBH II. This block from C. thermophilum CBH II contributes approximately 8.5.degree. C. to the stability of chimeras that contain it. Two of the 8 remaining blocks with p values 0.1 were found to make smaller stability contributions, of 1.2.degree. C. and 2.7.degree. C., whereas the other six decrease stability.

TABLE-US-00012 TABLE 9 T.sub.50 linear regression model parameters and p-values. Parameter values with p .ltoreq. 0.1, used to calculate the regression fit line of FIG. 1, appear in bold. Block effects are reported relative to a parent 1 (H. insolens CBH II) reference state with 16 parameters representing substitution of each of the 8 blocks from parents 2 and 3. Parameter Block Value p-value Parent1 62.8 0.00 B12 -0.9 0.35 B13 -3.5 0.00 B22 -1.7 0.06 B23 -1.1 0.25 B32 0.5 0.68 B33 1.2 0.10 B42 2.7 0.05 B43 0.0 0.99 B52 -1.3 0.10 B53 -0.6 0.50 B62 -3.5 0.02 B63 -0.7 0.37 B72 -3.8 0.05 B73 8.5 0.00 B82 0.0 1.00 B83 -5.6 0.00

[0158] Alignment of the B7P1 and B7P3 sequences (FIG. 10) shows that block 7 differs at 10 out of 56 amino acid positions in the H. insolens and C. thermophilum enzymes. In the background of the chimera with the highest T.sub.50 value, 21311131, each residue in B7P3 (segment 7 of parent 3 (SEQ ID NO:6)) was individually mutated to the corresponding residue in B7P1 (segment 7 of parent 1 (SEQ ID NO:2)) and determined T.sub.50 values for each of the point mutants was obtained. A mutation, S313C, markedly altered the chimera's thermostability: this single mutation reduced the T.sub.50 of 21311131 by approximately 10.degree. C. (FIG. 11).

[0159] To study the effect of the reverse mutation in different backgrounds, genes for the H. insolens and H. jecorina parent CBH IIs encoding the C313S substitution (C314S in H. insolens and C311S in H. jecorina) were constructed, expressed, and the enzymes' T.sub.50 values were determined. The stabilities of chimeras 11111131 and 22222232, in which the stabilizing B7P3 is substituted into the wildtype H. insolens and H. jecorina enzymes were also quantified. Both the B7P3 block substitution and the Cys-Ser point mutation markedly stabilized the parent CBH IIs; the largest effect was a .about.8.degree. C. increase in T.sub.50 for H. jecorina CBH II containing the C311S substitution (FIG. 12). The Cys-Ser mutation was also tested in two chimeras, 31311112 and 13231111, that did not contain B7P3 as well as in a homologous CBH II (from Phanerochaete chrysosporium) which was not in the recombination parent set. The P. chrysosporium CBH II catalytic domain is only 55-56% identical to the parent CBH II catalytic domains. All of these enzymes were stabilized by the Cys-Ser substitution; the P. chrysosporium CBH II was stabilized by a remarkable 10.degree. C. (FIG. 13).

[0160] Eight of the thermostable CBH II chimeras and the parent enzymes containing the equivalent C313S mutation were His6-tagged and purified so that their specific activities could be determined. As shown in Table 10A, the specific activities, as measured on amorphous cellulose (PASC) at 50.degree. C., for these chimeras and native enzymes containing the Cys-Ser mutation are similar to those of the wildtype parents. Thus the increased thermostability does not come at the expense of specific activity.

TABLE-US-00013 TABLE 10A Specific activity values (.mu.g glucose reducing sugar equivalent/(.mu.g CBH II enzyme .times. min .times. 10.sup.2)) for native, point mutant and selected thermostable chimeric CBH IIs. Error bars show standard errors, where standard error is defined as standard dev/sqrt (n), for three replicates. 2-hr reaction, 3 mg enzyme/g PASC, 50.degree. C., 25 mM sodium acetate, pH 4.8. Specific Activity .mu.g Reducing Sugar/(.mu.g CBH II Enzyme Enzyme .times. min) .times. 10.sup.2 Humicola insolens (Parent 1) 5.3 +/- 0.5 Hypocrea jecorina (Parent 2) 8.4 +/- 0.4 Chaetomium thermophilum (Parent 3) 4.8 +/- 0.3 Phanerochaete chyrsosporium 7.7 +/- 0.3 Humicola insolens C314S 5.3 +/- 0.9 Hypocrea jecorina C311S 7.8 +/- 0.5 Phanerochaete chyrsosporium C311S 8.5 +/- 0.1 HJPlus (Chimera 12222332) 9.6 +/- 0.8 Chimera 13111132 8.5 +/- 0.3 Chimera 22222232 7.7 +/- 0.3 Chimera 13311332 6.8 +/- 0.6 Chimera 13311331 6.2 +/- 0.3 Chimera 11111131 6.1 +/- 0.9 Chimera 13112332 5.6 +/- 0.4 Chimera 21311131 5.5 +/- 0.3 Chimera 11113132 5.3 +/- 0.5 Chimera 21333331 3.8 +/- 0.4

TABLE-US-00014 TABLE 10B total activity in both synthetic (SDCAA) and rich (YPD) expression culture media supernatants for H. jecorina and H. insolens wild type, C313S point mutant and B7P3 block susbstitution CBH IIs. Values presented are .mu.g glucose/mL cellulase activity assay per mL of expression culture supernatant CBH II equivalent added to cellulase activity assay. For SDCAA cultures, concentrated SDCAA culture supernatants were used and activity toward phosphoric acid swollen cellulose (1 mg/mL) at 50.degree. C. for 100 minutes in 50 mM sodium acetate, pH 4.8, was measured. YPD supernatant CBH II was concentrated by binding to avicel and activity toward avicel (15 mg/mL) at 55.degree. C. for 150 minutes in 50 mM sodium acetate, pH 4.8, was measured. SDCAA SDCAA SDCAA YPD YPD CBH II (1) (2) Mean YPD (1) (2) Mean H. jecorina 19 17 18 0.4 0.4 0.4 H. jeco C311S 50 43 47 6.1 5.6 5.9 H. jeco B7P3 35 33 34 3.9 3.6 3.8 H. insolens 73 83 78 6.2 6.0 6.1 H. inso C314S 100 97 98 8.8 8.0 8.4 H. inso B7P3 39 42 40 4.4 4.1 4.2

[0161] These same eight thermostable chimeras (T.sub.50 2-10.degree. C. higher than the most stable parent) were then tested for activity on crystalline cellulose during a 16-hour incubation over a range of temperatures, including temperatures where the parent enzymes exhibit little or no activity. FIG. 9a shows that 7 of 8 tested thermostable chimeras were maximally active toward Avicel at 60-65.degree. C., with all 8 chimeras retaining activity at 70.degree. C., the highest temperature tested. In contrast, the three parent CBH IIs show maximum activity at 50.degree. C. and are either completely or almost completely inactive at 70.degree. C. Additionally, the seven chimeras with increased optimum activity temperatures hydrolyze significantly more Avicel than any of the parent CBH II enzymes. As shown in FIG. 9b, similar behaviors are observed for the H. insolens and H. jecorina parents containing the Cys-Ser point mutation. The Cys-Ser point mutation also increased the Avicel hydrolysis and maximum operating temperature for the P. chrysosporium CBH II. The P3B7 block substitution, which was made in the H. insolens and H. jecorina parents, increased both the operating temperature and hydrolysis of the H. insolens CBH II but, despite increasing maximum operating temperature, did not improve overall cellulose hydrolysis by the H. jecorina enzyme.

[0162] Low (<1 mg/L) secretion of wildtype H. jecorina CBH II was observed from the heterologous S. cerevisiae expression host. The C311S mutation in the wildtype H. jecorina CBH II enzyme markedly increases total secreted CBH II activity (Table 11). In synthetic (SDCAA) medium, the C311S and B7P3 substitutions increase H. jecorina CBH II total secreted activity by a factor of two, while in rich (YPD) medium the activity increase is tenfold. For the H. insolens CBH II parent, which is expressed at much higher levels than the other two parent CBH IIs, the C314S mutation increased secreted activity by a factor of .about.1.5 whereas the B7P3 block substitution decreased it. Because the H. insolens and H. jecorina wildtype and Cys-Ser mutants all have similar specific activities (Table 10), the increase in total secreted cellulase activity is the result of improved secretion of the functional enzyme. A correlation between S. cerevisiae heterologous protein secretion and protein stability has been observed, suggesting that the increased secretion of the Cys-Ser mutant CBH IIs might reflect their higher stabilities.

TABLE-US-00015 TABLE 11 Specific activity values (.mu.g glucose reducing sugar equivalent/(.mu.g CBH II enzyme .times. min .times. 10.sup.2)) for native, point mutant and selected thermostable chimeric CBH IIs. Error bars show standard errors, where standard error is defined as standard dev/sqrt (n), for three replicates. 2-hr reaction, 3 mg enzyme/g PASC, 50.degree. C., 25 mM sodium acetate, pH 4.8. Specific Activity .mu.g Reducing Sugar/(.mu.g CBH II Enzyme Enzyme .times. min) .times. 10.sup.2 Humicola insolens (Parent 1) 5.3 +/- 0.5 Hypocrea jecorina (Parent 2) 8.4 +/- 0.4 Chaetomium thermophilum (Parent 3) 4.8 +/- 0.3 Phanerochaete chyrsosporium 7.7 +/- 0.3 Humicola insolens C314S 5.3 +/- 0.9 Hypocrea jecorina C311S 7.8 +/- 0.5 Phanerochaete chyrsosporium C311S 8.5 +/- 0.1 HJPlus (Chimera 12222332) 9.6 +/- 0.8 Chimera 13111132 8.5 +/- 0.3 Chimera 22222232 7.7 +/- 0.3 Chimera 13311332 6.8 +/- 0.6 Chimera 13311331 6.2 +/- 0.3 Chimera 11111131 6.1 +/- 0.9 Chimera 13112332 5.6 +/- 0.4 Chimera 21311131 5.5 +/- 0.3 Chimera 11113132 5.3 +/- 0.5 Chimera 21333331 3.8 +/- 0.4

[0163] To model the Cys-Ser mutation, the high-resolution H. insolens CBH II loon crystal structure was used. First, the hydrogen bond network was optimized with REDUCE. Cys314 was predicted to form a hydrogen bond to the carbonyl of Pro 339. To confirm this prediction, sidechain packing was optimized using the modeling platform SHARPEN. Ser314 is predicted to make the similar interactions to Cys314, resulting in stronger hydrogen bonding and a more favorable geometry (FIG. 14).

[0164] A number of effects might explain why the Cys-Ser mutation stabilizes a broad range of CBH IIs, including native CBH IIs and chimeras. Cys and Ser are similar (though not isosteric), and these two amino acids dominate sequence alignments at this position compared to other alternatives. The hydrogen bonding partners for this residue are backbone elements (the amide of Gly316 and the carbonyl of Pro339 and are therefore less likely to be dependent on third-party amino acid variations. Furthermore, the immediate neighboring side chains for this pocket (Asn283, Pro339, Phe345) are conserved among all four native CBH II cellulases studied.

[0165] The high-resolution (1.3 .ANG.) H. insolens crystal structure (pdb entry 1ocn6) shows that Cys314 is part of a hydrogen bonding network (FIG. 15). The increased hydrogen bonding capacity of Ser relative to Cys may suggest a role for stronger hydrogen bonding interactions in the stabilization. The crystal structure also suggests that Ser may be preferred for steric reasons. Specifically, when the Cys side chain is rebuilt with canonical bond angles, a 6.degree. bend is removed and Cys is pushed closer to the carbonyl of Pro339, creating an unfavorable steric interaction.

[0166] An alignment of the 196 protein sequences sharing the greatest identity to the H. jecorina CBH II. Fifty-four of the 250 most identical sequences were excluded from the alignment due to redundancy (i.e. point mutants for structural studies or >95% identical isoforms). There is a bias in favor of Ser311: 158 sequences have Ser, 20 have Ala, 10 have Cys, 5 have a deletion, and 3 have Gly. However, there are 42 other positions where the most frequent choice occurs with greater than twice the frequency of the H. jecorina amino acid.

[0167] The large stabilizing effect of the Cys-Ser mutation raises the possibility that Ser at this position is a global indicator of native cellulase thermostability. However, the T.sub.50 of 64.8.degree. C. for H. insolens CBH II, which features Cys at this position, is greater than that of the C. thermophilum CBH II (64.0.degree. C.), indicating that Ser is not the only stability determinant.

[0168] Thermostability is not the only property of interest for industrial cellulases. Specific activity, changes to cellulose binding, and effects on expression and product inhibition are all important as well. The chimeras and data herein demonstrate that recombination yields CBH II chimeras whose improved thermostability comes without cost to specific activity measured in short-time (e.g., 2-hour) cellulose hydrolysis assays. Similar observations were made for CBH IIs containing the thermostabilizing Cys-Ser mutation. In long-time hydrolysis assays, several of the CBH II chimeras and all three tested Cys-Ser mutant CBH IIs hydrolyzed more cellulose than the native CBH IIs. This superior performance is likely the result of having specific activity comparable to that of the parent CBH IIs along with greater thermostability that allows the enzyme to continue to function for longer time at the elevated temperatures. Because these assays were carried out with equal amounts of purified parent, chimera and Cys-Ser mutant enzymes, the observed high temperature hydrolysis improvements are not the result of increased secretion from the S. cerevisiae expression host. The thermostable chimeras and the Cys-Ser mutants may therefore prove to be useful components of enzyme formulations for cellulose degradation.

[0169] While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention(s).

Sequence CWU 1

1

10711083DNAHumicola insolensCDS(1)..(1083) 1ggt aac ccc ttt gaa ggt gtt cag ctg tgg gct aat aac tat tat aga 48Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 tct gag gta cat aca ctg gcc att ccg caa att aca gac ccc gcg ttg 96Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 cgt gcc gca gct agt gct gtg gct gag gtg cca agt ttt caa tgg ctg 144Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Gln Trp Leu 35 40 45 gac aga aat gta aca gtg gat act ttg ttg gta cag act ttg tca gaa 192Asp Arg Asn Val Thr Val Asp Thr Leu Leu Val Gln Thr Leu Ser Glu 50 55 60 atc cgt gag gcc aat caa gca ggt gct aat ccc caa tat gca gcg caa 240Ile Arg Glu Ala Asn Gln Ala Gly Ala Asn Pro Gln Tyr Ala Ala Gln 65 70 75 80 atc gtg gtc tat gat ctg ccc gat aga gac tgt gca gct gcc gcc tcg 288Ile Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser 85 90 95 aat ggt gaa tgg gca ata gcg aac aac ggt gta aac aat tac aaa gct 336Asn Gly Glu Trp Ala Ile Ala Asn Asn Gly Val Asn Asn Tyr Lys Ala 100 105 110 tac att aat aga att aga gag ata ttg ata agt ttt tcg gac gtt aga 384Tyr Ile Asn Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg 115 120 125 acg ata tta gtc att gag cca gat agt cta gct aat atg gtc aca aat 432Thr Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn 130 135 140 atg aat gtc ccg aag tgt tcc ggt gca gcc agc act tat agg gaa tta 480Met Asn Val Pro Lys Cys Ser Gly Ala Ala Ser Thr Tyr Arg Glu Leu 145 150 155 160 acc ata tat gca ctg aag caa ttg gat ctg cct cat gtc gct atg tac 528Thr Ile Tyr Ala Leu Lys Gln Leu Asp Leu Pro His Val Ala Met Tyr 165 170 175 atg gat gcc ggc cac gct gga tgg tta ggc tgg ccg gca aac att cag 576Met Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln 180 185 190 cca gcc gca gaa ttg ttt gcc aaa att tac gaa gat gct gga aag cct 624Pro Ala Ala Glu Leu Phe Ala Lys Ile Tyr Glu Asp Ala Gly Lys Pro 195 200 205 aga gca gtg aga ggt ctt gca act aat gtt gct aat tac aat gca tgg 672Arg Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp 210 215 220 tca gtt tca tcc cct cca cca tac aca agt cca aat cca aac tac gat 720Ser Val Ser Ser Pro Pro Pro Tyr Thr Ser Pro Asn Pro Asn Tyr Asp 225 230 235 240 gaa aag cat tat atc gaa gca ttc aga ccc tta tta gaa gcc cgt ggt 768Glu Lys His Tyr Ile Glu Ala Phe Arg Pro Leu Leu Glu Ala Arg Gly 245 250 255 ttc cca gcc caa ttt ata gtg gat cag gga aga tca ggt aag caa cca 816Phe Pro Ala Gln Phe Ile Val Asp Gln Gly Arg Ser Gly Lys Gln Pro 260 265 270 act ggc caa aag gag tgg ggg cat tgg tgt aat gct att ggc aca gga 864Thr Gly Gln Lys Glu Trp Gly His Trp Cys Asn Ala Ile Gly Thr Gly 275 280 285 ttt ggt atg aga cct act gct aat acc ggt cac cag tat gtg gat gct 912Phe Gly Met Arg Pro Thr Ala Asn Thr Gly His Gln Tyr Val Asp Ala 290 295 300 ttt gtt tgg gtt aaa ccg ggc ggt gaa tgc gac ggg acc agc gat act 960Phe Val Trp Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Thr 305 310 315 320 acg gcg gcc aga tat gat tat cat tgt ggt ctg gaa gat gca tta aaa 1008Thr Ala Ala Arg Tyr Asp Tyr His Cys Gly Leu Glu Asp Ala Leu Lys 325 330 335 cca gct cct gaa gcc ggc cag tgg ttc aac gaa tac ttc att caa ttg 1056Pro Ala Pro Glu Ala Gly Gln Trp Phe Asn Glu Tyr Phe Ile Gln Leu 340 345 350 ctt agg aac gct aac ccg ccc ttt taa 1083Leu Arg Asn Ala Asn Pro Pro Phe 355 360 2360PRTHumicola insolens 2Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Gln Trp Leu 35 40 45 Asp Arg Asn Val Thr Val Asp Thr Leu Leu Val Gln Thr Leu Ser Glu 50 55 60 Ile Arg Glu Ala Asn Gln Ala Gly Ala Asn Pro Gln Tyr Ala Ala Gln 65 70 75 80 Ile Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser 85 90 95 Asn Gly Glu Trp Ala Ile Ala Asn Asn Gly Val Asn Asn Tyr Lys Ala 100 105 110 Tyr Ile Asn Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg 115 120 125 Thr Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn 130 135 140 Met Asn Val Pro Lys Cys Ser Gly Ala Ala Ser Thr Tyr Arg Glu Leu 145 150 155 160 Thr Ile Tyr Ala Leu Lys Gln Leu Asp Leu Pro His Val Ala Met Tyr 165 170 175 Met Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln 180 185 190 Pro Ala Ala Glu Leu Phe Ala Lys Ile Tyr Glu Asp Ala Gly Lys Pro 195 200 205 Arg Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp 210 215 220 Ser Val Ser Ser Pro Pro Pro Tyr Thr Ser Pro Asn Pro Asn Tyr Asp 225 230 235 240 Glu Lys His Tyr Ile Glu Ala Phe Arg Pro Leu Leu Glu Ala Arg Gly 245 250 255 Phe Pro Ala Gln Phe Ile Val Asp Gln Gly Arg Ser Gly Lys Gln Pro 260 265 270 Thr Gly Gln Lys Glu Trp Gly His Trp Cys Asn Ala Ile Gly Thr Gly 275 280 285 Phe Gly Met Arg Pro Thr Ala Asn Thr Gly His Gln Tyr Val Asp Ala 290 295 300 Phe Val Trp Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Thr 305 310 315 320 Thr Ala Ala Arg Tyr Asp Tyr His Cys Gly Leu Glu Asp Ala Leu Lys 325 330 335 Pro Ala Pro Glu Ala Gly Gln Trp Phe Asn Glu Tyr Phe Ile Gln Leu 340 345 350 Leu Arg Asn Ala Asn Pro Pro Phe 355 360 31077DNAHypocrea jecorinaCDS(1)..(1077) 3ggt aat cca ttc gtt ggg gtg aca ccc tgg gcg aac gcc tat tat gct 48Gly Asn Pro Phe Val Gly Val Thr Pro Trp Ala Asn Ala Tyr Tyr Ala 1 5 10 15 tct gag gtt tca tcc cta gct att ccc tct tta aca ggt gca atg gct 96Ser Glu Val Ser Ser Leu Ala Ile Pro Ser Leu Thr Gly Ala Met Ala 20 25 30 aca gcc gcc gct gcc gtt gca aag gtc cct tcc ttc atg tgg ctg gat 144Thr Ala Ala Ala Ala Val Ala Lys Val Pro Ser Phe Met Trp Leu Asp 35 40 45 act ttg gac aaa acc ccc tta atg gaa caa acg ttg gct gat ata cgt 192Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile Arg 50 55 60 act gcg aat aaa aac ggc ggc aat tat gct gga caa ttt gtg gtt tat 240Thr Ala Asn Lys Asn Gly Gly Asn Tyr Ala Gly Gln Phe Val Val Tyr 65 70 75 80 gac ctg ccg gat aga gat tgt gct gca cta gcg agc aac ggg gag tac 288Asp Leu Pro Asp Arg Asp Cys Ala Ala Leu Ala Ser Asn Gly Glu Tyr 85 90 95 agc att gcg gat ggc ggt gtc gca aag tac aaa aac tat ata gat act 336Ser Ile Ala Asp Gly Gly Val Ala Lys Tyr Lys Asn Tyr Ile Asp Thr 100 105 110 atc agg caa ata gtt gtc gaa tac agt gat att cgt acg ctg ctt gta 384Ile Arg Gln Ile Val Val Glu Tyr Ser Asp Ile Arg Thr Leu Leu Val 115 120 125 atc gaa ccc gat tcc tta gcg aac ttg gta aca aat cta ggt act ccg 432Ile Glu Pro Asp Ser Leu Ala Asn Leu Val Thr Asn Leu Gly Thr Pro 130 135 140 aag tgt gcg aac gcg cag agt gct tat ctt gag tgc atc aat tat gca 480Lys Cys Ala Asn Ala Gln Ser Ala Tyr Leu Glu Cys Ile Asn Tyr Ala 145 150 155 160 gtc acc cag ttg aat ttg cca aac gtt gca atg tat ctt gat gct ggt 528Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala Gly 165 170 175 cat gcc ggg tgg ttg ggt tgg cca gca aat cag gat ccc gct gcg cag 576His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp Pro Ala Ala Gln 180 185 190 ctg ttt gca aat gtt tac aaa aat gcc tca agt cct aga gcg ctg agg 624Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro Arg Ala Leu Arg 195 200 205 ggt ctt gca aca aat gtt gct aat tac aac gga tgg aat att acc tca 672Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Gly Trp Asn Ile Thr Ser 210 215 220 ccc cca tca tac aca caa gga aat gct gtt tac aat gaa aaa ctt tat 720Pro Pro Ser Tyr Thr Gln Gly Asn Ala Val Tyr Asn Glu Lys Leu Tyr 225 230 235 240 att cat gcc att ggt cca ctg ctg gct aat cac gga tgg agt aat gcc 768Ile His Ala Ile Gly Pro Leu Leu Ala Asn His Gly Trp Ser Asn Ala 245 250 255 ttt ttc att aca gat caa ggg aga agt ggt aaa caa cct act gga caa 816Phe Phe Ile Thr Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly Gln 260 265 270 caa caa tgg ggt gac tgg tgt aat gtt atc ggt act ggg ttt ggc atc 864Gln Gln Trp Gly Asp Trp Cys Asn Val Ile Gly Thr Gly Phe Gly Ile 275 280 285 aga cca tca gcg aat acg ggt gat tca ttg ttg gac tca ttt gtt tgg 912Arg Pro Ser Ala Asn Thr Gly Asp Ser Leu Leu Asp Ser Phe Val Trp 290 295 300 gtt aaa ccc ggg ggt gaa tgt gat gga acg agt gat tct tct gct cca 960Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Ser Ser Ala Pro 305 310 315 320 agg ttc gat tct cat tgc gca tta cca gat gct ttg cag cca gca cct 1008Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln Pro Ala Pro 325 330 335 caa gca gga gct tgg ttc caa gct tat ttt gta caa tta ctg act aac 1056Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu Leu Thr Asn 340 345 350 gcc aat cct agt ttt cta taa 1077Ala Asn Pro Ser Phe Leu 355 4358PRTHypocrea jecorina 4Gly Asn Pro Phe Val Gly Val Thr Pro Trp Ala Asn Ala Tyr Tyr Ala 1 5 10 15 Ser Glu Val Ser Ser Leu Ala Ile Pro Ser Leu Thr Gly Ala Met Ala 20 25 30 Thr Ala Ala Ala Ala Val Ala Lys Val Pro Ser Phe Met Trp Leu Asp 35 40 45 Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile Arg 50 55 60 Thr Ala Asn Lys Asn Gly Gly Asn Tyr Ala Gly Gln Phe Val Val Tyr 65 70 75 80 Asp Leu Pro Asp Arg Asp Cys Ala Ala Leu Ala Ser Asn Gly Glu Tyr 85 90 95 Ser Ile Ala Asp Gly Gly Val Ala Lys Tyr Lys Asn Tyr Ile Asp Thr 100 105 110 Ile Arg Gln Ile Val Val Glu Tyr Ser Asp Ile Arg Thr Leu Leu Val 115 120 125 Ile Glu Pro Asp Ser Leu Ala Asn Leu Val Thr Asn Leu Gly Thr Pro 130 135 140 Lys Cys Ala Asn Ala Gln Ser Ala Tyr Leu Glu Cys Ile Asn Tyr Ala 145 150 155 160 Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala Gly 165 170 175 His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp Pro Ala Ala Gln 180 185 190 Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro Arg Ala Leu Arg 195 200 205 Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Gly Trp Asn Ile Thr Ser 210 215 220 Pro Pro Ser Tyr Thr Gln Gly Asn Ala Val Tyr Asn Glu Lys Leu Tyr 225 230 235 240 Ile His Ala Ile Gly Pro Leu Leu Ala Asn His Gly Trp Ser Asn Ala 245 250 255 Phe Phe Ile Thr Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly Gln 260 265 270 Gln Gln Trp Gly Asp Trp Cys Asn Val Ile Gly Thr Gly Phe Gly Ile 275 280 285 Arg Pro Ser Ala Asn Thr Gly Asp Ser Leu Leu Asp Ser Phe Val Trp 290 295 300 Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Ser Ser Ala Pro 305 310 315 320 Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln Pro Ala Pro 325 330 335 Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu Leu Thr Asn 340 345 350 Ala Asn Pro Ser Phe Leu 355 51077DNAChaetomium thermophiliumCDS(1)..(1077) 5ggt aac cct ttc agt ggt gtg cag tta tgg gct aat act tac tat tct 48Gly Asn Pro Phe Ser Gly Val Gln Leu Trp Ala Asn Thr Tyr Tyr Ser 1 5 10 15 tca gaa gtc cac acc tta gct atc cca agc tta agt cca gaa tta gcg 96Ser Glu Val His Thr Leu Ala Ile Pro Ser Leu Ser Pro Glu Leu Ala 20 25 30 gct aag gcg gcg aaa gta gct gaa gtg cca tca ttc caa tgg tta gat 144Ala Lys Ala Ala Lys Val Ala Glu Val Pro Ser Phe Gln Trp Leu Asp 35 40 45 aga aac gtg act gtg gat act ctg ttt tct ggt aca ctt gct gag ata 192Arg Asn Val Thr Val Asp Thr Leu Phe Ser Gly Thr Leu Ala Glu Ile 50 55 60 agg gcg gct aac caa agg gga gct aat cca cca tat gct ggc atc ttt 240Arg Ala Ala Asn Gln Arg Gly Ala Asn Pro Pro Tyr Ala Gly Ile Phe 65 70 75 80 gtg gtt tat gac ctt cct gat aga gat tgt gct gcc gct gca agc aat 288Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn 85 90 95 ggt gaa tgg agt ata gct aac aac ggt gct aac aac tat aag aga tat 336Gly Glu Trp Ser Ile Ala Asn Asn Gly Ala Asn Asn Tyr Lys Arg Tyr 100 105 110 atc gat aga att aga gaa ttg ttg att cag tac tca gat atc agg aca 384Ile Asp Arg Ile Arg Glu Leu Leu Ile Gln Tyr Ser Asp Ile Arg Thr 115 120 125 att ttg gtt att gaa cca gac agt cta gca aat atg gtt act aac atg 432Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met 130 135 140 aac gta caa aaa tgt tct aac gca gca tct acg tat aaa gaa ctg act 480Asn Val Gln Lys Cys Ser Asn Ala Ala Ser Thr Tyr Lys Glu Leu Thr 145 150 155 160 gtg tat gca ttg aaa cag ttg aac ttg cca cac gta gcc atg tat atg 528Val Tyr Ala Leu Lys Gln Leu Asn Leu Pro His Val Ala Met Tyr Met 165 170 175 gat gca ggt cac gcc ggc tgg tta ggc

