Beta-glucosidase I Variants With Improved Properties

Abstract

The present disclosure is generally directed to enzymes and in particular beta-glucosidase variants. Also described are nucleic acids encoding beta-glucosidase variants, compositions comprising beta-glucosidase variants, methods of using beta-glucosidase variants, and methods of identifying additional useful beta-glucosidase variants.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No. 13/510,902, a national phase filing under 35 U.S.C. 371 of PCT/US2010/057531, filed Nov. 19, 2010, which claims the benefit of U.S. provisional application Ser. No. 61/263,240 filed Nov. 20, 2009. Each of the above-referenced applications is hereby incorporated by reference in its entirety.

SEQUENCE LISTING

[0003] The content of the electronically sequence listing in ASCII text filed herewith (File Name: NB31435USCNT2_SeqList_ST25.txt; Size: 315,111 bytes; Date of creation: Aug. 22, 2016) is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

[0004] The present disclosure is generally directed to enzymes and in particular beta-glucosidase variants. Also described are nucleic acids encoding beta-glucosidase variants, compositions comprising beta-glucosidase variants, methods of using beta-glucosidase variants, and methods of identifying additional useful beta-glucosidase variants.

BACKGROUND

[0005] Cellulose and hemicellulose are the most abundant plant materials produced by photosynthesis. They can be degraded and used as an energy source by numerous microorganisms (e.g., bacteria, yeast and fungi) that produce extracellular enzymes capable of hydrolysis of the polymeric substrates to monomeric sugars (Aro et al., J. Biol. Chem., 276: 24309-24314, 2001). As the limits of non-renewable resources approach, the potential of cellulose to become a major renewable energy resource is enormous (Krishna et al., Bioresource Tech., 77: 193-196, 2001). The effective utilization of cellulose through biological processes is one approach to overcoming the shortage of foods, feeds, and fuels (Ohmiya et al., Biotechnol. Gen. Engineer Rev., 14: 365-414, 1997).

[0006] Cellulases are enzymes that hydrolyze cellulose (beta-1,4-glucan or beta D-glucosidic linkages) resulting in the formation of glucose, cellobiose, cellooligosaccharides, and the like. Cellulases have been traditionally divided into three major classes: endoglucanases (EC 3.2.1.4) ("EG"), exoglucanases or cellobiohydrolases (EC 3.2.1.91) ("CBH") and beta-glucosidases ([beta]-D-glucoside glucohydrolase; EC 3.2.1.21) ("BG") (Knowles et al., TIBTECH 5: 255-261, 1987; and Schulein, Methods Enzymol., 160: 234-243, 1988). Endoglucanases act mainly on the amorphous parts of the cellulose fiber, whereas cellobiohydrolases are also able to degrade crystalline cellulose (Nevalainen and Penttila, Mycota, 303-319, 1995). Thus, the presence of a cellobiohydrolase in a cellulase system is required for efficient solubilization of crystalline cellulose (Suurnakki et al., Cellulose, 7: 189-209, 2000). Beta-glucosidase acts to liberate D-glucose units from cellobiose, cello-oligosaccharides, and other glucosides (Freer, J. Biol. Chem., 268: 9337-9342, 1993).

[0007] Cellulases are known to be produced by a large number of bacteria, yeast and fungi. Certain fungi produce a complete cellulase system capable of degrading crystalline forms of cellulose, such that the cellulases are readily produced in large quantities via fermentation. Filamentous fungi play a special role since many yeast, such as Saccharomyces cerevisiae, lack the ability to hydrolyze cellulose (see, e.g., Wood et al., Methods in Enzymology, 160: 87-116, 1988).

[0008] The fungal cellulase classifications of CBH, EG and BG can be further expanded to include multiple components within each classification. For example, multiple CBHs, EGs and BGs have been isolated from a variety of fungal sources including Trichoderma reesei (also referred to as Hypocrea jecorina), which contains known genes for two CBHs, i.e., CBH I ("CBH1") and CBH II ("CBH2"), at least eight EGs, i.e., EG I, EG II, EG III, EGIV, EGV, EGVI, EGVII and EGVIII, and at least five BGs, i.e., BG1, BG2, BG3, BG4, BG5 and BG7 (Foreman et al. (2003), J. Biol. Chem. 278(34):31988-31997). EGIV, EGVI and EGVIII also have xyloglucanase activity.

[0009] In order to efficiently convert crystalline cellulose to glucose the complete cellulase system comprising components from each of the CBH, EG and BG classifications is required, with isolated components less effective in hydrolyzing crystalline cellulose (Filho et al., Can. J. Microbiol., 42:1-5, 1996). Endo-1,4-beta-glucanases (EG) and exo-cellobiohydrolases (CBH) catalyze the hydrolysis of cellulose to cellooligosaccharides (cellobiose as a main product), while beta-glucosidases (BGL) convert the oligosaccharides to glucose. A synergistic relationship has been observed between cellulase components from different classifications. In particular, the EG-type cellulases and CBH-type cellulases synergistically interact to efficiently degrade cellulose.

[0010] Although beta-glucosidase compositions have been previously described, there remains a need for new and improved beta-glucosidase compositions. Improved beta-glucosidase compositions find use for example in saccharifying biomass. Beta-glucosidases that exhibit desirable levels in one or more of expression, activity and stability are of particular interest.

SUMMARY

[0011] The present disclosure provides polypeptides having beta-glucosidase activity. The disclosure is based in part on beta-glucosidase variants. Also described are nucleic acids encoding beta-glucosidase variants, compositions comprising beta-glucosidase variants, methods of using beta-glucosidase variants, and methods of identifying additional useful beta-glucosidase variants. In one aspect of the invention, the beta-glucosidase variants are H. jecorina beta-glucosidase 1 (BGL1) variants.

[0012] Accordingly, in one aspect, the invention provides beta-glucosidase 1 (BGL1) variants having at least two improved activities over wild type BGL1 selected from the group consisting of: (a) pre-treated corn stover (PCS) hydrolysis activity, (b) cellobiase activity, (c) protein expression (HPLC), (d) beta-glucosidase activity measured by either a cellobiase activity in the presence of ammonia pretreated corncob (CC), or by a CC hydrolysis activity, (e) thermostability, (f) phosphoric acid swollen cellulose (PASC) hydrolysis activity, and (g) increased hydrolytic activity in the presence of glucose as compared to wild-type BGL1, wherein the BGL1 variant is any variant as shown in Tables 4-8, 3-2, 4-2 and 4-3.

[0013] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved (b) and (d) activities over wild type BGL1, wherein the BGL1 variant is L266A, I567E, S283F, S283P, T258E, T258I, T258K, T258Q, P536T, P536W, I532Y, Y530T, P607D, Q406M, Q406S, V602T, G300M, A630S, A630T, T180H, T180M, A450M, I444E, I444F, I444N, I444W, I444Y, V500Q, A633I, S482P, A667V, A485L, A485W, Y678R, V603G, L266C, I567F, S624P, P607L, G606I, G606K, G606L, G606M, G606Q, G606V, G605E, I444V, A633V, A655W, Y678H, V522Y, G554F, L266N, F556L, S550I, S550T, S550V, T258L, P536I, P536V, F392R, S624G, S624N, S624Q, S624T, A601M, A630V, N463S, A450F, A450T, A450V, A450W, I486V, S482I, Y678I, G427F, D564T, Q684C, Q684G, Y530S, Q684N, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, F260D, F260G, F260Q, P607G, N400S, F260W, Y530F, Q406D, G605C, N263T, P607I, A450P, T242H, A630Y, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, F260L, L293F, A633C, S312C, or N455D.

[0014] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved (b) and (e) activities over wild type BGL1, wherein the BGL1 variant is P607H, T011E, T011Y, N146E, I567V, N566G, A630K, Y639K, Q245N, K320Y, A347Y, P536Q, N369E, N369W, N369Y, N146A, N146Q, P607K, N369T, A655N, I671K, F260T, P607S, F260D, F260G, F260Q, P607G, N400S, P607F, P607I, A450P, T242H, T568E, A630Y, A655D, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, F260L, or L293F.

[0015] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved (b) and (f) activities over wild type BGL1, wherein the BGL1 variant is N261C, T258C, F392Q, S624E, P607C, P604M, A377Q, N461A, N461F, N461P, T436A, T436C, T436F, T436I, T436M, T436Q, T436Y, Q220C, A655L, T646H, Y678F, A468I, D177M, P661E, L266N, F556L, S550I, S550T, S550V, T258L, P536I, P536V, F392R, S624G, S624N, S624Q, S624T, A601M, A630V, N463S, A450F, A450T, A450V, A450W, I486V, S482I, Y678I, G427F, D564T, Q684C, Q684G, N566H, F556V, P604Y, L293V, A630G, N461C, N463T, D457C, Q220M, T221A, T221G, T221I, A655R, A468F, A468S, Q216I, D564V, P536Q, N369E, N369W, N369Y, T436E, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, F260W, Y530F, N461V, I671C, K206A, A450P, T242H, E170F, S507G, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, L293F, A633C, S312C or N455D.

[0016] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved (a) and (b) activities over wild type BGL1, wherein the BGL1 variant is I567Q, A565F, A565K, A565Q, A565V, F556E, F260I, P607E, G605R, G300C, A377C, A377D, S308C, N146H, N146S, A655C, A655G, P176L, T209I, L266C, I567F, S624P, P607L, G606I, G606K, G606L, G606M, G606Q, G606V, G605E, I444V, A633V, A655W, Y678H, V522Y, G554F, N566H, F556V, P604Y, L293V, A630G, N461C, N463T, D457C, Q220M, T221A, T221G, T221I, A655R, A468F, A468S, Q216I, D564V, A565C, A655N, I671K, F260T, P607S, Y639V, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, F260D, F260G, F260Q, P607G, N400S, P607F, Q406D, G605C, N263T, N461V|I671C, K206A, T568E, E170F, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, F260L, A633C, S312C, or N455D.

[0017] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved (b) and (c) activities over wild type BGL1, wherein the BGL1 variant is I567K, I567R, A565E, A565S, A565Y, F392Y, Q406H, Q406T, P604C, N038F, T568A, N461G, Y639L, Y639M, T243A, T243C, Q245H, Q245M, Q245T, T646A, T646C, I671F, I671L, I567V, N566G, A630K, Y639K, Q245N, K320Y, A347Y, A565C, Y639G, Y530F, N461V, I671C, K206A, T568E, A630Y, A655D, S507G, F260E, T568K, or F260L.

[0018] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved (a) and (d) activities over wild type BGL1, wherein the BGL1 variant is I567S, G606E, G606H, G606N, G606S, L293A, S308R, I444C, M201D, R542N, L266C, I567F, S624P, P607L, G606I, G606K, G606L, G606M, G606Q, G606V, G605E, I444V, A633V, A655W, Y678H, V522Y, G554F, N566F, L293M, Q220P, S692L, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, F260D, F260G, F260Q, P607G, N400S, Q406D, G605C, N263T, S308E, A338D, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, F260L, A633C, S312C, or N455D.

[0019] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved (e) and (g) activities over wild type BGL1, wherein the BGL1 variant is L266F, I567Y, A270R, S384C, A630W, E128R, N146M, N146V, N146W, L181F, V043C, Y639P, S507F, Q245P, G662C, A630H, V466T, N146A, N146Q, P607K, N369T, S384E, L181M, V043A, V043G, V043N, Q060D, A655Y, T242S, S474D, P607F, A630Y, S308E, A655D, or L293F.

[0020] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved (c) and (e) activities over wild type BGL1, wherein the BGL1 variant is N261E, N261K, N400A, V602K, L293I, N461S, D457A, V043Q, Q303N, K320S, G662D, F260A, S474R, I567V, N566G, A630K, Y639K, Q245N, K320Y, A347Y, A601D, S384E, L181M, V043A, V043G, V043N, Q060D, A655Y, T242S, S474D, D564T, T568E, A655D, A338D, F260E, T568K, or F260L.

[0021] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved (a) and (f) activities over wild type BGL1, wherein the BGL1 variant is N566L, N566P, N566W, A270K, A270N, F556H, F556K, P604N, N461D, N463E, K206G, A468Q, A468Y, N566F, N566H, F556V, P604Y, L293V, A630G, N461C, N463T, D457C, Q220M, T221A, T221G, T221I, A655R, A468F, A468S, Q216I, D564V, A468T, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, N461V|I671C, K206A, E170F, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, A633C, S312C, or N455D.

[0022] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved (a) and (c) activities over wild type BGL1, wherein the BGL1 variant is S283D, A270D, N146Y, F260A, S474R, A565C, K206S, D564T, N461V|I671C, K206A, T568E, A338D, F260E, T568K, or F260L.

[0023] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved (a) and (e) activities over wild type BGL1, wherein the BGL1 variant is F556G, F260S, P604E, P604V, N146D, Y639T, T221C, N473S, N583R, R645G, G662Y, F260A, S474R, A655N, I671K, F260T, P607S, S692L, D564T, F260D, F260G, F260Q, P607G, N400S, P607F, T568E, S308E, A338D, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, or F260L.

[0024] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved (c) and (d) activities over wild type BGL1, wherein the BGL1 variant is D259S, T243V, Y530F, A338D, or F260L.

[0025] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved (d) and (e) activities over wild type BGL1, wherein the BGL1 variant is S550Q, P607R, N400Q, V602F, A601G, A601L, L293K, Y575C, Y575R, A450Q, I486C, I486Y, A655S, Q245F, D329A, P536G, P607Q, A655Q, Y575A, Y575K, A630H, V466T, S692L, F260D, F260G, F260Q, P607G, N400S, P607I, A450P, T242H, S308E, A630Y, A338D, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, F260L, or L293F.

[0026] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved (d) and (f) activities over wild type BGL1, wherein the BGL1 variant is P536F, F392C, S624L, S624R, S624W, I486F, I486W, A667G, A667S, L266N, F556L, S550I, S550T, S550V, T258L, P536I, P536V, F392R, S624G, S624N, S624Q, S624T, A601M, A630V, N463S, A450F, A450T, A450V, A450W, I486V, S482I, Y678I, G427F, D564T, Q684C, Q684G, N566F, Y575A, Y575K, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, F260W, P607I, A450P, T242H, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, L293F, A633C, S312C, or N455D.

[0027] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved (c) and (g) activities over wild type BGL1, wherein the BGL1 variant is S384G, S384W, N038E, N038M, N038P, V043H, V043W, Y068E, Y068G, Y068M, L110C, L110G, L110Q, L110W, A655H, N264L, S384E, L181M, V043A, V043G, V043N, Q060D, A655Y, T242S, S474D, Y639G, K206S, A655D, or S507G.

[0028] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved (d) and (g) activities over wild type BGL1, wherein the BGL1 variant is G606D, Y068V, L293M, Q220P, A630H, V466T, Y530S, Q684N, F260W, Q406D, G605C, N263T, S308E, A630Y, L293F, A633C, S312C, or N455D.

[0029] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved (b) and (g) activities over wild type BGL1, wherein the BGL1 variant is A377I, N461Y, N146A, N146Q, P607K, N369T, T436E, Y639G, Y530S, Q684N, Y639V, F260W, P607F, Q406D, G605C, N263T, A630Y, A655D, E170F, S507G, L293F, A633C, S312C, or N455D.

[0030] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved (c) and (f) activities over wild type BGL1, wherein the BGL1 variant is K206D, A601D, Y530F, N461V|I671C, K206A, S507G, F260E, or T568K.

[0031] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved (e) and (f) activities over wild type BGL1, wherein the BGL1 variant is A468G, P536Q, N369E, N369W, N369Y, A601D, Y575A, Y575K, P607I, A450P, T242H, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, or L293F.

[0032] In another aspect, the invention provides BGL1 variants, as described above and throughout this specification, having improved (a) and (g) activities over wild type BGL1, wherein the BGL1 variant is R179V, L293M, Q220P, K206S, A468T, Y639V, P607F, Q406D, G605C, N263T, S308E, E170F, A633C, S312C, or N455D.

[0033] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two, or all three of (a), (b), and (d) over wild type BGL1, wherein the BGL1 variant is L266C, I567F, S624P, P607L, G606I, G606K, G606L, G606M, G606Q, G606V, G605E, I444V, A633V, A655W, Y678H, V522Y, G554F, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, F260D, F260G, F260Q, P607G, N400S, Q406D, G605C, N263T, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, F260L, A633C, S312C, or N455D.

[0034] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two or all three of (b), (d), and (f) over wild type BGL1, wherein the BGL1 variant is L266N, F556L, S550I, S550T, S550V, T258L, P536I, P536V, F392R, S624G, S624N, S624Q, S624T, A601M, A630V, N463S, A450F, A450T, A450V, A450W, I486V, S482I, Y678I, G427F, D564T, Q684C, Q684G, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, F260W, Y530F, P607I, A450P, T242H, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, A633C, S312C, N455D, or L293F.

[0035] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two or all three of (a), (c), and (e) over wild type BGL1, wherein the BGL1 variant is F260A, S474R, D564T, T568E, A338D, F260E, T568K, or F260L.

[0036] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two or all three of (b), (c), and (e) over wild type BGL1, wherein the BGL1 variant is I567V, N566G, A630K, Y639K, Q245N, K320Y, A347Y, T568E, A655D, F260E, T568K, or F260L.

[0037] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two or all three of (a), (d), and (f) over wild type BGL1, wherein the BGL1 variant is N566F, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, A633C, S312C, or N455D.