tgg ccc gct aat ata cag cct 576Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro 180 185 190 gcc gca gaa tta ttc gcg caa ata tac aga gac gct gga cgt ccg gct 624Ala Ala Glu Leu Phe Ala Gln Ile Tyr Arg Asp Ala Gly Arg Pro Ala 195 200 205 gcg gtc agg ggt ctt gcc act aac gtt gca aat tac aac gct tgg tca 672Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser 210 215 220 ata gcg agt cct cca tcg tac aca agc cct aac cca aac tac gat gag 720Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu 225 230 235 240 aag cat tac ata gaa gca ttt gct cct ttg ctt cgt aac caa ggt ttt 768Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly Phe 245 250 255 gat gca aag ttt atc gtc gat acc gga aga aac ggc aag cag ccg aca 816Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr 260 265 270 ggg cag cta gaa tgg ggg cac tgg tgc aat gtc aag ggt acg ggt ttc 864Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe 275 280 285 ggt gtt aga ccc acg gct aac act ggg cat gag ttg gtt gat gca ttc 912Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe 290 295 300 gtt tgg gta aaa ccc gga gga gag tca gac ggt act tct gat act agt 960Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Ser 305 310 315 320 gct gcc aga tac gat tac cac tgt ggc ctt tct gat gct ttg aca cca 1008Ala Ala Arg Tyr Asp Tyr His Cys Gly Leu Ser Asp Ala Leu Thr Pro 325 330 335 gcc cct gaa gcc ggg caa tgg ttc cag gcc tac ttc gaa caa cta ttg 1056Ala Pro Glu Ala Gly Gln Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu 340 345 350 att aac gca aac cca cca tag 1077Ile Asn Ala Asn Pro Pro 355 6358PRTChaetomium thermophilium 6Gly Asn Pro Phe Ser Gly Val Gln Leu Trp Ala Asn Thr Tyr Tyr Ser 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Ser Leu Ser Pro Glu Leu Ala 20 25 30 Ala Lys Ala Ala Lys Val Ala Glu Val Pro Ser Phe Gln Trp Leu Asp 35 40 45 Arg Asn Val Thr Val Asp Thr Leu Phe Ser Gly Thr Leu Ala Glu Ile 50 55 60 Arg Ala Ala Asn Gln Arg Gly Ala Asn Pro Pro Tyr Ala Gly Ile Phe 65 70 75 80 Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn 85 90 95 Gly Glu Trp Ser Ile Ala Asn Asn Gly Ala Asn Asn Tyr Lys Arg Tyr 100 105 110 Ile Asp Arg Ile Arg Glu Leu Leu Ile Gln Tyr Ser Asp Ile Arg Thr 115 120 125 Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met 130 135 140 Asn Val Gln Lys Cys Ser Asn Ala Ala Ser Thr Tyr Lys Glu Leu Thr 145 150 155 160 Val Tyr Ala Leu Lys Gln Leu Asn Leu Pro His Val Ala Met Tyr Met 165 170 175 Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro 180 185 190 Ala Ala Glu Leu Phe Ala Gln Ile Tyr Arg Asp Ala Gly Arg Pro Ala 195 200 205 Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser 210 215 220 Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu 225 230 235 240 Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly Phe 245 250 255 Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr 260 265 270 Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe 275 280 285 Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe 290 295 300 Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Ser 305 310 315 320 Ala Ala Arg Tyr Asp Tyr His Cys Gly Leu Ser Asp Ala Leu Thr Pro 325 330 335 Ala Pro Glu Ala Gly Gln Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu 340 345 350 Ile Asn Ala Asn Pro Pro 355 7267DNAArtificial SequenceCBD Linker 7gct agc tgc tca agc gtc tgg ggc caa tgt ggt ggc cag aat tgg tcg 48Ala Ser Cys Ser Ser Val Trp Gly Gln Cys Gly Gly Gln Asn Trp Ser 1 5 10 15 ggt ccg act tgc tgt gct tcc gga agc aca tgc gtc tac tcc aac gac 96Gly Pro Thr Cys Cys Ala Ser Gly Ser Thr Cys Val Tyr Ser Asn Asp 20 25 30 tat tac tcc cag tgt ctt ccc ggc gct gca agc tca agc tcg tcc acg 144Tyr Tyr Ser Gln Cys Leu Pro Gly Ala Ala Ser Ser Ser Ser Ser Thr 35 40 45 cgc gcc gcg tcg acg act tct cga gta tcc ccc aca aca tcc cgg tcg 192Arg Ala Ala Ser Thr Thr Ser Arg Val Ser Pro Thr Thr Ser Arg Ser 50 55 60 agc tcc gcg acg cct cca cct ggt tct act act acc aga gta cct cca 240Ser Ser Ala Thr Pro Pro Pro Gly Ser Thr Thr Thr Arg Val Pro Pro 65 70 75 80 gtc gga tcg gga acc gct acg tat tca 267Val Gly Ser Gly Thr Ala Thr Tyr Ser 85 889PRTArtificial SequenceSynthetic Construct 8Ala Ser Cys Ser Ser Val Trp Gly Gln Cys Gly Gly Gln Asn Trp Ser 1 5 10 15 Gly Pro Thr Cys Cys Ala Ser Gly Ser Thr Cys Val Tyr Ser Asn Asp 20 25 30 Tyr Tyr Ser Gln Cys Leu Pro Gly Ala Ala Ser Ser Ser Ser Ser Thr 35 40 45 Arg Ala Ala Ser Thr Thr Ser Arg Val Ser Pro Thr Thr Ser Arg Ser 50 55 60 Ser Ser Ala Thr Pro Pro Pro Gly Ser Thr Thr Thr Arg Val Pro Pro 65 70 75 80 Val Gly Ser Gly Thr Ala Thr Tyr Ser 85 921DNAArtificial SequenceOligonucleotide Primer CBH2L 9gctgaacgtg tcatcggtta c 211023DNAArtificial SequenceOligonucleotide Primer RSQ3080 10gcaacacctg gcaattcctt acc 231124DNAArtificial SequenceOligonucleotide Primer CBH2LPCR 11gctgaacgtg tcatcgttac ttag 2412356PRTPhanerochaete chrysosporium 12Asn Asn Pro Trp Thr Gly Phe Gln Ile Phe Leu Ser Pro Tyr Tyr Ala 1 5 10 15 Asn Glu Val Ala Ala Ala Ala Lys Gln Ile Thr Asp Pro Thr Leu Ser 20 25 30 Ser Lys Ala Ala Ser Val Ala Asn Ile Pro Thr Phe Thr Trp Leu Asp 35 40 45 Ser Val Ala Lys Ile Pro Asp Leu Gly Thr Tyr Leu Ala Ser Ala Ser 50 55 60 Ala Leu Gly Lys Ser Thr Gly Thr Lys Gln Leu Val Gln Ile Val Ile 65 70 75 80 Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Lys Ala Ser Asn Gly Glu 85 90 95 Phe Ser Ile Ala Asn Asn Gly Gln Ala Asn Tyr Glu Asn Tyr Ile Asp 100 105 110 Gln Ile Val Ala Gln Ile Gln Gln Phe Pro Asp Val Arg Val Val Ala 115 120 125 Val Ile Glu Pro Asp Ser Leu Ala Asn Leu Val Thr Asn Leu Asn Val 130 135 140 Gln Lys Cys Ala Asn Ala Lys Thr Thr Tyr Leu Ala Cys Val Asn Tyr 145 150 155 160 Ala Leu Thr Asn Leu Ala Lys Val Gly Val Tyr Met Tyr Met Asp Ala 165 170 175 Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Leu Ser Pro Ala Ala 180 185 190 Gln Leu Phe Thr Gln Val Trp Gln Asn Ala Gly Lys Ser Pro Phe Ile 195 200 205 Lys Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Leu Gln Ala Ala 210 215 220 Ser Pro Asp Pro Ile Thr Gln Gly Asn Pro Asn Tyr Asp Glu Ile His 225 230 235 240 Tyr Ile Asn Ala Leu Ala Pro Leu Leu Gln Gln Ala Gly Trp Asp Ala 245 250 255 Thr Phe Ile Val Asp Gln Gly Arg Ser Gly Val Gln Asn Ile Arg Gln 260 265 270 Gln Trp Gly Asp Trp Cys Asn Ile Lys Gly Ala Gly Phe Gly Thr Arg 275 280 285 Pro Thr Thr Asn Thr Gly Ser Gln Phe Ile Asp Ser Ile Val Trp Val 290 295 300 Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asn Ser Ser Ser Pro Arg 305 310 315 320 Tyr Asp Ser Thr Cys Ser Leu Pro Asp Ala Ala Gln Pro Ala Pro Glu 325 330 335 Ala Gly Thr Trp Phe Gln Ala Tyr Phe Gln Thr Leu Val Ser Ala Ala 340 345 350 Asn Pro Pro Leu 355 13358PRTArtificial SequenceChimeric CBH II polypeptide 13Gly Asn Pro Phe Ser Gly Val Gln Leu Trp Ala Asn Thr Tyr Tyr Ser 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Ser Leu Ser Pro Glu Leu Ala 20 25 30 Ala Lys Ala Ala Lys Val Ala Glu Val Pro Ser Phe Met Trp Leu Asp 35 40 45 Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile Arg 50 55 60 Thr Ala Asn Lys Asn Gly Ala Asn Pro Pro Tyr Ala Gly Ile Phe Val 65 70 75 80 Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly 85 90 95 Glu Trp Ser Ile Ala Asn Asn Gly Ala Asn Asn Tyr Lys Arg Tyr Ile 100 105 110 Asp Arg Ile Arg Glu Leu Leu Ile Gln Tyr Ser Asp Ile Arg Thr Ile 115 120 125 Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met Asn 130 135 140 Val Gln Lys Cys Ser Asn Ala Ala Ser Thr Tyr Lys Glu Leu Thr Val 145 150 155 160 Tyr Ala Leu Lys Gln Leu Asn Leu Pro His Val Ala Met Tyr Met Asp 165 170 175 Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro Ala 180 185 190 Ala Glu Leu Phe Ala Gln Ile Tyr Arg Asp Ala Gly Arg Pro Ala Ala 195 200 205 Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser Val 210 215 220 Ser Ser Pro Pro Pro Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu Lys 225 230 235 240 His Tyr Ile Glu Ala Phe Arg Pro Leu Leu Glu Ala Arg Gly Phe Pro 245 250 255 Ala Gln Phe Ile Val Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly 260 265 270 Gln Lys Glu Trp Gly His Trp Cys Asn Ala Ile Gly Thr Gly Phe Gly 275 280 285 Met Arg Pro Thr Ala Asn Thr Gly His Gln Tyr Val Asp Ala Phe Val 290 295 300 Trp Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Thr Ser Ala 305 310 315 320 Ala Arg Tyr Asp Tyr His Cys Gly Leu Ser Asp Ala Leu Thr Pro Ala 325 330 335 Pro Glu Ala Gly Gln Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu Ile 340 345 350 Asn Ala Asn Pro Pro Phe 355 14360PRTArtificial SequenceChimeric CBH II polypeptide 14Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Gln Trp Leu 35 40 45 Asp Arg Asn Val Thr Val Asp Thr Leu Phe Ser Gly Thr Leu Ala Glu 50 55 60 Ile Arg Ala Ala Asn Gln Arg Gly Ala Asn Pro Gln Tyr Ala Ala Gln 65 70 75 80 Ile Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser 85 90 95 Asn Gly Glu Trp Ala Ile Ala Asn Asn Gly Val Asn Asn Tyr Lys Ala 100 105 110 Tyr Ile Asn Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg 115 120 125 Thr Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn 130 135 140 Met Asn Val Pro Lys Cys Ser Gly Ala Ala Ser Thr Tyr Arg Glu Leu 145 150 155 160 Thr Ile Tyr Ala Leu Lys Gln Leu Asp Leu Pro His Val Ala Met Tyr 165 170 175 Met Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln 180 185 190 Pro Ala Ala Glu Leu Phe Ala Lys Ile Tyr Glu Asp Ala Gly Lys Pro 195 200 205 Arg Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp 210 215 220 Ser Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp 225 230 235 240 Glu Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly 245 250 255 Phe Asp Ala Gln Phe Ile Val Asp Gln Gly Arg Ser Gly Lys Gln Pro 260 265 270 Thr Gly Gln Lys Glu Trp Gly His Trp Cys Asn Ala Ile Gly Thr Gly 275 280 285 Phe Gly Met Arg Pro Thr Ala Asn Thr Gly His Gln Tyr Val Asp Ala 290 295 300 Phe Val Trp Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Thr 305 310 315 320 Ser Ala Ala Arg Tyr Asp Tyr His Cys Gly Leu Ser Asp Ala Leu Thr 325 330 335 Pro Ala Pro Glu Ala Gly Gln Trp Phe Gln Ala Tyr Phe Glu Gln Leu 340 345 350 Leu Ile Asn Ala Asn Pro Pro Phe 355 360 15361PRTArtificial SequenceChimeric CBH II Polypeptide 15Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Gln Trp Leu 35 40 45 Asp Arg Asn Val Thr Val Asp Thr Leu Leu Val Gln Thr Leu Ser Glu 50 55 60 Ile Arg Glu Ala Asn Gln Ala Gly Ala Asn Pro Pro Tyr Ala Gly Ile 65 70 75 80 Phe Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser 85 90 95 Asn Gly Glu Trp Ser Ile Ala Asn Asn Gly Ala Asn Asn Tyr Lys Arg 100 105 110 Tyr Ile Asp Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg 115 120 125 Thr Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn 130 135 140 Met Asn Val Pro Lys Cys Ser Gly Ala Ala Ser Thr Tyr Lys Glu Leu 145 150 155 160 Thr Val Tyr Ala Leu Lys Gln Leu Asn Leu Pro His Val Ala Met Tyr 165 170 175 Met Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln 180 185 190 Pro Ala Ala Glu Leu Phe Ala Gln Ile Tyr Arg Asp Ala Gly Arg Pro 195

200 205 Ala Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp 210 215 220 Ser Val Ser Ser Pro Pro Pro Tyr Thr Ser Pro Asn Pro Asn Tyr Asp 225 230 235 240 Glu Lys His Tyr Ile Glu Ala Phe Arg Pro Leu Leu Glu Ala Arg Gly 245 250 255 Phe Pro Asn Ala Phe Phe Ile Thr Asp Gln Gly Arg Ser Gly Lys Gln 260 265 270 Pro Thr Gly Gln Gln Gln Trp Gly Asp Trp Cys Asn Val Ile Gly Thr 275 280 285 Gly Phe Gly Ile Arg Pro Ser Ala Asn Thr Gly Asp Ser Leu Leu Asp 290 295 300 Ser Phe Val Trp Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp 305 310 315 320 Thr Thr Ala Ala Arg Tyr Asp Tyr His Cys Gly Leu Glu Asp Ala Leu 325 330 335 Lys Pro Ala Pro Glu Ala Gly Gln Trp Phe Asn Glu Tyr Phe Ile Gln 340 345 350 Leu Leu Arg Asn Ala Asn Pro Pro Phe 355 360 16359PRTArtificial SequenceChimeric CBH II Polypeptide 16Gly Asn Pro Phe Val Gly Val Thr Pro Trp Ala Asn Ala Tyr Tyr Ala 1 5 10 15 Ser Glu Val Ser Ser Leu Ala Ile Pro Ser Leu Thr Gly Ala Met Ala 20 25 30 Thr Ala Ala Ala Ala Val Ala Lys Val Pro Ser Phe Gln Trp Leu Asp 35 40 45 Arg Asn Val Thr Val Asp Thr Leu Leu Val Gln Thr Leu Ser Glu Ile 50 55 60 Arg Glu Ala Asn Gln Ala Gly Ala Asn Pro Gln Tyr Ala Ala Gln Ile 65 70 75 80 Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn 85 90 95 Gly Glu Trp Ala Ile Ala Asn Asn Gly Val Asn Asn Tyr Lys Ala Tyr 100 105 110 Ile Asn Arg Ile Arg Glu Leu Leu Ile Gln Tyr Ser Asp Ile Arg Thr 115 120 125 Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met 130 135 140 Asn Val Gln Lys Cys Ser Asn Ala Ala Ser Thr Tyr Arg Glu Leu Thr 145 150 155 160 Ile Tyr Ala Leu Lys Gln Leu Asp Leu Pro His Val Ala Met Tyr Met 165 170 175 Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro 180 185 190 Ala Ala Glu Leu Phe Ala Lys Ile Tyr Glu Asp Ala Gly Lys Pro Arg 195 200 205 Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser 210 215 220 Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu 225 230 235 240 Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly Phe 245 250 255 Asp Ala Gln Phe Ile Val Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr 260 265 270 Gly Gln Lys Glu Trp Gly His Trp Cys Asn Ala Ile Gly Thr Gly Phe 275 280 285 Gly Met Arg Pro Thr Ala Asn Thr Gly His Gln Tyr Val Asp Ala Phe 290 295 300 Val Trp Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Thr Thr 305 310 315 320 Ala Ala Arg Tyr Asp Tyr His Cys Gly Leu Glu Asp Ala Leu Lys Pro 325 330 335 Ala Pro Glu Ala Gly Gln Trp Phe Asn Glu Tyr Phe Ile Gln Leu Leu 340 345 350 Arg Asn Ala Asn Pro Pro Phe 355 17357PRTArtificial SequenceChimeric CBH II Polypeptide 17Gly Asn Pro Phe Ser Gly Val Gln Leu Trp Ala Asn Thr Tyr Tyr Ser 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Ser Leu Ser Pro Glu Leu Ala 20 25 30 Ala Lys Ala Ala Lys Val Ala Glu Val Pro Ser Phe Gln Trp Leu Asp 35 40 45 Arg Asn Val Thr Val Asp Thr Leu Leu Val Gln Thr Leu Ser Glu Ile 50 55 60 Arg Glu Ala Asn Gln Ala Gly Gly Asn Tyr Ala Gly Gln Phe Val Val 65 70 75 80 Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Leu Ala Ser Asn Gly Glu 85 90 95 Tyr Ser Ile Ala Asp Gly Gly Val Ala Lys Tyr Lys Asn Tyr Ile Asp 100 105 110 Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg Thr Ile Leu 115 120 125 Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met Asn Val 130 135 140 Pro Lys Cys Ser Gly Ala Ala Ser Ala Tyr Leu Glu Cys Ile Asn Tyr 145 150 155 160 Ala Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala 165 170 175 Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp Pro Ala Ala 180 185 190 Gln Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro Arg Ala Leu 195 200 205 Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser Val Ser 210 215 220 Ser Pro Pro Pro Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu Lys His 225 230 235 240 Tyr Ile Glu Ala Phe Arg Pro Leu Leu Glu Ala Arg Gly Phe Pro Ala 245 250 255 Gln Phe Ile Val Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly Gln 260 265 270 Lys Glu Trp Gly His Trp Cys Asn Ala Ile Gly Thr Gly Phe Gly Met 275 280 285 Arg Pro Thr Ala Asn Thr Gly His Gln Tyr Val Asp Ala Phe Val Trp 290 295 300 Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Thr Thr Ala Ala 305 310 315 320 Arg Tyr Asp Tyr His Cys Gly Leu Glu Asp Ala Leu Lys Pro Ala Pro 325 330 335 Glu Ala Gly Gln Trp Phe Asn Glu Tyr Phe Ile Gln Leu Leu Arg Asn 340 345 350 Ala Asn Pro Pro Phe 355 18357PRTArtificial SequenceChimeric CBH II Polypeptide 18Gly Asn Pro Phe Val Gly Val Thr Pro Trp Ala Asn Ala Tyr Tyr Ala 1 5 10 15 Ser Glu Val Ser Ser Leu Ala Ile Pro Ser Leu Thr Gly Ala Met Ala 20 25 30 Thr Ala Ala Ala Ala Val Ala Lys Val Pro Ser Phe Gln Trp Leu Asp 35 40 45 Arg Asn Val Thr Val Asp Thr Leu Phe Ser Gly Thr Leu Ala Glu Ile 50 55 60 Arg Ala Ala Asn Gln Arg Gly Gly Asn Tyr Ala Gly Gln Phe Val Val 65 70 75 80 Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Leu Ala Ser Asn Gly Glu 85 90 95 Tyr Ser Ile Ala Asp Gly Gly Val Ala Lys Tyr Lys Asn Tyr Ile Asp 100 105 110 Arg Ile Arg Glu Leu Leu Ile Gln Tyr Ser Asp Ile Arg Thr Ile Leu 115 120 125 Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met Asn Val 130 135 140 Gln Lys Cys Ser Asn Ala Ala Ser Thr Tyr Lys Glu Leu Thr Val Tyr 145 150 155 160 Ala Leu Lys Gln Leu Asn Leu Pro His Val Ala Met Tyr Met Asp Ala 165 170 175 Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro Ala Ala 180 185 190 Glu Leu Phe Ala Gln Ile Tyr Arg Asp Ala Gly Arg Pro Ala Ala Val 195 200 205 Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser Val Ser 210 215 220 Ser Pro Pro Pro Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu Lys His 225 230 235 240 Tyr Ile Glu Ala Phe Arg Pro Leu Leu Glu Ala Arg Gly Phe Pro Ala 245 250 255 Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr Gly Gln 260 265 270 Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe Gly Val 275 280 285 Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe Val Trp 290 295 300 Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Ser Ala Ala 305 310 315 320 Arg Tyr Asp Tyr His Cys Gly Leu Ser Asp Ala Leu Thr Pro Ala Pro 325 330 335 Glu Ala Gly Gln Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu Ile Asn 340 345 350 Ala Asn Pro Pro Phe 355 19360PRTArtificial SequenceChimeric CBH II Polypeptide 19Gly Asn Pro Phe Ser Gly Val Gln Leu Trp Ala Asn Thr Tyr Tyr Ser 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Ser Leu Ser Pro Glu Leu Ala 20 25 30 Ala Lys Ala Ala Lys Val Ala Glu Val Pro Ser Phe Gln Trp Leu Asp 35 40 45 Arg Asn Val Thr Val Asp Thr Leu Leu Val Gln Thr Leu Ser Glu Ile 50 55 60 Arg Glu Ala Asn Gln Ala Gly Ala Asn Pro Pro Tyr Ala Gly Ile Phe 65 70 75 80 Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn 85 90 95 Gly Glu Trp Ser Ile Ala Asn Asn Gly Ala Asn Asn Tyr Lys Arg Tyr 100 105 110 Ile Asp Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg Thr 115 120 125 Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met 130 135 140 Asn Val Pro Lys Cys Ser Gly Ala Ala Ser Thr Tyr Arg Glu Leu Thr 145 150 155 160 Ile Tyr Ala Leu Lys Gln Leu Asp Leu Pro His Val Ala Met Tyr Met 165 170 175 Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro 180 185 190 Ala Ala Glu Leu Phe Ala Lys Ile Tyr Glu Asp Ala Gly Lys Pro Arg 195 200 205 Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser 210 215 220 Val Ser Ser Pro Pro Pro Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu 225 230 235 240 Lys His Tyr Ile Glu Ala Phe Arg Pro Leu Leu Glu Ala Arg Gly Phe 245 250 255 Pro Ala Gln Phe Ile Val Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr 260 265 270 Gly Gln Lys Glu Trp Gly His Trp Cys Asn Ala Ile Gly Thr Gly Phe 275 280 285 Gly Met Arg Pro Thr Ala Asn Thr Gly His Gln Tyr Val Asp Ala Phe 290 295 300 Val Trp Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Ser Ser 305 310 315 320 Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln Pro 325 330 335 Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu Leu 340 345 350 Thr Asn Ala Asn Pro Ser Phe Leu 355 360 20356PRTArtificial SequenceChimeric CBH II Polypeptide 20Gly Asn Pro Phe Val Gly Val Thr Pro Trp Ala Asn Ala Tyr Tyr Ala 1 5 10 15 Ser Glu Val Ser Ser Leu Ala Ile Pro Ser Leu Thr Gly Ala Met Ala 20 25 30 Thr Ala Ala Ala Ala Val Ala Lys Val Pro Ser Phe Met Trp Leu Asp 35 40 45 Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile Arg 50 55 60 Thr Ala Asn Lys Asn Gly Gly Asn Tyr Ala Gly Gln Phe Val Val Tyr 65 70 75 80 Asp Leu Pro Asp Arg Asp Cys Ala Ala Leu Ala Ser Asn Gly Glu Tyr 85 90 95 Ser Ile Ala Asp Gly Gly Val Ala Lys Tyr Lys Asn Tyr Ile Asp Arg 100 105 110 Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg Thr Ile Leu Val 115 120 125 Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met Asn Val Pro 130 135 140 Lys Cys Ser Gly Ala Ala Ser Ala Tyr Leu Glu Cys Ile Asn Tyr Ala 145 150 155 160 Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala Gly 165 170 175 His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp Pro Ala Ala Gln 180 185 190 Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro Arg Ala Leu Arg 195 200 205 Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Gly Trp Asn Ile Thr Ser 210 215 220 Pro Pro Ser Tyr Thr Gln Gly Asn Ala Val Tyr Asn Glu Lys Leu Tyr 225 230 235 240 Ile His Ala Ile Gly Pro Leu Leu Ala Asn His Gly Trp Ser Ala Lys 245 250 255 Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr Gly Gln Leu 260 265 270 Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe Gly Val Arg 275 280 285 Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe Val Trp Val 290 295 300 Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Thr Ala Ala Arg 305 310 315 320 Tyr Asp Tyr His Cys Gly Leu Glu Asp Ala Leu Lys Pro Ala Pro Glu 325 330 335 Ala Gly Gln Trp Phe Asn Glu Tyr Phe Ile Gln Leu Leu Arg Asn Ala 340 345 350 Asn Pro Pro Phe 355 21358PRTArtificial SequenceChimeric CBH II Polypeptide 21Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Gln Trp Leu 35 40 45 Asp Arg Asn Val Thr Val Asp Thr Leu Phe Ser Gly Thr Leu Ala Glu 50 55 60 Ile Arg Ala Ala Asn Gln Arg Gly Gly Asn Tyr Ala Gly Gln Phe Val 65 70 75 80 Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Leu Ala Ser Asn Gly 85 90 95 Glu Tyr Ser Ile Ala Asp Gly Gly Val Ala Lys Tyr Lys Asn Tyr Ile 100 105 110 Asp Arg Ile Arg Glu Leu Leu Ile Gln Tyr Ser Asp Ile Arg Thr Ile 115 120 125 Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met Asn 130 135 140 Val Gln Lys Cys Ser Asn Ala Ala Ser Thr Tyr Arg Glu Leu Thr Ile 145 150 155 160 Tyr Ala Leu Lys Gln Leu Asp Leu Pro His Val Ala Met Tyr Met Asp 165 170 175 Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro Ala 180 185 190 Ala Glu Leu Phe Ala Lys Ile Tyr Glu Asp Ala Gly Lys Pro Arg Ala 195 200 205 Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser Val 210 215 220 Ser Ser Pro Pro Pro Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu Lys 225 230 235 240 His Tyr Ile Glu Ala Phe Arg Pro Leu Leu Glu Ala Arg Gly Phe Pro 245 250 255 Ala Gln Phe Ile Val Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly 260 265 270 Gln Lys Glu Trp Gly His Trp Cys Asn Ala Ile Gly Thr Gly Phe Gly 275 280 285 Met Arg Pro Thr Ala Asn Thr Gly His Gln Tyr Val Asp Ala Phe Val 290 295 300 Trp Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Thr Thr Ala 305 310

315 320 Ala Arg Tyr Asp Tyr His Cys Gly Leu Glu Asp Ala Leu Lys Pro Ala 325 330 335 Pro Glu Ala Gly Gln Trp Phe Asn Glu Tyr Phe Ile Gln Leu Leu Arg 340 345 350 Asn Ala Asn Pro Pro Phe 355 22357PRTArtificial SequenceChimeric CBH II Polypeptide 22Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Met Trp Leu 35 40 45 Asp Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile 50 55 60 Arg Thr Ala Asn Lys Asn Gly Gly Asn Tyr Ala Gly Gln Phe Val Val 65 70 75 80 Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Leu Ala Ser Asn Gly Glu 85 90 95 Tyr Ser Ile Ala Asp Gly Gly Val Ala Lys Tyr Lys Asn Tyr Ile Asp 100 105 110 Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg Thr Ile Leu 115 120 125 Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met Asn Val 130 135 140 Pro Lys Cys Ser Gly Ala Ala Ser Thr Tyr Lys Glu Leu Thr Val Tyr 145 150 155 160 Ala Leu Lys Gln Leu Asn Leu Pro His Val Ala Met Tyr Met Asp Ala 165 170 175 Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro Ala Ala 180 185 190 Glu Leu Phe Ala Gln Ile Tyr Arg Asp Ala Gly Arg Pro Ala Ala Val 195 200 205 Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser Val Ser 210 215 220 Ser Pro Pro Pro Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu Lys His 225 230 235 240 Tyr Ile Glu Ala Phe Arg Pro Leu Leu Glu Ala Arg Gly Phe Pro Ala 245 250 255 Gln Phe Ile Val Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly Gln 260 265 270 Lys Glu Trp Gly His Trp Cys Asn Ala Ile Gly Thr Gly Phe Gly Met 275 280 285 Arg Pro Thr Ala Asn Thr Gly His Gln Tyr Val Asp Ala Phe Val Trp 290 295 300 Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Thr Thr Ala Ala 305 310 315 320 Arg Tyr Asp Tyr His Cys Gly Leu Glu Asp Ala Leu Lys Pro Ala Pro 325 330 335 Glu Ala Gly Gln Trp Phe Asn Glu Tyr Phe Ile Gln Leu Leu Arg Asn 340 345 350 Ala Asn Pro Pro Phe 355 23359PRTArtificial SequenceChimeric CBH II Polypeptide 23Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Met Trp Leu 35 40 45 Asp Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile 50 55 60 Arg Thr Ala Asn Lys Asn Gly Ala Asn Pro Gln Tyr Ala Ala Gln Ile 65 70 75 80 Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn 85 90 95 Gly Glu Trp Ala Ile Ala Asn Asn Gly Val Asn Asn Tyr Lys Ala Tyr 100 105 110 Ile Asn Arg Ile Arg Glu Leu Leu Ile Gln Tyr Ser Asp Ile Arg Thr 115 120 125 Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met 130 135 140 Asn Val Gln Lys Cys Ser Asn Ala Ala Ser Thr Tyr Lys Glu Leu Thr 145 150 155 160 Val Tyr Ala Leu Lys Gln Leu Asn Leu Pro His Val Ala Met Tyr Met 165 170 175 Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro 180 185 190 Ala Ala Glu Leu Phe Ala Gln Ile Tyr Arg Asp Ala Gly Arg Pro Ala 195 200 205 Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser 210 215 220 Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu 225 230 235 240 Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly Phe 245 250 255 Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr 260 265 270 Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe 275 280 285 Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe 290 295 300 Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Ser 305 310 315 320 Ala Ala Arg Tyr Asp Tyr His Cys Gly Leu Ser Asp Ala Leu Thr Pro 325 330 335 Ala Pro Glu Ala Gly Gln Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu 340 345 350 Ile Asn Ala Asn Pro Pro Phe 355 24360PRTArtificial SequenceChimeric CBH II Polypeptide 24Gly Asn Pro Phe Ser Gly Val Gln Leu Trp Ala Asn Thr Tyr Tyr Ser 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Ser Leu Ser Pro Glu Leu Ala 20 25 30 Ala Lys Ala Ala Lys Val Ala Glu Val Pro Ser Phe Gln Trp Leu Asp 35 40 45 Arg Asn Val Thr Val Asp Thr Leu Phe Ser Gly Thr Leu Ala Glu Ile 50 55 60 Arg Ala Ala Asn Gln Arg Gly Ala Asn Pro Gln Tyr Ala Ala Gln Ile 65 70 75 80 Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn 85 90 95 Gly Glu Trp Ala Ile Ala Asn Asn Gly Val Asn Asn Tyr Lys Ala Tyr 100 105 110 Ile Asn Arg Ile Arg Glu Leu Leu Ile Gln Tyr Ser Asp Ile Arg Thr 115 120 125 Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met 130 135 140 Asn Val Gln Lys Cys Ser Asn Ala Ala Ser Thr Tyr Lys Glu Leu Thr 145 150 155 160 Val Tyr Ala Leu Lys Gln Leu Asn Leu Pro His Val Ala Met Tyr Met 165 170 175 Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro 180 185 190 Ala Ala Glu Leu Phe Ala Gln Ile Tyr Arg Asp Ala Gly Arg Pro Ala 195 200 205 Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser 210 215 220 Val Ser Ser Pro Pro Pro Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu 225 230 235 240 Lys His Tyr Ile Glu Ala Phe Arg Pro Leu Leu Glu Ala Arg Gly Phe 245 250 255 Pro Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr 260 265 270 Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe 275 280 285 Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe 290 295 300 Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser Ser 305 310 315 320 Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln Pro 325 330 335 Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu Leu 340 345 350 Thr Asn Ala Asn Pro Ser Phe Leu 355 360 25360PRTArtificial SequenceChimeric CBH II Polypeptide 25Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Gln Trp Leu 35 40 45 Asp Arg Asn Val Thr Val Asp Thr Leu Leu Val Gln Thr Leu Ser Glu 50 55 60 Ile Arg Glu Ala Asn Gln Ala Gly Ala Asn Pro Pro Tyr Ala Gly Ile 65 70 75 80 Phe Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser 85 90 95 Asn Gly Glu Trp Ser Ile Ala Asn Asn Gly Ala Asn Asn Tyr Lys Arg 100 105 110 Tyr Ile Asp Arg Ile Arg Glu Leu Leu Ile Gln Tyr Ser Asp Ile Arg 115 120 125 Thr Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn 130 135 140 Met Asn Val Gln Lys Cys Ser Asn Ala Ala Ser Ala Tyr Leu Glu Cys 145 150 155 160 Ile Asn Tyr Ala Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr 165 170 175 Leu Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp 180 185 190 Pro Ala Ala Gln Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro 195 200 205 Arg Ala Leu Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp 210 215 220 Ser Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp 225 230 235 240 Glu Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly 245 250 255 Phe Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro 260 265 270 Thr Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly 275 280 285 Phe Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala 290 295 300 Phe Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr 305 310 315 320 Ser Ala Ala Arg Tyr Asp Tyr His Cys Gly Leu Ser Asp Ala Leu Thr 325 330 335 Pro Ala Pro Glu Ala Gly Gln Trp Phe Gln Ala Tyr Phe Glu Gln Leu 340 345 350 Leu Ile Asn Ala Asn Pro Pro Phe 355 360 26359PRTArtificial SequenceChimeric CBH II Polypeptide 26Gly Asn Pro Phe Val Gly Val Thr Pro Trp Ala Asn Ala Tyr Tyr Ala 1 5 10 15 Ser Glu Val Ser Ser Leu Ala Ile Pro Ser Leu Thr Gly Ala Met Ala 20 25 30 Thr Ala Ala Ala Ala Val Ala Lys Val Pro Ser Phe Gln Trp Leu Asp 35 40 45 Arg Asn Val Thr Val Asp Thr Leu Phe Ser Gly Thr Leu Ala Glu Ile 50 55 60 Arg Ala Ala Asn Gln Arg Gly Ala Asn Pro Pro Tyr Ala Gly Ile Phe 65 70 75 80 Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn 85 90 95 Gly Glu Trp Ser Ile Ala Asn Asn Gly Ala Asn Asn Tyr Lys Arg Tyr 100 105 110 Ile Asp Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg Thr 115 120 125 Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met 130 135 140 Asn Val Pro Lys Cys Ser Gly Ala Ala Ser Thr Tyr Arg Glu Leu Thr 145 150 155 160 Ile Tyr Ala Leu Lys Gln Leu Asp Leu Pro His Val Ala Met Tyr Met 165 170 175 Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro 180 185 190 Ala Ala Glu Leu Phe Ala Lys Ile Tyr Glu Asp Ala Gly Lys Pro Arg 195 200 205 Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser 210 215 220 Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu 225 230 235 240 Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly Phe 245 250 255 Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr 260 265 270 Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe 275 280 285 Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe 290 295 300 Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Ser 305 310 315 320 Ala Ala Arg Tyr Asp Tyr His Cys Gly Leu Ser Asp Ala Leu Thr Pro 325 330 335 Ala Pro Glu Ala Gly Gln Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu 340 345 350 Ile Asn Ala Asn Pro Pro Phe 355 27358PRTArtificial SequenceChimeric CBH II Polypeptide 27Gly Asn Pro Phe Ser Gly Val Gln Leu Trp Ala Asn Thr Tyr Tyr Ser 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Ser Leu Ser Pro Glu Leu Ala 20 25 30 Ala Lys Ala Ala Lys Val Ala Glu Val Pro Ser Phe Gln Trp Leu Asp 35 40 45 Arg Asn Val Thr Val Asp Thr Leu Phe Ser Gly Thr Leu Ala Glu Ile 50 55 60 Arg Ala Ala Asn Gln Arg Gly Gly Asn Tyr Ala Gly Gln Phe Val Val 65 70 75 80 Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Leu Ala Ser Asn Gly Glu 85 90 95 Tyr Ser Ile Ala Asp Gly Gly Val Ala Lys Tyr Lys Asn Tyr Ile Asp 100 105 110 Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg Thr Ile Leu 115 120 125 Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met Asn Val 130 135 140 Pro Lys Cys Ser Gly Ala Ala Ser Thr Tyr Lys Glu Leu Thr Val Tyr 145 150 155 160 Ala Leu Lys Gln Leu Asn Leu Pro His Val Ala Met Tyr Met Asp Ala 165 170 175 Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro Ala Ala 180 185 190 Glu Leu Phe Ala Gln Ile Tyr Arg Asp Ala Gly Arg Pro Ala Ala Val 195 200 205 Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser Ile Ala 210 215 220 Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu Lys His 225 230 235 240 Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly Phe Asp Ala 245 250 255 Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr Gly Gln 260 265 270 Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe Gly Val 275 280 285 Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe Val Trp 290 295 300 Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser Ser Ala Pro 305 310 315 320 Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln Pro Ala Pro 325 330 335 Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu Leu Thr Asn 340 345 350 Ala Asn Pro Ser Phe Leu 355 28361PRTArtificial SequenceChimeric CBH II Polypeptide 28Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Gln Trp Leu 35 40 45 Asp Arg Asn Val Thr Val Asp Thr Leu Phe Ser Gly Thr Leu