[0038] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two or all three of (a), (b), and (f) over wild type BGL1, wherein the BGL1 variant is N566H, F556V, P604Y, L293V, A630G, N461C, N463T, D457C, Q220M, T221A, T221G, T221I, A655R, A468F, A468S, Q216I, D564V, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, N461V, I671C, K206A, E170F, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, A633C, S312C, or N455D.

[0039] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two or all three of (a), (b), and (c) over wild type BGL1, wherein the BGL1 variant is A565C, N461V, I671C, K206A, T568E, F260E, T568K, or F260L.

[0040] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two or all three of (b), (d), and (e) over wild type BGL1, wherein the BGL1 variant is P536G, P607Q, A655Q, F260D, F260G, F260Q, P607G, N400S, P607I, A450P, T242H, A630Y, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, F260L, or L293F.

[0041] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two or all three of (b), (e), and (f) over wild type BGL1, wherein the BGL1 variant is P536Q, N369E, N369W, N369Y, P607I, A450P, T242H, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, or L293F.

[0042] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two or all three of (c), (e), and (f) over wild type BGL1, wherein the BGL1 variant is A601D, F260E or T568K.

[0043] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two or all three of (a), (d), and (g) over wild type BGL1, wherein the BGL1 variant is L293M, Q220P, Q406D, G605C, N263T, S308E, A633C, S312C, or N455D.

[0044] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two or all three of (d), (e), and (f) over wild type BGL1, wherein the BGL1 variant is Y575A, Y575K, P607I, A450P, T242H, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, or L293F.

[0045] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two or all three of (d), (e), and (g) over wild type BGL1, wherein the BGL1 variant is A630H, V466T, S308E, A630Y, or L293F.

[0046] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two or all three of (b), (e), and (g) over wild type BGL1, wherein the BGL1 variant is N146A, N146Q, P607K, N369T, P607F, A630Y, A655D, or L293F.

[0047] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two or all three of (c), (e), and (g) over wild type BGL1, wherein the BGL1 variant is S384E, L181M, V043A, V043G, V043N, Q060D, A655Y, T242S, S474D, or A655D.

[0048] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two or all three of (b), (f), and (g) over wild type BGL1, wherein the BGL1 variant is T436E, F260W, E170F, S507G, L293F, A633C, S312C, or N455D.

[0049] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two or all three of (b), (c), and (g) over wild type BGL1, wherein the BGL1 variant is Y639G, P607F, A655D, or S507G.

[0050] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two or all three of (a), (b), and (e) over wild type BGL1, wherein the BGL1 variant is A655N, I671K, F260T, P607S, F260D, F260G, F260Q, P607G, N400S, P607F, T568E, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, or F260L.

[0051] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two or all three of (a), (c), and (g) over wild type BGL1, wherein the BGL1 variant is K206S or P607F. In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two or all three of (b), (d), and (g) over wild type BGL1, wherein the BGL1 variant is Y530S, Q684N, F260W, Q406D, G605C, N263T, A630Y, L293F, A633C, S312C, or N455D. In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two or all three of (a), (f), and (g) over wild type BGL1, wherein the BGL1 variant is A468T, E170F, A633C, S312C, or N455D. In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two or all three of (a), (d), and (e) over wild type BGL1, wherein the BGL1 variant is S692L, F260D, F260G, F260Q, P607G, N400S, S308E, A338D, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, or F260L.

[0052] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two or all three of (a), (b), and (g) over wild type BGL1, wherein the BGL1 variant is Y639V.

[0053] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two, any three, or all four of (a), (b), (d) and (f) activities over wild type BGL1, wherein the BGL1 variant is A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, or S683W.

[0054] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two, any three, or all four of (a), (b), (d) and (e) over wild type BGL1, wherein the BGL1 variant is F260D, F260G, F260Q, P607G, or N400S. In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two, any three, or all four of (b), (d), (f) and (g) over wild type BGL1, wherein the BGL1 variant is F260W, L293F, A633C, S312C, or N455D.

[0055] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two, any three, or all four of (b), (c), (d) and (f) over wild type BGL1, wherein the BGL1 variant is Y530F.

[0056] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two, any three, or all four of (a), (b), (e) and (g) over wild type BGL1, wherein the BGL1 variant is P607F.

[0057] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two, any three, or all four of (a), (b), (d) and (g) over wild type BGL1, wherein the BGL1 variant is Q406D, G605C, N263T, A633C, S312C, or N455D. In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two, any three, or all four of (a), (b), (c) and (f) over wild type BGL1, wherein the BGL1 variant is N461V, I671C, K206A, F260E or T568K. In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two, any three, or all four of (b), (d), (e) and (f) over wild type BGL1, wherein the BGL1 variant is P607I, A450P, T242H, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, or L293F. In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two, any three, or all four of (a), (b), (c) and (e) over wild type BGL1, wherein the BGL1 variant is T568E, F260E, T568K, or F260L.

[0058] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two, any three, or all four of (a), (d), (e) and (g) over wild type BGL1, wherein the BGL1 variant is S308E.

[0059] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two, any three, or all four of (b), (d), (e) and (g) over wild type BGL1, wherein the BGL1 variant is A630Y or L293F. In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two, any three, or all four of (b), (c), (e) and (g) over wild type BGL1, wherein the BGL1 variant is A655D.

[0060] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two, any three, or all four of (a), (b), (f) and (g) over wild type BGL1, wherein the BGL1 variant is E170F, A633C, S312C, or N455D. In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two, any three, or all four of (a), (c), (d) and (e) over wild type BGL1, wherein the BGL1 variant is A338D or F260L. In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two, any three, or all four of (b), (c), (f) and (g) over wild type BGL1, wherein the BGL1 variant is S507G.

[0061] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two, any three, any four, or all five of (a), (b), (c), (e) and (f) over wild type BGL1, wherein the BGL1 variant is F260E or T568K. In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two, any three, any four, or all five of (a), (b), (d), (e) and (f) over wild type BGL1, wherein the BGL1 variant is P536C, A630Q, D215S, G372A, G547A, F611A, G662C, or G662F. In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two, any three, any four, or all five of (a), (b), (c), (d) and (e) over wild type BGL1, wherein the BGL1 variant is F260L. In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two, any three, any four, or all five of (b), (d), (e), (f) and (g) over wild type BGL1, wherein the BGL1 variant is L293F. In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected from any two, any three, any four, or all five of (a), (b), (d), (f) and (g) over wild type BGL1, wherein the BGL1 variant is A633C, S312C, or N455D.

[0062] The invention also provides for BGL1 variants having at least two improved activities over wild type BGL1 selected from the group consisting of: (a) pre-treated corn stover (PCS) hydrolysis activity, (b) cellobiase activity, (c) protein expression, (d) beta-glucosidase activity as measured by an ammonia pretreated corncob (CC) hydrolysis activity, (e) thermostability, (f) phosphoric acid swollen cellulose (PASC) hydrolysis activity, and (g) hydrolytic activity in the presence of glucose, wherein the BGL1 variant comprises two or more substitutions from selected from those listed in Table 5-1.