Ala Glu 50 55 60 Ile Arg Ala Ala Asn Gln Arg Gly Ala Asn Pro Pro Tyr Ala Gly Ile 65 70 75 80 Phe Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser 85 90 95 Asn Gly Glu Trp Ser Ile Ala Asn Asn Gly Ala Asn Asn Tyr Lys Arg 100 105 110 Tyr Ile Asp Arg Ile Arg Glu Leu Leu Ile Gln Tyr Ser Asp Ile Arg 115 120 125 Thr Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn 130 135 140 Met Asn Val Gln Lys Cys Ser Asn Ala Ala Ser Thr Tyr Lys Glu Leu 145 150 155 160 Thr Val Tyr Ala Leu Lys Gln Leu Asn Leu Pro His Val Ala Met Tyr 165 170 175 Met Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln 180 185 190 Pro Ala Ala Glu Leu Phe Ala Gln Ile Tyr Arg Asp Ala Gly Arg Pro 195 200 205 Ala Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Gly Trp 210 215 220 Asn Ile Thr Ser Pro Pro Ser Tyr Thr Gln Gly Asn Ala Val Tyr Asn 225 230 235 240 Glu Lys Leu Tyr Ile His Ala Ile Gly Pro Leu Leu Ala Asn His Gly 245 250 255 Trp Ser Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro 260 265 270 Thr Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly 275 280 285 Phe Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala 290 295 300 Phe Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser 305 310 315 320 Ser Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln 325 330 335 Pro Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu 340 345 350 Leu Thr Asn Ala Asn Pro Ser Phe Leu 355 360 29357PRTArtificial SequenceChimeric CBH II Polypeptide 29Gly Asn Pro Phe Val Gly Val Thr Pro Trp Ala Asn Ala Tyr Tyr Ala 1 5 10 15 Ser Glu Val Ser Ser Leu Ala Ile Pro Ser Leu Thr Gly Ala Met Ala 20 25 30 Thr Ala Ala Ala Ala Val Ala Lys Val Pro Ser Phe Met Trp Leu Asp 35 40 45 Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile Arg 50 55 60 Thr Ala Asn Lys Asn Gly Gly Asn Tyr Ala Gly Gln Phe Val Val Tyr 65 70 75 80 Asp Leu Pro Asp Arg Asp Cys Ala Ala Leu Ala Ser Asn Gly Glu Tyr 85 90 95 Ser Ile Ala Asp Gly Gly Val Ala Lys Tyr Lys Asn Tyr Ile Asp Arg 100 105 110 Ile Arg Glu Leu Leu Ile Gln Tyr Ser Asp Ile Arg Thr Ile Leu Val 115 120 125 Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met Asn Val Gln 130 135 140 Lys Cys Ser Asn Ala Ala Ser Ala Tyr Leu Glu Cys Ile Asn Tyr Ala 145 150 155 160 Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala Gly 165 170 175 His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp Pro Ala Ala Gln 180 185 190 Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro Arg Ala Leu Arg 195 200 205 Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser Val Ser Ser 210 215 220 Pro Pro Pro Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu Lys His Tyr 225 230 235 240 Ile Glu Ala Phe Arg Pro Leu Leu Glu Ala Arg Gly Phe Pro Ala Lys 245 250 255 Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr Gly Gln Leu 260 265 270 Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe Gly Val Arg 275 280 285 Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe Val Trp Val 290 295 300 Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser Ser Ala Pro Arg 305 310 315 320 Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln Pro Ala Pro Gln 325 330 335 Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu Leu Thr Asn Ala 340 345 350 Asn Pro Ser Phe Leu 355 30361PRTArtificial SequenceChimeric CBH II Polypeptide 30Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Gln Trp Leu 35 40 45 Asp Arg Asn Val Thr Val Asp Thr Leu Leu Val Gln Thr Leu Ser Glu 50 55 60 Ile Arg Glu Ala Asn Gln Ala Gly Ala Asn Pro Gln Tyr Ala Ala Gln 65 70 75 80 Ile Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser 85 90 95 Asn Gly Glu Trp Ala Ile Ala Asn Asn Gly Val Asn Asn Tyr Lys Ala 100 105 110 Tyr Ile Asn Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg 115 120 125 Thr Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn 130 135 140 Met Asn Val Pro Lys Cys Ser Gly Ala Ala Ser Thr Tyr Lys Glu Leu 145 150 155 160 Thr Val Tyr Ala Leu Lys Gln Leu Asn Leu Pro His Val Ala Met Tyr 165 170 175 Met Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln 180 185 190 Pro Ala Ala Glu Leu Phe Ala Gln Ile Tyr Arg Asp Ala Gly Arg Pro 195 200 205 Ala Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp 210 215 220 Ser Val Ser Ser Pro Pro Pro Tyr Thr Ser Pro Asn Pro Asn Tyr Asp 225 230 235 240 Glu Lys His Tyr Ile Glu Ala Phe Arg Pro Leu Leu Glu Ala Arg Gly 245 250 255 Phe Pro Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro 260 265 270 Thr Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly 275 280 285 Phe Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala 290 295 300 Phe Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser 305 310 315 320 Ser Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln 325 330 335 Pro Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu 340 345 350 Leu Thr Asn Ala Asn Pro Ser Phe Leu 355 360 31359PRTArtificial SequenceChimeric CBH II Polypeptide 31Gly Asn Pro Phe Val Gly Val Thr Pro Trp Ala Asn Ala Tyr Tyr Ala 1 5 10 15 Ser Glu Val Ser Ser Leu Ala Ile Pro Ser Leu Thr Gly Ala Met Ala 20 25 30 Thr Ala Ala Ala Ala Val Ala Lys Val Pro Ser Phe Gln Trp Leu Asp 35 40 45 Arg Asn Val Thr Val Asp Thr Leu Leu Val Gln Thr Leu Ser Glu Ile 50 55 60 Arg Glu Ala Asn Gln Ala Gly Ala Asn Pro Pro Tyr Ala Gly Ile Phe 65 70 75 80 Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn 85 90 95 Gly Glu Trp Ser Ile Ala Asn Asn Gly Ala Asn Asn Tyr Lys Arg Tyr 100 105 110 Ile Asp Arg Ile Arg Glu Leu Leu Ile Gln Tyr Ser Asp Ile Arg Thr 115 120 125 Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met 130 135 140 Asn Val Gln Lys Cys Ser Asn Ala Ala Ser Thr Tyr Lys Glu Leu Thr 145 150 155 160 Val Tyr Ala Leu Lys Gln Leu Asn Leu Pro His Val Ala Met Tyr Met 165 170 175 Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro 180 185 190 Ala Ala Glu Leu Phe Ala Gln Ile Tyr Arg Asp Ala Gly Arg Pro Ala 195 200 205 Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser 210 215 220 Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu 225 230 235 240 Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly Phe 245 250 255 Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr 260 265 270 Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe 275 280 285 Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe 290 295 300 Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Thr 305 310 315 320 Ala Ala Arg Tyr Asp Tyr His Cys Gly Leu Glu Asp Ala Leu Lys Pro 325 330 335 Ala Pro Glu Ala Gly Gln Trp Phe Asn Glu Tyr Phe Ile Gln Leu Leu 340 345 350 Arg Asn Ala Asn Pro Pro Phe 355 32359PRTArtificial SequenceChimeric CBH II Polypeptide 32Gly Asn Pro Phe Val Gly Val Thr Pro Trp Ala Asn Ala Tyr Tyr Ala 1 5 10 15 Ser Glu Val Ser Ser Leu Ala Ile Pro Ser Leu Thr Gly Ala Met Ala 20 25 30 Thr Ala Ala Ala Ala Val Ala Lys Val Pro Ser Phe Gln Trp Leu Asp 35 40 45 Arg Asn Val Thr Val Asp Thr Leu Leu Val Gln Thr Leu Ser Glu Ile 50 55 60 Arg Glu Ala Asn Gln Ala Gly Ala Asn Pro Pro Tyr Ala Gly Ile Phe 65 70 75 80 Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn 85 90 95 Gly Glu Trp Ser Ile Ala Asn Asn Gly Ala Asn Asn Tyr Lys Arg Tyr 100 105 110 Ile Asp Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg Thr 115 120 125 Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met 130 135 140 Asn Val Pro Lys Cys Ser Gly Ala Ala Ser Thr Tyr Arg Glu Leu Thr 145 150 155 160 Ile Tyr Ala Leu Lys Gln Leu Asp Leu Pro His Val Ala Met Tyr Met 165 170 175 Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro 180 185 190 Ala Ala Glu Leu Phe Ala Lys Ile Tyr Glu Asp Ala Gly Lys Pro Arg 195 200 205 Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser 210 215 220 Val Ser Ser Pro Pro Pro Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu 225 230 235 240 Lys His Tyr Ile Glu Ala Phe Arg Pro Leu Leu Glu Ala Arg Gly Phe 245 250 255 Pro Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr 260 265 270 Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe 275 280 285 Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe 290 295 300 Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Thr 305 310 315 320 Ala Ala Arg Tyr Asp Tyr His Cys Gly Leu Glu Asp Ala Leu Lys Pro 325 330 335 Ala Pro Glu Ala Gly Gln Trp Phe Asn Glu Tyr Phe Ile Gln Leu Leu 340 345 350 Arg Asn Ala Asn Pro Pro Phe 355 33359PRTArtificial SequenceChimeric CBH II Polypeptide 33Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Met Trp Leu 35 40 45 Asp Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile 50 55 60 Arg Thr Ala Asn Lys Asn Gly Ala Asn Pro Pro Tyr Ala Gly Ile Phe 65 70 75 80 Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn 85 90 95 Gly Glu Trp Ser Ile Ala Asn Asn Gly Ala Asn Asn Tyr Lys Arg Tyr 100 105 110 Ile Asp Arg Ile Arg Glu Leu Leu Ile Gln Tyr Ser Asp Ile Arg Thr 115 120 125 Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met 130 135 140 Asn Val Gln Lys Cys Ser Asn Ala Ala Ser Ala Tyr Leu Glu Cys Ile 145 150 155 160 Asn Tyr Ala Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu 165 170 175 Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp Pro 180 185 190 Ala Ala Gln Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro Arg 195 200 205 Ala Leu Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser 210 215 220 Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu 225 230 235 240 Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly Phe 245 250 255 Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr 260 265 270 Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe 275 280 285 Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe 290 295 300 Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Thr 305 310 315 320 Ala Ala Arg Tyr Asp Tyr His Cys Gly Leu Glu Asp Ala Leu Lys Pro 325 330 335 Ala Pro Glu Ala Gly Gln Trp Phe Asn Glu Tyr Phe Ile Gln Leu Leu 340 345 350 Arg Asn Ala Asn Pro Pro Phe 355 34361PRTArtificial SequenceChimeric CBH II Polypeptide 34Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Gln Trp Leu 35 40 45 Asp Arg Asn Val Thr Val Asp Thr Leu Phe Ser Gly Thr Leu Ala Glu 50 55 60 Ile Arg Ala Ala Asn Gln Arg Gly Ala Asn Pro Gln Tyr Ala Ala Gln 65 70 75 80 Ile Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser 85 90 95 Asn Gly Glu Trp Ala Ile Ala Asn Asn Gly Val Asn Asn Tyr Lys Ala 100 105 110 Tyr Ile Asn Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg 115 120 125 Thr Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn 130 135 140 Met Asn Val Pro Lys Cys Ser Gly Ala Ala Ser Ala Tyr Leu Glu Cys 145 150 155

160 Ile Asn Tyr Ala Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr 165 170 175 Leu Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp 180 185 190 Pro Ala Ala Gln Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro 195 200 205 Arg Ala Leu Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp 210 215 220 Ser Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp 225 230 235 240 Glu Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly 245 250 255 Phe Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro 260 265 270 Thr Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly 275 280 285 Phe Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala 290 295 300 Phe Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser 305 310 315 320 Ser Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln 325 330 335 Pro Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu 340 345 350 Leu Thr Asn Ala Asn Pro Ser Phe Leu 355 360 35358PRTArtificial SequenceChimeric CBH II Polypeptide 35Gly Asn Pro Phe Val Gly Val Thr Pro Trp Ala Asn Ala Tyr Tyr Ala 1 5 10 15 Ser Glu Val Ser Ser Leu Ala Ile Pro Ser Leu Thr Gly Ala Met Ala 20 25 30 Thr Ala Ala Ala Ala Val Ala Lys Val Pro Ser Phe Met Trp Leu Asp 35 40 45 Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile Arg 50 55 60 Thr Ala Asn Lys Asn Gly Ala Asn Pro Pro Tyr Ala Gly Ile Phe Val 65 70 75 80 Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly 85 90 95 Glu Trp Ser Ile Ala Asn Asn Gly Ala Asn Asn Tyr Lys Arg Tyr Ile 100 105 110 Asp Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg Thr Ile 115 120 125 Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met Asn 130 135 140 Val Pro Lys Cys Ser Gly Ala Ala Ser Thr Tyr Arg Glu Leu Thr Ile 145 150 155 160 Tyr Ala Leu Lys Gln Leu Asp Leu Pro His Val Ala Met Tyr Met Asp 165 170 175 Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro Ala 180 185 190 Ala Glu Leu Phe Ala Lys Ile Tyr Glu Asp Ala Gly Lys Pro Arg Ala 195 200 205 Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser Ile 210 215 220 Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu Lys 225 230 235 240 His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly Phe Asp 245 250 255 Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr Gly 260 265 270 Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe Gly 275 280 285 Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe Val 290 295 300 Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Thr Ala 305 310 315 320 Ala Arg Tyr Asp Tyr His Cys Gly Leu Glu Asp Ala Leu Lys Pro Ala 325 330 335 Pro Glu Ala Gly Gln Trp Phe Asn Glu Tyr Phe Ile Gln Leu Leu Arg 340 345 350 Asn Ala Asn Pro Pro Phe 355 36360PRTArtificial SequenceChimeric CBH II Polypeptide 36Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Met Trp Leu 35 40 45 Asp Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile 50 55 60 Arg Thr Ala Asn Lys Asn Gly Ala Asn Pro Gln Tyr Ala Ala Gln Ile 65 70 75 80 Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn 85 90 95 Gly Glu Trp Ala Ile Ala Asn Asn Gly Val Asn Asn Tyr Lys Ala Tyr 100 105 110 Ile Asn Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg Thr 115 120 125 Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met 130 135 140 Asn Val Pro Lys Cys Ser Gly Ala Ala Ser Thr Tyr Arg Glu Leu Thr 145 150 155 160 Ile Tyr Ala Leu Lys Gln Leu Asp Leu Pro His Val Ala Met Tyr Met 165 170 175 Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro 180 185 190 Ala Ala Glu Leu Phe Ala Lys Ile Tyr Glu Asp Ala Gly Lys Pro Arg 195 200 205 Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser 210 215 220 Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu 225 230 235 240 Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly Phe 245 250 255 Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr 260 265 270 Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe 275 280 285 Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe 290 295 300 Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser Ser 305 310 315 320 Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln Pro 325 330 335 Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu Leu 340 345 350 Thr Asn Ala Asn Pro Ser Phe Leu 355 360 37360PRTArtificial SequenceChimeric CBH II Polypeptide 37Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Met Trp Leu 35 40 45 Asp Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile 50 55 60 Arg Thr Ala Asn Lys Asn Gly Ala Asn Pro Gln Tyr Ala Ala Gln Ile 65 70 75 80 Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn 85 90 95 Gly Glu Trp Ala Ile Ala Asn Asn Gly Val Asn Asn Tyr Lys Ala Tyr 100 105 110 Ile Asn Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg Thr 115 120 125 Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met 130 135 140 Asn Val Pro Lys Cys Ser Gly Ala Ala Ser Ala Tyr Leu Glu Cys Ile 145 150 155 160 Asn Tyr Ala Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu 165 170 175 Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp Pro 180 185 190 Ala Ala Gln Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro Arg 195 200 205 Ala Leu Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser 210 215 220 Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu 225 230 235 240 Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly Phe 245 250 255 Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr 260 265 270 Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe 275 280 285 Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe 290 295 300 Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser Ser 305 310 315 320 Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln Pro 325 330 335 Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu Leu 340 345 350 Thr Asn Ala Asn Pro Ser Phe Leu 355 360 38359PRTArtificial SequenceChimeric CBH II Polypeptide 38Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Met Trp Leu 35 40 45 Asp Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile 50 55 60 Arg Thr Ala Asn Lys Asn Gly Ala Asn Pro Gln Tyr Ala Ala Gln Ile 65 70 75 80 Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn 85 90 95 Gly Glu Trp Ala Ile Ala Asn Asn Gly Val Asn Asn Tyr Lys Ala Tyr 100 105 110 Ile Asn Arg Ile Arg Glu Leu Leu Ile Gln Tyr Ser Asp Ile Arg Thr 115 120 125 Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met 130 135 140 Asn Val Gln Lys Cys Ser Asn Ala Ala Ser Thr Tyr Arg Glu Leu Thr 145 150 155 160 Ile Tyr Ala Leu Lys Gln Leu Asp Leu Pro His Val Ala Met Tyr Met 165 170 175 Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro 180 185 190 Ala Ala Glu Leu Phe Ala Lys Ile Tyr Glu Asp Ala Gly Lys Pro Arg 195 200 205 Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser 210 215 220 Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu 225 230 235 240 Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly Phe 245 250 255 Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr 260 265 270 Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe 275 280 285 Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe 290 295 300 Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Thr 305 310 315 320 Ala Ala Arg Tyr Asp Tyr His Cys Gly Leu Glu Asp Ala Leu Lys Pro 325 330 335 Ala Pro Glu Ala Gly Gln Trp Phe Asn Glu Tyr Phe Ile Gln Leu Leu 340 345 350 Arg Asn Ala Asn Pro Pro Phe 355 39360PRTArtificial SequenceChimeric CBH II Polypeptide 39Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Met Trp Leu 35 40 45 Asp Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile 50 55 60 Arg Thr Ala Asn Lys Asn Gly Ala Asn Pro Gln Tyr Ala Ala Gln Ile 65 70 75 80 Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn 85 90 95 Gly Glu Trp Ala Ile Ala Asn Asn Gly Val Asn Asn Tyr Lys Ala Tyr 100 105 110 Ile Asn Arg Ile Arg Glu Leu Leu Ile Gln Tyr Ser Asp Ile Arg Thr 115 120 125 Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met 130 135 140 Asn Val Gln Lys Cys Ser Asn Ala Ala Ser Thr Tyr Arg Glu Leu Thr 145 150 155 160 Ile Tyr Ala Leu Lys Gln Leu Asp Leu Pro His Val Ala Met Tyr Met 165 170 175 Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro 180 185 190 Ala Ala Glu Leu Phe Ala Lys Ile Tyr Glu Asp Ala Gly Lys Pro Arg 195 200 205 Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser 210 215 220 Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu 225 230 235 240 Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly Phe 245 250 255 Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr 260 265 270 Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe 275 280 285 Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe 290 295 300 Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser Ser 305 310 315 320 Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln Pro 325 330 335 Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu Leu 340 345 350 Thr Asn Ala Asn Pro Ser Phe Leu 355 360 40360PRTArtificial SequenceChimeric CBH II Polypeptide 40Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Met Trp Leu 35 40 45 Asp Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile 50 55 60 Arg Thr Ala Asn Lys Asn Gly Ala Asn Pro Pro Tyr Ala Gly Ile Phe 65 70 75 80 Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn 85 90 95 Gly Glu Trp Ser Ile Ala Asn Asn Gly Ala Asn Asn Tyr Lys Arg Tyr 100 105 110 Ile Asp Arg Ile Arg Glu Leu Leu Ile Gln Tyr Ser Asp Ile Arg Thr 115 120 125 Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met 130 135 140 Asn Val Gln Lys Cys Ser Asn Ala Ala Ser Ala Tyr Leu Glu Cys Ile 145 150 155 160 Asn Tyr Ala Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu 165 170 175 Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp Pro 180 185 190 Ala Ala Gln Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro Arg 195 200 205 Ala Leu Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser 210 215 220 Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu 225 230 235 240 Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly Phe 245 250 255 Asp Ala Lys Phe Ile Val Asp Thr

Gly Arg Asn Gly Lys Gln Pro Thr 260 265 270 Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe 275 280 285 Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe 290 295 300 Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser Ser 305 310 315 320 Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln Pro 325 330 335 Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu Leu 340 345 350 Thr Asn Ala Asn Pro Ser Phe Leu 355 360 41359PRTArtificial SequenceChimeric CBH II Polypeptide 41Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Met Trp Leu 35 40 45 Asp Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile 50 55 60 Arg Thr Ala Asn Lys Asn Gly Ala Asn Pro Gln Tyr Ala Ala Gln Ile 65 70 75 80 Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn 85 90 95 Gly Glu Trp Ala Ile Ala Asn Asn Gly Val Asn Asn Tyr Lys Ala Tyr 100 105 110 Ile Asn Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg Thr 115 120 125 Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met 130 135 140 Asn Val Pro Lys Cys Ser Gly Ala Ala Ser Thr Tyr Arg Glu Leu Thr 145 150 155 160 Ile Tyr Ala Leu Lys Gln Leu Asp Leu Pro His Val Ala Met Tyr Met 165 170 175 Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro 180 185 190 Ala Ala Glu Leu Phe Ala Lys Ile Tyr Glu Asp Ala Gly Lys Pro Arg 195 200 205 Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser 210 215 220 Val Ser Ser Pro Pro Pro Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu 225 230 235 240 Lys His Tyr Ile Glu Ala Phe Arg Pro Leu Leu Glu Ala Arg Gly Phe 245 250 255 Pro Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr 260 265 270 Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe 275 280 285 Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe 290 295 300 Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Thr 305 310 315 320 Ala Ala Arg Tyr Asp Tyr His Cys Gly Leu Glu Asp Ala Leu Lys Pro 325 330 335 Ala Pro Glu Ala Gly Gln Trp Phe Asn Glu Tyr Phe Ile Gln Leu Leu 340 345 350 Arg Asn Ala Asn Pro Pro Phe 355 42360PRTArtificial SequenceChimeric CBH II Polypeptide 42Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Met Trp Leu 35 40 45 Asp Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile 50 55 60 Arg Thr Ala Asn Lys Asn Gly Ala Asn Pro Pro Tyr Ala Gly Ile Phe 65 70 75 80 Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn 85 90 95 Gly Glu Trp Ser Ile Ala Asn Asn Gly Ala Asn Asn Tyr Lys Arg Tyr 100 105 110 Ile Asp Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg Thr 115 120 125 Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met 130 135 140 Asn Val Pro Lys Cys Ser Gly Ala Ala Ser Thr Tyr Arg Glu Leu Thr 145 150 155 160 Ile Tyr Ala Leu Lys Gln Leu Asp Leu Pro His Val Ala Met Tyr Met 165 170 175 Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro 180 185 190 Ala Ala Glu Leu Phe Ala Lys Ile Tyr Glu Asp Ala Gly Lys Pro Arg 195 200 205 Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser 210 215 220 Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu 225 230 235 240 Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly Phe 245 250 255 Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr 260 265 270 Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe 275 280 285 Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe 290 295 300 Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser Ser 305 310 315 320 Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln Pro 325 330 335 Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu Leu 340 345 350 Thr Asn Ala Asn Pro Ser Phe Leu 355 360 43360PRTArtificial SequenceChimeric CBH II Polypeptide 43Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Gln Trp Leu 35 40 45 Asp Arg Asn Val Thr Val Asp Thr Leu Phe Ser Gly Thr Leu Ala Glu 50 55 60 Ile Arg Ala Ala Asn Gln Arg Gly Ala Asn Pro Pro Tyr Ala Gly Ile 65 70 75 80 Phe Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser 85 90 95 Asn Gly Glu Trp Ser Ile Ala Asn Asn Gly Ala Asn Asn Tyr Lys Arg 100 105 110 Tyr Ile Asp Arg Ile Arg Glu Leu Leu Ile Gln Tyr Ser Asp Ile Arg 115 120 125 Thr Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn 130 135 140 Met Asn Val Gln Lys Cys Ser Asn Ala Ala Ser Ala Tyr Leu Glu Cys 145 150 155 160 Ile Asn Tyr Ala Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr 165 170 175 Leu Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp 180 185 190 Pro Ala Ala Gln Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro 195 200 205 Arg Ala Leu Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp 210 215 220 Ser Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp 225 230 235 240 Glu Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly 245 250 255 Phe Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro 260 265 270 Thr Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly 275 280 285 Phe Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala 290 295 300 Phe Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr 305 310 315 320 Thr Ala Ala Arg Tyr Asp Tyr His Cys Gly Leu Glu Asp Ala Leu Lys 325 330 335 Pro Ala Pro Glu Ala Gly Gln Trp Phe Asn Glu Tyr Phe Ile Gln Leu 340 345 350 Leu Arg Asn Ala Asn Pro Pro Phe 355 360 44359PRTArtificial SequenceChimeric CBH II Polypeptide 44Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Met Trp Leu 35 40 45 Asp Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile 50 55 60 Arg Thr Ala Asn Lys Asn Gly Ala Asn Pro Gln Tyr Ala Ala Gln Ile 65 70 75 80 Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn 85 90 95 Gly Glu Trp Ala Ile Ala Asn Asn Gly Val Asn Asn Tyr Lys Ala Tyr 100 105 110 Ile Asn Arg Ile Arg Glu Leu Leu Ile Gln Tyr Ser Asp Ile Arg Thr 115 120 125 Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met 130 135 140 Asn Val Gln Lys Cys Ser Asn Ala Ala Ser Ala Tyr Leu Glu Cys Ile 145 150 155 160 Asn Tyr Ala Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu 165 170 175 Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp Pro 180 185 190 Ala Ala Gln Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro Arg 195 200 205 Ala Leu Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser 210 215 220 Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu 225 230 235 240 Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly Phe 245 250 255 Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr 260 265 270 Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe 275 280 285 Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe 290 295 300 Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Thr 305 310 315 320 Ala Ala Arg Tyr Asp Tyr His Cys Gly Leu Glu Asp Ala Leu Lys Pro 325 330 335 Ala Pro Glu Ala Gly Gln Trp Phe Asn Glu Tyr Phe Ile Gln Leu Leu 340 345 350 Arg Asn Ala Asn Pro Pro Phe 355 45360PRTArtificial SequenceChimeric CBH II Polypeptide 45Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Met Trp Leu 35 40 45 Asp Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile 50 55 60 Arg Thr Ala Asn Lys Asn Gly Ala Asn Pro Gln Tyr Ala Ala Gln Ile 65 70 75 80 Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn 85 90 95 Gly Glu Trp Ala Ile Ala Asn Asn Gly Val Asn Asn Tyr Lys Ala Tyr 100 105 110 Ile Asn Arg Ile Arg Glu Leu Leu Ile Gln Tyr Ser Asp Ile Arg Thr 115 120 125 Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met 130 135 140 Asn Val Gln Lys Cys Ser Asn Ala Ala Ser Ala Tyr Leu Glu Cys Ile 145 150 155 160 Asn Tyr Ala Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu 165 170 175 Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp Pro 180 185 190 Ala Ala Gln Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro Arg 195 200 205 Ala Leu Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser 210 215 220 Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu 225 230 235 240 Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly Phe 245 250 255 Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr 260 265 270 Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe 275 280 285 Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe 290 295 300 Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser Ser 305 310 315 320 Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln Pro 325 330 335 Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu Leu 340 345 350 Thr Asn Ala Asn Pro Ser Phe Leu 355 360 46361PRTArtificial SequenceChimeric CBH II Polypeptide 46Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Gln Trp Leu 35 40 45 Asp Arg Asn Val Thr Val Asp Thr Leu Phe Ser Gly Thr Leu Ala Glu 50 55 60 Ile Arg Ala Ala Asn Gln Arg Gly Ala Asn Pro Pro Tyr Ala Gly Ile 65 70 75 80 Phe Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser 85 90 95 Asn Gly Glu Trp Ser Ile Ala Asn Asn Gly Ala Asn Asn Tyr Lys Arg 100 105 110 Tyr Ile Asp Arg Ile Arg Glu Leu Leu Ile Gln Tyr Ser Asp Ile Arg 115 120 125 Thr Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn 130 135 140 Met Asn Val Gln Lys Cys Ser Asn Ala Ala Ser Ala Tyr Leu Glu Cys 145 150 155 160 Ile Asn Tyr Ala Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr 165 170 175 Leu Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp 180 185 190 Pro Ala Ala Gln Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro 195 200 205 Arg Ala Leu Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp 210 215 220 Ser Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp 225 230 235 240 Glu Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly 245 250 255 Phe Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro 260 265 270 Thr Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly 275 280 285 Phe Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala 290 295 300 Phe Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser 305 310 315 320 Ser Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln 325 330 335 Pro Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu 340 345 350 Leu Thr Asn Ala Asn Pro Ser Phe Leu 355 360

47360PRTArtificial SequenceChimeric CBH II Polypeptide 47Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Met Trp Leu 35 40 45 Asp Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile 50 55 60 Arg Thr Ala Asn Lys Asn Gly Ala Asn Pro Gln Tyr Ala Ala Gln Ile 65 70 75 80 Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn 85 90 95 Gly Glu Trp Ala Ile Ala Asn Asn Gly Val Asn Asn Tyr Lys Ala Tyr 100 105 110 Ile Asn Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg Thr 115 120 125 Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met 130 135 140 Asn Val Pro Lys Cys Ser Gly Ala Ala Ser Ala Tyr Leu Glu Cys Ile 145 150 155 160 Asn Tyr Ala Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu 165 170 175 Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp Pro 180 185 190 Ala Ala Gln Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro Arg 195 200 205 Ala Leu Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser 210 215 220 Val Ser Ser Pro Pro Pro Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu 225 230 235 240 Lys His Tyr Ile Glu Ala Phe Arg Pro Leu Leu Glu Ala Arg Gly Phe 245 250 255 Pro Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr 260 265 270 Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe 275 280 285 Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe 290 295 300 Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser Ser 305 310 315 320 Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln Pro 325 330 335 Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu Leu 340 345 350 Thr Asn Ala Asn Pro Ser Phe Leu 355 360 48361PRTArtificial SequenceChimeric CBH II Polypeptide 48Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Gln Trp Leu 35 40 45 Asp Arg Asn Val Thr Val Asp Thr Leu Phe Ser Gly Thr Leu Ala Glu 50 55 60 Ile Arg Ala Ala Asn Gln Arg Gly Ala Asn Pro Pro Tyr Ala Gly Ile 65 70 75 80 Phe Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser 85 90 95 Asn Gly Glu Trp Ser Ile Ala Asn Asn Gly Ala Asn Asn Tyr Lys Arg 100 105 110 Tyr Ile Asp Thr Ile Arg Gln Ile Val Val Glu Tyr Ser Asp Ile Arg 115 120 125 Thr Leu Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Leu Val Thr Asn 130 135 140 Leu Gly Thr Pro Lys Cys Ala Asn Ala Gln Ser Ala Tyr Leu Glu Cys 145 150 155 160 Ile Asn Tyr Ala Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr 165 170 175 Leu Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp 180 185 190 Pro Ala Ala Gln Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro 195 200 205 Arg Ala Leu Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp 210 215 220 Ser Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp 225 230 235 240 Glu Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly 245 250 255 Phe Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro 260 265 270 Thr Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly 275 280 285 Phe Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala 290 295 300 Phe Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser 305 310 315 320 Ser Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln 325 330 335 Pro Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu 340 345 350 Leu Thr Asn Ala Asn Pro Ser Phe Leu 355 360 49361PRTArtificial SequenceChimeric CBH II Polypeptide 49Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Gln Trp Leu 35 40 45 Asp Arg Asn Val Thr Val Asp Thr Leu Phe Ser Gly Thr Leu Ala Glu 50 55 60 Ile Arg Ala Ala Asn Gln Arg Gly Ala Asn Pro Gln Tyr Ala Ala Gln 65 70 75 80 Ile Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser 85 90 95 Asn Gly Glu Trp Ala Ile Ala Asn Asn Gly Val Asn Asn Tyr Lys Ala 100 105 110 Tyr Ile Asn Arg Ile Arg Glu Leu Leu Ile Gln Tyr Ser Asp Ile Arg 115 120 125 Thr Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn 130 135 140 Met Asn Val Gln Lys Cys Ser Asn Ala Ala Ser Thr Tyr Arg Glu Leu 145 150 155 160 Thr Ile Tyr Ala Leu Lys Gln Leu Asp Leu Pro His Val Ala Met Tyr 165 170 175 Met Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln 180 185 190 Pro Ala Ala Glu Leu Phe Ala Lys Ile Tyr Glu Asp Ala Gly Lys Pro 195 200 205 Arg Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp 210 215 220 Ser Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp 225 230 235 240 Glu Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly 245 250 255 Phe Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro 260 265 270 Thr Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly 275 280 285 Phe Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala 290 295 300 Phe Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser 305 310 315 320 Ser Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln 325 330 335 Pro Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu 340 345 350 Leu Thr Asn Ala Asn Pro Ser Phe Leu 355 360 50360PRTArtificial SequenceChimeric CBH II Polypeptide 50Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Met Trp Leu 35 40 45 Asp Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile 50 55 60 Arg Thr Ala Asn Lys Asn Gly Ala Asn Pro Pro Tyr Ala Gly Ile Phe 65 70 75 80 Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn 85 90 95 Gly Glu Trp Ser Ile Ala Asn Asn Gly Ala Asn Asn Tyr Lys Arg Tyr 100 105 110 Ile Asp Arg Ile Arg Glu Leu Leu Ile Gln Tyr Ser Asp Ile Arg Thr 115 120 125 Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met 130 135 140 Asn Val Gln Lys Cys Ser Asn Ala Ala Ser Thr Tyr Arg Glu Leu Thr 145 150 155 160 Ile Tyr Ala Leu Lys Gln Leu Asp Leu Pro His Val Ala Met Tyr Met 165 170 175 Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro 180 185 190 Ala Ala Glu Leu Phe Ala Lys Ile Tyr Glu Asp Ala Gly Lys Pro Arg 195 200 205 Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser 210 215 220 Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu 225 230 235 240 Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly Phe 245 250 255 Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr 260 265 270 Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe 275 280 285 Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe 290 295 300 Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser Ser 305 310 315 320 Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln Pro 325 330 335 Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu Leu 340 345 350 Thr Asn Ala Asn Pro Ser Phe Leu 355 360 51361PRTArtificial SequenceChimeric CBH II Polypeptide 51Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Gln Trp Leu 35 40 45 Asp Arg Asn Val Thr Val Asp Thr Leu Phe Ser Gly Thr Leu Ala Glu 50 55 60 Ile Arg Ala Ala Asn Gln Arg Gly Ala Asn Pro Pro Tyr Ala Gly Ile 65 70 75 80 Phe Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser 85 90 95 Asn Gly Glu Trp Ser Ile Ala Asn Asn Gly Ala Asn Asn Tyr Lys Arg 100 105 110 Tyr Ile Asp Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg 115 120 125 Thr Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn 130 135 140 Met Asn Val Pro Lys Cys Ser Gly Ala Ala Ser Ala Tyr Leu Glu Cys 145 150 155 160 Ile Asn Tyr Ala Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr 165 170 175 Leu Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp 180 185 190 Pro Ala Ala Gln Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro 195 200 205 Arg Ala Leu Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp 210 215 220 Ser Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp 225 230 235 240 Glu Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly 245 250 255 Phe Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro 260 265 270 Thr Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly 275 280 285 Phe Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala 290 295 300 Phe Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser 305 310 315 320 Ser Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln 325 330 335 Pro Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu 340 345 350 Leu Thr Asn Ala Asn Pro Ser Phe Leu 355 360 52361PRTArtificial SequenceChimeric CBH II Polypeptide 52Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Gln Trp Leu 35 40 45 Asp Arg Asn Val Thr Val Asp Thr Leu Leu Val Gln Thr Leu Ser Glu 50 55 60 Ile Arg Glu Ala Asn Gln Ala Gly Ala Asn Pro Gln Tyr Ala Ala Gln 65 70 75 80 Ile Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser 85 90 95 Asn Gly Glu Trp Ala Ile Ala Asn Asn Gly Val Asn Asn Tyr Lys Ala 100 105 110 Tyr Ile Asn Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg 115 120 125 Thr Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn 130 135 140 Met Asn Val Pro Lys Cys Ser Gly Ala Ala Ser Thr Tyr Lys Glu Leu 145 150 155 160 Thr Val Tyr Ala Leu Lys Gln Leu Asn Leu Pro His Val Ala Met Tyr 165 170 175 Met Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln 180 185 190 Pro Ala Ala Glu Leu Phe Ala Gln Ile Tyr Arg Asp Ala Gly Arg Pro 195 200 205 Ala Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp 210 215 220 Ser Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp 225 230 235 240 Glu Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly 245 250 255 Phe Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro 260 265 270 Thr Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly 275 280 285 Phe Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala 290 295 300 Phe Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser 305 310 315 320 Ser Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln 325 330 335 Pro Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu 340 345 350 Leu Thr Asn Ala Asn Pro Ser Phe Leu 355 360 53361PRTArtificial SequenceChimeric CBH II Polypeptide 53Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Gln Trp Leu 35 40 45 Asp Arg Asn Val Thr Val Asp Thr Leu Phe Ser Gly Thr Leu Ala Glu 50 55 60 Ile Arg Ala Ala Asn Gln Arg Gly Ala Asn Pro Gln Tyr Ala Ala Gln 65 70 75 80 Ile Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser 85 90