[0063] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are (a) and (c) and the substitutions are: L167W|D225Q, T242S|S312Y, D178K|A338K|S474D|G662L, K345E|N369T|G372A|K428N|P661L|S683W, D177M|D225Q|D564V|Q684G, and D178N|N264K|A338D|S474R|G662K, D177M|D564T|Q626F|Q684A, K428N|S683W, K345E|K428N|S683W, Q226Y|G372A|V603G|T666C, L167W|D177M|Q626F, L167W|D177M|D225Q|D564V|Q684G, D177M|D225Q|D564T|Q684N, D177M|Q626F|Q684R, N238W|R265P|K656R, N264M|R265P (optionally also G662F), N264L|A338I|S474R|G662D, L167W|D225Q|D564V|Q626F|Q684N, D177M|D225Q|D564T|Q684A, D177M|D225Q|D564V|Q626F|Q684N, K345E|N369E|G372A|P661E, N369T|P661L|S683W, R265M|K560S, N369T|G372A|P661L|S683W, P176L|Q226W|K320Y|R363E, K345E|N369T|G372A|P661E|S683W, K320S|R363E, E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C, T242S|T666C, Q226Y|T666C, Q216E|S312K|S692K, P176L|G662F, P176L|Q226W|Q316T|K320Y|R363E, P176L|Q226W|K320S|R363E|G662F, P176L|Q226W|Q316T|K320Y|V522Y, P176L|Q226W|K320Y|R363E|V522Y, Q226W|K320Y|R363E, Q316T|K320Y|R363E|G662F, Q226W|Q316T|R363E|V522Y|G662F, P176L|K320S|R363E|G662C, R363E|G547A|G662C, Q226W|K320S|G662C, P176L|Q226W|Q316T|K320Y|R363E|G662F, P176L|Q226W|Q316T|K320S|G662F, P176L|Q316T|K320S|R363E|V522Y|G662C, K320Y|R363E|G662C, K345E|N369E|P661L, E170F|V603G, K345E|N369E|G372A|S683W, N369E|S683W, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0064] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are (b) and (f) and the substitutions are: L167W|D177M|D225Q|Q626F|Q684G, L167W|D177M|D564V|Q684G, D215S|S312Y, E170F|S312Y|N369Y, L167W|D225Q|Q626F|Q684R, L167W|D564T|Q626F, P176L|Q226W|Q316T|K320S|V522Y|G662C, R363E|V522Y|G662F, Q316T|K320S|V522Y|G662F, Q226W|K320Y|V522Y, Q316T|K320S|V522Y, and Q226W|K320S|R363E|V522Y|G662F, L167W|D177M|D225Q|D564V, D177M|D225Q|D564T|Q684A, D177M|D225Q|D564V|Q626F|Q684N, L167W|D177M|Q626F|Q684G, L167W|D177M|D564V|Q626F|Q684A, P176L|K320S|V522Y|G662C, K345E|N369T|G372A|P661E|S683W, K320S|R363E, E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C, T242S|T666C, Q226Y|T666C, Q216E|S312K|S692K, P176L|G662F, P176L|Q226W|Q316T|K320Y|R363E, P176L|Q226W|K320S|R363E|G662F, P176L|Q226W|Q316T|K320Y|V522Y, P176L|Q226W|K320Y|R363E|V522Y, Q226W|K320Y|R363E, Q316T|K320Y|R363E|G662F, Q226W|Q316T|R363E|V522Y|G662F, P176L|K320S|R363E|G662C, R363E|G547A|G662C, Q226W|K320S|G662C, P176L|Q226W|Q316T|K320Y|R363E|G662F, P176L|Q226W|Q316T|K320S|G662F, P176L|Q316T|K320S|R363E|V522Y|G662C, K320Y|R363E|G662C, E170F|Q226Y|N369Y|G372A|P661F, and L167W|D177M|D564T|Q626F|Q684G, K345E|N369E|G372A|S683W, N369E|S683W, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0065] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are (e) and (g) and the substitutions are: L167W|D225Q|Q626F|Q684D, L167W|D225Q|Q684N, L167W|D225Q|D564T|, Q626F|Q684C, Q626F|Q684D, N264M|R265P|N369I|D370W, R179V|N238F|D370W, R179V|N238F|K656R, R179V|N264M|D370W, R179V|N238F|R265M, R179V|R265P|D370W|K656R, R179V|N238W|N264M|R265M|N369I, R179V|N369I|D370W|K656R, R179V|N264M|R265P|K656R, R179V|R265M|N369I, R179V|N264M|R265M|D370W|K656R, R179V|N264M|R265M|N369I, R179V|N238W|N264M, N238W|N264M|R265M|D370W, R179V|N238W|R265P|D370W, R179V|N238W|N264M|D370W|K656R, N264M|R265P, R265P|D370W (optionally also G662F), R179V|N264M|R265P|N369I|D370W, R265M|N369I, R179V|R265M|D370W, N238W|N264M|R265P, R179V|N238W|N264M|R265P, N264M|N369I, N238F|R265M|N369I, N263C|K345E|N369E|G372A|K428N|P661E|S683W, N263C|K345E|N369T|G372A|K428N|P661E|S683W, N263C|K345E|N369E|G372A, N263C|P661L|S683W, N263C|K345E|N369T|G372A|K428N, K345E|G372A|K428N|P661E, E170F|Q226Y|N369Y|G372A, Q226Y|T242S|G372A|P661F, Q216E|T282I|S312D|S692K, Q216I|T282K|S312K|A622K, P176L|Q316T|G662C, Q226W|Q316T|V522Y|G662F, P176L|Q316T, A347Y|R542N, N238F|N264M|R265M|N369I, L167W|D225Q|D564V|Q626F|Q684N, E170F|V603G, L167W|D177M|D564T|Q684N, K345E|N369E|G372A|S683W, N369E|S683W, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0066] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are (d) and (f) and the substitutions are: L167W|D177M|D564V|Q684R, L167W|D225Q|D564V, D177M|D225Q|D564T|Q626F|Q684N, L167W|Q626F, D225Q|D564V|Q626F|Q684R, D177M|D225Q|D564V|Q684R, Q226W|K320Y, P176L|V522Y, R363E|G662C, L167W|D225Q|Q626F|Q684R, L167W|D564T|Q626F, P176L|Q226W|Q316T|K320S|V522Y|G662C, R363E|V522Y|G662F, Q316T|K320S|V522Y|G662F, Q226W|K320Y|V522Y, Q316T|K320S|V522Y, Q226W|K320S|R363E|V522Y|G662F, L167W|D177M|D564T|Q626F|Q684N, L167W|Q626F|Q684D, L167W|D177M|D564T|Q684R, L167W|D177M|D225Q|Q684D, R179V|R265P|N369I, Q316T|K320Y|R363E|V522Y|G662F, L167W|D177M|Q626F, L167W|D177M|D225Q|D564V|Q684G, D177M|D225Q|D564T|Q684N, D177M|Q626F|Q684R, L167W|D177M|Q626F|Q684G, L167W|D177M|D564V|Q626F|Q684A, P176L|K320S|V522Y|G662C, K345E|N369T|G372A|P661E|S683W, K320S|R363E, E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C, T242S|T666C, Q226Y|T666C, Q216E|S312K|S692K, P176L|G662F, P176L|Q226W|Q316T|K320Y|R363E, P176L|Q226W|K320S|R363E|G662F, P176L|Q226W|Q316T|K320Y|V522Y, P176L|Q226W|K320Y|R363E|V522Y, Q226W|K320Y|R363E, Q316T|K320Y|R363E|G662F, Q226W|Q316T|R363E|V522Y|G662F, P176L|K320S|R363E|G662C, R363E|G547A|G662C, Q226W|K320S|G662C, P176L|Q226W|Q316T|K320Y|R363E|G662F, P176L|Q226W|Q316T|K320S|G662F, P176L|Q316T|K320S|R363E|V522Y|G662C, K320Y|R363E|G662C, E170F|Q226Y|N369Y|G372A|P661F, L167W|D177M|D564T|Q626F|Q684G, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0067] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are (b) and (e) and the substitutions are: K345E|N369E|K428N|P661L, Q316T|K320Y|V522Y, N369T|G372A|P661L|S683W, P176L|Q226W|K320Y|R363E, P176L|K320S|V522Y|G662C, K345E|N369E|P661L, L167W|D177M|D564T|Q684N, K345E|N369E|G372A|S683W, N369E|S683W, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0068] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are (d) and (e) and the substitutions are: N263C|K345E|N369E|P661L, N238F|N264M|R265M|N369I, P176L|K320S|V522Y|G662C, K345E|N369E|P661L, E170F|V603G, L167W|D177M|D564T|Q684N, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0069] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are (b) and (g) and the substitutions are: E170F|T242S|N369Y|G372A|V603G|T666C, E170F|Q226Y|N369Y|V603G|T666C, E170F|Q226Y|S312Y, L167W|D177M|D225Q|D564V, L167W|D177M|Q626F|Q684G, L167W|D177M|D564V|Q626F|Q684A, E170F|Q226Y|N369Y|G372A|P661F, L167W|D177M|D564T|Q626F|Q684G, L167W|D177M|D564T|Q684N, K345E|N369E|G372A|S683W, N369E|S683W, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0070] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are (d) and (g) and the substitutions are: D178I|Q303E1A338I, Q316T|K320Y|G662F, L167W D177M|D564T|Q626F|Q684N, L167W|Q626F|Q684D, L167W|D177M|D564T|Q684R, L167W|D177M|D225Q|Q684D, R179V|R265P|N369I, Q316T|K320Y|R363E|V522Y|G662F, N238F|N264M|R265M|N369I, N238W|R265P|K656R, N264M|R265P, (optionally also G662F), N264L|A338I|S474R|G662D, L167W|D177M|Q626F|Q684G, and L167W|D177M|D564V|Q626F|Q684A, E170F|V603G, E170F|Q226Y|N369Y|G372A|P661F, L167W|D177M|D564T|Q626F|Q684G, L167W|D177M|D564T|Q684N, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0071] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two or all three of (b), (d), and (f) and the substitutions are: L167W|D225Q|Q626F|Q684R, L167W|D564T|Q626F, P176L|Q226W|Q316T|K320S|V522Y|G662C, R363E|V522Y|G662F, Q316T|K320S|V522Y|G662F, Q226W|K320Y|V522Y, Q316T|K320S|V522Y, Q226W|K320S|R363E|V522Y|G662F, L167W|D177M|Q626F|Q684G, L167W|D177M|D564V|Q626F|Q684A, P176L|K320S|V522Y|G662C, K345E|N369T|G372A|P661E|S683W, K320S|R363E, E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C, T242S|T666C, Q226Y|T666C, Q216E|S312K|S692K, P176L|G662F, P176L|Q226W|Q316T|K320Y|R363E, P176L|Q226W|K320S|R363E|G662F, P176L|Q226W|Q316T|K320Y|V522Y, P176L|Q226W|K320Y|R363E|V522Y, Q226W|K320Y|R363E, Q316T|K320Y|R363E|G662F, Q226W|Q316T|R363E|V522Y|G662F, P176L|K320S|R363E|G662C, R363E|G547A|G662C, Q226W|K320S|G662C, P176L|Q226W|Q316T|K320Y|R363E|G662F, P176L|Q226W|Q316T|K320S|G662F, P176L|Q316T|K320S|R363E|V522Y|G662C, K320Y|R363E|G662C, E170F|Q226Y|N369Y|G372A|P661F, L167W|D177M|D564T|Q626F|Q684G, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0072] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two or all three of (d), (f), and (g) and the substitutions are: L167W|D177M|D564T|Q626F|Q684N, L167W|Q626F|Q684D, L167W|D177M|D564T|Q684R, L167W|D177M|D225Q|Q684D, R179V|R265P|N369I, Q316T|K320Y|R363E|V522Y|G662F, L167W|D177M|Q626F|Q684G, L167W|D177M|D564V|Q626F|Q684A, E170F|Q226Y|N369Y|G372A|P661F, L167W|D177M|D564T|Q626F|Q684G, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0073] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two or all three of (a), (c), and (e) and the substitutions are: K345E|N369T|G372A|K428N|P661L|S683W, L167W|D225Q|D564V|Q626F|Q684N, N369T|G372A|P661L|S683W, P176L|Q226W|K320Y|R363E, K345E|N369E|P661L, E170F|V603G, K345E|N369E|G372A|S683W, N369E|S683W, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0074] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two or all three of (b), (f), and (g) and the substitutions are: L167W|D177M|D225Q|D564V, L167W|D177M|Q626F|Q684G, L167W|D177M|D564V|Q626F|Q684A, E170F|Q226Y|N369Y|G372A|P661F, L167W|D177M|D564T|Q626F|Q684G, K345E|N369E|G372A|S683W, N369E|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0075] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two or all three of (a), (c), and (d) and the substitutions are: D177M|D225Q|D564V|Q684G, D178N|N264K|A338D|S474R|G662K, L167W|D177M|Q626F, L167W|D177M|D225Q|D564V|Q684G, D177M|D225Q|D564T|Q684N, D177M|Q626F|Q684R, N238W|R265P|K656R, N264M|R265P (optionally also G662F), N264L|A338I|S474R|G662D, K345E|N369E|G372A|P661E, N369T|P661L|S683W, K345E|N369T|G372A|P661E|S683W, K320S|R363E, E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C, T242S|T666C, Q226Y|T666C, Q216E|S312K|S692K, P176L|G662F, P176L|Q226W|Q316T|K320Y|R363E, P176L|Q226W|K320S|R363E|G662F, P176L|Q226W|Q316T|K320Y|V522Y, P176L|Q226W|K320Y|R363E|V522Y, Q226W|K320Y|R363E, Q316T|K320Y|R363E|G662F, Q226W|Q316T|R363E|V522Y|G662F, P176L|K320S|R363E|G662C, R363E|G547A|G662C, Q226W|K320S|G662C, P176L|Q226W|Q316T|K320Y|R363E|G662F, P176L|Q226W|Q316T|K320S|G662F, P176L|Q316T|K320S|R363E|V522Y|G662C, K320Y|R363E|G662C, K345E|N369E|P661L, E170F|V603G, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0076] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two or all three of (a), (c), and (g) and the substitutions are: D177M|D564T|Q626F|Q684A, N238W|R265P|K656R, N264M|R265P (optionally also G662F), N264L|A338I|S474R|G662D, |D225Q|D564V|Q626F|Q684N, R265M|K560S, E170F|V603G, K345E|N369E|G372A|S683W, N369E|S683W, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0077] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two or all three of (d), (e), and (g) and the substitutions are: N238F|N264M|R265M|N369I, E170F|V603G, L167W|D177M|D564T|Q684N, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0078] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two or all three of (a), (b), and (c) and the substitutions are: K428N|S683W, K345E|K428N|S683W, Q226Y|G372A|V603G|T666C, D177M|D225Q|D564T|Q684A, D177M|D225Q|D564V|Q626F|Q684N, K345E|N369E|G372A|P661E, N369T|P661L|S683W, N369T|G372A|P661L|S683W, P176L|Q226W|K320Y|R363E, K345E|N369T|G372A|P661E|S683W, K320S|R363E, E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C, T242S|T666C, Q226Y|T666C, Q216E|S312K|S692K, P176L|G662F, P176L|Q226W|Q316T|K320Y|R363E, P176L|Q226W|K320S|R363E|G662F, P176L|Q226W|Q316T|K320Y|V522Y, P176L|Q226W|K320Y|R363E|V522Y, Q226W|K320Y|R363E, Q316T|K320Y|R363E|G662F, Q226W|Q316T|R363E|V522Y|G662F, P176L|K320S|R363E|G662C, R363E|G547A|G662C, Q226W|K320S|G662C, P176L|Q226W|Q316T|K320Y|R363E|G662F, P176L|Q226W|Q316T|K320S|G662F, P176L|Q316T|K320S|R363E|V522Y|G662C, K320Y|R363E|G662C, K345E|N369E|P661L, K345E|N369E|G372A|S683W, N369E|S683W, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0079] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three or all four of (a), (c), (d), and (f) and the substitutions are: L167W|D177M|Q626F, L167W|D177M|D225Q|D564V|Q684G, D177M|D225Q|D564T|Q684N, D177M|Q626F|Q684R, K345E|N369T|G372A|P661E|S683W, K320S|R363E, E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C, T242S|T666C, Q226Y|T666C, Q216E|S312K|S692K, P176L|G662F, P176L|Q226W|Q316T|K320Y|R363E, P176L|Q226W|K320S|R363E|G662F, P176L|Q226W|Q316T|K320Y|V522Y, P176L|Q226W|K320Y|R363E|V522Y, Q226W|K320Y|R363E, Q316T|K320Y|R363E|G662F, Q226W|Q316T|R363E|V522Y|G662F, P176L|K320S|R363E|G662C, R363E|G547A|G662C, Q226W|K320S|G662C, P176L|Q226W|Q316T|K320Y|R363E|G662F, P176L|Q226W|Q316T|K320S|G662F, P176L|Q316T|K320S|R363E|V522Y|G662C, K320Y|R363E|G662C, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0080] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are (a), (c), (d), and (g) and the substitutions are: N238W|R265P|K656R, N264M|R265P (optionally also G662F), N264L|A338I|S474R|G662DE170F|V603G, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0081] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three or all four of (a), (c), (e), and (g) and the substitutions are: L167W|D225Q|D564V|Q626F|Q684N, E170F|V603G, K345E|N369E|G372A|S683W, N369E|S683W, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0082] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three or all four of (a), (b), (c), and (f) and the substitutions are: D177M|D225Q|D564T|Q684A, D177M|D225Q|D564V|Q626F|Q684N, K345E|N369T|G372A|P661E|S683W, K320S|R363E, E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C, T242S|T666C, Q226Y|T666C, Q216E|S312K|S692K, P176L|G662F, P176L|Q226W|Q316T|K320Y|R363E, P176L|Q226W|K320S|R363E|G662F, P176L|Q226W|Q316T|K320Y|V522Y, P176L|Q226W|K320Y|R363E|V522Y, Q226W|K320Y|R363E, Q316T|K320Y|R363E|G662F, Q226W|Q316T|R363E|V522Y|G662F, P176L|K320S|R363E|G662C, R363E|G547A|G662C, Q226W|K320S|G662C, P176L|Q226W|Q316T|K320Y|R363E|G662F, P176L|Q226W|Q316T|K320S|G662F, P176L|Q316T|K320S|R363E|V522Y|G662C, K320Y|R363E|G662C, K345E|N369E|G372A|S683W, N369E|S683W, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0083] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three or all four of (a), (b), (c), and (d) and the substitutions are: K345E|N369E|G372A|P661E, N369T|P661L|S683W, K345E|N369T|G372A|P661E|S683W, K320S|R363E, E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C, T242S|T666C, Q226Y|T666C, Q216E|S312K|S692K, P176L|G662F, P176L|Q226W|Q316T|K320Y|R363E, P176L|Q226W|K320S|R363E|G662F, P176L|Q226W|Q316T|K320Y|V522Y, P176L|Q226W|K320Y|R363E|V522Y, Q226W|K320Y|R363E, Q316T|K320Y|R363E|G662F, Q226W|Q316T|R363E|V522Y|G662F, P176L|K320S|R363E|G662C, R363E|G547A|G662C, Q226W|K320S|G662C, P176L|Q226W|Q316T|K320Y|R363E|G662F, P176L|Q226W|Q316T|K320S|G662F, P176L|Q316T|K320S|R363E|V522Y|G662C, K320Y|R363E|G662C, K345E|N369E|P661L, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0084] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three or all four of (b), (d), (f), and (g) and the substitutions are: L167W|D177M|Q626F|Q684G, L167W|D177M|D564V|Q626F|Q684A, E170F|Q226Y|N369Y|G372A|P661F, L167W|D177M|D564T|Q626F|Q684G, K345E|N369E|G372A|S683W, N369E|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0085] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three or all four of (a), (c), (f), and (g) and the substitutions are: R265M|K560S, K345E|N369E|G372A|S683W, N369E|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0086] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three or all four of (a), (b), (c), and (e) and the substitutions are: N369T|G372A|P661L|S683W, P176L|Q226W|K320Y|R363E, K345E|N369E|P661L, K345E|N369E|G372A|S683W, N369E|S683W, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0087] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three or all four of (b), (d), (e), and (f) and the substitutions are: P176L|K320S|V522Y|G662C, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0088] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, any four, or all five of (a), (b), (c), (d), and (f) and the substitutions are: K345E|N369T|G372A|P661E|S683W, K320S|R363E, E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C, T242S|T666C, Q226Y|T666C, Q216E|S312K|S692K, P176L|G662F, P176L|Q226W|Q316T|K320Y|R363E, P176L|Q226W|K320S|R363E|G662F, P176L|Q226W|Q316T|K320Y|V522Y, P176L|Q226W|K320Y|R363E|V522Y, Q226W|K320Y|R363E, Q316T|K320Y|R363E|G662F, Q226W|Q316T|R363E|V522Y|G662F, P176L|K320S|R363E|G662C, R363E|G547A|G662C, Q226W|K320S|G662C, P176L|Q226W|Q316T|K320Y|R363E|G662F, P176L|Q226W|Q316T|K320S|G662F, P176L|Q316T|K320S|R363E|V522Y|G662C, K320Y|R363E|G662C, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0089] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, any four, or all five of (a), (b), (c), (d) and (e) and the substitutions are: K345E|N369E|P661L, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0090] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, any four, or all five of (a), (c), (d), (e) and (g) and the substitutions are: E170F|V603G, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, and P176L|Q226W|G547A|G662C.