95 Asn Gly Glu Trp Ala Ile Ala Asn Asn Gly Val Asn Asn Tyr Lys Ala 100 105 110 Tyr Ile Asn Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg 115 120 125 Thr Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn 130 135 140 Met Asn Val Pro Lys Cys Ser Gly Ala Ala Ser Thr Tyr Lys Glu Leu 145 150 155 160 Thr Val Tyr Ala Leu Lys Gln Leu Asn Leu Pro His Val Ala Met Tyr 165 170 175 Met Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln 180 185 190 Pro Ala Ala Glu Leu Phe Ala Gln Ile Tyr Arg Asp Ala Gly Arg Pro 195 200 205 Ala Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp 210 215 220 Ser Val Ser Ser Pro Pro Pro Tyr Thr Ser Pro Asn Pro Asn Tyr Asp 225 230 235 240 Glu Lys His Tyr Ile Glu Ala Phe Arg Pro Leu Leu Glu Ala Arg Gly 245 250 255 Phe Pro Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro 260 265 270 Thr Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly 275 280 285 Phe Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala 290 295 300 Phe Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser 305 310 315 320 Ser Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln 325 330 335 Pro Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu 340 345 350 Leu Thr Asn Ala Asn Pro Ser Phe Leu 355 360 54361PRTArtificial SequenceChimeric CBH II Polypeptide 54Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Gln Trp Leu 35 40 45 Asp Arg Asn Val Thr Val Asp Thr Leu Leu Val Gln Thr Leu Ser Glu 50 55 60 Ile Arg Glu Ala Asn Gln Ala Gly Ala Asn Pro Gln Tyr Ala Ala Gln 65 70 75 80 Ile Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser 85 90 95 Asn Gly Glu Trp Ala Ile Ala Asn Asn Gly Val Asn Asn Tyr Lys Ala 100 105 110 Tyr Ile Asn Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg 115 120 125 Thr Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn 130 135 140 Met Asn Val Pro Lys Cys Ser Gly Ala Ala Ser Ala Tyr Leu Glu Cys 145 150 155 160 Ile Asn Tyr Ala Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr 165 170 175 Leu Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp 180 185 190 Pro Ala Ala Gln Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro 195 200 205 Arg Ala Leu Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp 210 215 220 Ser Val Ser Ser Pro Pro Pro Tyr Thr Ser Pro Asn Pro Asn Tyr Asp 225 230 235 240 Glu Lys His Tyr Ile Glu Ala Phe Arg Pro Leu Leu Glu Ala Arg Gly 245 250 255 Phe Pro Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro 260 265 270 Thr Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly 275 280 285 Phe Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala 290 295 300 Phe Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser 305 310 315 320 Ser Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln 325 330 335 Pro Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu 340 345 350 Leu Thr Asn Ala Asn Pro Ser Phe Leu 355 360 55360PRTArtificial SequenceChimeric CBH II Polypeptide 55Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Met Trp Leu 35 40 45 Asp Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile 50 55 60 Arg Thr Ala Asn Lys Asn Gly Ala Asn Pro Gln Tyr Ala Ala Gln Ile 65 70 75 80 Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn 85 90 95 Gly Glu Trp Ala Ile Ala Asn Asn Gly Val Asn Asn Tyr Lys Ala Tyr 100 105 110 Ile Asn Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg Thr 115 120 125 Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met 130 135 140 Asn Val Pro Lys Cys Ser Gly Ala Ala Ser Thr Tyr Lys Glu Leu Thr 145 150 155 160 Val Tyr Ala Leu Lys Gln Leu Asn Leu Pro His Val Ala Met Tyr Met 165 170 175 Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro 180 185 190 Ala Ala Glu Leu Phe Ala Gln Ile Tyr Arg Asp Ala Gly Arg Pro Ala 195 200 205 Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser 210 215 220 Val Ser Ser Pro Pro Pro Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu 225 230 235 240 Lys His Tyr Ile Glu Ala Phe Arg Pro Leu Leu Glu Ala Arg Gly Phe 245 250 255 Pro Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr 260 265 270 Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe 275 280 285 Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe 290 295 300 Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser Ser 305 310 315 320 Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln Pro 325 330 335 Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu Leu 340 345 350 Thr Asn Ala Asn Pro Ser Phe Leu 355 360 56361PRTArtificial SequenceChimeric CBH II Polypeptide 56Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Gln Trp Leu 35 40 45 Asp Arg Asn Val Thr Val Asp Thr Leu Phe Ser Gly Thr Leu Ala Glu 50 55 60 Ile Arg Ala Ala Asn Gln Arg Gly Ala Asn Pro Gln Tyr Ala Ala Gln 65 70 75 80 Ile Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser 85 90 95 Asn Gly Glu Trp Ala Ile Ala Asn Asn Gly Val Asn Asn Tyr Lys Ala 100 105 110 Tyr Ile Asn Arg Ile Arg Glu Leu Leu Ile Gln Tyr Ser Asp Ile Arg 115 120 125 Thr Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn 130 135 140 Met Asn Val Gln Lys Cys Ser Asn Ala Ala Ser Ala Tyr Leu Glu Cys 145 150 155 160 Ile Asn Tyr Ala Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr 165 170 175 Leu Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp 180 185 190 Pro Ala Ala Gln Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro 195 200 205 Arg Ala Leu Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp 210 215 220 Ser Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp 225 230 235 240 Glu Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly 245 250 255 Phe Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro 260 265 270 Thr Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly 275 280 285 Phe Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala 290 295 300 Phe Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser 305 310 315 320 Ser Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln 325 330 335 Pro Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu 340 345 350 Leu Thr Asn Ala Asn Pro Ser Phe Leu 355 360 57361PRTArtificial SequenceChimeric CBH II Polypeptide 57Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Gln Trp Leu 35 40 45 Asp Arg Asn Val Thr Val Asp Thr Leu Leu Val Gln Thr Leu Ser Glu 50 55 60 Ile Arg Glu Ala Asn Gln Ala Gly Ala Asn Pro Gln Tyr Ala Ala Gln 65 70 75 80 Ile Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser 85 90 95 Asn Gly Glu Trp Ala Ile Ala Asn Asn Gly Val Asn Asn Tyr Lys Ala 100 105 110 Tyr Ile Asn Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg 115 120 125 Thr Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn 130 135 140 Met Asn Val Pro Lys Cys Ser Gly Ala Ala Ser Thr Tyr Arg Glu Leu 145 150 155 160 Thr Ile Tyr Ala Leu Lys Gln Leu Asp Leu Pro His Val Ala Met Tyr 165 170 175 Met Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln 180 185 190 Pro Ala Ala Glu Leu Phe Ala Lys Ile Tyr Glu Asp Ala Gly Lys Pro 195 200 205 Arg Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp 210 215 220 Ser Val Ser Ser Pro Pro Pro Tyr Thr Ser Pro Asn Pro Asn Tyr Asp 225 230 235 240 Glu Lys His Tyr Ile Glu Ala Phe Arg Pro Leu Leu Glu Ala Arg Gly 245 250 255 Phe Pro Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro 260 265 270 Thr Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly 275 280 285 Phe Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala 290 295 300 Phe Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser 305 310 315 320 Ser Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln 325 330 335 Pro Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu 340 345 350 Leu Thr Asn Ala Asn Pro Ser Phe Leu 355 360 58361PRTArtificial SequenceChimeric CBH II Polypeptide 58Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Gln Trp Leu 35 40 45 Asp Arg Asn Val Thr Val Asp Thr Leu Phe Ser Gly Thr Leu Ala Glu 50 55 60 Ile Arg Ala Ala Asn Gln Arg Gly Ala Asn Pro Pro Tyr Ala Gly Ile 65 70 75 80 Phe Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser 85 90 95 Asn Gly Glu Trp Ser Ile Ala Asn Asn Gly Ala Asn Asn Tyr Lys Arg 100 105 110 Tyr Ile Asp Arg Ile Arg Glu Leu Leu Ile Gln Tyr Ser Asp Ile Arg 115 120 125 Thr Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn 130 135 140 Met Asn Val Gln Lys Cys Ser Asn Ala Ala Ser Thr Tyr Arg Glu Leu 145 150 155 160 Thr Ile Tyr Ala Leu Lys Gln Leu Asp Leu Pro His Val Ala Met Tyr 165 170 175 Met Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln 180 185 190 Pro Ala Ala Glu Leu Phe Ala Lys Ile Tyr Glu Asp Ala Gly Lys Pro 195 200 205 Arg Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp 210 215 220 Ser Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp 225 230 235 240 Glu Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly 245 250 255 Phe Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro 260 265 270 Thr Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly 275 280 285 Phe Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala 290 295 300 Phe Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser 305 310 315 320 Ser Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln 325 330 335 Pro Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu 340 345 350 Leu Thr Asn Ala Asn Pro Ser Phe Leu 355 360 59361PRTArtificial SequenceChimeric CBH II Polypeptide 59Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Gln Trp Leu 35 40 45 Asp Arg Asn Val Thr Val Asp Thr Leu Phe Ser Gly Thr Leu Ala Glu 50 55 60 Ile Arg Ala Ala Asn Gln Arg Gly Ala Asn Pro Gln Tyr Ala Ala Gln 65 70 75 80 Ile Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser 85 90 95 Asn Gly Glu Trp Ala Ile Ala Asn Asn Gly Val Asn Asn Tyr Lys Ala 100 105 110 Tyr Ile Asn Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg 115 120 125 Thr Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn 130 135 140 Met Asn Val Pro Lys Cys Ser Gly Ala Ala Ser Thr Tyr Arg Glu Leu 145 150 155 160 Thr Ile Tyr Ala Leu Lys Gln Leu Asp Leu Pro His Val Ala Met Tyr 165 170 175 Met Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln 180 185 190 Pro Ala Ala Glu Leu Phe Ala

Lys Ile Tyr Glu Asp Ala Gly Lys Pro 195 200 205 Arg Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp 210 215 220 Ser Val Ser Ser Pro Pro Pro Tyr Thr Ser Pro Asn Pro Asn Tyr Asp 225 230 235 240 Glu Lys His Tyr Ile Glu Ala Phe Arg Pro Leu Leu Glu Ala Arg Gly 245 250 255 Phe Pro Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro 260 265 270 Thr Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly 275 280 285 Phe Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala 290 295 300 Phe Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser 305 310 315 320 Ser Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln 325 330 335 Pro Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu 340 345 350 Leu Thr Asn Ala Asn Pro Ser Phe Leu 355 360 60358PRTArtificial SequenceChimeric CBH II Polypeptide 60Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Met Trp Leu 35 40 45 Asp Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile 50 55 60 Arg Thr Ala Asn Lys Asn Gly Gly Asn Tyr Ala Gly Gln Phe Val Val 65 70 75 80 Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Leu Ala Ser Asn Gly Glu 85 90 95 Tyr Ser Ile Ala Asp Gly Gly Val Ala Lys Tyr Lys Asn Tyr Ile Asp 100 105 110 Thr Ile Arg Gln Ile Val Val Glu Tyr Ser Asp Ile Arg Thr Leu Leu 115 120 125 Val Ile Glu Pro Asp Ser Leu Ala Asn Leu Val Thr Asn Leu Gly Thr 130 135 140 Pro Lys Cys Ala Asn Ala Gln Ser Ala Tyr Leu Glu Cys Ile Asn Tyr 145 150 155 160 Ala Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala 165 170 175 Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp Pro Ala Ala 180 185 190 Gln Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro Arg Ala Leu 195 200 205 Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser Val Ser 210 215 220 Ser Pro Pro Pro Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu Lys His 225 230 235 240 Tyr Ile Glu Ala Phe Arg Pro Leu Leu Glu Ala Arg Gly Phe Pro Ala 245 250 255 Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr Gly Gln 260 265 270 Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe Gly Val 275 280 285 Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe Val Trp 290 295 300 Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser Ser Ala Pro 305 310 315 320 Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln Pro Ala Pro 325 330 335 Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu Leu Thr Asn 340 345 350 Ala Asn Pro Ser Phe Leu 355 61358PRTArtificial SequenceChimeric CBH II Polypeptide 61Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Met Trp Leu 35 40 45 Asp Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile 50 55 60 Arg Thr Ala Asn Lys Asn Gly Gly Asn Tyr Ala Gly Gln Phe Val Val 65 70 75 80 Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Leu Ala Ser Asn Gly Glu 85 90 95 Tyr Ser Ile Ala Asp Gly Gly Val Ala Lys Tyr Lys Asn Tyr Ile Asp 100 105 110 Thr Ile Arg Gln Ile Val Val Glu Tyr Ser Asp Ile Arg Thr Leu Leu 115 120 125 Val Ile Glu Pro Asp Ser Leu Ala Asn Leu Val Thr Asn Leu Gly Thr 130 135 140 Pro Lys Cys Ala Asn Ala Gln Ser Ala Tyr Leu Glu Cys Ile Asn Tyr 145 150 155 160 Ala Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala 165 170 175 Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp Pro Ala Ala 180 185 190 Gln Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro Arg Ala Leu 195 200 205 Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser Ile Ala 210 215 220 Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu Lys His 225 230 235 240 Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly Phe Asp Ala 245 250 255 Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr Gly Gln 260 265 270 Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly Phe Gly Val 275 280 285 Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala Phe Val Trp 290 295 300 Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser Ser Ala Pro 305 310 315 320 Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln Pro Ala Pro 325 330 335 Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu Leu Thr Asn 340 345 350 Ala Asn Pro Ser Phe Leu 355 62361PRTArtificial SequenceChimeric CBH II Polypeptide 62Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Gln Trp Leu 35 40 45 Asp Arg Asn Val Thr Val Asp Thr Leu Phe Ser Gly Thr Leu Ala Glu 50 55 60 Ile Arg Ala Ala Asn Gln Arg Gly Ala Asn Pro Pro Tyr Ala Gly Ile 65 70 75 80 Phe Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser 85 90 95 Asn Gly Glu Trp Ser Ile Ala Asn Asn Gly Ala Asn Asn Tyr Lys Arg 100 105 110 Tyr Ile Asp Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg 115 120 125 Thr Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn 130 135 140 Met Asn Val Pro Lys Cys Ser Gly Ala Ala Ser Thr Tyr Arg Glu Leu 145 150 155 160 Thr Ile Tyr Ala Leu Lys Gln Leu Asp Leu Pro His Val Ala Met Tyr 165 170 175 Met Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln 180 185 190 Pro Ala Ala Glu Leu Phe Ala Lys Ile Tyr Glu Asp Ala Gly Lys Pro 195 200 205 Arg Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp 210 215 220 Ser Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp 225 230 235 240 Glu Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly 245 250 255 Phe Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro 260 265 270 Thr Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly 275 280 285 Phe Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala 290 295 300 Phe Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser 305 310 315 320 Ser Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln 325 330 335 Pro Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu 340 345 350 Leu Thr Asn Ala Asn Pro Ser Phe Leu 355 360 63360PRTArtificial SequenceChimeric CBH II Polypeptide 63Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg 1 5 10 15 Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu 20 25 30 Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser Phe Gln Trp Leu 35 40 45 Asp Arg Asn Val Thr Val Asp Thr Leu Phe Ser Gly Thr Leu Ala Glu 50 55 60 Ile Arg Ala Ala Asn Gln Arg Gly Ala Asn Pro Pro Tyr Ala Gly Ile 65 70 75 80 Phe Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser 85 90 95 Asn Gly Glu Trp Ser Ile Ala Asn Asn Gly Ala Asn Asn Tyr Lys Arg 100 105 110 Tyr Ile Asp Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg 115 120 125 Thr Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn 130 135 140 Met Asn Val Pro Lys Cys Ser Gly Ala Ala Ser Thr Tyr Arg Glu Leu 145 150 155 160 Thr Ile Tyr Ala Leu Lys Gln Leu Asp Leu Pro His Val Ala Met Tyr 165 170 175 Met Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln 180 185 190 Pro Ala Ala Glu Leu Phe Ala Lys Ile Tyr Glu Asp Ala Gly Lys Pro 195 200 205 Arg Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp 210 215 220 Ser Ile Ala Ser Pro Pro Ser Tyr Thr Ser Pro Asn Pro Asn Tyr Asp 225 230 235 240 Glu Lys His Tyr Ile Glu Ala Phe Ala Pro Leu Leu Arg Asn Gln Gly 245 250 255 Phe Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn Gly Lys Gln Pro 260 265 270 Thr Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val Lys Gly Thr Gly 275 280 285 Phe Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu Leu Val Asp Ala 290 295 300 Phe Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr 305 310 315 320 Thr Ala Ala Arg Tyr Asp Tyr His Cys Gly Leu Glu Asp Ala Leu Lys 325 330 335 Pro Ala Pro Glu Ala Gly Gln Trp Phe Asn Glu Tyr Phe Ile Gln Leu 340 345 350 Leu Arg Asn Ala Asn Pro Pro Phe 355 360 64447PRTTrichoderma reesei 64Gln Ala Cys Ser Ser Val Trp Gly Gln Cys Gly Gly Gln Asn Trp Ser 1 5 10 15 Gly Pro Thr Cys Cys Ala Ser Gly Ser Thr Cys Val Tyr Ser Asn Asp 20 25 30 Tyr Tyr Ser Gln Cys Leu Pro Gly Ala Ala Ser Ser Ser Ser Ser Thr 35 40 45 Arg Ala Ala Ser Thr Thr Ser Arg Val Ser Pro Thr Thr Ser Arg Ser 50 55 60 Ser Ser Ala Thr Pro Pro Pro Gly Ser Thr Thr Thr Arg Val Pro Pro 65 70 75 80 Val Gly Ser Gly Thr Ala Thr Tyr Ser Gly Asn Pro Phe Val Gly Val 85 90 95 Thr Pro Trp Ala Asn Ala Tyr Tyr Ala Ser Glu Val Ser Ser Leu Ala 100 105 110 Ile Pro Ser Leu Thr Gly Ala Met Ala Thr Ala Ala Ala Ala Val Ala 115 120 125 Lys Val Pro Ser Phe Met Trp Leu Asp Thr Leu Asp Lys Thr Pro Leu 130 135 140 Met Glu Gln Thr Leu Ala Asp Ile Arg Thr Ala Asn Lys Asn Gly Gly 145 150 155 160 Asn Tyr Ala Gly Gln Phe Val Val Tyr Asp Leu Pro Asp Arg Asp Cys 165 170 175 Ala Ala Leu Ala Ser Asn Gly Glu Tyr Ser Ile Ala Asp Gly Gly Val 180 185 190 Ala Lys Tyr Lys Asn Tyr Ile Asp Thr Ile Arg Gln Ile Val Val Glu 195 200 205 Tyr Ser Asp Ile Arg Thr Leu Leu Val Ile Glu Pro Asp Ser Leu Ala 210 215 220 Asn Leu Val Thr Asn Leu Gly Thr Pro Lys Cys Ala Asn Ala Gln Ser 225 230 235 240 Ala Tyr Leu Glu Cys Ile Asn Tyr Ala Val Thr Gln Leu Asn Leu Pro 245 250 255 Asn Val Ala Met Tyr Leu Asp Ala Gly His Ala Gly Trp Leu Gly Trp 260 265 270 Pro Ala Asn Gln Asp Pro Ala Ala Gln Leu Phe Ala Asn Val Tyr Lys 275 280 285 Asn Ala Ser Ser Pro Arg Ala Leu Arg Gly Leu Ala Thr Asn Val Ala 290 295 300 Asn Tyr Asn Gly Trp Asn Ile Thr Ser Pro Pro Ser Tyr Thr Gln Gly 305 310 315 320 Asn Ala Val Tyr Asn Glu Lys Leu Tyr Ile His Ala Ile Gly Pro Leu 325 330 335 Leu Ala Asn His Gly Trp Ser Asn Ala Phe Phe Ile Thr Asp Gln Gly 340 345 350 Arg Ser Gly Lys Gln Pro Thr Gly Gln Gln Gln Trp Gly Asp Trp Cys 355 360 365 Asn Val Ile Gly Thr Gly Phe Gly Ile Arg Pro Ser Ala Asn Thr Gly 370 375 380 Asp Ser Leu Leu Asp Ser Phe Val Trp Val Lys Pro Gly Gly Glu Cys 385 390 395 400 Asp Gly Thr Ser Asp Ser Ser Ala Pro Arg Phe Asp Ser His Cys Ala 405 410 415 Leu Pro Asp Ala Leu Gln Pro Ala Pro Gln Ala Gly Ala Trp Phe Gln 420 425 430 Ala Tyr Phe Val Gln Leu Leu Thr Asn Ala Asn Pro Ser Phe Leu 435 440 445 65453PRTHumicola insolens 65Ala Ser Cys Ala Pro Thr Trp Gly Gln Cys Gly Gly Ile Gly Phe Asn 1 5 10 15 Gly Pro Thr Cys Cys Gln Ser Gly Ser Thr Cys Val Lys Gln Asn Asp 20 25 30 Trp Tyr Ser Gln Cys Leu Pro Gly Ser Gln Val Thr Thr Thr Ser Thr 35 40 45 Thr Ser Thr Ser Ser Ser Ser Thr Thr Ser Arg Ala Thr Ser Thr Thr 50 55 60 Ser Thr Gly Gly Val Thr Ser Ile Thr Thr Ala Pro Thr Arg Thr Val 65 70 75 80 Thr Ile Pro Gly Gly Ala Thr Thr Thr Ala Ser Tyr Asn Gly Asn Pro 85 90 95 Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg Ser Glu Val 100 105 110 His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu Arg Ala Ala 115 120 125 Ala Ser Ala Val Ala Glu Val Pro Ser Phe Gln Trp Leu Asp Arg Asn 130 135 140 Val Thr Val Asp Thr Leu Leu Val Glu Thr Leu Ser Glu Ile Arg Ala 145 150 155 160 Ala Asn Gln Ala Gly Ala Asn Pro Pro Tyr Ala Ala Gln Ile Val Val 165 170 175 Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly Glu 180 185 190 Trp Ala Ile Ala Asn Asn Gly Ala Asn Asn Tyr Lys Gly Tyr Ile Asn 195 200 205 Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg Thr Ile Leu 210

215 220 Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met Asn Val 225 230 235 240 Ala Lys Cys Ser Gly Ala Ala Ser Thr Tyr Arg Glu Leu Thr Ile Tyr 245 250 255 Ala Leu Lys Gln Leu Asp Leu Pro His Val Ala Met Tyr Met Asp Ala 260 265 270 Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro Ala Ala 275 280 285 Glu Leu Phe Ala Lys Ile Tyr Glu Asp Ala Gly Lys Pro Arg Ala Val 290 295 300 Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser Ile Ser 305 310 315 320 Ser Pro Pro Pro Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu Lys His 325 330 335 Tyr Ile Glu Ala Phe Arg Pro Leu Leu Glu Ala Arg Gly Phe Pro Ala 340 345 350 Gln Phe Ile Val Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly Gln 355 360 365 Lys Glu Trp Gly His Trp Cys Asn Ala Ile Gly Thr Gly Phe Gly Met 370 375 380 Arg Pro Thr Ala Asn Thr Gly His Gln Tyr Val Asp Ala Phe Val Trp 385 390 395 400 Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Thr Thr Ala Ala 405 410 415 Arg Tyr Asp Tyr His Cys Gly Leu Glu Asp Ala Leu Lys Pro Ala Pro 420 425 430 Glu Ala Gly Gln Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu Arg Asn 435 440 445 Ala Asn Pro Pro Phe 450 66440PRTPhanerochaete chrysosporium 66Ala Ser Ser Glu Trp Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly Pro 1 5 10 15 Thr Thr Cys Val Ser Gly Thr Thr Cys Thr Val Leu Asn Pro Tyr Tyr 20 25 30 Ser Gln Cys Leu Pro Gly Ser Ala Val Thr Thr Thr Ser Val Ile Thr 35 40 45 Ser His Ser Ser Ser Val Ser Ser Val Ser Ser His Ser Gly Ser Ser 50 55 60 Thr Ser Thr Ser Ser Pro Thr Gly Pro Thr Gly Thr Asn Pro Pro Pro 65 70 75 80 Pro Pro Ser Ala Asn Asn Pro Trp Thr Gly Phe Gln Ile Phe Leu Ser 85 90 95 Pro Tyr Tyr Ala Asn Glu Val Ala Ala Ala Ala Lys Gln Ile Thr Asp 100 105 110 Pro Thr Leu Ser Ser Lys Ala Ala Ser Val Ala Asn Ile Pro Thr Phe 115 120 125 Thr Trp Leu Asp Ser Val Ala Lys Ile Pro Asp Leu Gly Thr Tyr Leu 130 135 140 Ala Ser Ala Ser Ala Leu Gly Lys Ser Thr Gly Thr Lys Gln Leu Val 145 150 155 160 Gln Ile Val Ile Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Lys Ala 165 170 175 Ser Asn Gly Glu Phe Ser Ile Ala Asn Asn Gly Gln Ala Asn Tyr Glu 180 185 190 Asn Tyr Ile Asp Gln Ile Val Ala Gln Ile Gln Gln Phe Pro Asp Val 195 200 205 Arg Val Val Ala Val Ile Glu Pro Asp Ser Leu Ala Asn Leu Val Thr 210 215 220 Asn Leu Asn Val Gln Lys Cys Ala Asn Ala Lys Thr Thr Tyr Leu Ala 225 230 235 240 Cys Val Asn Tyr Ala Leu Thr Asn Leu Ala Lys Val Gly Val Tyr Met 245 250 255 Tyr Met Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Leu 260 265 270 Ser Pro Ala Ala Gln Leu Phe Thr Gln Val Trp Gln Asn Ala Gly Lys 275 280 285 Ser Pro Phe Ile Lys Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala 290 295 300 Leu Gln Ala Ala Ser Pro Asp Pro Ile Thr Gln Gly Asn Pro Asn Tyr 305 310 315 320 Asp Glu Ile His Tyr Ile Asn Ala Leu Ala Pro Leu Leu Gln Gln Ala 325 330 335 Gly Trp Asp Ala Thr Phe Ile Val Asp Gln Gly Arg Ser Gly Val Gln 340 345 350 Asn Ile Arg Gln Gln Trp Gly Asp Trp Cys Asn Ile Lys Gly Ala Gly 355 360 365 Phe Gly Thr Arg Pro Thr Thr Asn Thr Gly Ser Gln Phe Ile Asp Ser 370 375 380 Ile Val Trp Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asn Ser 385 390 395 400 Ser Ser Pro Arg Tyr Asp Ser Thr Cys Ser Leu Pro Asp Ala Ala Gln 405 410 415 Pro Ala Pro Glu Ala Gly Thr Trp Phe Gln Ala Tyr Phe Gln Thr Leu 420 425 430 Val Ser Ala Ala Asn Pro Pro Leu 435 440 67447PRTTrichoderma reesei 67Gln Ala Cys Ser Ser Val Trp Gly Gln Cys Gly Gly Gln Asn Trp Ser 1 5 10 15 Gly Pro Thr Cys Cys Ala Ser Gly Ser Thr Cys Val Tyr Ser Asn Asp 20 25 30 Tyr Tyr Ser Gln Cys Leu Pro Gly Ala Ala Ser Ser Ser Ser Ser Thr 35 40 45 Arg Ala Ala Ser Thr Thr Ser Arg Val Ser Pro Thr Thr Ser Arg Ser 50 55 60 Ser Ser Ala Thr Pro Pro Pro Gly Ser Thr Thr Thr Arg Val Pro Pro 65 70 75 80 Val Gly Ser Gly Thr Ala Thr Tyr Ser Gly Asn Pro Phe Val Gly Val 85 90 95 Thr Pro Trp Ala Asn Ala Tyr Tyr Ala Ser Glu Val Ser Ser Leu Ala 100 105 110 Ile Pro Ser Leu Thr Gly Ala Met Ala Thr Ala Ala Ala Ala Val Ala 115 120 125 Lys Val Pro Ser Phe Met Trp Leu Asp Thr Leu Asp Lys Thr Pro Leu 130 135 140 Met Glu Gln Thr Leu Ala Asp Ile Arg Thr Ala Asn Lys Asn Gly Gly 145 150 155 160 Asn Tyr Ala Gly Gln Phe Val Val Tyr Asp Leu Pro Asp Arg Asp Cys 165 170 175 Ala Ala Leu Ala Ser Asn Gly Glu Tyr Ser Ile Ala Asp Gly Gly Val 180 185 190 Ala Lys Tyr Lys Asn Tyr Ile Asp Thr Ile Arg Gln Ile Val Val Glu 195 200 205 Tyr Ser Asp Ile Arg Thr Leu Leu Val Ile Glu Pro Asp Ser Leu Ala 210 215 220 Asn Leu Val Thr Asn Leu Gly Thr Pro Lys Cys Ala Asn Ala Gln Ser 225 230 235 240 Ala Tyr Leu Glu Cys Ile Asn Tyr Ala Val Thr Gln Leu Asn Leu Pro 245 250 255 Asn Val Ala Met Tyr Leu Asp Ala Gly His Ala Gly Trp Leu Gly Trp 260 265 270 Pro Ala Asn Gln Asp Pro Ala Ala Gln Leu Phe Ala Asn Val Tyr Lys 275 280 285 Asn Ala Ser Ser Pro Arg Ala Leu Arg Gly Leu Ala Thr Asn Val Ala 290 295 300 Asn Tyr Asn Gly Trp Asn Ile Thr Ser Pro Pro Ser Tyr Thr Gln Gly 305 310 315 320 Asn Ala Val Tyr Asn Glu Lys Leu Tyr Ile His Ala Ile Gly Pro Leu 325 330 335 Leu Ala Asn His Gly Trp Ser Asn Ala Phe Phe Ile Thr Asp Gln Gly 340 345 350 Arg Ser Gly Lys Gln Pro Thr Gly Gln Gln Gln Trp Gly Asp Trp Cys 355 360 365 Asn Val Ile Gly Thr Gly Phe Gly Ile Arg Pro Ser Ala Asn Thr Gly 370 375 380 Asp Ser Leu Leu Asp Ser Phe Val Trp Val Lys Pro Gly Gly Glu Cys 385 390 395 400 Asp Gly Thr Ser Asp Ser Ser Ala Pro Arg Phe Asp Pro His Cys Ala 405 410 415 Leu Pro Asp Ala Leu Gln Pro Ala Pro Gln Ala Gly Ala Trp Phe Gln 420 425 430 Ala Tyr Phe Val Gln Leu Leu Thr Asn Ala Asn Pro Ser Phe Leu 435 440 445 68447PRTTrichoderma reesei 68Gln Ala Cys Ser Ser Val Trp Gly Gln Cys Gly Gly Gln Asn Trp Ser 1 5 10 15 Gly Pro Thr Cys Cys Ala Ser Gly Ser Thr Cys Val Tyr Ser Asn Asp 20 25 30 Tyr Tyr Ser Gln Cys Leu Pro Gly Ala Ala Ser Ser Ser Ser Ser Thr 35 40 45 Arg Ala Ala Ser Thr Thr Ser Arg Val Ser Pro Thr Thr Ser Arg Ser 50 55 60 Ser Ser Ala Thr Pro Pro Pro Gly Ser Thr Thr Thr Arg Val Pro Pro 65 70 75 80 Val Glu Ser Gly Thr Ala Thr Tyr Ser Gly Asn Pro Phe Val Gly Val 85 90 95 Thr Pro Trp Ala Asn Ala Tyr Tyr Ala Ser Glu Val Ser Ser Leu Ala 100 105 110 Ile Pro Ser Leu Thr Gly Ala Met Ala Thr Ala Ala Ala Ala Val Ala 115 120 125 Lys Val Pro Ser Phe Met Trp Leu Asp Thr Leu Asp Lys Thr Pro Leu 130 135 140 Met Glu Gln Thr Leu Ala Asp Ile Arg Thr Ala Asn Lys Asn Gly Gly 145 150 155 160 Asn Tyr Ala Gly Gln Phe Val Val Tyr Asp Leu Pro Asp Arg Asp Cys 165 170 175 Ala Ala Leu Ala Ser Asn Gly Glu Tyr Ser Ile Ala Asp Gly Gly Val 180 185 190 Ala Lys Tyr Lys Asn Tyr Ile Asp Thr Ile Arg Gln Ile Val Val Glu 195 200 205 Tyr Ser Asp Ile Arg Thr Leu Leu Val Ile Glu Pro Asp Ser Leu Ala 210 215 220 Asn Leu Val Thr Asn Leu Ser Thr Pro Lys Cys Ala Asn Ala Gln Ser 225 230 235 240 Ala Tyr Leu Glu Cys Ile Asn Tyr Ala Val Thr Gln Leu Asn Leu Pro 245 250 255 Asn Val Ala Met Tyr Leu Asp Ala Gly His Ala Gly Trp Leu Gly Trp 260 265 270 Pro Ala Asn Gln Asp Pro Ala Ala Gln Leu Phe Ala Asn Val Tyr Lys 275 280 285 Asn Ala Ser Ser Pro Arg Ala Leu Arg Gly Leu Ala Thr Asn Val Ala 290 295 300 Ser Tyr Asn Gly Trp Asn Ile Thr Ser Pro Pro Ser Tyr Thr Gln Gly 305 310 315 320 Asn Ala Val Tyr Asn Glu Lys Leu Tyr Ile His Ala Ile Gly Pro Leu 325 330 335 Leu Ala Asn His Gly Trp Ser Asn Ala Phe Phe Ile Thr Asp Gln Gly 340 345 350 Arg Ser Gly Lys Gln Pro Thr Gly Gln Gln Gln Trp Gly Asp Trp Cys 355 360 365 Asn Val Ile Gly Thr Gly Phe Gly Ile Arg Pro Ser Ala Asn Thr Gly 370 375 380 Asp Ser Leu Leu Asp Ser Phe Val Trp Val Lys Pro Gly Gly Glu Cys 385 390 395 400 Asp Gly Thr Ser Asp Ser Ser Ala Pro Gln Phe Asp Pro His Cys Ala 405 410 415 Leu Pro Asp Ala Leu Gln Pro Ala Pro Gln Ala Gly Ala Trp Phe Gln 420 425 430 Ala Tyr Phe Val Gln Leu Leu Thr Asn Ala Asn Pro Ser Phe Leu 435 440 445 69447PRTTrichoderma reesei 69Gln Ala Cys Ser Ser Val Trp Gly Gln Cys Gly Gly Gln Asn Trp Ser 1 5 10 15 Gly Pro Thr Cys Cys Ala Ser Gly Ser Thr Cys Val Tyr Ser Asn Asp 20 25 30 Tyr Tyr Ser Gln Cys Leu Pro Gly Ala Ala Ser Ser Ser Ser Ser Thr 35 40 45 Arg Ala Ala Ser Thr Thr Ser Arg Val Ser Pro Thr Thr Ser Arg Ser 50 55 60 Ser Ser Ala Thr Pro Pro Pro Gly Ser Thr Thr Thr Arg Val Pro Pro 65 70 75 80 Val Gly Ser Gly Thr Ala Thr Tyr Ser Gly Asn Pro Phe Val Gly Val 85 90 95 Thr Pro Trp Ala Asn Ala Tyr Tyr Ala Ser Glu Val Ser Ser Leu Ala 100 105 110 Ile Pro Ser Leu Thr Gly Ala Met Ala Thr Ala Ala Ala Ala Val Ala 115 120 125 Lys Val Pro Ser Phe Met Trp Leu Asp Thr Leu Asp Lys Thr Pro Leu 130 135 140 Met Glu Gln Thr Leu Ala Asp Ile Arg Thr Ala Asn Lys Asn Gly Gly 145 150 155 160 Asn Tyr Ala Gly Gln Phe Val Val Tyr Asp Leu Pro Asp Arg Asp Cys 165 170 175 Ala Ala Leu Ala Ser Asn Gly Glu Tyr Ser Ile Ala Asp Gly Gly Val 180 185 190 Ala Lys Tyr Lys Asn Tyr Ile Asp Thr Ile Arg Gln Ile Val Val Glu 195 200 205 Tyr Ser Asp Ile Arg Thr Leu Leu Val Ile Glu Pro Asp Ser Leu Ala 210 215 220 Asn Leu Val Thr Asn Leu Ser Thr Pro Lys Cys Ala Asn Ala Gln Ser 225 230 235 240 Ala Tyr Leu Glu Cys Ile Asn Tyr Ala Val Thr Gln Leu Asn Leu Pro 245 250 255 Asn Val Ala Met Tyr Leu Asp Ala Gly His Ala Gly Trp Leu Gly Trp 260 265 270 Pro Ala Asn Gln Asp Pro Ala Ala Gln Leu Phe Ala Asn Val Tyr Lys 275 280 285 Asn Ala Ser Ser Pro Arg Ala Leu Arg Gly Leu Ala Thr Asn Val Ala 290 295 300 Asn Tyr Asn Gly Trp Asn Ile Thr Ser Pro Pro Ser Tyr Thr Gln Gly 305 310 315 320 Asn Ala Val Tyr Asn Glu Lys Leu Tyr Ile His Ala Ile Gly Pro Leu 325 330 335 Leu Ala Asn His Gly Trp Ser Asn Ala Phe Phe Ile Thr Asp Gln Gly 340 345 350 Arg Ser Gly Lys Gln Pro Thr Gly Gln Gln Gln Trp Gly Asp Trp Cys 355 360 365 Asn Val Ile Gly Thr Gly Phe Gly Ile Arg Pro Ser Ala Asn Thr Gly 370 375 380 Asp Ser Leu Leu Asp Ser Phe Val Trp Val Lys Pro Gly Gly Glu Cys 385 390 395 400 Asp Gly Thr Ser Asp Ser Ser Ala Pro Arg Phe Asp Pro His Cys Ala 405 410 415 Leu Pro Asp Ala Leu Gln Pro Ala Pro Gln Ala Gly Ala Trp Phe Gln 420 425 430 Ala Tyr Phe Val Gln Leu Leu Thr Asn Ala Asn Pro Ser Phe Leu 435 440 445 70447PRTTrichoderma reesei 70Gln Ala Cys Ser Ser Val Trp Gly Gln Cys Gly Gly Gln Asn Trp Ser 1 5 10 15 Gly Pro Thr Cys Cys Ala Ser Gly Ser Thr Cys Val Tyr Ser Asn Asp 20 25 30 Tyr Tyr Ser Gln Cys Leu Pro Gly Ala Ala Ser Ser Ser Ser Ser Thr 35 40 45 Arg Ala Ala Ser Thr Thr Ser Arg Val Ser Pro Thr Thr Ser Arg Ser 50 55 60 Ser Ser Ala Thr Pro Pro Pro Gly Ser Thr Thr Thr Arg Val Pro Pro 65 70 75 80 Val Gly Ser Gly Thr Ala Thr Tyr Ser Gly Asn Pro Phe Val Gly Val 85 90 95 Thr Pro Trp Ala Asn Ala Tyr Tyr Ala Ser Glu Val Ser Ser Leu Ala 100 105 110 Ile Pro Ser Leu Thr Gly Ala Met Ala Thr Ala Ala Ala Ala Val Ala 115 120 125 Lys Val Pro Ser Phe Met Trp Leu Asp Thr Leu Asp Lys Thr Pro Leu 130 135 140 Met Glu Gln Thr Leu Ala Asp Ile Arg Thr Ala Asn Lys Asn Gly Gly 145 150 155 160 Asn Tyr Ala Gly Gln Phe Val Val Tyr Asp Leu Pro Asp Arg Asp Cys 165 170 175 Ala Ala Leu Ala Ser Asn Gly Glu Tyr Ser Ile Ala Asp Gly Gly Val 180 185 190 Ala Lys Tyr Lys Asn Tyr Ile Asp Thr Ile Arg Gln Ile Val Val Glu 195 200 205 Tyr Ser Asp Ile Arg Thr Leu Leu Val Ile Glu Pro Asp Ser Leu Ala 210 215 220 Asn Leu Val Thr Asn Leu Gly Thr Pro Lys Cys Ala Asn Ala Gln Ser 225 230 235 240 Ala Tyr Leu Glu Cys Ile Asn Tyr Ala Val Thr Gln Leu Asn Leu Pro 245 250 255 Asn Val Ala Met Tyr Leu Asp Ala Gly His Ala Gly Trp Leu Gly Trp 260 265

270 Pro Ala Asn Gln Asp Pro Ala Ala Gln Leu Phe Ala Asn Val Tyr Lys 275 280 285 Asn Ala Ser Ser Pro Arg Ala Leu Arg Gly Leu Ala Thr Asn Val Ala 290 295 300 Ser Tyr Asn Gly Trp Asn Ile Thr Ser Pro Pro Ser Tyr Thr Gln Gly 305 310 315 320 Asn Ala Val Tyr Asn Glu Lys Leu Tyr Ile His Ala Ile Gly Pro Leu 325 330 335 Leu Ala Asn His Gly Trp Ser Asn Ala Phe Phe Ile Thr Asp Gln Gly 340 345 350 Arg Ser Gly Lys Gln Pro Thr Gly Gln Gln Gln Trp Gly Asp Trp Cys 355 360 365 Asn Val Ile Gly Thr Gly Phe Gly Ile Arg Pro Ser Ala Asn Thr Gly 370 375 380 Asp Ser Leu Leu Asp Ser Phe Val Trp Val Lys Pro Gly Gly Glu Cys 385 390 395 400 Asp Gly Thr Ser Asp Ser Ser Ala Pro Arg Phe Asp Pro His Cys Ala 405 410 415 Leu Pro Asp Ala Leu Gln Pro Ala Pro Gln Ala Gly Ala Trp Phe Gln 420 425 430 Ala Tyr Phe Val Gln Leu Leu Thr Asn Ala Asn Pro Ser Phe Leu 435 440 445 71447PRTTrichoderma reesei 71Gln Ala Cys Ser Ser Val Trp Gly Gln Cys Gly Gly Gln Asn Trp Ser 1 5 10 15 Gly Pro Thr Cys Cys Ala Ser Gly Ser Thr Cys Val Tyr Ser Asn Asp 20 25 30 Tyr Tyr Ser Gln Cys Leu Pro Gly Ala Ala Ser Ser Ser Ser Ser Thr 35 40 45 Arg Ala Ala Ser Thr Thr Ser Arg Val Ser Pro Thr Thr Ser Arg Ser 50 55 60 Ser Ser Ala Thr Pro Pro Pro Gly Ser Thr Thr Thr Arg Val Pro Pro 65 70 75 80 Val Gly Ser Gly Thr Ala Thr Tyr Ser Gly Asn Pro Phe Val Gly Val 85 90 95 Thr Pro Trp Ala Asn Ala Tyr Tyr Ala Ser Glu Val Ser Ser Leu Ala 100 105 110 Ile Pro Ser Leu Thr Gly Ala Met Ala Thr Ala Ala Ala Ala Val Ala 115 120 125 Lys Val Pro Ser Phe Met Trp Leu Asp Thr Leu Asp Lys Thr Pro Leu 130 135 140 Met Glu Gln Thr Leu Ala Asp Ile Arg Thr Ala Asn Lys Asn Gly Gly 145 150 155 160 Asn Tyr Ala Gly Gln Phe Val Val Tyr Asp Leu Pro Asp Arg Asp Cys 165 170 175 Ala Ala Leu Ala Ser Asn Gly Glu Tyr Ser Ile Ala Asp Gly Gly Val 180 185 190 Ala Lys Tyr Lys Asn Tyr Ile Asp Thr Ile Arg Gln Ile Val Val Glu 195 200 205 Tyr Ser Asp Ile Arg Thr Leu Leu Val Ile Glu Pro Asp Ser Leu Ala 210 215 220 Asn Leu Val Thr Asn Leu Gly Thr Pro Lys Cys Ala Asn Ala Gln Ser 225 230 235 240 Ala Tyr Leu Glu Cys Ile Asn Tyr Ala Val Thr Gln Leu Asn Leu Pro 245 250 255 Asn Val Ala Met Tyr Leu Asp Ala Gly His Ala Gly Trp Leu Gly Trp 260 265 270 Pro Ala Asn Gln Asp Pro Ala Ala Gln Leu Phe Ala Asn Val Tyr Lys 275 280 285 Asn Ala Ser Ser Pro Arg Ala Leu Arg Gly Leu Ala Thr Asn Val Ala 290 295 300 Asn Tyr Asn Gly Trp Asn Ile Thr Ser Pro Pro Ser Tyr Thr Gln Gly 305 310 315 320 Asn Ala Val Tyr Asn Glu Lys Leu Tyr Ile His Ala Ile Gly Pro Leu 325 330 335 Leu Ala Asn His Gly Trp Ser Asn Ala Phe Phe Ile Thr Asp Gln Gly 340 345 350 Arg Ser Gly Lys Gln Pro Thr Gly Gln Gln Gln Trp Gly Asp Trp Cys 355 360 365 Asn Val Ile Gly Thr Gly Phe Gly Ile Arg Pro Ser Ala Asn Thr Gly 370 375 380 Asp Ser Leu Leu Asp Ser Phe Val Trp Val Lys Pro Gly Gly Glu Cys 385 390 395 400 Asp Gly Thr Ser Asp Ser Ser Ala Pro Gln Phe Asp Pro His Cys Ala 405 410 415 Leu Pro Asp Ala Leu Gln Pro Ala Pro Gln Ala Gly Ala Trp Phe Gln 420 425 430 Ala Tyr Phe Val Gln Leu Leu Thr Asn Ala Asn Pro Ser Phe Leu 435 440 445 72447PRTTrichoderma reesei 72Gln Ala Cys Ser Ser Val Trp Gly Gln Cys Gly Gly Gln Asn Trp Ser 1 5 10 15 Gly Pro Thr Cys Cys Ala Ser Gly Ser Thr Cys Val Tyr Ser Asn Asp 20 25 30 Tyr Tyr Ser Gln Cys Leu Pro Gly Ala Ala Ser Ser Ser Ser Ser Thr 35 40 45 Arg Ala Ala Ser Thr Thr Ser Arg Val Ser Pro Thr Thr Ser Arg Ser 50 55 60 Ser Ser Ala Thr Pro Pro Pro Gly Ser Thr Thr Thr Arg Val Pro Pro 65 70 75 80 Val Gly Ser Gly Thr Ala Thr Tyr Ser Gly Asn Pro Phe Val Gly Val 85 90 95 Thr Pro Trp Ala Asn Ala Tyr Tyr Ala Ser Glu Val Ser Ser Leu Ala 100 105 110 Ile Pro Ser Leu Thr Gly Ala Met Ala Thr Ala Ala Ala Ala Val Ala 115 120 125 Lys Val Pro Ser Phe Met Trp Leu Asp Thr Leu Asp Lys Thr Pro Leu 130 135 140 Met Glu Gln Thr Leu Ala Asp Ile Arg Thr Ala Asn Lys Asn Gly Gly 145 150 155 160 Asn Tyr Ala Gly Gln Phe Val Val Tyr Asp Leu Pro Asp Arg Asp Cys 165 170 175 Ala Ala Leu Ala Ser Asn Gly Glu Tyr Ser Ile Ala Asp Gly Gly Val 180 185 190 Ala Lys Tyr Lys Asn Tyr Ile Asp Thr Ile Arg Gln Ile Val Val Glu 195 200 205 Tyr Ser Asp Ile Arg Thr Leu Leu Val Ile Glu Pro Asp Ser Leu Ala 210 215 220 Asn Leu Val Thr Asn Leu Ser Thr Pro Lys Cys Ala Asn Ala Gln Ser 225 230 235 240 Ala Tyr Leu Glu Cys Ile Asn Tyr Ala Val Thr Gln Leu Asn Leu Pro 245 250 255 Asn Val Ala Met Tyr Leu Asp Ala Gly His Ala Gly Trp Leu Gly Trp 260 265 270 Pro Ala Asn Gln Asp Pro Ala Ala Gln Leu Phe Ala Asn Val Tyr Lys 275 280 285 Asn Ala Ser Ser Pro Arg Ala Leu Arg Gly Leu Ala Thr Asn Val Ala 290 295 300 Ser Tyr Asn Gly Trp Asn Ile Thr Ser Pro Pro Ser Tyr Thr Gln Gly 305 310 315 320 Asn Ala Val Tyr Asn Glu Lys Leu Tyr Ile His Ala Ile Gly Pro Leu 325 330 335 Leu Ala Asn His Gly Trp Ser Asn Ala Phe Phe Ile Thr Asp Gln Gly 340 345 350 Arg Ser Gly Lys Gln Pro Thr Gly Gln Gln Gln Trp Gly Asp Trp Cys 355 360 365 Asn Val Ile Gly Thr Gly Phe Gly Ile Arg Pro Ser Ala Asn Thr Gly 370 375 380 Asp Ser Leu Leu Asp Ser Phe Val Trp Val Lys Pro Gly Gly Glu Cys 385 390 395 400 Asp Gly Thr Ser Asp Ser Ser Ala Pro Arg Phe Asp Pro His Cys Ala 405 410 415 Leu Pro Asp Ala Leu Gln Pro Ala Pro Gln Ala Gly Ala Trp Phe Gln 420 425 430 Ala Tyr Phe Val Gln Leu Leu Thr Asn Ala Asn Pro Ser Phe Leu 435 440 445 73447PRTTrichoderma reesei 73Gln Ala Cys Ser Ser Val Trp Gly Gln Cys Gly Gly Gln Asn Trp Ser 1 5 10 15 Gly Pro Thr Cys Cys Ala Ser Gly Ser Thr Cys Val Tyr Ser Asn Asp 20 25 30 Tyr Tyr Ser Gln Cys Leu Pro Gly Ala Ala Ser Ser Ser Ser Ser Thr 35 40 45 Arg Ala Ala Ser Thr Thr Ser Arg Val Ser Pro Thr Thr Ser Arg Ser 50 55 60 Ser Ser Ala Thr Pro Pro Pro Gly Ser Thr Thr Thr Arg Val Pro Pro 65 70 75 80 Val Gly Ser Gly Thr Ala Thr Tyr Ser Gly Asn Pro Phe Val Gly Val 85 90 95 Thr Pro Trp Ala Asn Ala Tyr Tyr Ala Ser Glu Val Ser Ser Leu Ala 100 105 110 Ile Pro Ser Leu Thr Gly Ala Met Ala Thr Ala Ala Ala Ala Val Ala 115 120 125 Lys Val Pro Ser Phe Met Trp Leu Asp Thr Leu Asp Lys Thr Pro Leu 130 135 140 Met Glu Gln Thr Leu Ala Asp Ile Arg Thr Ala Asn Lys Asn Gly Gly 145 150 155 160 Asn Tyr Ala Gly Gln Phe Val Val Tyr Asp Leu Pro Asp Arg Asp Cys 165 170 175 Ala Ala Leu Ala Ser Asn Gly Glu Tyr Ser Ile Ala Asp Gly Gly Val 180 185 190 Ala Lys Tyr Lys Asn Tyr Ile Asp Thr Ile Arg Gln Ile Val Val Glu 195 200 205 Tyr Ser Asp Ile Arg Thr Leu Leu Val Ile Glu Pro Asp Ser Leu Ala 210 215 220 Asn Leu Val Thr Asn Leu Ser Thr Pro Lys Cys Ala Asn Ala Gln Ser 225 230 235 240 Ala Tyr Leu Glu Cys Ile Asn Tyr Ala Val Thr Gln Leu Asn Leu Pro 245 250 255 Asn Val Ala Met Tyr Leu Asp Ala Gly His Ala Gly Trp Leu Gly Trp 260 265 270 Pro Ala Asn Gln Asp Pro Ala Ala Gln Leu Phe Ala Asn Val Tyr Lys 275 280 285 Asn Ala Ser Ser Pro Arg Ala Leu Arg Gly Leu Ala Thr Asn Val Ala 290 295 300 Asn Tyr Asn Gly Trp Asn Ile Thr Ser Pro Pro Ser Tyr Thr Gln Gly 305 310 315 320 Asn Ala Val Tyr Asn Glu Lys Leu Tyr Ile His Ala Ile Gly Pro Leu 325 330 335 Leu Ala Asn His Gly Trp Ser Asn Ala Phe Phe Ile Thr Asp Gln Gly 340 345 350 Arg Ser Gly Lys Gln Pro Thr Gly Gln Gln Gln Trp Gly Asp Trp Cys 355 360 365 Asn Val Ile Gly Thr Gly Phe Gly Ile Arg Pro Ser Ala Asn Thr Gly 370 375 380 Asp Ser Leu Leu Asp Ser Phe Val Trp Val Lys Pro Gly Gly Glu Cys 385 390 395 400 Asp Gly Thr Ser Asp Ser Ser Ala Pro Gln Phe Asp Pro His Cys Ala 405 410 415 Leu Pro Asp Ala Leu Gln Pro Ala Pro Gln Ala Gly Ala Trp Phe Gln 420 425 430 Ala Tyr Phe Val Gln Leu Leu Thr Asn Ala Asn Pro Ser Phe Leu 435 440 445 74447PRTTrichoderma reesei 74Gln Ala Cys Ser Ser Val Trp Gly Gln Cys Gly Gly Gln Asn Trp Ser 1 5 10 15 Gly Pro Thr Cys Cys Ala Ser Gly Ser Thr Cys Val Tyr Ser Asn Asp 20 25 30 Tyr Tyr Ser Gln Cys Leu Pro Gly Ala Ala Ser Ser Ser Ser Ser Thr 35 40 45 Arg Ala Ala Ser Thr Thr Ser Arg Val Ser Pro Thr Thr Ser Arg Ser 50 55 60 Ser Ser Ala Thr Pro Pro Pro Gly Ser Thr Thr Thr Arg Val Pro Pro 65 70 75 80 Val Gly Ser Gly Thr Ala Thr Tyr Ser Gly Asn Pro Phe Val Gly Val 85 90 95 Thr Pro Trp Ala Asn Ala Tyr Tyr Ala Ser Glu Val Ser Ser Leu Ala 100 105 110 Ile Pro Ser Leu Thr Gly Ala Met Ala Thr Ala Ala Ala Ala Val Ala 115 120 125 Lys Val Pro Ser Phe Met Trp Leu Asp Thr Leu Asp Lys Thr Pro Leu 130 135 140 Met Glu Gln Thr Leu Ala Asp Ile Arg Thr Ala Asn Lys Asn Gly Gly 145 150 155 160 Asn Tyr Ala Gly Gln Phe Val Val Tyr Asp Leu Pro Asp Arg Asp Cys 165 170 175 Ala Ala Leu Ala Ser Asn Gly Glu Tyr Ser Ile Ala Asp Gly Gly Val 180 185 190 Ala Lys Tyr Lys Asn Tyr Ile Asp Thr Ile Arg Gln Ile Val Val Glu 195 200 205 Tyr Ser Asp Ile Arg Thr Leu Leu Val Ile Glu Pro Asp Ser Leu Ala 210 215 220 Asn Leu Val Thr Asn Leu Gly Thr Pro Lys Cys Ala Asn Ala Gln Ser 225 230 235 240 Ala Tyr Leu Glu Cys Ile Asn Tyr Ala Val Thr Gln Leu Asn Leu Pro 245 250 255 Asn Val Ala Met Tyr Leu Asp Ala Gly His Ala Gly Trp Leu Gly Trp 260 265 270 Pro Ala Asn Gln Asp Pro Ala Ala Gln Leu Phe Ala Asn Val Tyr Lys 275 280 285 Asn Ala Ser Ser Pro Arg Ala Leu Arg Gly Leu Ala Thr Asn Val Ala 290 295 300 Ser Tyr Asn Gly Trp Asn Ile Thr Ser Pro Pro Ser Tyr Thr Gln Gly 305 310 315 320 Asn Ala Val Tyr Asn Glu Lys Leu Tyr Ile His Ala Ile Gly Pro Leu 325 330 335 Leu Ala Asn His Gly Trp Ser Asn Ala Phe Phe Ile Thr Asp Gln Gly 340 345 350 Arg Ser Gly Lys Gln Pro Thr Gly Gln Gln Gln Trp Gly Asp Trp Cys 355 360 365 Asn Val Ile Gly Thr Gly Phe Gly Ile Arg Pro Ser Ala Asn Thr Gly 370 375 380 Asp Ser Leu Leu Asp Ser Phe Val Trp Val Lys Pro Gly Gly Glu Cys 385 390 395 400 Asp Gly Thr Ser Asp Ser Ser Ala Pro Gln Phe Asp Pro His Cys Ala 405 410 415 Leu Pro Asp Ala Leu Gln Pro Ala Pro Gln Ala Gly Ala Trp Phe Gln 420 425 430 Ala Tyr Phe Val Gln Leu Leu Thr Asn Ala Asn Pro Ser Phe Leu 435 440 445 75447PRTTrichoderma reesei 75Gln Ala Cys Ser Ser Val Trp Gly Gln Cys Gly Gly Gln Asn Trp Ser 1 5 10 15 Gly Pro Thr Cys Cys Ala Ser Gly Ser Thr Cys Val Tyr Ser Asn Asp 20 25 30 Tyr Tyr Ser Gln Cys Leu Pro Gly Ala Ala Ser Ser Ser Ser Ser Thr 35 40 45 Arg Ala Ala Ser Thr Thr Ser Arg Val Ser Pro Thr Thr Ser Arg Ser 50 55 60 Ser Ser Ala Thr Pro Pro Pro Gly Ser Thr Thr Thr Arg Val Pro Pro 65 70 75 80 Val Gly Ser Gly Thr Ala Thr Tyr Ser Gly Asn Pro Phe Val Gly Val 85 90 95 Thr Pro Trp Ala Asn Ala Tyr Tyr Ala Ser Glu Val Ser Ser Leu Ala 100 105 110 Ile Pro Ser Leu Thr Gly Ala Met Ala Thr Ala Ala Ala Ala Val Ala 115 120 125 Lys Val Pro Ser Phe Met Trp Leu Asp Thr Leu Asp Lys Thr Pro Leu 130 135 140 Met Glu Gln Thr Leu Ala Asp Ile Arg Thr Ala Asn Lys Asn Gly Gly 145 150 155 160 Asn Tyr Ala Gly Gln Phe Val Val Tyr Asp Leu Pro Asp Arg Asp Cys 165 170 175 Ala Ala Leu Ala Ser Asn Gly Glu Tyr Ser Ile Ala Asp Gly Gly Val 180 185 190 Ala Lys Tyr Lys Asn Tyr Ile Asp Thr Ile Arg Gln Ile Val Val Glu 195 200 205 Tyr Ser Asp Ile Arg Thr Leu Leu Val Ile Glu Pro Asp Ser Leu Ala 210 215 220 Asn Leu Val Thr Asn Leu Ser Thr Pro Lys Cys Ala Asn Ala Gln Ser 225 230 235 240 Ala Tyr Leu Glu Cys Ile Asn Tyr Ala Val Thr Gln Leu Asn Leu Pro 245 250 255 Asn Val Ala Met Tyr Leu Asp Ala Gly His Ala Gly Trp Leu Gly Trp 260 265 270 Pro Ala Asn Gln Asp Pro Ala Ala Gln Leu Phe Ala Asn Val Tyr Lys 275 280 285 Asn Ala Ser Ser Pro Arg Ala Leu Arg Gly Leu Ala Thr Asn Val Ala 290 295 300 Ser Tyr Asn Gly Trp Asn Ile Thr Ser Pro Pro Ser Tyr Thr Gln Gly 305 310 315 320 Asn Ala Val Tyr Asn Glu Lys Leu Tyr Ile