[0091] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, any four, or all five of (a), (b), (d), (f) and (g) and the substitutions are: E170F|Q226Y|N369Y|G372A|P661F, L167W|D177M|D564T|Q626F|Q684G, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0092] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, any four, or all five of (a), (b), (d), (e) and (g) and the substitutions are: L167W|D177M|D564T|Q684N, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0093] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, any four, any five or all six of (a), (b), (c), (e), (f) and (g) and the substitutions are: K345E|N369E|G372A|S683W, N369E|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0094] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, any four, any five or all six of (a), (b), (c), (d), (e) and (g) and the substitutions are: G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0095] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, any four, any five or all six of (a), (b), (c), (d), (e) and (f) and the substitutions are: K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0096] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, any four, any five, any six or all seven of (a), (b), (c), (d), (e), (f) and (g) and the substitutions are: N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0097] The present disclosure provides a beta-glucosidase variant, wherein the variant is a mature form having beta-glucosidase activity and comprising a mutation, wherein when the mutation is a single mutation it is not at a position selected from the group consisting of: 37, 61, 125, 129, 132, 133, 158, 159, 166, 177, 236, 237, 238, 240, 252, 314, 444, and 449, and wherein the positions are numbered by correspondence with the amino acid sequence of a reference beta-glucosidase 1 (BGL1) set forth as SEQ ID NO:3. In some embodiments, the mutation is a substitution. In other embodiments, the mutation is a deletion or an insertion. In some preferred embodiments, the mutation does not consist of a substitution selected from the group consisting of: W37A, W37G, W37N, W37D, D61N, D61A, R125K, R125A, K158G, H159A, H159S, E166Q, D177E, D236G, D236N, D236E, W237F, W237A, W237L, W237C, and W237P. In some preferred embodiments, the substitution results in a beta-glucosidase variant with improvements in one or more of expression, activity and stability, in comparison to the reference BGL1.