His Ala Ile Gly Pro Leu 325 330 335 Leu Ala Asn His Gly Trp Ser Asn Ala Phe Phe Ile Thr Asp Gln Gly 340 345 350 Arg Ser Gly Lys Gln Pro Thr Gly Gln Gln Gln Trp Gly Asp Trp Cys 355 360 365 Asn Val Ile Gly Thr Gly Phe Gly Ile Arg Pro Ser Ala Asn Thr Gly 370 375 380 Asp Ser Leu Leu Asp Ser Phe Val Trp Val Lys Pro Gly Gly Glu Cys 385 390 395 400 Asp Gly Thr Ser Asp Ser Ser Ala Pro Gln Phe Asp Pro His Cys Ala 405 410 415 Leu Pro Asp Ala Leu Gln Pro Ala Pro Gln Ala Gly Ala Trp Phe Gln 420 425 430 Ala Tyr Phe Val Gln Leu Leu Thr Asn Ala Asn Pro Ser Phe Leu 435 440 445 76453PRTHumicola insolens 76Ala Ser Cys Ala Pro Thr Trp Gly Gln Cys Gly Gly Ile Gly Phe Asn 1 5 10 15 Gly Pro Thr Cys Cys Gln Ser Gly Ser Thr Cys Val Lys Gln Asn Asp 20 25 30 Trp Tyr Ser Gln Cys Leu Pro Gly Ser Gln Val Thr Thr Thr Ser Thr 35 40 45 Thr Ser Thr Ser Ser Ser Ser Thr Thr Ser Arg Ala Thr Ser Thr Thr 50 55 60 Ser Thr Gly Gly Val Thr Ser Ile Thr Thr Ala Pro Thr Arg Thr Val 65 70 75 80 Thr Ile Pro Gly Gly Ala Thr Thr Thr Ala Ser Tyr Asn Gly Asn Pro 85 90 95 Phe Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg Ser Glu Val 100 105 110 His Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu Arg Ala Ala 115 120 125 Ala Ser Ala Val Ala Glu Val Pro Ser Phe Gln Trp Leu Asp Arg Asn 130 135 140 Val Thr Val Asp Thr Leu Leu Val Glu Thr Leu Ser Glu Ile Arg Ala 145 150 155 160 Ala Asn Gln Ala Gly Ala Asn Pro Pro Tyr Ala Ala Gln Ile Val Val 165 170 175 Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly Glu 180 185 190 Trp Ala Ile Ala Asn Asn Gly Ala Asn Asn Tyr Lys Gly Tyr Ile Asn 195 200 205 Arg Ile Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg Thr Ile Leu 210 215 220 Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met Asn Val 225 230 235 240 Ala Lys Cys Ser Gly Ala Ala Ser Thr Tyr Arg Glu Leu Thr Ile Tyr 245 250 255 Ala Leu Lys Gln Leu Asp Leu Pro His Val Ala Met Tyr Met Asp Ala 260 265 270 Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro Ala Ala 275 280 285 Glu Leu Phe Ala Lys Ile Tyr Glu Asp Ala Gly Lys Pro Arg Ala Val 290 295 300 Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser Ile Ser 305 310 315 320 Ser Pro Pro Pro Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu Lys His 325 330 335 Tyr Ile Glu Ala Phe Arg Pro Leu Leu Glu Ala Arg Gly Phe Pro Ala 340 345 350 Gln Phe Ile Val Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly Gln 355 360 365 Lys Glu Trp Gly His Trp Cys Asn Ala Ile Gly Thr Gly Phe Gly Met 370 375 380 Arg Pro Thr Ala Asn Thr Gly His Gln Tyr Val Asp Ala Phe Val Trp 385 390 395 400 Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Thr Thr Ala Ala 405 410 415 Arg Tyr Asp Pro His Cys Gly Leu Glu Asp Ala Leu Lys Pro Ala Pro 420 425 430 Glu Ala Gly Gln Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu Arg Asn 435 440 445 Ala Asn Pro Pro Phe 450 77440PRTPhanerochaete chrysosporium 77Ala Ser Ser Glu Trp Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly Pro 1 5 10 15 Thr Thr Cys Val Ser Gly Thr Thr Cys Thr Val Leu Asn Pro Tyr Tyr 20 25 30 Ser Gln Cys Leu Pro Gly Ser Ala Val Thr Thr Thr Ser Val Ile Thr 35 40 45 Ser His Ser Ser Ser Val Ser Ser Val Ser Ser His Ser Gly Ser Ser 50 55 60 Thr Ser Thr Ser Ser Pro Thr Gly Pro Thr Gly Thr Asn Pro Pro Pro 65 70 75 80 Pro Pro Ser Ala Asn Asn Pro Trp Thr Gly Phe Gln Ile Phe Leu Ser 85 90 95 Pro Tyr Tyr Ala Asn Glu Val Ala Ala Ala Ala Lys Gln Ile Thr Asp 100 105 110 Pro Thr Leu Ser Ser Lys Ala Ala Ser Val Ala Asn Ile Pro Thr Phe 115 120 125 Thr Trp Leu Asp Ser Val Ala Lys Ile Pro Asp Leu Gly Thr Tyr Leu 130 135 140 Ala Ser Ala Ser Ala Leu Gly Lys Ser Thr Gly Thr Lys Gln Leu Val 145 150 155 160 Gln Ile Val Ile Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Lys Ala 165 170 175 Ser Asn Gly Glu Phe Ser Ile Ala Asn Asn Gly Gln Ala Asn Tyr Glu 180 185 190 Asn Tyr Ile Asp Gln Ile Val Ala Gln Ile Gln Gln Phe Pro Asp Val 195 200 205 Arg Val Val Ala Val Ile Glu Pro Asp Ser Leu Ala Asn Leu Val Thr 210 215 220 Asn Leu Asn Val Gln Lys Cys Ala Asn Ala Lys Thr Thr Tyr Leu Ala 225 230 235 240 Cys Val Asn Tyr Ala Leu Thr Asn Leu Ala Lys Val Gly Val Tyr Met 245 250 255 Tyr Met Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Leu 260 265 270 Ser Pro Ala Ala Gln Leu Phe Thr Gln Val Trp Gln Asn Ala Gly Lys 275 280 285 Ser Pro Phe Ile Lys Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala 290 295 300 Leu Gln Ala Ala Ser Pro Asp Pro Ile Thr Gln Gly Asn Pro Asn Tyr 305 310 315 320 Asp Glu Ile His Tyr Ile Asn Ala Leu Ala Pro Leu Leu Gln Gln Ala 325 330 335 Gly Trp Asp Ala Thr Phe Ile Val Asp Gln Gly Arg Ser Gly Val Gln 340 345 350 Asn Ile Arg Gln Gln Trp Gly Asp Trp Cys Asn Ile Lys Gly Ala Gly 355 360 365 Phe Gly Thr Arg Pro Thr Thr Asn Thr Gly Ser Gln Phe Ile Asp Ser 370 375 380 Ile Val Trp Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asn Ser 385 390 395 400 Ser Ser Pro Arg Tyr Asp Pro Thr Cys Ser Leu Pro Asp Ala Ala Gln 405 410 415 Pro Ala Pro Glu Ala Gly Thr Trp Phe Gln Ala Tyr Phe Gln Thr Leu 420 425 430 Val Ser Ala Ala Asn Pro Pro Leu 435 440 78459PRTTrichoderma reesei 78Asp Tyr Lys Asp Asp Asp Asp Lys Glu Phe Leu Glu Ala Ser Cys Ser 1 5 10 15 Ser Val Trp Gly Gln Cys Gly Gly Gln Asn Trp Ser Gly Pro Thr Cys 20 25 30 Cys Ala Ser Gly Ser Thr Cys Val Tyr Ser Asn Asp Tyr Tyr Ser Gln 35 40 45 Cys Leu Pro Gly Ala Ala Ser Ser Ser Ser Ser Thr Arg Ala Ala Ser 50 55 60 Thr Thr Ser Arg Val Ser Pro Thr Thr Ser Arg Ser Ser Ser Ala Thr 65 70 75 80 Pro Pro Pro Gly Ser Thr Thr Thr Arg Val Pro Pro Val Gly Ser Gly 85 90 95 Thr Ala Thr Tyr Ser Gly Asn Pro Phe Val Gly Val Thr Pro Trp Ala 100 105 110 Asn Ala Tyr Tyr Ala Ser Glu Val Ser Ser Leu Ala Ile Pro Ser Leu 115 120 125 Thr Gly Ala Met Ala Thr Ala Ala Ala Ala Val Ala Lys Val Pro Ser 130 135 140 Phe Met Trp Leu Asp Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr 145 150 155 160 Leu Ala Asp Ile Arg Thr Ala Asn Lys Asn Gly Gly Asn Tyr Ala Gly 165 170 175 Gln Phe Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Leu Ala 180 185 190 Ser Asn Gly Glu Tyr Ser Ile Ala Asp Gly Gly Val Ala Lys Tyr Lys 195 200 205 Asn Tyr Ile Asp Thr Ile Arg Gln Ile Val Val Glu Tyr Ser Asp Ile 210 215 220 Arg Thr Leu Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Leu Val Thr 225 230 235 240 Asn Leu Gly Thr Pro Lys Cys Ala Asn Ala Gln Ser Ala Tyr Leu Glu 245 250 255 Cys Ile Asn Tyr Ala Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met 260 265 270 Tyr Leu Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln 275 280 285 Asp Pro Ala Ala Gln Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser 290 295 300 Pro Arg Ala Leu Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Gly 305 310 315 320 Trp Asn Ile Thr Ser Pro Pro Ser Tyr Thr Gln Gly Asn Ala Val Tyr 325 330 335 Asn Glu Lys Leu Tyr Ile His Ala Ile Gly Pro Leu Leu Ala Asn His 340 345 350 Gly Trp Ser Asn Ala Phe Phe Ile Thr Asp Gln Gly Arg Ser Gly Lys 355 360 365 Gln Pro Thr Gly Gln Gln Gln Trp Gly Asp Trp Cys Asn Val Ile Gly 370 375 380 Thr Gly Phe Gly Ile Arg Pro Ser Ala Asn Thr Gly Asp Ser Leu Leu 385 390 395 400 Asp Ser Phe Val Trp Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser 405 410 415 Asp Ser Ser Ala Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala 420 425 430 Leu Gln Pro Ala Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val 435 440 445 Gln Leu Leu Thr Asn Ala Asn Pro Ser Phe Leu 450 455 79484PRTPodospora anserina 79Met Ala Lys Arg Leu Leu Leu Thr Ala Ala Leu Ala Ala Thr Thr Leu 1 5 10 15 Ala Ala Pro Val Ile Glu Glu Arg Gln Asn Cys Gly Ser Val Trp Ser 20 25 30 Gln Cys Gly Gly Gln Gly Trp Thr Gly Ala Thr Cys Cys Ala Ser Gly 35 40 45 Ser Thr Cys Val Ala Gln Asn Gln Trp Tyr Ser Gln Cys Leu Pro Gly 50 55 60 Ser Gln Val Thr Thr Thr Ala Gln Ala Pro Ser Ser Thr Arg Thr Thr 65 70 75 80 Thr Ser Ser Ser Ser Arg Pro Thr Ser Ser Ser Ile Ser Thr Ser Ala 85 90 95 Val Asn Val Pro Thr Thr Thr Thr Ser Ala Gly Ala Ser Val Thr Val 100 105 110 Pro Pro Gly Gly Gly Ala Ser Ser Thr Ala Ser Tyr Ser Gly Asn Pro 115 120 125 Phe Leu Gly Val Gln Gln Trp Ala Asn Ser Tyr Tyr Ser Ser Glu Val 130 135 140 His Thr Leu Ala Ile Pro Ser Leu Thr Gly Pro Met Ala Thr Lys Ala 145 150 155 160 Ala Ala Val Ala Lys Val Pro Ser Phe Gln Trp Met Asp Arg Asn Val 165 170 175 Thr Val Asp Thr Leu Phe Ser Gly Thr Leu Ala Asp Ile Arg Ala Ala 180 185 190 Asn Arg Ala Gly Ala Asn Pro Pro Tyr Ala Gly Ile Phe Val Val Tyr 195 200 205 Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly Glu Trp 210 215 220 Ala Ile Ala Asp Gly Gly Ala Ala Lys Tyr Lys Ala Tyr Ile Asp Arg 225 230 235 240 Ile Arg His His Leu Val Gln Tyr Ser Asp Ile Arg Thr Ile Leu Val 245 250 255 Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met Asn Val Pro 260 265 270 Lys Cys Gln Gly Ala Ala Asn Thr Tyr Lys Glu Leu Thr Val Tyr Ala 275 280 285 Leu Lys Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala Gly 290 295 300 His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gly Pro Ala Ala Glu 305 310 315 320 Leu Phe Ala Gly Ile Tyr Lys Asp Ala Gly Arg Pro Thr Ser Leu Arg 325 330 335 Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Gly Trp Ser Leu Ser Ser 340 345 350 Ala Pro Ser Tyr Thr Thr Pro Asn Pro Asn Phe Asp Glu Lys Arg Phe 355 360 365 Val Gln Ala Phe Ser Pro Leu Leu Thr Ala Ala Gly Phe Pro Ala His 370 375 380 Phe Ile Thr Asp Thr Gly Arg Ser Gly Lys Gln Pro Thr Gly Gln Leu 385 390 395 400 Glu Trp Gly His Trp Cys Asn Ala Ile Gly Thr Gly Phe Gly Pro Arg 405 410 415 Pro Thr Thr Asp Thr Gly Leu Asp Ile Glu Asp Ala Phe Val Trp Ile 420 425 430 Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Thr Thr Ala Ala Arg 435 440 445 Tyr Asp His His Cys Gly Phe Ala Asp Ala Leu Lys Pro Ala Pro Glu 450 455 460 Ala Gly Gln Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu Thr Asn Ala 465 470 475 480 Asn Pro Pro Phe 80485PRTChaetomium Globosum 80Met Ala Ala Lys Leu Phe Leu Ala Ala Ala Leu Ala Ala Thr Ala Leu 1 5 10 15 Ala Ala Pro Val Val Glu Glu Arg Gln Asn Cys Ala Thr Leu Trp Gly 20 25 30 Gln Cys Gly Gly Asn Gly Trp Asn Gly Ala Thr Cys Cys Ala Ser Gly 35 40 45 Ser Thr Cys Thr Lys Gln Asn Asp Trp Tyr Ser Gln Cys Leu Pro Gly 50 55 60 Gly Ala Val Thr Thr Pro Gly Thr Thr Thr Lys Pro Thr Ser Thr Ser 65 70 75 80 Thr Ser Thr Ser Thr Ser Ser Arg Ser Thr Ser Thr Ser Gln Gly Gly 85 90 95 Gly Val Ser Ser Ser Thr Ser Ser Pro Pro Val Val Thr Asn Pro Pro 100 105 110 Thr Ser Ile Pro Gly Gly Ala Ser Ser Thr Ala Ser Tyr Thr Gly Asn 115 120 125 Pro Phe Ser Gly Val Gln Met Trp Ala Asn Asp Tyr Tyr Arg Ser Glu 130 135 140 Val His Thr Leu Ala Met Pro Ser Leu Thr Gly Ala Met Ala Thr Lys 145 150 155 160 Ala Ala Lys Val Ala Glu Val Pro Ser Tyr Gln Trp Met Asp Arg Asn 165 170 175 Val Thr Val Asp Thr Leu Phe Ser Gly Thr Leu Ala Gln Ile Arg Ala 180 185 190 Ala Asn Gln Ala Gly Ala Ser Pro Pro Tyr Ala Gly Ile Phe Val Val 195 200 205 Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly Glu 210 215 220 Trp Ser Ile Ala Asn Gly Gly Ala Ala Asn Tyr Lys Ala Tyr Ile Lys 225 230 235 240 Arg Ile Arg Glu Leu Ile Ile Gln Tyr Ser Asp Ile Arg Met Leu Leu 245 250 255 Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met Gly Val 260 265 270 Ala Lys Cys Ala Gly Ala Ala Ser Thr Tyr Lys Glu Leu Thr Ile His 275 280 285 Ala Leu Lys Glu Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala 290 295 300 Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro Ala Ala 305 310 315 320 Asp Leu Phe Ala Thr Leu Tyr Lys Asp Ala Gly Arg Pro Ala Ala Val

325 330 335 Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser Val Ser 340 345 350 Ser Ala Pro Ala Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu Lys His 355 360 365 Tyr Val Glu Ala Phe Ser Pro Leu Leu Thr Ala Ala Gly Phe Pro Ala 370 375 380 His Phe Ile Thr Asp Thr Gly Arg Ser Gly Lys Gln Pro Thr Gly Gln 385 390 395 400 Leu Glu Trp Gly His Trp Cys Asn Ala Val Gly Thr Gly Phe Gly Gln 405 410 415 Arg Pro Ser Ala Asn Thr Gly His Asp Leu Leu Asp Ala Phe Val Trp 420 425 430 Ile Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Thr Thr Ala Ala 435 440 445 Arg Tyr Asp His Asn Cys Gly Leu Ala Asp Ala Leu Lys Pro Ala Pro 450 455 460 Glu Ala Gly Gln Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu Thr Asn 465 470 475 480 Ala Asn Pro Pro Phe 485 81487PRTMagnaporthe grisea 81Met Ala Ser Lys Leu Phe Leu Ala Ala Ala Leu Leu Gln Gly Ala Leu 1 5 10 15 Ser Ser Pro Leu Ala Val Glu Glu Arg Gln Ala Cys Ala Ala Gln Trp 20 25 30 Gly Gln Cys Gly Gly Gln Asp Tyr Thr Gly Pro Thr Cys Cys Gln Ser 35 40 45 Gly Ser Thr Cys Val Val Ser Asn Gln Trp Tyr Ser Gln Cys Leu Pro 50 55 60 Gly Ser Ser Asn Pro Thr Thr Thr Ser Arg Thr Ser Thr Ser Ser Ser 65 70 75 80 Ser Ser Thr Ser Arg Thr Ser Ser Ser Thr Ser Arg Pro Pro Ser Ser 85 90 95 Val Pro Thr Thr Pro Thr Ser Val Pro Pro Thr Ile Thr Thr Thr Pro 100 105 110 Thr Thr Thr Pro Thr Gly Gly Ser Gly Pro Gly Thr Thr Ala Ser Phe 115 120 125 Thr Gly Asn Pro Phe Ala Gly Val Asn Leu Phe Pro Asn Lys Phe Tyr 130 135 140 Ser Ser Glu Val His Thr Leu Ala Ile Pro Ser Leu Thr Gly Ser Leu 145 150 155 160 Val Ala Lys Ala Ser Ala Val Ala Gln Val Pro Ser Phe Gln Trp Leu 165 170 175 Asp Ile Ala Ala Lys Val Glu Thr Leu Met Pro Gly Ala Leu Ala Asp 180 185 190 Val Arg Ala Ala Asn Ala Ala Gly Gly Asn Tyr Ala Ala Gln Leu Val 195 200 205 Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly 210 215 220 Glu Phe Ser Ile Ala Asp Gly Gly Val Val Lys Tyr Lys Ala Tyr Ile 225 230 235 240 Asp Ala Ile Arg Lys Gln Leu Leu Ala Tyr Ser Asp Val Arg Thr Ile 245 250 255 Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met Gly 260 265 270 Val Pro Lys Cys Ala Gly Ala Lys Asp Ala Tyr Leu Glu Cys Thr Ile 275 280 285 Tyr Ala Val Lys Gln Leu Asn Leu Pro His Val Ala Met Tyr Leu Asp 290 295 300 Gly Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Leu Gln Pro Ala 305 310 315 320 Ala Asp Leu Phe Gly Lys Leu Tyr Ala Asp Ala Gly Lys Pro Ser Gln 325 330 335 Leu Arg Gly Met Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Asp Leu 340 345 350 Thr Thr Ala Pro Ser Tyr Thr Thr Pro Asn Pro Asn Phe Asp Glu Lys 355 360 365 Lys Tyr Ile Ser Ala Phe Ala Pro Leu Leu Ala Ala Lys Gly Trp Ser 370 375 380 Ala His Phe Ile Ile Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly 385 390 395 400 Gln Lys Glu Trp Gly His Trp Cys Asn Gln Gln Gly Val Gly Phe Gly 405 410 415 Arg Arg Pro Ser Ala Asn Thr Gly Ser Glu Leu Ala Asp Ala Phe Val 420 425 430 Trp Ile Lys Pro Gly Gly Glu Cys Asp Gly Val Ser Asp Pro Thr Ala 435 440 445 Pro Arg Phe Asp His Phe Cys Gly Thr Asp Tyr Gly Ala Met Ser Asp 450 455 460 Ala Pro Gln Ala Gly Gln Trp Phe Gln Lys Tyr Phe Glu Met Leu Leu 465 470 475 480 Thr Asn Ala Asn Pro Pro Leu 485 82421PRTSclerotinia sclerotiorum 82Met Gly Leu Lys Asn Val Leu Leu Ala Ala Ala Ala Val Ala Pro Thr 1 5 10 15 Val Tyr Ala Gln Gly Ala Gly Tyr Ser Gln Cys Gly Gly Gln Gly Trp 20 25 30 Ser Gly Ala Thr Thr Cys Val Ser Gly Phe Thr Cys Thr Tyr Thr Asn 35 40 45 Glu Tyr Tyr Ser Gln Cys Leu Pro Gly Ser Gly Gly Gly Ala Ser Ser 50 55 60 Ser Arg Pro Thr Thr Thr Ala Pro Thr Thr Ile Val Thr Ser Thr Lys 65 70 75 80 Ala Ser Thr Thr Thr Gly Ser Ser Ala Thr Thr Thr Ala Ala Pro Ala 85 90 95 Ala Gly Asn Pro Phe Val Gly Lys Ala Leu Tyr Val Asn Pro Tyr Tyr 100 105 110 Ala Ser Glu Ile Ser Ala Ser Ala Ile Pro Ser Leu Thr Gly Ala Met 115 120 125 Ala Thr Lys Ala Ala Ala Val Ala Lys Val Pro Thr Phe Phe Trp Leu 130 135 140 Asp Thr Ala Asp Lys Val Pro Thr Met Gly Thr Tyr Leu Ser Asn Ile 145 150 155 160 Arg Ala Leu Asn Lys Ala Gly Ala Asn Pro Pro Val Ala Gly Thr Phe 165 170 175 Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn 180 185 190 Gly Glu Tyr Ser Ile Ala Asn Asn Gly Val Ala Asn Tyr Lys Ala Tyr 195 200 205 Ile Asp Ser Ile Val Thr Ile Leu Lys Asn Tyr Ser Asp Thr Ser Val 210 215 220 Ile Leu Ile Ile Val Asp Leu Pro Asn Val Ser Met Tyr Leu Asp Ala 225 230 235 240 Gly His Ala Gly Trp Leu Gly Trp Ser Ala Asn Ile Gly Pro Ala Ala 245 250 255 Gln Leu Phe Gly Gln Val Tyr Lys Ala Ala Gly Ser Pro Ser Gln Val 260 265 270 Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Thr Ser Ser 275 280 285 Ser Cys Pro Ser Tyr Thr Ser Gly Asp Ser Asn Cys Asn Glu Lys Leu 290 295 300 Tyr Ile Asn Ala Leu Ala Pro Leu Leu Thr Ala Gln Gly Phe Pro Ala 305 310 315 320 His Phe Ile Met Asp Thr Gly Arg Asn Gly Val Gln Pro Thr Ala Gln 325 330 335 Gln Ala Trp Gly Asp Trp Cys Asn Val Ile Gly Thr Gly Phe Gly Val 340 345 350 Arg Pro Thr Thr Asn Thr Gly Asp Ala Leu Glu Asp Ala Phe Val Trp 355 360 365 Val Lys Pro Gly Gly Glu Ala Asp Gly Thr Ser Asn Thr Thr Ala Ala 370 375 380 Arg Tyr Asp Phe His Cys Gly Leu Ser Asp Ala Leu Gln Pro Ala Pro 385 390 395 400 Glu Ala Gly Thr Trp Phe Gln Ala Tyr Phe Ala Gln Leu Leu Thr Asn 405 410 415 Ala Asn Pro Ser Phe 420 83449PRTPhaeosphaeria nodorum 83Met Lys Ala Val Ser Phe Leu Ala Val Ala Ala Leu Ala Pro Ala Ile 1 5 10 15 Lys Ala Gln Ala Ser Leu Tyr Gln Gln Cys Gly Gly Thr Gly Phe Ser 20 25 30 Gly Ser Thr Thr Cys Val Ser Gly Ala Tyr Cys Ser Lys Val Asn Asp 35 40 45 Ser Ala Thr Ser Ala Ala Pro Ala Pro Thr Thr Phe Lys Thr Ser Lys 50 55 60 Thr Val Gly Ser Pro Ala Thr Gly Ser Ser Thr Thr Gly Ser Ser Ala 65 70 75 80 Thr Gly Thr Ala Ser Pro Gly Asp Gly Ser Asn Pro Leu Lys Gly Lys 85 90 95 Asn Phe Tyr Ala Asn Ser Tyr Tyr Ala Ser Glu Ile Asn Asn Leu Ala 100 105 110 Ala Pro Ser Leu Val Ala Ala Gly Asn Ala Ala Leu Ala Ala Lys Ala 115 120 125 Ser Asn Val Ala Lys Val Gly Thr Phe Tyr Trp Leu Asp Val Arg Ala 130 135 140 Lys Val Pro Ile Ile Ser Thr Phe Ala Lys Asp Val Gln Lys Arg Asn 145 150 155 160 Ala Ala Gly Ala Asn Glu Val Leu Pro Leu Val Val Tyr Asp Leu Pro 165 170 175 Glu Arg Asp Cys Ala Ala Leu Ala Ser Asn Gly Glu Leu Ser Leu Ala 180 185 190 Asn Asn Gly Thr Ala Leu Tyr Gln Glu Tyr Ile Asp Met Ile Ala Ala 195 200 205 Gln Ile Lys Gln Phe Pro Asp Val Thr Phe Leu Leu Val Val Glu Pro 210 215 220 Asp Ser Leu Ala Asn Leu Val Thr Asn Leu Asn Val Ala Lys Cys Ala 225 230 235 240 Asn Ala Ala Thr Ala Tyr Lys Thr Leu Thr Ala Tyr Ala Ile Lys Thr 245 250 255 Leu Asn Leu Lys Asn Val Ile Met Tyr Leu Asp Ala Gly His Ala Gly 260 265 270 Trp Leu Gly Trp Thr Ala Asn Ile Glu Pro Ala Ala Glu Leu Phe Gly 275 280 285 Ala Leu Tyr Lys Ser Ala Gly Ser Pro Ala Ala Val Arg Gly Leu Val 290 295 300 Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser Ile Ala Thr Cys Pro Ser 305 310 315 320 Tyr Thr Gln Gly Asn Thr Asn Cys Asp Glu Lys Arg Tyr Val Asn Ala 325 330 335 Leu Ala Pro Leu Leu Val Lys Asn Gly Phe Pro Ala His Phe Leu Thr 340 345 350 Asp Thr Gly Arg Asn Gly Val Gln Pro Thr Lys Gln Gln Ala Trp Gly 355 360 365 Asp Trp Cys Asn Val Ile Gly Thr Gly Phe Gly Ile Arg Pro Ser Ser 370 375 380 Thr Thr Asp Asp Pro Leu Leu Asp Ala Tyr Val Trp Val Lys Pro Gly 385 390 395 400 Gly Glu Gly Asp Gly Thr Ser Asp Thr Ser Ala Val Arg Tyr Asp Ala 405 410 415 His Cys Gly Tyr Ala Asp Ala Leu Lys Pro Ala Pro Glu Ala Gly Ser 420 425 430 Trp Phe Gln Ala Tyr Phe Val Gln Leu Leu Ser Asn Ala Ser Pro Ala 435 440 445 Phe 84438PRTAgaricus bisporus 84Met Phe Lys Phe Ala Ala Leu Leu Ala Leu Ala Ser Leu Val Pro Gly 1 5 10 15 Phe Val Gln Ala Gln Ser Pro Val Trp Gly Gln Cys Gly Gly Asn Gly 20 25 30 Trp Thr Gly Pro Thr Thr Cys Ala Ser Gly Ser Thr Cys Val Lys Gln 35 40 45 Asn Asp Phe Tyr Ser Gln Cys Leu Pro Asn Asn Gln Ala Pro Pro Ser 50 55 60 Thr Thr Thr Gln Pro Gly Thr Thr Pro Pro Ala Thr Thr Thr Ser Gly 65 70 75 80 Gly Thr Gly Pro Thr Ser Gly Ala Gly Asn Pro Tyr Thr Gly Lys Thr 85 90 95 Val Trp Leu Ser Pro Phe Tyr Ala Asp Glu Val Ala Gln Ala Ala Ala 100 105 110 Asp Ile Ser Asn Pro Ser Leu Ala Thr Lys Ala Ala Ser Val Ala Lys 115 120 125 Ile Pro Thr Phe Thr Trp Phe Asp Thr Val Ala Lys Val Pro Asp Leu 130 135 140 Gly Gly Tyr Leu Ala Asp Ala Gln Ser Lys Asn Gln Leu Val Gln Ile 145 150 155 160 Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Leu Ala Ser Asn 165 170 175 Gly Glu Phe Ser Leu Ala Asn Asp Gly Leu Asn Lys Tyr Lys Asn Tyr 180 185 190 Val Asp Gln Ile Ala Ala Gln Ile Lys Gln Phe Pro Asp Val Ser Val 195 200 205 Val Ala Val Ile Glu Pro Asp Ser Leu Ala Asn Leu Val Thr Asn Leu 210 215 220 Asn Val Gln Lys Cys Ala Asn Ala Gln Ser Ala Tyr Lys Glu Gly Val 225 230 235 240 Ile Tyr Ala Ile Gln Lys Leu Asp Ala Val Gly Val Thr Met Tyr Ile 245 250 255 Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Leu Ser Pro 260 265 270 Ala Ala Gln Leu Phe Ala Gln Ile Tyr Arg Asp Ala Gly Ser Pro Arg 275 280 285 Asn Leu Arg Gly Ile Ala Thr Asn Val Ala Asn Phe Asn Ala Leu Arg 290 295 300 Ala Ser Ser Pro Asp Pro Ile Thr Gln Gly Asn Ser Asn Tyr Asp Glu 305 310 315 320 Ile His Tyr Ile Glu Ala Leu Ala Pro Met Leu Ser Asn Ala Gly Phe 325 330 335 Pro Ala His Phe Ile Val Asp Gln Gly Arg Ser Gly Val Gln Asn Ile 340 345 350 Arg Asp Gln Trp Gly Asp Trp Cys Asn Val Lys Gly Ala Gly Phe Gly 355 360 365 Gln Arg Pro Thr Thr Asn Thr Gly Ser Ser Leu Ile Asp Ala Ile Val 370 375 380 Trp Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Ser Ser Ser 385 390 395 400 Pro Arg Phe Asp Ser His Cys Ser Leu Ser Asp Ala His Gln Pro Ala 405 410 415 Pro Glu Ala Gly Thr Trp Phe Gln Ala Tyr Phe Glu Thr Leu Val Ala 420 425 430 Asn Ala Asn Pro Ala Leu 435 85443PRTLentinula edodes 85Met Lys Ile Thr Ser Thr Gly Leu Leu Ala Leu Ser Ser Leu Leu Pro 1 5 10 15 Phe Ala Leu Gly Gln Ser Gln Leu Tyr Ala Gln Cys Gly Gly Ile Gly 20 25 30 Trp Ser Gly Ala Thr Thr Cys Val Ser Gly Ala Thr Cys Thr Val Val 35 40 45 Asn Ala Tyr Tyr Ser Gln Cys Leu Pro Gly Ser Ala Ser Ala Pro Pro 50 55 60 Thr Ser Thr Ser Ser Ile Gly Thr Gly Thr Thr Thr Ser Ser Ala Pro 65 70 75 80 Gly Ser Thr Gly Thr Thr Thr Pro Ala Ala Gly Asn Pro Phe Thr Glu 85 90 95 Gln Ile Tyr Leu Ser Pro Tyr Tyr Ala Asn Glu Ile Ala Ala Ala Val 100 105 110 Thr Gln Ile Ser Asp Pro Thr Thr Ala Ala Ala Ala Ala Lys Val Ala 115 120 125 Asn Ile Pro Thr Phe Ile Trp Leu Asp Gln Val Ala Lys Val Pro Asp 130 135 140 Leu Gly Thr Tyr Leu Ala Asp Ala Ser Ala Lys Gln Lys Ser Glu Gly 145 150 155 160 Lys Asn Tyr Leu Val Gln Ile Val Val Tyr Asp Leu Pro Asp Arg Asp 165 170 175 Cys Ala Ala Leu Ala Ser Asn Gly Glu Phe Thr Ile Ala Asp Asn Gly 180 185 190 Glu Ala Asn Tyr His Asp Tyr Ile Asp Gln Ile Val Ala Gln Ile Lys 195 200 205 Gln Tyr Pro Asp Val His Val Val Ala Val Ile Glu Pro Asp Ser Leu 210 215 220 Ala Asn Leu Val Thr Asn Leu Ser Val Ala Lys Cys Ala Asn Ala Gln 225 230 235 240 Thr Thr Tyr Leu Glu Cys Val Thr Tyr Ala Met Gln Gln Leu Ser Ala 245 250 255 Val Gly Val Thr Met Tyr Leu Asp Ala Gly His Ala Gly Trp Leu Gly 260 265 270 Trp Pro Ala Asn Leu Ser Pro Ala Ala Gln Leu Phe Thr Ser Leu Tyr 275 280 285 Ser Asn Ala Gly Ser Pro Ser Gly Val Arg Gly Leu Ala Thr Asn Val 290 295 300 Ala Asn Tyr Asn Ala Leu Val Ala Thr Thr Pro Asp Pro Ile Thr Gln 305 310 315 320 Gly Asp Pro Asn Tyr Asp Glu Met Leu Tyr Ile Glu Ala Leu Ala Pro 325 330 335 Leu Leu

Gly Ser Phe Pro Ala His Phe Ile Val Asp Gln Gly Arg Ser 340 345 350 Gly Val Gln Asp Ile Arg Gln Gln Trp Gly Asp Trp Cys Asn Val Leu 355 360 365 Gly Ala Gly Phe Gly Thr Gln Pro Thr Thr Asn Thr Gly Ser Ser Leu 370 375 380 Ile Asp Ser Ile Val Trp Val Lys Pro Gly Gly Glu Cys Asp Gly Thr 385 390 395 400 Ser Asn Thr Ser Ser Pro Arg Tyr Asp Ala His Cys Gly Leu Pro Asp 405 410 415 Ala Thr Pro Asn Ala Pro Glu Ala Gly Thr Trp Phe Gln Ala Tyr Phe 420 425 430 Glu Thr Leu Val Glu Lys Ala Asn Pro Pro Leu 435 440 86460PRTConiophora puteana 86Met Phe Lys Phe Ala Ala Leu Ser Ala Phe Val Ala Leu Val Pro Leu 1 5 10 15 Leu Val Asn Ala Gln Val Ala Ala Tyr Gly Gln Cys Gly Gly Gln Asp 20 25 30 Trp Thr Gly Ala Thr Ala Cys Ala Ser Gly Thr Ala Cys Thr Lys Val 35 40 45 Asn Asp Tyr Tyr Tyr Gln Cys Leu Pro Gly Ser Ser Gly Ser Ser Val 50 55 60 Ser Gly Gly Ser Gly Ser Gly Ser Thr Ser Ala Pro Ser Pro Thr Ser 65 70 75 80 Thr Val Pro Thr Ser Thr Ser Ser Ala Ser Thr Ala Pro Ser Ser Thr 85 90 95 Ser Thr Ser Ser Ala Ala Ser Ser Asp Asn Pro Tyr Thr Gly Tyr Gln 100 105 110 Ile Phe Leu Asn Pro Glu Tyr Ala Ser Glu Val Gln Ala Ala Ile Pro 115 120 125 Ser Ile Thr Asp Ser Ala Val Ala Ala Lys Ala Leu Lys Val Ala Glu 130 135 140 Val Pro Val Phe Phe Trp Leu Asp Gln Val Ala Lys Val Pro Asp Leu 145 150 155 160 Glu Thr Tyr Leu Ala Ala Ala Asp Lys Gln Gly Lys Ser Ser Gly Gln 165 170 175 Lys Gln Leu Leu Gln Ile Val Val Tyr Asp Leu Pro Asp Arg Asp Cys 180 185 190 Ala Ala Asn Ala Ser Asn Gly Glu Phe Ser Ile Ser Asp Asp Gly Gln 195 200 205 Ala Lys Tyr Glu Asn Tyr Ile Asp Gln Ile Val Ala Ile Val Lys Lys 210 215 220 Tyr Pro Asp Val Arg Val Val Ala Val Val Glu Pro Asp Ser Met Gly 225 230 235 240 Asn Leu Val Thr Asn Met Asp Leu Pro Lys Cys Ser Ala Ala Ala Pro 245 250 255 Thr Tyr Lys Thr Cys Ile Asn Tyr Ala Ile Ala Gln Leu Ser Ser Ala 260 265 270 Gly Val Tyr Met Tyr Val Asp Ala Gly His Ala Gly Trp Leu Gly Trp 275 280 285 Pro Asn Asn Leu Ala Pro Ala Ala Gln Leu Phe Gly Glu Leu Tyr Glu 290 295 300 Thr Ser Gly Lys Ser Ala Tyr Phe Arg Gly Leu Ala Thr Asn Val Ala 305 310 315 320 Asn Tyr Asn Ala Leu Asn Thr Ser Ser Pro Asp Pro Cys Thr Gln Asn 325 330 335 Ala Pro Asn Tyr Asp Glu Met Leu Tyr Ile Asn Ala Leu Ser Pro Leu 340 345 350 Leu Gln Gln Gln Gly Phe Ser Ala Gln Phe Ile Val Asp Gln Gly Arg 355 360 365 Ser Gly Val Gln Asn Ile Arg Asn Ala Trp Gly Asp Trp Cys Asn Ile 370 375 380 Lys Gly Ala Gly Phe Gly Ile Arg Pro Thr Thr Asp Thr Gly Ser Pro 385 390 395 400 Leu Ile Asp Ser Ile Val Trp Val Lys Pro Gly Gly Glu Cys Asp Gly 405 410 415 Thr Ser Asn Ser Ser Ala Pro Arg Tyr Asp Ser Thr Cys Ser Leu Ser 420 425 430 Asp Ser Leu Gln Pro Ala Pro Glu Ala Gly Thr Trp Phe Gln Gln Tyr 435 440 445 Phe Glu Ala Leu Val Thr Asn Ala Val Pro Ser Leu 450 455 460 87442PRTVolvariella volvacea 87Met Ser Arg Phe Ser Ala Leu Thr Ala Leu Leu Leu Ser Leu Pro Leu 1 5 10 15 Leu Ala Ile Ala Gln Ser Pro Leu Tyr Gly Gln Cys Gly Gly Asn Gly 20 25 30 Trp Thr Gly Pro Lys Thr Cys Val Ser Gly Ala Thr Cys Thr Val Ile 35 40 45 Asn Asp Trp Tyr Trp Gln Cys Leu Pro Gly Asn Gly Pro Thr Ser Ser 50 55 60 Ser Pro Thr Ser Thr Pro Thr Thr Thr Thr Thr Thr Gly Gly Pro Gln 65 70 75 80 Pro Thr Val Pro Ala Ala Gly Asn Pro Tyr Thr Gly Tyr Glu Ile Tyr 85 90 95 Leu Ser Pro Tyr Tyr Ala Ala Glu Ala Gln Ala Ala Ala Ala Gln Ile 100 105 110 Ser Asp Ala Thr Gln Lys Ala Lys Ala Leu Lys Val Ala Gln Ile Pro 115 120 125 Thr Phe Thr Trp Phe Asp Val Ile Ala Lys Thr Ser Thr Leu Gly Asp 130 135 140 Tyr Leu Ala Glu Ala Ser Ala Leu Gly Lys Ser Ser Gly Lys Lys Tyr 145 150 155 160 Leu Val Gln Ile Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala 165 170 175 Leu Ala Ser Asn Gly Glu Phe Ser Ile Ala Asn Asn Gly Leu Asn Asn 180 185 190 Tyr Lys Gly Tyr Ile Asp Gln Leu Val Ala Gln Ile Lys Lys Tyr Pro 195 200 205 Asp Val Arg Val Val Ala Val Ile Glu Pro Asp Ser Leu Ala Asn Leu 210 215 220 Val Thr Asn Leu Asn Val Ser Lys Cys Ala Asn Ala Gln Thr Ala Tyr 225 230 235 240 Lys Ala Gly Val Thr Tyr Ala Leu Gln Gln Leu Asn Ser Val Gly Val 245 250 255 Tyr Met Tyr Leu Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala 260 265 270 Asn Leu Asn Pro Ala Ala Gln Leu Phe Ser Gln Leu Tyr Arg Asp Ala 275 280 285 Gly Ser Pro Gln Tyr Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr 290 295 300 Asn Ala Leu Ser Ala Ser Ser Pro Asp Pro Val Thr Gln Gly Asn Pro 305 310 315 320 Asn Tyr Asp Glu Leu His Tyr Ile Asn Ala Leu Ala Pro Ala Leu Gln 325 330 335 Ser Gly Gly Phe Pro Ala His Phe Ile Val Asp Gln Gly Arg Ser Gly 340 345 350 Val Gln Asn Ile Arg Gln Gln Trp Gly Asp Trp Cys Asn Val Lys Gly 355 360 365 Ala Gly Phe Gly Gln Arg Pro Thr Leu Ser Thr Gly Ser Ser Leu Ile 370 375 380 Asp Ala Ile Val Trp Ile Lys Pro Gly Gly Glu Cys Asp Gly Thr Thr 385 390 395 400 Asn Thr Ser Ser Pro Arg Tyr Asp Ser His Cys Gly Leu Ser Asp Ala 405 410 415 Thr Pro Asn Ala Pro Glu Ala Gly Gln Trp Phe Gln Ala Tyr Phe Glu 420 425 430 Thr Leu Val Arg Asn Ala Ser Pro Pro Leu 435 440 88389PRTConiophora puteana 88Met Phe Ser Pro Val Val Leu Gly Ala Leu Ala Ala Leu Leu Pro Ala 1 5 10 15 Ala Val Gln Ala Met Pro Ala Ser Thr Gln Ala Arg Ala Ala Asp Ala 20 25 30 Thr Ala Asn Pro Tyr Thr Gly Tyr Thr Ile Phe Lys Asn Pro Glu Tyr 35 40 45 Val Ala Glu Val Gln Ala Ala Val Gln Gln Ile Ser Asp Ser Ser Leu 50 55 60 Ala Ser Ala Ala Ala Gly Val Glu Asp Val Pro Val Phe Phe Trp Leu 65 70 75 80 Asp Gln Val Ala Lys Val Pro Asn Leu Thr Thr Tyr Leu Ala Ala Ala 85 90 95 Asp Ala Glu Ala Lys Ser Ser Gly Ser Gln Gln Leu Phe Gln Ile Val 100 105 110 Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly 115 120 125 Glu Phe Ser Ile Ser Asp Asn Gly Gln Ala Asn Tyr Glu Asn Tyr Ile 130 135 140 Asp Gln Ile Val Ala Ser Ile Lys Gln Tyr Pro Asp Val Arg Val Val 145 150 155 160 Ala Val Val Glu Pro Asp Ser Met Ala Asn Leu Val Thr Asn Leu Ser 165 170 175 Val Gln Lys Cys Ala Asp Ala Glu Ser Thr Tyr Lys Thr Cys Val Ala 180 185 190 Tyr Ala Ile Glu Gln Leu Ala Thr Val Gly Val Tyr Met Tyr Leu Asp 195 200 205 Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Leu Ser Pro Ala 210 215 220 Ala Glu Leu Phe Ala Gln Met Tyr Ser Thr Thr Gly Ser Ser Pro Tyr 225 230 235 240 Phe Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ser Leu Thr Thr 245 250 255 Asp Ser Pro Asp Pro Ile Thr Ser Gly Asp Ser Asn Tyr Asp Glu Leu 260 265 270 Leu Tyr Ile Glu Ala Leu Ser Pro Leu Leu Val Asp Asn Gly Phe Pro 275 280 285 Ala Gln Phe Ile Val Glu Gln Ala Arg Ser Gly Val Gln Asn Ile Arg 290 295 300 Ser Ala Trp Gly Asp Trp Cys Asn Val Lys Gly Ala Gly Phe Gly Leu 305 310 315 320 Arg Pro Ser Thr Asp Thr Pro Ser Ser Leu Ile Asp Ser Ile Val Trp 325 330 335 Val Lys Pro Gly Gly Glu Ala Asp Gly Thr Ser Asn Ser Ser Ala Ala 340 345 350 Arg Tyr Asp Tyr His Cys Ser Leu Ser Asp Ala Leu Gln Pro Ala Pro 355 360 365 Glu Ala Gly Thr Trp Phe Gln Thr Tyr Phe Glu Asp Leu Val Ser Gly 370 375 380 Ala Asn Pro Ala Phe 385 89454PRTCoprinopsis cinerea okayama 89Met Leu Lys Gly Ser Lys Phe Phe Ala Leu Ser Leu Ala Leu Leu Pro 1 5 10 15 Ala Leu Val Gln Ala Gln Arg Pro Leu Tyr Ala Gln Cys Gly Gly Thr 20 25 30 Gly Trp Thr Gly Glu Thr Thr Cys Val Ser Gly Ala Val Cys Glu Val 35 40 45 Ile Asn Gln Trp Tyr His Gln Cys Leu Pro Gly Ser Asn Gln Pro Gln 50 55 60 Pro Pro Val Thr Thr Gln Pro Pro Val Val Val Pro Thr Thr Ser Gln 65 70 75 80 Pro Pro Val Val Val Pro Thr Asn Pro Pro Gly Gly Thr Pro Val Pro 85 90 95 Ser Thr Gly Asn Pro Phe Glu Gly Tyr Asp Ile Tyr Leu Ser Pro Tyr 100 105 110 Tyr Ala Glu Glu Val Glu Ala Ala Ala Ala Met Ile Asp Asp Pro Val 115 120 125 Leu Lys Ala Lys Ala Leu Lys Val Lys Glu Ile Pro Thr Phe Ile Trp 130 135 140 Phe Asp Val Val Arg Lys Thr Pro Asp Leu Gly Arg Tyr Leu Ala Asp 145 150 155 160 Ala Thr Ala Ile Gln Gln Arg Thr Gly Arg Lys Gln Leu Val Gln Ile 165 170 175 Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn 180 185 190 Gly Glu Phe Ser Leu Ala Asp Gly Gly Met Glu Lys Tyr Lys Asp Tyr 195 200 205 Val Asp Arg Leu Ala Ser Glu Ile Arg Lys Tyr Pro Asp Val Arg Ile 210 215 220 Val Ala Val Ile Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met 225 230 235 240 Asn Val Ala Lys Cys Arg Gly Ala Glu Ala Ala Tyr Lys Glu Gly Val 245 250 255 Ile Tyr Ala Leu Arg Gln Leu Ser Ala Leu Gly Val Tyr Ser Tyr Val 260 265 270 Asp Ala Gly His Ala Gly Trp Leu Gly Trp Asn Ala Asn Leu Ala Pro 275 280 285 Ser Ala Arg Leu Phe Ala Gln Ile Tyr Lys Asp Ala Gly Arg Ser Ala 290 295 300 Phe Ile Arg Gly Leu Ala Thr Asn Val Ser Asn Tyr Asn Ala Leu Ser 305 310 315 320 Ala Thr Thr Arg Asp Pro Val Thr Gln Gly Asn Asp Asn Tyr Asp Glu 325 330 335 Leu Arg Phe Ile Asn Ala Leu Ala Pro Leu Leu Arg Asn Glu Gly Trp 340 345 350 Asp Ala Lys Phe Ile Val Asp Gln Gly Arg Ser Gly Val Gln Asn Ile 355 360 365 Arg Gln Glu Trp Gly Asn Trp Cys Asn Val Tyr Gly Ala Gly Phe Gly 370 375 380 Met Arg Pro Thr Leu Asn Thr Pro Ser Ser Ala Ile Asp Ala Ile Val 385 390 395 400 Trp Ile Lys Pro Gly Gly Glu Ala Asp Gly Thr Ser Asp Thr Ser Ala 405 410 415 Pro Arg Tyr Asp Thr His Cys Gly Lys Ser Asp Ser His Lys Pro Ala 420 425 430 Pro Glu Ala Gly Thr Trp Phe Gln Glu Tyr Phe Val Asn Leu Val Lys 435 440 445 Asn Ala Asn Pro Pro Leu 450 90361PRTMoniliophthora perniciosa 90Ile Pro Gly Ser Asp Pro Gly Asn Pro Gly Pro Thr Ser Ser Ser Thr 1 5 10 15 Leu Ser Ser Thr Ala Ala Pro Pro Thr Asn Thr Gln Ser Pro Val Glu 20 25 30 Asp Asn Pro Tyr Thr Gly Tyr Thr Ile Tyr Leu Ser Pro Tyr Tyr Ala 35 40 45 Asp Glu Ile Asp Ala Ala Ala Ala Lys Ile Thr Asp Pro Thr Leu Lys 50 55 60 Val Gln Ala Leu Lys Val Lys Glu Ile Pro Thr Phe Ile Trp Phe Asp 65 70 75 80 Thr Thr Ala Lys Leu Ser Thr Leu Glu Pro Tyr Leu Lys Asp Ala Ser 85 90 95 Ala Lys Gly Lys Ala Glu Gly Lys Lys Tyr Leu Leu Gln Ile Val Val 100 105 110 Tyr Thr Leu Pro Glu Arg Asp Cys Ala Ala Leu Ala Ser Asn Gly Glu 115 120 125 Leu Ser Ile Asp Asn Gly Gly Glu Val Lys Ser Arg Glu Tyr Ile Asp 130 135 140 Thr Met Val Ala Thr Ile Lys Lys Tyr Pro Asp Val Arg Val Val Ala 145 150 155 160 Val Val Glu Pro Asp Ser Leu Ala Asn Leu Val Thr Asn Leu Asn Val 165 170 175 Gln Lys Cys Ser Lys Ala Gln Thr Ile Tyr Lys Thr Ser Thr Gln Tyr 180 185 190 Ala Leu Lys Gln Leu Asp Thr Ala Gly Val Tyr Met Tyr Leu Asp Ala 195 200 205 Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Leu Thr Pro Thr Ala 210 215 220 Gln Leu Phe Gln Gln Val Trp Gln Asp Ala Gly Ser Pro Lys Phe Val 225 230 235 240 Arg Gly Leu Ala Thr Asn Val Ala Asn Phe Asn Ala Leu Arg Ala Ala 245 250 255 Ser Pro Asp Pro Val Thr Ser Gln Asn Pro Asn Tyr Asp Glu Ile His 260 265 270 Tyr Ile Glu Gly Arg Ala Gly Gln Gln Asn Leu Arg Lys Glu Trp Gly 275 280 285 Asp Trp Cys Asn Val Lys Gly Ala Gly Phe Gly Thr Arg Pro Thr Thr 290 295 300 Asn Thr Gly Ser Ser Leu Ile Asp Ser Ile Val Trp Val Lys Pro Gly 305 310 315 320 Gly Glu Ser Ala Arg Phe Asp Ala Lys Cys Val Ser Ala Ser Ser His 325 330 335 Val Pro Ala Pro Glu Ala Gly Thr Trp Phe Gln Glu Tyr Phe Glu Ala 340 345 350 Leu Val Arg Asn Ala Asn Pro Ala Leu 355 360 91460PRTPiromyces rhizinflatus 91Met Lys Phe Ser Thr Leu Ile Gly Thr Leu Phe Ala Thr Gly Ala Leu 1 5 10 15 Ala Ser Ser Cys His Arg Asp Tyr Pro Cys Cys Asn Asp Cys Asn Val 20 25 30 Val Tyr Gln Asp Trp Glu Arg Asp Trp Gly Val Leu Asn Gly Gln Glu 35 40 45 Trp Cys Phe Ile Asp Lys Asn Arg Cys Asn Gly Gly Gly Tyr Cys Lys 50 55 60 Phe Glu Ser Leu Gly Tyr Pro Cys Cys Asn Gly

Cys Asp Val Tyr Tyr 65 70 75 80 Thr Asp Asn Asp Gly Arg Trp Gly Val Glu Asn Gly Asn Trp Cys Gly 85 90 95 Ile Arg Asp Asp Lys Cys Asn Gly Tyr Gln Gln Pro Arg Thr Thr Thr 100 105 110 Thr Thr Arg Thr Thr Thr Arg Thr Thr Thr Thr Gln Arg Pro Val Gln 115 120 125 Thr Asn Val Ser Asp Asn Phe Phe Glu Asn Thr Leu Tyr Ser Asn Phe 130 135 140 Lys Phe Gln Gly Glu Val Gln Ser Ser Ile Gln Lys Leu Ser Gly Asp 145 150 155 160 Met Ala Lys Lys Ala Glu Lys Val Lys Tyr Val Pro Thr Ala Val Trp 165 170 175 Leu Ala Trp Glu Gly Ala Pro Arg Glu Val Pro Gln Tyr Leu Asp Asp 180 185 190 Ala Gly Ser Lys Thr Val Val Phe Val Leu Tyr Met Ile Pro Thr Arg 195 200 205 Asp Cys Asn Ala Asn Ala Ser Val Gly Gly Ser Ala Thr Leu Glu Lys 210 215 220 Tyr Lys Gly Tyr Ile Asp Asn Ile Tyr Asn Thr Phe Asn Gln Tyr Pro 225 230 235 240 Asn Ser Lys Ile Val Met Ile Leu Glu Pro Asp Thr Ile Gly Asn Leu 245 250 255 Val Thr Ala Asn Asn Ala Asn Cys Met Asn Val Gln Asn Leu His Lys 260 265 270 Gln Gly Leu Ala Tyr Ala Ile Ser Lys Phe Gly Thr Gln Lys Asn Val 275 280 285 Arg Val Tyr Leu Asp Ala Ala His Gly Ala Trp Leu Ser Ser His Ala 290 295 300 Asp Lys Thr Ala Gln Val Ile Lys Glu Ile Leu Asn Asn Ala Gly Ser 305 310 315 320 Gly Lys Leu Arg Gly Ile Thr Thr Asn Val Ser Asn Tyr Gln Thr Val 325 330 335 Asn Asp Glu Tyr Ser Tyr Gln Met Arg Leu Asn Ser Ala Leu Gln Asn 340 345 350 Leu Gly Val Arg Asp Leu His Tyr Ile Ile Asp Thr Ser Arg Asn Gly 355 360 365 Ala Asn Ile Ala Gln Gln Phe Asn Gln Ser Gly Thr Trp Cys Asn Phe 370 375 380 Lys Gly Ala Gly Leu Gly Ala Arg Pro Gln Ala Asn Pro Asp Ser Ser 385 390 395 400 Lys Pro Leu Leu Asp Ala Tyr Met Trp Ile Lys Thr Pro Gly Glu Ala 405 410 415 Asp Gly Ser Ser Ser Gly Ser Arg Ala Asp Pro Val Cys Gly Arg Trp 420 425 430 Asp Ser Leu Gln Gly Ala Pro Asp Ala Gly Ser Trp Phe His Asp Tyr 435 440 445 Phe Val Met Leu Leu Gln Asn Ala Asn Pro Pro Phe 450 455 460 92518PRTPiromyces rhizinflatus 92Met Lys Phe Ile Val Cys Ala Ser Ile Leu Ser Leu Gly Leu Phe Lys 1 5 10 15 Phe Ala Asp Ala Ala Cys Ala Gly Pro Tyr Ala Gln Cys Gly Gly Asn 20 25 30 Asn Phe Asn Gly Glu Asn Cys Cys Gln Ser Gly Tyr Lys Cys Val Ala 35 40 45 Ile Asn Glu Trp Tyr Ser Gln Cys Gln Glu Gly Ala Ala Glu Pro Glu 50 55 60 Pro Val Pro Gln Ser Ser Ala Ala Asp Asp Gln Trp Asn Asn Asn Asn 65 70 75 80 Gln Trp Asn Asn Asn Gln Gln Asn Asn Gln Trp Asn Asn Pro Trp Asp 85 90 95 Asn Asn Asn Asn Gln Trp Asn Asn Asn Gln Trp Asn Asn Asn Gln Gln 100 105 110 Asn Asn Gln Trp Asn Asn Asn Gln Gln Gln Asn Asn Gln Trp Asp Asn 115 120 125 Asn Asn Gln Trp Asn Asn Asn Gln Trp Asn Asn Asn Gln Gln Gln Asn 130 135 140 Asn Gln Gln Gln Asn Asn Gln Gln Gln Asn Asn Gln Trp Asn Asn Asn 145 150 155 160 Asn Gln Trp Asn Asn Asn Gln Gln Gln Asn Asn Gln Gln Gln Asn Asn 165 170 175 Gln Ala Pro Ala Gln Ser Asn Gly Gly Gly Ala Ser Gly Ser Ser Gln 180 185 190 Asn Phe Phe Thr Asn Glu Ile Tyr Ala Asn Pro Arg Phe Ile Glu Glu 195 200 205 Ile Asp Ser Ser Ile Pro Lys Leu Thr Pro Glu Leu Ala Ala Lys Ala 210 215 220 Glu Lys Val Lys Gln Val Pro Thr Ala Val Trp Leu Ala Trp Asp Gly 225 230 235 240 Ala Pro Gly Glu Val Glu Gly His Leu Lys Ala Ala Gly Ser Lys Thr 245 250 255 Val Val Phe Ile Leu Tyr Met Ile Pro Thr Arg Asp Cys Asn Ser Asn 260 265 270 Ala Ser Ala Gly Gly Ala Ser Asp Leu Asn Lys Tyr Lys Gly Tyr Val 275 280 285 Asp Asp Ile Ala Gly Thr Ile Lys Ser His Pro Glu Ser Lys Val Val 290 295 300 Met Ile Val Glu Pro Asp Thr Leu Gly Asn Leu Val Thr Gly Ser Ser 305 310 315 320 Glu Ala Cys Lys Asn Val His Ser Leu His Lys Ser Ala Leu Ser Tyr 325 330 335 Ala Val Asn Val Phe Gly Ala Met Ser Asn Val Ser Val Tyr Leu Asp 340 345 350 Ala Ala His Gly Lys Trp Leu Gly Gly Ser Thr Asp Lys Val Ala Ser 355 360 365 Val Leu Lys Glu Ile Leu Asp Asn Ala Pro Asn Gly Lys Ile Arg Gly 370 375 380 Leu Ser Thr Asn Val Ser Asn Tyr Gln Pro Leu Ser Ser Glu Ala Gln 385 390 395 400 Tyr His Gln Gly Leu Ser Ser Ala Leu Ala Ala Val Gly Tyr Pro Gly 405 410 415 Met Lys Phe Val Val Asp Thr Gly Arg Asn Gly Val Asp Val Ser Ser 420 425 430 Thr Phe Ser Ile Asn Glu Thr Trp Cys Asn Phe Val Gly Thr Gly Phe 435 440 445 Gly Glu Arg Pro Gln Gly Asn Pro Ser Gly Tyr Pro Leu Leu Asp Ala 450 455 460 Phe Met Trp Leu Lys Thr Pro Gly Glu Ala Asp Gly Ser Ala Thr Gly 465 470 475 480 Ser Arg Ala Asp Pro Val Cys Ala Arg Gln Asp Ser Leu Gln Gly Ala 485 490 495 Pro Asp Ala Gly Gln Trp Phe His Glu Tyr Phe Val Gln Leu Leu Glu 500 505 510 Asn Ala Lys Pro Gly Phe 515 93376PRTPiromyces rhizinflatus 93Glu Thr Leu Pro Gln Gln Ser Asn Ser Ala Lys Thr Leu Pro Gln Gln 1 5 10 15 Ser Asp Ser Ala Lys Thr Ile Pro Gln Pro Thr Ser Ala Glu Ser Gln 20 25 30 Thr Ser Lys Thr Leu Pro Gln Thr Gly Gly Ser Gly Asn Gly Ser Ser 35 40 45 Gln Asn Phe Phe Leu Asn Glu Ile Tyr Ala Asn Pro Lys Phe Ile Glu 50 55 60 Glu Val Glu Asp Ser Ile Glu Lys Leu Thr Pro Glu Leu Gln Ala Lys 65 70 75 80 Ala Glu Lys Val Lys Asp Val Pro Thr Ala Val Trp Leu Ala Trp Asp 85 90 95 Gly Ser Pro Gly Glu Val Glu Gly His Leu Val Ala Ala Gly Ser Lys 100 105 110 Thr Val Val Phe Leu Leu Tyr Met Ile Pro Thr Arg Asp Cys Asn Ser 115 120 125 Asn Ala Ser Ala Gly Gly Ala Ala Ser Leu Asp Lys Tyr Lys Gly Tyr 130 135 140 Ile Asp Asp Ile Ser Asn Thr Ile Lys Ser His Pro Glu Ser Lys Val 145 150 155 160 Val Met Val Val Glu Pro Asp Thr Leu Gly Asn Leu Val Thr Gly Asn 165 170 175 Ser Glu Ala Cys Lys Asn Val His Thr Leu His Lys Asn Ala Leu Ser 180 185 190 Tyr Ala Val Asp Val Phe Gly Ala Met Ser Asn Val Ser Val Tyr Leu 195 200 205 Asp Ala Ala His Gly Met Trp Leu Gly Pro His Thr Asp Lys Val Ala 210 215 220 Ser Val Ile Lys Glu Ile Leu Asn Asn Ala Pro Asn Gly Lys Ile Arg 225 230 235 240 Gly Leu Ser Thr Asn Val Ser Asn Tyr Gln Pro Val Ser Ser Glu Tyr 245 250 255 Gln Tyr His Gln Lys Leu Ala Ala Ser Leu Ala Ala Val Gly Val Asn 260 265 270 Asp Val His Phe Ile Val Asp Thr Gly Arg Ser Gly Val Asp Val Thr 275 280 285 Glu Thr Phe Ser Lys Gln Gln Thr Trp Cys Asn Phe Ile Gly Ala Gly 290 295 300 Leu Gly Pro Arg Pro Gln Gly Asn Pro Asp Ala Ser Met Pro Leu Leu 305 310 315 320 Asp Ala Tyr Met Trp Leu Lys Thr Pro Gly Glu Ala Asp Gly Ser Ala 325 330 335 Val Gly Asp Arg Ala Asp Pro Val Cys Ser His Glu Asp Ser Leu Gln 340 345 350 Val Ala Pro Asp Ala Gly Gln Trp Phe His Asp Tyr Phe Val Leu Leu 355 360 365 Leu Lys Asn Ala Asn Pro Pro Phe 370 375 94463PRTPiromyces rhizinflatus 94Met Lys Phe Leu Ser Leu Ile Gly Thr Leu Phe Ala Thr Gly Ala Leu 1 5 10 15 Ala Ser Gln Cys His Pro Asn Trp Pro Cys Cys Leu Asn Cys Asp Val 20 25 30 Val Tyr Gln Asp Gly Glu Gly Asp Trp Gly Val Leu Asn Asn Asp Trp 35 40 45 Cys Phe Ile Ser Lys Ser Arg Cys Gly Asn Asn Asn Asn Asn Asn Asn 50 55 60 Gly Tyr Cys Lys Phe Gln Ser Leu Gly Tyr Pro Cys Cys Ser Gly Cys 65 70 75 80 Gln Val Val Tyr Thr Asp Gly Asp Gly Asn Trp Gly Val Glu Asn Gly 85 90 95 Asn Trp Cys Gly Ile Arg Asp Glu Gln Cys Gly Gly Gly Asn Asn Trp 100 105 110 Gln Gln Pro Thr Thr Thr Arg Thr Thr Thr Arg Thr Thr Thr Arg Thr 115 120 125 Gln Asn Asn Arg Pro Thr Ser Asp Asn Phe Phe Asp Asn Thr Leu Tyr 130 135 140 Ser Asn Tyr Lys Phe Gln Asp Glu Val Gln Ser Ser Ile Asn Lys Leu 145 150 155 160 Ser Gly Gln Met Ala Glu Lys Ala Lys Lys Val Lys Tyr Val Pro Thr 165 170 175 Ala Ala Trp Leu Ala Trp Ser Gly Ala Pro Asp Glu Val Pro Arg Tyr 180 185 190 Leu Gln Glu Ala Gly Ser Asp Thr Val Val Phe Val Leu Tyr Met Ile 195 200 205 Pro Thr Arg Asp Cys Asn Ala Asn Ala Ser Ala Gly Gly Ser Ala Asn 210 215 220 Leu Asp Thr Tyr Lys Gly Tyr Val Asn Lys Ile Tyr Asn Thr Ile Asn 225 230 235 240 Gln Tyr Pro Asn Ser Arg Ile Val Met Ile Leu Glu Pro Asp Thr Ile 245 250 255 Gly Asn Leu Val Thr Ala Asn Asn Gln Asn Cys Gln Asn Val Gln Asn 260 265 270 Leu His Lys Asn Ala Leu Ala Tyr Ala Ile Ser Lys Phe Gly Thr Gln 275 280 285 Ser Asn Val Ser Val Tyr Leu Asp Ala Ala His Gly Ala Trp Leu Ser 290 295 300 Pro His Ala Asp Lys Thr Ala Gln Val Ile Lys Glu Ile Leu Ser Lys 305 310 315 320 Ala Gly Asn Gly Lys Ile Arg Gly Ile Ser Thr Asn Val Ser Asn Tyr 325 330 335 Gln Thr Ile Asp Asp Glu Tyr Asn Tyr His Gln Arg Leu Asn Ser Ala 340 345 350 Leu Gln Asn Leu Gly Val Ser Asn Met His Phe Ile Val Asp Thr Ser 355 360 365 Arg Asn Gly Ala Asn Ile Ala Ser Gln Phe Asn Gln Ser Gly Thr Trp 370 375 380 Cys Asn Phe Lys Gly Ala Gly Leu Gly Gln Arg Pro Lys Gly Ser Pro 385 390 395 400 Asp Pro Ser Lys Pro Leu Leu Asp Ala Tyr Met Trp Ile Lys Thr Pro 405 410 415 Gly Glu Ala Asp Gly Ser Ser Ser Gly Ala Arg Ala Asp Pro Val Cys 420 425 430 Gly Arg Trp Asp Ser Leu Gln Gly Ala Pro Asp Ala Gly Ser Trp Phe 435 440 445 His Asp Tyr Phe Val Met Leu Leu Gln Asn Ala Asn Pro Ser Phe 450 455 460 95510PRTNeocallimastix frontalis 95Met Lys Phe Ile Val Phe Ala Ser Ile Leu Ser Ser Gly Ile Ile Lys 1 5 10 15 Leu Ser Asn Ala Ala Cys Gly Gly Pro Tyr Ala Gln Cys Gly Gly Ser 20 25 30 Gly Phe Ser Gly Glu Ala Cys Cys Gln Asp Gly Tyr Lys Cys Val Ala 35 40 45 Met Asn Glu Trp Tyr Ser Gln Cys Gln Ala Gly Ser Asp Ala Pro Ala 50 55 60 Asn Ser Ala Ala Pro Val Asn Ser Ala Val Gly Asn Asp Asn Asn Asn 65 70 75 80 Asn Asn Asn Gln Trp Asn Asn Gln Trp Asn Asn Asn Gly Trp Asn Trp 85 90 95 Gly Asn Gly Asn Asn Glu Ala Gln Asn Pro Trp Asn Asn Asn Gly Trp 100 105 110 Ser Trp Glu Gly Gly Asn Asn Gly Asn Gln Asp Gln Asn Gln Trp Asp 115 120 125 Asn Asn Gly Trp Pro Trp Gly Gly Asn Asn Gly Asn Gln Gly Pro Val 130 135 140 Gln Val Asn Ile Gly Glu Asn Asn Asn Gln Asn Gln Asn Pro Ala Asn 145 150 155 160 Asp Ala Pro Ala Pro Pro Pro Ala Gln Gly Gln Ala Pro Ala Pro Ala 165 170 175 Pro Ala Ala Ala Ala Gly Gly Ser Gly Ser Ser Gln Asn Phe Phe Gln 180 185 190 Asn Glu Ile Tyr Ala Asn Pro Lys Phe Ile Glu Glu Val Asp Ser Ser 195 200 205 Ile Ala Lys Leu Asp Gly Glu Leu Lys Ala Lys Ala Glu Lys Val Lys 210 215 220 Ser Val Pro Thr Ala Val Trp Leu Ala Trp Asp Gly Ala Pro Gly Glu 225 230 235 240 Val Ala Gln His Leu Glu Ala Ala Gly Ser Lys Thr Val Val Phe Ile 245 250 255 Met Tyr Met Ile Pro Thr Arg Asp Cys Asn Ala Asn Ala Ser Ala Gly 260 265 270 Gly Ala Ser Asn Leu Gln Thr Tyr Lys Gly Tyr Val Asp Ser Ile Ser 275 280 285 Asn Thr Ile Lys Lys Tyr Pro Asn Ser Lys Val Val Met Ile Leu Glu 290 295 300 Pro Asp Thr Leu Gly Asn Leu Val Thr Ala Asn Ser Glu Asn Cys Lys 305 310 315 320 Asn Val His Gln Leu His Lys Asp Ala Leu Ser Tyr Gly Val Asn Val 325 330 335 Phe Gly Ser Met Ser Asn Val Ser Val Tyr Leu Asp Ala Ala His Gly 340 345 350 Ala Trp Leu Gly Asp Ser Thr Asp Lys Val Ala Ala Val Val Lys Glu 355 360 365 Ile Leu Ser Asn Ala Pro Asn Gly Lys Ile Arg Gly Leu Ser Thr Asn 370 375 380 Ile Ser Asn Tyr Gln Pro Val Asp Ser Glu Tyr Gly Tyr His Gln Lys 385 390 395 400 Leu Ala Ser Ala Leu Ser Ala Ala Gly Tyr Pro Asp Met His Phe Val 405 410 415 Val Asp Thr Gly Arg Asp Gly Val Ala Ile Ser Ser Gly Thr Trp Cys 420 425 430 Asn Leu Ile Gly Thr Gly Phe Gly Glu Arg Pro Lys Gly Asn Pro Asn 435 440 445 Pro Gly Met Pro Leu Leu Asp Ala Tyr Met Trp Leu Lys Thr Pro Gly 450 455 460 Glu Ala Asp Gly Ser Ser Thr Gly Ala Arg Ala Asp Pro Val Cys Ala 465 470 475 480 Lys Ser Asp Ser Leu Pro Gly Ala Pro Asp Ala Gly Gln Trp Phe His 485 490 495 Asp Tyr Phe Val Gln Leu Leu Lys Asn Ala Lys Pro Ala Phe 500 505 510 96459PRTOrpinomyces sp. PC-2 96Met Lys Phe Ser Thr Val Leu Ala Thr Leu Phe Ala Thr Gly Ala Leu 1 5 10 15 Ala Ser Glu Cys His Trp Gln Tyr Pro Cys Cys Lys Asp Cys Thr Val 20 25