[0098] In addition, the present disclosure provides a beta-glucosidase variant, wherein the variant is a mature form having beta-glucosidase activity and comprising a substitution at one or more positions selected from the group consisting of: 22, 24, 25, 26, 27, 28, 33, 35, 36, 37, 50, 51, 52, 61, 67, 91, 92, 93, 99, 100, 125, 158, 159, 163, 164, 165, 166, 167, 168, 169, 170, 176, 177, 178, 179, 194, 196, 199, 204, 208, 209, 214, 215, 216, 224, 225, 226, 236, 237, 238, 242, 248, 249, 263, 264, 265, 276, 277, 278, 279, 282, 284, 287, 291, 301, 302, 303, 306, 312, 313, 316, 320, 324, 328, 329, 334, 335, 336, 337, 338, 339, 344, 345, 347, 361, 363, 369, 370, 371, 372, 374, 375, 380, 381, 382, 396, 397, 398, 399, 402, 409, 410, 411, 420, 426, 427, 428, 441, 445, 446, 447, 448, 449, 452, 453, 454, 455, 460, 467, 473, 474, 475, 489, 490, 492, 496, 497, 498, 521, 522, 534, 542, 547, 548, 553, 554, 555, 560, 561, 563, 564, 570, 571, 581, 583, 586, 591, 603, 611, 612, 622, 626, 627, 638, 642, 643, 645, 649, 650, 656, 660, 661, 662, 663, 666, 672, 673, 674, 675, 680, 681, 682, 683, 684, 685, 692, 702, and 705, wherein the positions are numbered by correspondence with the amino acid sequence of a reference beta-glucosidase 1 (BGL1) set forth as SEQ ID NO:3. In some embodiments, the variant comprises a further substitution at one or more positions selected from the group consisting of: 37, 61, 158, 159, 166, 236, 237, 238, and 449, wherein the positions are numbered by correspondence with the amino acid sequence of a reference beta-glucosidase 1 (BGL1) set forth as SEQ ID NO:3. In some embodiments, the further substitution is selected from the group consisting of: W037A, D061N, K158G, H159A or S, E166Q, D236G, and W237P. Moreover, in some embodiments, the substitution at one or more positions is selected from the group consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25 positions. In some embodiments, the variant is derived from a parent beta-glucosidase selected from the group consisting of Hypocrea jecorina BGL1 (TrireBGL1), Hansenula anomala BGL (HananBglu), Piromyces sp BGL (PirspBglu), Coccidioides immitis BGL (CocimBglu), Saccharomycopsis fibuligera BGL2 (SacfiBglu2), Saccharomycopsis fibuligera BGL1 (SacfiBglu1), Septoria lycopersici BGL (SeplyBglu), Kuraishia capsulata BGL (KurcaBglu), Trichoderma reesei BGL7 (TrireBGL7), Uromyces fabae BGL (UrofaBglu), Aspergillus terreus BGL (AspteBglu), Chaetomium globosum BGL (ChaglBglu), Trichoderma reesei BGL3 (TrireBGL3), Penicillium brasilianum BGL (PenbrBGL), Periconia sp. BGL (PerspBglu), Phaeosphaeria avenaria BGL (PhaavBglu), Aspergillus fumigatus BGL (AspfuBGL), Aspergillus oryzae BGL 1 (AsporBGL1), Aspergillus aculeatus BGL1 (AspacBGL1), Aspergillus niger BGL (AspniBGL), Talaromyces emersonii BGL (TalemBglu), and Thermoascus aurentiacus BGL (TheauBGL). In some preferred embodiments, the variant is derived from a parent beta-glucosidase whose amino acid sequence is at least 75% (80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) identical to a member of the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24. In some preferred embodiments, the variant comprises from one to fifty-nine of the conserved residues selected from the group consisting of A16, K28, G34, G44, C58, D61, R67, E100, G105, L110, P112, G124, R125, E128, D133, P134, L136, G147, Q149, K158, H159, R169, 5173, D178, P188, F189, M201, Y204, N208, K224, F229, G231, D236, W237, G250, D252, M253, M255, P256, R284, D287, R291, K335, N336, L341, P342, G385, P395, E441, D452, V478, L518, Y559, F562, F573, G574, G576, L577, and L651, wherein the positions are numbered by correspondence with the amino acid sequence of a reference beta-glucosidase 1 (BGL1) set forth as SEQ ID NO:3. In a subset of these embodiments, the variant comprises E441 and D452. In preferred embodiments, the substitution results in a beta-glucosidase variant with improvements in one or more of expression, activity and stability, when compared to the reference BGL1.