30 Tyr Tyr Thr Asp Thr Glu Gly Lys Trp Gly Val Leu Asn Asn Asp Trp 35 40 45 Cys Met Ile Asp Asn Arg Arg Cys Ser Ser Asn Asn Asn Asn Cys Ser 50 55 60 Ser Ser Ile Thr Ser Gln Gly Tyr Pro Cys Cys Ser Asn Asn Asn Cys 65 70 75 80 Lys Val Glu Tyr Thr Asp Asn Asp Gly Lys Trp Gly Val Glu Asn Asn 85 90 95 Asn Trp Cys Gly Ile Ser Asn Ser Cys Gly Gly Gly Gln Gln Gln Gln 100 105 110 Pro Thr Gln Pro Thr Gln Pro Thr Gln Pro Gln Gln Pro Thr Gln Pro 115 120 125 Ser Ser Asp Asn Phe Phe Glu Asn Glu Ile Tyr Ser Asn Tyr Lys Phe 130 135 140 Gln Gly Glu Val Asp Ile Ser Ile Lys Lys Leu Asn Gly Asp Leu Lys 145 150 155 160 Ala Lys Ala Glu Lys Val Lys Tyr Val Pro Thr Ala Val Trp Leu Ala 165 170 175 Trp Asp Gly Ala Pro Gln Glu Val Pro Arg Tyr Leu Gln Glu Ala Gly 180 185 190 Asn Lys Thr Val Val Phe Val Leu Tyr Met Ile Pro Thr Arg Asp Cys 195 200 205 Gly Ala Asn Ala Ser Ala Gly Gly Ser Ala Thr Ile Asp Lys Tyr Lys 210 215 220 Gly Tyr Ile Asn Asn Ile Tyr Asn Thr Ser Asn Gln Tyr Lys Asn Ser 225 230 235 240 Lys Ile Val Met Ile Leu Glu Pro Asp Thr Ile Gly Asn Leu Val Thr 245 250 255 Asn Asn Asn Asp Asn Cys Arg Asn Val Arg Asn Met His Lys Gln Ala 260 265 270 Leu Ser Tyr Ala Ile Ser Lys Phe Gly Thr Gln Ser His Val Lys Val 275 280 285 Tyr Leu Asp Ala Ala His Gly Ala Trp Leu Asn Gln Tyr Ala Asp Gln 290 295 300 Thr Ala Asn Val Ile Lys Glu Ile Leu Asn Asn Ala Gly Ser Gly Lys 305 310 315 320 Leu Arg Gly Ile Ser Thr Asn Val Ser Asn Tyr Gln Ser Ile Glu Ser 325 330 335 Glu Tyr Lys Tyr His Gln Asn Leu Asn Arg Ala Leu Glu Ser Lys Gly 340 345 350 Val Arg Gly Leu Lys Phe Ile Val Asp Thr Ser Arg Asn Gly Ala Asn 355 360 365 Val Glu Gly Ala Phe Asn Ala Ser Gly Thr Trp Cys Asn Phe Lys Gly 370 375 380 Ala Gly Leu Gly Gln Arg Pro Lys Gly Asn Pro Asn Pro Gly Ser Met 385 390 395 400 Pro Leu Leu Asp Ala Tyr Met Trp Ile Lys Thr Pro Gly Glu Ala Asp 405 410 415 Gly Ser Ser Gln Gly Ser Arg Ala Asp Pro Val Cys Ala Arg Gly Asp 420 425 430 Ser Leu Gln Gly Ala Pro Asp Ala Gly Ser Trp Phe His Glu Tyr Phe 435 440 445 Thr Met Leu Ile Gln Asn Ala Asn Pro Pro Phe 450 455 97345PRTPiromyces rhizinflatus 97Thr Ala Val Trp Leu Ala Trp Asp Gly Ala Pro Gly Glu Val Glu Gly 1 5 10 15 His Leu Lys Asn Ala Gly Asp Lys Thr Ile Val Phe Ile Leu Tyr Met 20 25 30 Ile Pro Thr Arg Asp Cys Asn Ser Lys Ala Ser Ala Gly Gly Ala Ser 35 40 45 Asn Leu Lys Lys Tyr Gln Gly Tyr Val Asp Ser Ile Ala Asn Thr Ile 50 55 60 Ser Lys His Pro Glu Ser Lys Val Val Met Val Ile Glu Pro Asp Thr 65 70 75 80 Leu Gly Asn Leu Ile Thr Gly Glu Thr Glu Glu Cys Lys Thr Val His 85 90 95 Thr Leu His Lys Asp Ala Leu Ala Tyr Ala Val Asn Val Phe Gly Asp 100 105 110 Met Ser Asn Val Ser Ala Tyr Leu Asp Ala Ala His Gly Lys Trp Leu 115 120 125 Gly Trp Ala Ala Gly Lys Thr Ala Ala Val Ile Lys Glu Ile Leu Asp 130 135 140 Asn Ala Pro Asn Gly Asn Ile Arg Gly Phe Ser Thr Asn Val Ser Asn 145 150 155 160 Tyr Gln Pro Ile Glu Ser Glu Tyr Glu Tyr His Glu Lys Leu Asn Ala 165 170 175 Ala Leu Glu Asp Leu Gly Ile Thr Gly Lys Lys Phe Ile Val Asp Thr 180 185 190 Gly Arg Ser Gly Val Asp Val Thr Glu Glu Phe Asn Leu Asn Gln Thr 195 200 205 Trp Cys Asn Leu Ile Tyr Ala Gly Leu Gly Glu Pro Ser Arg Gly Ser 210 215 220 Pro Asp Pro Glu Lys Phe Pro Leu Leu Asp Ala Tyr Phe Trp Leu Lys 225 230 235 240 Pro Pro Gly Glu Ala Asp Gly Ser Asp Thr Gly Ser Arg Ala Asp Pro 245 250 255 Val Cys Gly Arg Glu Asp Ser Phe Pro Gly Ala Pro Asp Ala Gly Ser 260 265 270 Trp Phe Ser Glu Tyr Phe Ala Ser Met Leu Glu Lys Ser Pro Phe Tyr 275 280 285 Gly Glu Gly Ile Glu Glu Glu Pro Glu Glu Pro Glu Glu Pro Leu Cys 290 295 300 Ser Gln Lys Phe Leu Asp Gln Gly Tyr Gln Cys Cys Ser Gln Cys Gly 305 310 315 320 Thr Ile Tyr Tyr Val Asp Asp Ala Gly Asn Trp Gly Val Glu Asn Asn 325 330 335 Glu Trp Cys Gly Leu Pro Glu Asp Cys 340 345 98457PRTMicromonospora cellulolyticum 98Val Ala Ile Leu Ser Ala Arg Arg Arg Ser Ala Ala Ile Ser Val Thr 1 5 10 15 Ala Val Ala Gly Leu Ala Ala Ala Gly Val Leu Arg Val Gly Gly Val 20 25 30 Ala Gly Thr Val Ser Gly Ser Leu Tyr Arg Asp Pro Ser Ser Ala Val 35 40 45 Val Arg Trp Val Ala Ala Asn Pro Gly Asp Phe Arg Ala Ala Val Ile 50 55 60 Arg Glu Lys Ile Ala Ser Gln Pro Gln Ala Arg Trp Tyr Ala Asn Phe 65 70 75 80 Asn Pro Ser Thr Ile Gln Ser Glu Val Ser Ala Phe Ile Gly Ala Ala 85 90 95 Asn Ser Ala Gln Gln Ile Pro Val Leu Ser Val Tyr Glu Ile Thr Asn 100 105 110 Arg Asp Cys Gly Gly Ala His Ala Gly Gly Ala Pro Asp Leu Asn Gln 115 120 125 Tyr Gln Thr Trp Val Ser Asn Phe Ala Arg Gly Leu Gly Asn Gln Thr 130 135 140 Val Leu Ile Ile Leu Glu Thr Asp Ser Leu Ala Leu Gln Thr Cys Leu 145 150 155 160 Ser Thr Ser Glu Leu Asn Ala Arg Asn Gln Ala Leu Ser Thr Ala Thr 165 170 175 Gln Thr Ile Lys Ser Ala Asn Pro Asn Ala Lys Val Tyr Leu Asp Gly 180 185 190 Gly His Ser Thr Trp Asn Ser Ala Asn Asp Thr Ala Asn Arg Leu Arg 195 200 205 Ala Ala Gly Val Gln Tyr Ala Asp Gly Phe Phe Thr Asn Val Ser Asn 210 215 220 Phe Asn Pro Thr Ser Ser Glu Ala Asn Phe Gly Arg Ala Val Ile Ser 225 230 235 240 Ala Leu Asn Gly Met Gly Ile Ser Gly Lys Arg Gln Val Ile Asp Thr 245 250 255 Ser Arg Asn Gly Gly Ala Ala Gly Asp Trp Cys Ala Asp Asp Asn Thr 260 265 270 Asp Arg Arg Ile Gly Gln Tyr Pro Thr Thr Asn Thr Gly Asp Ala Asn 275 280 285 Ile Asp Ala Tyr Leu Trp Val Lys Pro Pro Gly Glu Ala Asp Gly Cys 290 295 300 Ala Thr Arg Gly Ser Phe Gln Pro Asp Leu Ala Phe Ser Leu Ala Asn 305 310 315 320 Gly Val Pro Asn Pro Pro Thr Thr Ala Pro Pro Thr Thr Asn Arg Ala 325 330 335 Asp Asp Arg Pro Pro Thr Thr Ala Pro Pro Thr Thr Asp Thr Pro Thr 340 345 350 Thr Ala Pro Pro Thr Thr Pro Pro Pro Ala Gly Asn Gly Leu Ser Ala 355 360 365 Ser Val Ala Ile Thr Gln Trp Asn Gly Gly Phe Thr Ala Ser Val Asn 370 375 380 Val Thr Ala Gly Ser Ala Ile Asn Gly Trp Thr Val Thr Val Ala Leu 385 390 395 400 Pro Gly Gly Ala Ala Ile Thr Gly Thr Trp Asn Ala Gln Ala Ser Gly 405 410 415 Thr Ser Gly Thr Val Arg Phe Thr Asn Val Gly Tyr Asn Gly Gln Val 420 425 430 Gly Ala Gly Gln Thr Thr Asn Phe Gly Phe Gln Gly Thr Gly Thr Gly 435 440 445 Gln Gly Ala Thr Ala Thr Cys Ala Ala 450 455 99193PRTOrpinomyces sp. PC-2 99Thr Leu His Lys Asn Ala Leu Ser Tyr Ala Val Asn Val Phe Gly Ser 1 5 10 15 Met Lys Asn Val Ser Val Tyr Leu Asp Ala Ala His Gly Met Trp Leu 20 25 30 Ser Ala Val Ala Asp Lys Thr Ala Ala Val Ile Lys Glu Val Leu Asp 35 40 45 Asn Ala Pro Asn Gly Lys Ile Arg Gly Leu Ser Thr Asn Ile Ser Asn 50 55 60 Tyr Gln Pro Val Tyr Ser Glu Tyr Lys Tyr His Glu Lys Leu Ser Ala 65 70 75 80 Glu Leu Glu Lys Leu Gly Val Ser Asp Ile His Phe Ile Val Asp Thr 85 90 95 Gly Arg Asn Gly Val Asp Ile Thr Glu Thr Phe Ser Lys Thr Gln Thr 100 105 110 Trp Cys Asn Phe Val Gly Thr Gly Phe Gly Glu Arg Pro Gln Gly Asn 115 120 125 Pro Asp Pro Val Lys Met Pro Leu Leu Asp Ala Tyr Met Trp Leu Lys 130 135 140 Thr Pro Gly Glu Ala Asp Gly Ser Asp Thr Gly Ser Arg Ala Asp Pro 145 150 155 160 Val Cys Ala Arg Glu Asp Ser Leu Pro Gly Ser Pro Asp Ala Gly Gln 165 170 175 Trp Phe His Asp Tyr Phe Val Gln Leu Leu Glu Asn Ala Asn Pro Ala 180 185 190 Phe 100458PRTCatenulispora acidiphila 100Met Val Ala Thr Gln Leu Gly Gly Val Ala Met Ala Gly Thr Ile Ala 1 5 10 15 Ser Gly Thr Gln Phe Tyr Ala Asp Pro Asn Ser Gln Val Val Lys Trp 20 25 30 Asp Ala Ala Asn Pro Gly Asp Ala Arg Glu Pro Ala Ile Ala Ser Gln 35 40 45 Ile Ala Ser Val Ser Gln Gly Ile Trp Phe Ser Asn Tyr Arg Pro Ser 50 55 60 Thr Val Gln Ser Asp Val Ser Ala Val Thr Thr Ala Ala Ala Ala Ala 65 70 75 80 Gly Lys Thr Pro Val Leu Val Val Tyr Glu Ile Pro Asn Arg Asp Cys 85 90 95 Gly Gly Ala Ser Ala Gly Gly Ala Pro Asp Ile Ser Ser Tyr Glu Asn 100 105 110 Tyr Ile Gln Ser Phe Ala Asn Gly Leu Gly Ser His Gln Val Ile Val 115 120 125 Ile Leu Glu Pro Asp Ser Leu Ala Leu Gln Thr Cys Leu Ser Ser Gln 130 135 140 Gln Ala Thr Asp Arg Asp Asn Ala Ile Ala Phe Ala Gly Ala His Leu 145 150 155 160 Lys Ser Ala Asp Pro Ala Ala Lys Val Tyr Leu Asp Ala Gly His Ser 165 170 175 Ser Trp Asn Ser Pro Ser Ala Gln Ala Ala Ala Leu Asn Ala Ala Gly 180 185 190 Val Lys Thr Ser Ser Asp Gly Ile Phe Ser Asn Val Ser Asn Phe Gln 195 200 205 Thr Thr Ala Ser Glu Val Ser Tyr Asp Lys Gln Val Leu Ala Ala Leu 210 215 220 Gly Ser Pro Ser Asn Leu His Ile Val Val Asp Thr Ser Arg Asn Gly 225 230 235 240 Asn Gly Pro Ala Gly Ser Ala Trp Cys Asp Pro Ser Gly Arg Ala Leu 245 250 255 Gly Gln Ala Pro Thr Ala Asn Thr Gly Asp Ala Ala Val Asp Ala Phe 260 265 270 Leu Trp Ile Lys Pro Pro Gly Glu Ala Asp Gly Cys Ala Asp Ala Ala 275 280 285 Gly Thr Phe Asp Pro Ala Leu Ala Tyr Ala Leu Ile Thr Asn Gly Gly 290 295 300 Gly Pro Pro Pro Thr Ser Pro Ser Ser Thr Pro Ser Thr Thr Pro Ser 305 310 315 320 Thr Thr Pro Ser Thr Thr Pro Ser Thr Thr Pro Ser Thr Thr Pro Ser 325 330 335 Thr Thr Pro Ser Thr Pro Pro Ser Thr Thr Pro Ser Ser Pro Pro Ser 340 345 350 Ser Ser Pro Ala Gly Cys Gln Val Thr Tyr Thr Arg Thr Asn Glu Trp 355 360 365 Ala Gly Gly Phe Thr Ala Asn Val Ser Ile Thr Ser Ser Lys Ala Leu 370 375 380 Ser Ser Trp Thr Val Gly Phe Thr Tyr Gly Gly Asp Gln Gln Ile Thr 385 390 395 400 Asn Ser Trp Asn Gly Asn His Thr Gln Ser Gly Arg Asn Val Thr Leu 405 410 415 Thr Ser Leu Ser Tyr Asn Gly Ser Ile Gly Ala Gly Gln Thr Leu Thr 420 425 430 Gly Val Gly Val Gln Gly Thr Trp Thr Ser Ser Asp Ala Ala Pro Ser 435 440 445 Ala Phe Thr Leu Asn Gly Val Ala Cys His 450 455 101422PRTActinosynnema mirum 101Met Ala Gly Ala Ala Val Leu Val Thr Gly Gly Gly Gln Thr Ala Thr 1 5 10 15 Ala Ala Asp Ser Ala Phe Tyr Thr Asp Pro Gly Ser Ser Ser Ala Arg 20 25 30 Trp Val Ala Ala Asn Pro Asn Asp Ser Arg Ala Ala Val Ile Arg Asp 35 40 45 Arg Val Ala Ser Val Pro Gln Ala Lys Trp Phe Thr Thr Thr Asn Thr 50 55 60 Ser Thr Val Arg Ser Glu Val Ser Ala Phe Val Gly Ala Ala Ala Ser 65 70 75 80 Ala Gly Lys Ile Pro Ile Leu Val Val Tyr Asn Ile Pro Asn Arg Asp 85 90 95 Cys Gly Gly Ala Ser Gly Gly Gly Ala Pro Ser His Gln Ala Tyr Arg 100 105 110 Ala Trp Val Asp Glu Val Ala Ala Gly Leu Gly Gly Arg Pro Ala Ser 115 120 125 Ile Ile Leu Glu Pro Asp Val Leu Pro Ile Met Ser Asn Cys Gln Ser 130 135 140 Ala Asp Gln Gln Asn Gln Thr Lys Ala Ser Met Ser Tyr Ala Gly Arg 145 150 155 160 Lys Leu Lys Ser Gly Ser Gly Gln Ala Lys Val Tyr Phe Asp Ile Gly 165 170 175 Asn Ser Asp Trp Leu Ala Pro Ala Glu Ala Ala Asn Arg Leu Arg Gly 180 185 190 Ala Asp Val Ser Gly Ser Ser Asp Gly Ile Ala Ser Asn Val Ser Asn 195 200 205 Tyr Arg Ala Thr Gln Ala Glu Val Ser Tyr Thr Lys Ala Ile Leu Asn 210 215 220 Ala Leu Gly Asp Gly Arg Leu Lys Ala Val Ile Asp Thr Ser Arg Asn 225 230 235 240 Gly Asn Gly Pro Leu Gly Ser Glu Trp Cys Asp Pro Pro Gly Arg Ala 245 250 255 Ile Gly Thr Pro Ser Thr Lys Asn Thr Gly Asp Ser Gln Ile Asp Ala 260 265 270 Phe Leu Trp Val Lys Ile Val Gly Glu Ala Asp Gly Cys Ile Ala Ser 275 280 285 Ala Gly Gln Phe Val Pro Gln Arg Ala Tyr Asp Leu Ala Val Ala Ala 290 295 300 Gly Pro Val Pro Thr Thr Thr Thr Thr Thr Pro Gly Gly Asn Pro Gly 305 310 315 320 Gly Gly Cys Ala Val Thr His Arg Val Val Ser Gln Trp Asn Gly Gly 325 330 335 Phe Thr Gly Glu Val Val Val Glu Asn Arg Gly Pro Ala Ile Ser Ser 340 345 350 Trp Thr Leu Glu Phe Ser Ala Pro Gly Val Thr Val Thr Gln Gly Trp 355 360 365 Asn Gly Thr Trp Thr Asp Thr Gly Asp Gly Val Arg Val Val Asn Thr 370 375 380 Ala Trp Asn Gly Ala Leu Ala Ser Gly Gly Arg Val Thr Ala Gly Tyr 385 390 395

400 Asn Ala Asn Tyr Gly Gly Gly Ala Pro Pro Phe Ser Ser Pro Thr Leu 405 410 415 Asn Gly Ala Ala Cys Ser 420 102444PRTActinosynnema mirum 102Met Ala Ala Gly Ala Leu Ser Ser Ala Leu Val Ala Ala Ala Thr Ala 1 5 10 15 Ile Ala Thr Gly Thr Ala Ser Pro Ala Ala Val Ala Ala Asp Ser Glu 20 25 30 Phe Tyr Ser Asp Pro Ala Thr Ser Ala Ala Arg Trp Val Ala Ala Asn 35 40 45 Pro Asn Asp Ser Arg Ala Ala Val Ile Arg Asp Arg Val Ala Ser Val 50 55 60 Pro Gln Ala Lys Trp Phe Thr Thr Thr Asn Thr Ser Thr Ile Arg Ala 65 70 75 80 Glu Val Asp Ala His Thr Ser Ala Ala Ala Ser Ala Gly Lys Thr Pro 85 90 95 Ile Leu Val Val Tyr Asn Ile Pro Asn Arg Asp Cys Gly Gly Ala Ser 100 105 110 Gly Gly Gly Ala Pro Ser His Gly Ala Tyr Arg Gln Trp Val Asp Gln 115 120 125 Phe Ala Ala Gly Leu Ala Gly Arg Pro Ala Ala Ile Ile Leu Glu Pro 130 135 140 Asp Val Leu Pro Ile Met Ser Thr Cys Gln Ser Ala Ser Gln Gln Ala 145 150 155 160 Glu Thr Arg Ala Ser Met Ala Tyr Ala Gly Lys Ala Leu Lys Ala Ala 165 170 175 Ser Ser Gln Ala Lys Val Tyr Phe Asp Ile Gly His Ser Ala Trp Leu 180 185 190 Thr Pro Ala Glu Ala Ala Asn Arg Leu Arg Ala Ala Glu Val Ser Thr 195 200 205 Ser Ala Asp Gly Ile Ala Thr Asn Val Ser Asn Tyr Arg Arg Thr Ala 210 215 220 Asp Glu Val Ala Phe Ala Lys Ala Thr Leu Asn Ala Leu Gly Asp Gly 225 230 235 240 Arg Leu Lys Ala Val Val Asp Thr Ser Arg Asn Gly Asn Gly Pro Leu 245 250 255 Gly Ser Glu Trp Cys Asp Pro Pro Gly Arg Ala Ile Gly Thr Pro Ser 260 265 270 Thr Arg Asn Thr Gly Asp Pro Gln Ile Asp Ala Phe Leu Trp Val Lys 275 280 285 Ile Pro Gly Glu Ala Asp Gly Cys Ile Ala Gly Ala Gly Gln Phe Val 290 295 300 Pro Gln Arg Ala Tyr Asp Met Ala Val Ala Ala Gly Pro Ala Pro Thr 305 310 315 320 Thr Thr Thr Thr Thr Thr Thr Thr Thr Arg Val Thr Thr Thr Thr Thr 325 330 335 Thr Pro Pro Pro Asn Gly Ala Ala Cys Val Val Arg His Arg Val Val 340 345 350 Ser Ser Trp Ser Gly Gly His Thr Gly Glu Val Val Ile Glu Asn Arg 355 360 365 Gly Pro Ala Leu Gln Asn Trp Thr Leu Glu Phe Ser Ala Pro Gly Val 370 375 380 Ala Val Ser Gln Gly Trp Asn Gly Thr Trp Thr Asp Leu Gly Asp Thr 385 390 395 400 Val Arg Val Thr Ser Ala Ser Trp Asn Gly Gly Ile Ala Thr Gly Gly 405 410 415 Thr Ala Thr Thr Gly Tyr Ser Ala Ser Phe Ser Gly Gly Thr Pro Pro 420 425 430 Phe Thr Ser Pro Val Leu Asn Gly Thr Ala Cys Ala 435 440 103426PRTNocardiopsis dassonvillei 103Met Ser Ser Val Ser Ala Leu Ala Leu Gly Thr Ala Leu Leu Ala Thr 1 5 10 15 Ala Pro Ala Ser Ala Ala Asp Ser Glu Phe Tyr Val Asn Pro Asn Thr 20 25 30 Ser Ala Ala Val Trp Val Glu Glu Asn Pro Asn Asp Pro Arg Ala Asp 35 40 45 Val Ile Arg Asp Arg Ile Ala Ser Val Ala Gln Ala Thr Trp Phe Thr 50 55 60 Gln Tyr Asn Pro Ala Glu Val Arg Asp Asp Val Asp Ala Val Val Ser 65 70 75 80 Ala Ala Asp Ala Gln Gly Gln Thr Pro Ile Leu Val Val Tyr Asn Ile 85 90 95 Pro Gly Arg Asp Cys Gly Asn His Ser Gly Gly Gly Ala Pro Ser His 100 105 110 Asp Ala Tyr Arg Ala Trp Val Asp Glu Val Ala Ala Gly Leu Glu Gly 115 120 125 Arg Ser Ala Thr Ile Val Leu Glu Pro Asp Ala Leu Pro Leu Val Ser 130 135 140 Gly Cys Ser Asp Pro Ser Glu Leu Leu Asp Ser Met Ala Tyr Ala Gly 145 150 155 160 Lys Ala Leu Met Glu Gly Ser Ser Glu Ala Arg Val Tyr Phe Asp Ile 165 170 175 Gly Asn Ser Ala Trp Leu Asp Pro Gln Glu Ala Ala Gly Leu Leu Asn 180 185 190 Gly Ala Asp Val Ala Asn Ser Ala His Gly Val Ala Thr Asn Thr Ser 195 200 205 Asn Tyr Asn Trp Thr His Asp Glu Val Ala Phe Ala Glu Ala Val Ile 210 215 220 Ala Ala Thr Gly Val Pro Gly Leu Gly Ala Val Ile Asp Thr Ser Arg 225 230 235 240 Asn Gly Asn Gly Pro Ala Pro Gln Asn Glu Trp Cys Asp Pro Pro Gly 245 250 255 Arg Met Ile Gly Arg Pro Ser Thr Thr Asp Thr Gly Asn Pro Leu Ile 260 265 270 Asp Ala Phe Ile Trp Thr Lys Leu Pro Gly Glu Ala Asp Gly Cys Ile 275 280 285 Ala Pro Ala Gly Gln Phe Val Pro Gln Ala Ala Tyr Asp Met Ala Val 290 295 300 Asn Ala Pro Glu Tyr Pro Thr Asp Pro Gly Glu Pro Thr Asp Pro Glu 305 310 315 320 Glu Pro Thr Asp Pro Pro Glu Gly Glu Gly Cys Thr Ala Asp Tyr Arg 325 330 335 Val Val Ser Glu Trp Gly Asn Gly Phe Gln Ala Ala Val Thr Val Thr 340 345 350 Ala Glu Asp Ser Leu Ser Gly Trp Thr Val Thr Trp Thr Tyr Ala Asp 355 360 365 Gly Gln Arg Phe Ser Gln Gly Trp Asn Ala Glu Phe Ser Ser Ser Gly 370 375 380 Ser Arg Val Thr Ala Ser Asp Leu Gly Trp Asn Gly Thr Leu Ser Ala 385 390 395 400 Gly Gly Ser Thr Glu Phe Gly Phe Thr Gly Thr His Gly Gly Ser Asn 405 410 415 Gly Val Pro Glu Val Thr Cys Ser Ala Ala 420 425 104286PRTThermobifida fusca 104Asn Asp Ser Pro Phe Tyr Val Asn Pro Asn Met Ser Ser Ala Glu Trp 1 5 10 15 Val Arg Asn Asn Pro Asn Asp Pro Arg Thr Pro Val Ile Arg Asp Arg 20 25 30 Ile Ala Ser Val Pro Gln Gly Thr Trp Phe Ala His His Asn Pro Gly 35 40 45 Gln Ile Thr Gly Gln Val Asp Ala Leu Met Ser Ala Ala Gln Ala Ala 50 55 60 Gly Lys Ile Pro Ile Leu Val Val Ser Asn Ala Pro Gly Arg Asp Cys 65 70 75 80 Gly Asn His Ser Ser Gly Gly Ala Pro Ser His Ser Ala Tyr Arg Ser 85 90 95 Trp Ile Asp Glu Phe Ala Ala Gly Leu Lys Asn Arg Pro Ala Tyr Ile 100 105 110 Ile Val Glu Pro Asp Leu Ile Ser Leu Met Ser Ser Cys Met Gln His 115 120 125 Val Gln Gln Glu Val Leu Glu Thr Met Ala Tyr Ala Gly Lys Ala Leu 130 135 140 Lys Ala Gly Ser Ser Gln Ala Arg Ile Tyr Phe Asp Ala Gly His Ser 145 150 155 160 Ala Trp His Ser Pro Ala Gln Met Ala Ser Trp Leu Gln Gln Ala Asp 165 170 175 Ile Ser Asn Ser Ala His Gly Ile Ala Thr Asn Thr Ser Asn Tyr Arg 180 185 190 Trp Thr Ala Asp Glu Val Ala Tyr Ala Lys Ala Val Leu Ser Ala Ile 195 200 205 Gly Asn Pro Ser Leu Arg Ala Val Ile Asp Thr Ser Arg Asn Gly Asn 210 215 220 Gly Pro Ala Gly Asn Glu Trp Cys Asp Pro Ser Gly Arg Ala Ile Gly 225 230 235 240 Thr Pro Ser Thr Thr Asn Thr Gly Asp Pro Met Ile Asp Ala Phe Leu 245 250 255 Trp Ile Lys Leu Pro Gly Glu Ala Asp Gly Cys Ile Ala Gly Ala Gly 260 265 270 Gln Phe Val Pro Gln Ala Ala Tyr Glu Met Ala Ile Ala Ala 275 280 285 105448PRTMicromonospora sp. ATCC 39149 105Met Ala Ala Ser Ala Leu Thr Ala Ala Val Ala Val Ser Ile Leu Gly 1 5 10 15 Gly Thr Pro Ala Ser Ala Ala Asp Ser Ala Phe Tyr Val Asp Pro Gln 20 25 30 Ala Ser Ala Ala Arg Trp Val Ala Ala Asn Pro Gly Asp Trp Arg Ala 35 40 45 Ala Val Ile Arg Asp Arg Ile Ala Ala Val Pro Gln Gly Arg Trp Phe 50 55 60 Thr Thr Thr Asn Thr Ser Thr Val Arg Ser Glu Val Asp Gln Phe Val 65 70 75 80 Gly Ala Ala Ala Ala Ala Gly Lys Val Pro Ile Met Val Val Tyr Asn 85 90 95 Ile Pro Asn Arg Asp Cys Ser Gly Ala Ser Gly Gly Gly Ala Pro Ser 100 105 110 His Thr Ala Tyr Arg Gln Trp Val Asp Gln Val Ala Ala Gly Leu Ala 115 120 125 Gly Arg Pro Ala Thr Ile Val Leu Glu Pro Asp Val Leu Pro Ile Met 130 135 140 Thr Asn Cys Gln Asn Ala Ser Gln Gln Ala Glu Thr Arg Ala Ser Met 145 150 155 160 Ala Tyr Ala Gly Lys Lys Leu Lys Ser Gly Ser Ala Gln Ala Lys Val 165 170 175 Tyr Phe Asp Ala Gly Asn Ser Ala Trp Leu Ala Pro Ala Glu Ile Ala 180 185 190 Ser Arg Leu Asn Gly Ala Asp Ile Ala Asn Ser Ala Asp Gly Ile Ser 195 200 205 Leu Asn Val Ser Asn Tyr Arg Thr Thr Ala Glu Ser Val Ser Tyr Ala 210 215 220 Lys Gln Val Ile Ala Ala Thr Gly Val Ser Arg Leu Lys Ala Val Ile 225 230 235 240 Asp Thr Ser Arg Asn Gly Asn Gly Pro Leu Gly Ser Glu Trp Cys Asp 245 250 255 Pro Pro Gly Arg Ala Ile Gly Thr Pro Ser Thr Thr Ala Thr Gly Asp 260 265 270 Ser Ala Ile Ala Ala Tyr Leu Trp Val Lys Leu Pro Gly Glu Ala Asp 275 280 285 Gly Cys Ile Ala Pro Ala Gly Gln Phe Val Pro Gln Arg Ala Tyr Asp 290 295 300 Leu Ala Ile Ala Ala Gly Pro Val Pro Thr Thr Ala Pro Pro Thr Thr 305 310 315 320 Ala Pro Pro Thr Thr Ala Pro Pro Thr Thr Ala Pro Pro Thr Thr Ala 325 330 335 Pro Pro Thr Thr Pro Pro Asn Gly Ala Cys Lys Val Thr Phe Thr Pro 340 345 350 Asn Thr Trp Ser Gly Gly Phe Thr Ala Glu Leu Arg Val Thr Asn Gly 355 360 365 Gly Ser Ala Leu Asn Gly Trp Ser Leu Ser Phe Gly Phe Gly Ser Gly 370 375 380 Ser Gly Val Arg Leu Thr Ser Gly Trp Asn Gly Glu Trp Ser Gln Asn 385 390 395 400 Gly Asp Val Phe Leu Val Arg Asn Ala Ala Trp Asn Gly Asn Leu Pro 405 410 415 Ala Gly Gly Thr Leu Ser Val Gly Phe Gln Gly Thr Phe Ser Gly Ala 420 425 430 Ser Leu Pro Thr Ala Val Gly Phe Thr Leu Asn Gly Ser Arg Cys Asn 435 440 445 106456PRTThermobispora bispora 106Met Ser Arg Ile Arg Arg Phe Leu Ala Thr Ala Leu Ala Ala Ala Thr 1 5 10 15 Ala Gly Val Gly Ala Ile Val Thr Ala Ile Ala Ser Ala Gly Pro Ala 20 25 30 His Ala Tyr Asp Ser Pro Phe Tyr Val Asp Pro Gln Ser Asn Ala Ala 35 40 45 Lys Trp Val Ala Ala Asn Pro Asn Asp Pro Arg Thr Pro Val Ile Arg 50 55 60 Asp Arg Ile Ala Ala Val Pro Thr Gly Arg Trp Phe Ala Asn Tyr Asn 65 70 75 80 Pro Ser Thr Val Arg Ala Glu Val Asp Ala Tyr Val Gly Ala Ala Ala 85 90 95 Ala Ala Gly Lys Ile Pro Ile Met Val Val Tyr Ala Met Pro Asn Arg 100 105 110 Asp Cys Gly Gly Pro Ser Ala Gly Gly Ala Pro Asn His Thr Ala Tyr 115 120 125 Arg Ala Trp Ile Asp Glu Ile Ala Ala Gly Leu Arg Asn Arg Pro Ala 130 135 140 Val Ile Ile Leu Glu Pro Asp Ala Leu Pro Ile Met Thr Asn Cys Met 145 150 155 160 Ser Pro Ser Glu Gln Ala Glu Val Gln Ala Ser Ala Val Gly Ala Gly 165 170 175 Lys Lys Phe Lys Ala Ala Ser Ser Gln Ala Lys Val Tyr Phe Asp Ala 180 185 190 Gly His Asp Ala Trp Val Pro Ala Asp Glu Met Ala Ser Arg Leu Arg 195 200 205 Gly Ala Asp Ile Ala Asn Ser Ala Asp Gly Ile Ala Leu Asn Val Ser 210 215 220 Asn Tyr Arg Tyr Thr Ser Gly Leu Ile Ser Tyr Ala Lys Ser Val Leu 225 230 235 240 Ser Ala Ile Gly Ala Ser His Leu Arg Ala Val Ile Asp Thr Ser Arg 245 250 255 Asn Gly Asn Gly Pro Leu Gly Ser Glu Trp Cys Asp Pro Pro Gly Arg 260 265 270 Ala Thr Gly Thr Trp Ser Thr Thr Asp Thr Gly Asp Pro Ala Ile Asp 275 280 285 Ala Phe Leu Trp Ile Lys Pro Pro Gly Glu Ala Asp Gly Cys Ile Ala 290 295 300 Thr Pro Gly Val Phe Val Pro Asp Arg Ala Tyr Glu Leu Ala Met Asn 305 310 315 320 Ala Ala Pro Pro Thr Tyr Ser Pro Ser Pro Thr Pro Ser Thr Pro Ser 325 330 335 Pro Ser Pro Ser Gln Ser Asp Pro Gly Ser Pro Ser Pro Ser Pro Ser 340 345 350 Gln Pro Pro Ala Gly Arg Ala Cys Glu Ala Thr Tyr Ala Leu Val Asn 355 360 365 Gln Trp Pro Gly Gly Phe Gln Ala Glu Val Thr Val Lys Asn Thr Gly 370 375 380 Ser Ser Pro Ile Asn Gly Trp Thr Val Gln Trp Thr Leu Pro Ser Gly 385 390 395 400 Gln Ser Ile Thr Gln Leu Trp Asn Gly Asp Leu Ser Thr Ser Gly Ser 405 410 415 Asn Val Thr Val Arg Asn Val Ser Trp Asn Gly Asn Val Pro Ala Gly 420 425 430 Gly Ser Thr Ser Phe Gly Phe Leu Gly Ser Gly Thr Gly Gln Leu Ser 435 440 445 Ser Ser Ile Thr Cys Ser Ala Ser 450 455 1075PRTArtificial SequenceConsensus sequence 107Gly Glu Xaa Asp Gly 1 5

Claims

1. (canceled)

2. A recombinant polypeptide selected from the group consisting of a, polypeptide comprising a sequence that is (a) identical to SEQ ID NO:67 and having a C400S mutation; (b) is at least 85% identical to SEQ ID NO:68 and having a C400S mutation; (c) is at least 85% identical to SEQ ID NO:69 and having a C400S mutation; (d) is at least 85% identical to SEQ ID NO:70 and having a C400S mutation; (e) is at least 85% identical to SEQ ID NO:71 and having a C400S mutation; (f) is at least 85% identical to SEQ ID NO:72 and having a C400S mutation; (d) is at least 85% identical to SEQ ID NO:73 and having a C400S mutation; (h) is at least 85% identical to SEQ ID NO:74 and having a C400S mutation; (i) is at least 85% identical to SEQ ID NO:75 and having a C400S mutation; (j) is at least 85% identical to SEQ ID NO:76 and having a C407S mutation; (k) is at least 85% identical to SEQ ID NO:77 and having a C394S mutation; and (l) is at least 85% identical to SEQ ID NO:78 and having a C412S, wherein the foregoing polypeptides have cellulase activity and improved thermostability compared to their corresponding parental (wild-type) protein lacking a Cys.fwdarw.Ser mutation.

3. The recombinant polypeptide of claim 2, wherein the polypeptide has from 1-30 conservative amino acid substitutions except at the position identified below wherein a C.fwdarw.S substitution is present: SEQ ID NO:67 comprising a C400S; SEQ ID NO:68 comprising a C400S; SEQ ID NO:69 comprising a C400S; SEQ ID NO:70 comprising a C400S; SEQ ID NO:71 comprising a C400S; SEQ ID NO:72 comprising a C400S; SEQ ID NO:73 comprising a C400S; SEQ ID NO:74 comprising a C400S; SEQ ID NO:75 comprising a C400S; SEQ ID NO:76 comprising a C407S; SEQ ID NO:77 comprising a C394S, or SEQ ID NO:78 comprising a C412S.

4-7. (canceled)

8. A polynucleotide encoding a polypeptide of claim 2.

9. A vector comprising a polynucleotide of claim 8.

10. A host cell comprising the polynucleotide of claim 8.

11. A host cell comprising the vector of claim 9.

12. An enzymatic preparation comprising a polypeptide of claim 2.

13. An enzymatic preparation comprising a polypeptide produced by a host cell of claim 10.

14. A method of treating a biomass comprising cellulose, the method comprising contacting the biomass with a polypeptide of claim 2.

15. A method of treating a biomass comprising cellulose, the method comprising contacting the biomass with an enzymatic preparation of claim 12.