[0099] Also provided by the present disclosure is a beta-glucosidase variant comprising a substitution, wherein the substitution comprises one or more of the group consisting of: K022 to A, E, F, G, H, I, P, Q, R, S, V, W, or Y; N024 to A, C, D, E, F, G, K, L, M, P, Q, R, S, T, V, or Y; L025 to A, D, F, G, I, K, N, Q, R, S, T, V, W, or Y; Q026 to C, D, E, G, H, I, K, L, P, R, S, T, V, W, or Y; D027 to A, C, E, L, M, Q, S, T, or V; K028 to L, M, N, S, or V; 5033 to C, G, or T; V035 to C, E, G, H, K, L, N, P, Q, R, S, T, W, or Y; G036 to C, D, E, F, I, K, N, R, S, W, or Y; W037 to E, F, H, I, M, S, V, or Y; S050 to A, C, F, G, I, K, L, M, N, P, R, T, V, or Y; K051 to A, C, D, E, G, H, I, L, M, N, Q, R, S, T, or V, 1052 to A, D, F, K, M, N, P, Q, S, T, or V; D061 to E, G, or P; R067 to A, C, D, E, F, G, I, L, M, N, P, Q, S, T, V, W, or Y; R091 to A, C, D, E, F, G, H, I, K, L, N, Q, S, T, V, W, or Y; E092 to A, C, D, F, H, I, K, L, M, N, Q, R, T, V, or Y; R093 to A, C, D, E, F, G, H, K, L, M, Q, S, T, V, or W; E099 to A, D, F, I, K, M, N, W, or Y; E100 to A, G, I, K, L, M, N, Q, S, T, or Y; R125 to A, or D; K158 to A, C, H, or T; H159 to C, E, G, N, W, or Y; N163 to A, H, or S; E164 to G, or S; Q165 to C, D, F, G, H, I, K, L, M, N, R, S, T, V, W, or Y; E166 to D, F, K, L, N, P, R, S, T, or Y; L167 to A, C, D, E, F, G, M, N, Q, R, S, V, W, or Y; N168 to A, D, E, G, H, Q, R, T, or Y; R169 to A, C, D, E, F, H, K, Q, S, or T; E170 to A, D, F, I, K, L, M, P, V, W, or Y; P176 to A, D, E, F, G, H, K, L, M, Q, R, S, T, V, W, or Y; D177 to A, C, E, F, G, H, K, L, M, N, Q, R, V, W, or Y; D178 to A, C, E, K, N, P, Q, R, S, T, W, or Y; R179 to A, C, G, I, K, M, Q, S, T, V, or W; Q194 to A, C, E, F, G, H, K, L, M, R, T, W, or Y; N196 to E, G, H, L, M, P, Q, R, or T; S199 to A, G, N, T, or V; Y204 to A, E, F, G, H, I, K, M, P, Q, R, S, T, V, or W; N208 to K, or R; T209 to C, D, E, G, H, I, K, L, M, Q, R, S, V, W, or Y; E214 to A, C, D, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; D215 to A, C, E, F, G, H, L, M, N, Q, S, V, or W; Q216 to A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, W, or Y; K224 to H, R, or V; D225 to A, C, E, F, G, H, I, L, M, Q, S, T, V, W, or Y; Q226 to A, C, D, E, F, H, I, K, L, M, N, R, S, T, V, W, or Y; D236 to A, P, Q, S, or T; W237 to H, I, K, M, R, S, T, or Y; N238 to A, C, D, E, F, G, M, P, S, T, or W; T242 to A, C, E, F, G, H, I, K, L, M, N, Q, R, S, V, W, or Y; N248 to A, C, F, G, L, T, W, or Y; S249 to A, G, I, M, or V; N263 to A, C, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, or Y; N264 to A, C, D, E, G, H, K, L, M, Q, R, S, T, V, or Y; R265 to A, E, F, G, I, K, L, M, N, P, Q, S, T, V, or Y; N276 to A, C, F, K, M, or Q; S277 to A, C, D, E, F, G, H, I, M, N, P, Q, R, W, or Y; N278 to A, C, D, F, G, H, I, L, M, Q, R, S, T, V, W, or Y; Q279 to C, D, E, G, H, I, K, N, S, T, V, or Y; T282 to C, D, G, H, K, L, N, P, R, S, or V; R284 to H, M, or N; D287 to C, E, F, G, H, I, K, L, M, N, S, V, W, or Y; Q301 to A, E, G, K, L, N, R, S, T, or V; D302 to A, C, E, F, G, K, L, M, N, P, S, T, W, or Y; Q303 to A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; Y306 to A, C, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, or W; S312 to A, C, D, G, I, K, L, M, N, Q, R, T, V, W, or Y; R313 to A, C, D, E, G, K, L, N, S, V, or W; Q316 to A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; K320 to A, C, E, G, H, L, M, N, P, Q, R, S, T, or Y; R324 to C, D, E, F, H, I, K, L, M, Q, V, W, or Y; R328 to C, E, F, G, I, K, L, M, Q, S, T, V, or Y; D329 to A, E, F, G, H, M, N, Q, S, T, or Y; L334 to A, C, F, M, T, V, or W; K335 to A, D, F, G, H, I, L, M, N, R, S, T, V, or W; N336 to A, C, G, H, L, M, Q, R, S, T, V, or Y; D337 to A, C, E, G, H, K, L, M, N, R, S, T, V, W, or Y; A338 to C, D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; N339 to D, E, G, H, I, K, L, P, Q, R, V, or Y; K344 to D, E, F, G, I, L, M, N, P, Q, R, S, T, or V; K345 to A, D, E, F, G, H, N, P, Q, R, S, T, V, W, or Y; A347 to D, F, H, I, K, L, M, P, Q, R, S, or Y; H361 to A, C, D, E, G, K, L, M, N, P, S, T, or Y; R363 to A, C, E, G, K, L, M, N, Q, S, T, V, W, or Y; N369 to A, C, D, E, F, I, L, M, R, S, T, V, W, or Y; D370 to E, F, G, Q, S, W, or Y; K371 to A, D, F, G, H, L, N, Q, R, S, T, V, or W; G372 to A, C, D, E, K, L, M, N, S, T, V, W, or Y; D374 to A, C, F, G, I, L, M, N, Q, R, S, T, V, W, or Y; D375 to A, C, E, H, I, R, V, or W; M380 to E, F, G, I, L, N, Q, S, T, V, or Y; G381 to H; W382 to F, N, or Y; Y396 to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, or W; D397 to A, C, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; A398 to C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; I399 to A, C, D, E, F, G, L, M, Q, S, T, V, W, or Y; R402 to A, C, E, F, G, I, L, P, Q, S, V, W, or Y; Q409 to C, D, G, H, I, or V; V410 to A, C, F, G, H, I, L, N, R, S, T, W, or Y; T411 to D, E, F, G, H, I, K, L, N, Q, R, S, V, or Y; S420 to A, C, D, G, H, K, N, Q, T, V, or Y; R426 to A, E, F, I, K, L, M, N, P, Q, S, T, W, or Y; G427 to C, D, E, F, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; K428 to A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; E441 to A, C, D, or G; T445 to A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, V, or Y; V446 to A, C, K, Q, or R; E447 to A, K, L, N, S, V, W, or Y; G448 to A, C, D, E, F, H, K, L, M, N, Q, R, S, T, V, or Y; N449 to A, C, E, F, G, H, K, L, M, P, R, T, V, or W; D452 to N; R453 to A, E, L, M, Q, or S; N454 to A, F, G, K, L, M, R, S, T, or V; N455 to A, C, D, E, F, G, H, I, L, M, S, T, V, W, or Y; H460 to A, C, D, E, F, G, I, K, L, M, N, Q, R, S, W, or Y; Q467 to A, C, D, E, H, K, N, P, S, V, W, or Y; N473 to A, C, E, F, G, H, K, L, M, P, Q, R, S, T, V, or W; S474 to A, C, D, E, F, G, I, K, L, M, N, P, Q, R, T, V, or Y; N475 to I, K, L, M, P, Q, R, S, T, V, W, or Y; E489 to D, or N; Q490 to A, C, E, F, G, H, K, L, P, R, S, T, V, W, or Y; L492 to A, D, F, H, I, M, N, Q, R, T, W, or Y; Q496 to A, G, K, N, P, S, T, V, or W; V497 to A, C, I, M, N, or T; K498 to A, C, E, F, G, H, I, L, M, N, Q, R, S, T, V, or Y; D521 to A, C, E, F, G, H, I, K, L, M, P, R, S, T, V, W, or Y; V522 to A, C, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; K534 to C, D, E, F, G, H, I, N, Q, R, S, T, or V; R542 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; G547 to A, C, E, F, K, L, N, P, Q, R, T, V, or Y; S548 to C, E, F, H, I, L, M, N, Q, R, T, V, W, or Y; E553 to D, I, K, N, Q, W, or Y; G554 to A, C, D, F, H, K, L, M, Q, R, S, T, V, or W; L555 to A, C, D, E, F, G, H, I, K, M, N, P, Q, T, V, W, or Y; K560 to A, C, E, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; H561 to A, C, D, E, F, G, I, M, N, Q, S, T, V, or W; D563 to A, C, E, F, I, L, M, Q, R, S, T, V, W, or Y; D564 to A, C, E, F, G, K, L, M, N, Q, R, S, T, V, or Y; R570 to A, C, D, E, G, H, I, M, N, Q, S, T, or V; Y571 to H, M, N, R, or W; K581 to A, C, D, E, F, G, H, I, L, M, N, P, R, S, T, V, W, or Y; N583 to A, C, D, E, F, G, H, I, K, L, M, P, R, S, T, V, W, or Y; R586 to D, E, F, G, H, L, N, P, V, W, or Y; S591 to C, D, F, G, H, I, K, M, P, Q, or V; V603 to A, C, D, E, F, G, H, L, M, N, P, Q, R, S, T, W, or Y; F611 to A, C, D, G, I, K, L, M, N, R, S, T, V, W, or Y; Q612 to C, D, F, G, H, I, K, L, M, R, S, V, or W; A622 to D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; Q626 to E, F, G, H, I, L, M, T, or V; V627 to D, K, P, Q, R, S, or Y; T638 to A, D, E, F, G, I, K, L, M, P, Q, R, S, V, W, or Y; S642 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; A643 to C, E, F, G, H, K, L, M, N, Q, R, S, T, V, W, or Y; R645 to A, D, E, F, G, H, I, K, L, M, P, Q, S, T, V, W, or Y; K649 to A, C, F, I, L, M, N, Q, S, T, W, or Y; Q650 to A, C, D, E, F, G, H, I, K, L, M, N, R, T, V, or Y; K656 to R; T660 to C, D, E, F, G, H, I, K, M, N, P, Q, R, S, V, W, or Y; P661 to A, C, D, E, F, G, H, I, K, L, M, Q, R, S, T, V, or W; G662 to A, C, D, E, F, H, I, K, L, M, N, Q, R, S, T, W, or Y; Q663 to A, C, D, E, F, G, H, I, K, L, M, N, R, S, V, or W; T666 to A, C, D, E, F, G, H, K, L, N, R, S, V, W, or Y; R672 to C, D, E, F, G, H, I, K, L, M, N, T, V, W, or Y; R673 to A, C, E, F, G, H, I, K, L, M, N, Q, S, T, V, or W; R674 to K, L, M, Q, T, V, or Y; D675 to C, E, H, L, S, or Y; D680 to A, C, E, F, H, I, K, L, M, N, Q, R, S, V, W, or Y; T681 to A, G, H, K, L, M, N, P, Q, R, S, V, W, or Y; A682 to C, E, I, L, M, N, P, S, W, or Y; S683 to A, C, D, E, F, G, I, K, L, M, P, Q, R, V, or W; Q684 to A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, or T; K685 to A, E, F, G, I, L, M, N, Q, R, S, T, V, W, or Y; 5692 to C, E, H, I, K, L, M, N, P, Q, T, V, or W; R702 to C, D, F, G, H, I, K, L, M, N, Q, S, T, V, or W; and R705 to C, F, H, I, L, M, P, S, T, V, or W, wherein the positions are numbered by correspondence with the amino acid sequence of a reference beta-glucosidase 1 (BGL1) set forth as SEQ ID NO:3. As described in the experimental section, exemplary beta-glucosidase variants have a PI greater than_1 for at least one of the following properties: expression (HPLC), CNPGase activity, thermostability, reduced glucose inhibition, cellobiase activity at pH 5, cellobiase activity at pH 6, cellobiase activity in the presence of ammonium pretreated corncob, and hydrolysis of acid pretreated corn stover.

[0100] The present disclosure further provides a beta-glucosidase variant comprising a substitution, wherein the substitution comprises one or more of the group consisting of: K022A, K022E, K022S, K022W, N024A, N024D, N024E, N024L, N024P, L025W, V035S, V035W, W037E, W037F, W037H, W037S, W037Y, K051A, D061E, D061G, D061P, R067G, R067L, R067M, R067P, R067T, R067V, R067Y, R091I, R091T, R091Y, E092K, E092L, E092T, R125A, R125D, K158A, K158C, H159C, H159E, H159G, H159N, H159W, H159Y, N163A, N163H, N163S, L167W, R169A, R169C, R169D, R169E, R169K, E170A, E170K, E170L, E170P, E170W, E170Y, P176A, P176D, P176G, D177C, D177G, D177K, D177N, D178A, D178E, D178P, D178T, D178W, Q194A, Q194Y, S199A, Y204A, Y204E, Y204G, Y204H, Y204I, Y204K, Y204P, Y204Q, Y204R, Y204S, Y204T, Y204V, Y204W, Q216D, Q216E, Q216N, Q216R, D225C, Q226A, D236A, D236P, D236Q, D236S, D236T, W237H, W237I, W237K, W237M, W237R, W237S, W237T, T242S, N248A, N248C, S249A, N264D, N264E, N264H, N264L, N264R, N264S, N264V, N264Y, R265A, R265G, R265Y, S277A, S277D, N278A, N278D, T282D, T282N, T282R, T282V, Q303A, Q303E, Q303N, Y306A, Y306E, Y306F, Y306L, Y306W, S312A, S312D, S312G, S312I, S312N, S312R, R313D, R313E, Q316A, Q316D, Q316F, K320A, K320H, K320N, K320S, K320Y, K335L, K335S, K335T, A338D, A338E, A338G, A338N, A338R, A347Y, R363A, R363G, R363K, R363M, R363V, D370E, D370Q, K371A, K371H, D374A, Y396A, D397N, I399L, S420A, S420D, G427E, G427S, K428A, E441A, E441C, E441D, E441G, V446A, E447A, E447N, G448A, G448D, G448F, G448M, G448N, G448R, G448S, G448T, G448Y, N454A, N473S, S474D, S474G, S474K, S474N, S474R, S474T, S474V, S474Y, E489D, D521A, K534Q, R542A, R542D, G547E, G547L, G547P, S548E, S548F, S548H, S548L, K560H, N583D, R586D, V603L, V603M, V603Q, V603S, Q612D, Q612G, Q612K, Q612V, A622L, A622W, A622Y, Q626I, Q626L, Q626T, Q626V, T638D, S642D, A643M, K649A, or K649W; Q650D; G662D, G662E, G662L, G662S, or G662T; Q663D, or Q663G; T666A, R673N, R673W, S683K, Q684D, Q684F, Q684H, Q684K, Q684L, Q684M, Q684R, Q684S, and Q684T, wherein the substitution consists of no more than a single replacement at each of the positions, and wherein the positions are numbered by correspondence with the amino acid sequence of a reference beta-glucosidase 1 (BGL1) set forth as SEQ ID NO:3. As described in the experimental section, exemplary beta-glucosidase variants have improved expression levels (e.g., PI greater than_1).

[0101] Also, the present disclosure provides a beta-glucosidase variant comprising a substitution, wherein the substitution comprises one or more of the group consisting of: K022F, K022G, K022H, K022I, K022P, K022Q, K022R, K022V, K022Y, N024C, N024F, N024G, N024K, N024M, N024Q, N024R, N024S, N024T, N024V, N024Y, L025A, L025D, L025F, L025G, L025I, L025K, L025N, L025Q, L025R, L025S, L025T, L025V, L025Y, Q026C, Q026D, Q026E, Q026G, Q026H, Q026I, Q026K, Q026L, Q026P, Q026R, Q026S, Q026T, Q026V, Q026W, Q026Y, D027A, D027C, D027E, D027L, D027M, D027Q, D027S, D027T, D027V, K028L, K028M, K028S, K028V, S033C, S033G, S033T, V035C, V035E, V035G, V035H, V035K, V035L, V035N, V035P, V035Q, V035R, V035T, V035Y, G036C, G036D, G036E, G036K, G036N, G036R, G036S, W037M, W037V, S050A, S050C, S050F, S050G, S050I, S050K, S050L, S050M, S050N, S050P, S050R, S050T, S050V, S050Y, K051C, K051D, K051E, K051G, K051H, K051I, K051L, K051M, K051N, K051Q, K051R, K051S, K051T, K051V, I052A, I052F, I052M, I052P, I052S, I052T, I052V, R067A, R067C, R067D, R067E, R067F, R067I, R067N, R067Q, R067S, R067W, R091A, R091D, R091E, R091F, R091G, R091H, R091K, R091L, R091N, R091Q, R091S, R091V, R091W, E092A, E092C, E092D, E092F, E092H, E092I, E092M, E092N, E092Q, E092R, E092V, E092Y, R093A, R093C, R093D, R093E, R093F, R093H, R093K, R093L, R093M, R093Q, R093S, R093T, R093V, R093W, E099A, E099D, E099F, E099I, E099K, E099M, E099N, E099W, E099Y, E100A, E100G, E100I, E100K, E100L, E100M, E100N, E100Q, E100S, E100T, E100Y, K158H, K158T, E164G, E164S, Q165C, Q165D, Q165F, Q165G, Q165H, Q165I, Q165K, Q165L, Q165M, Q165N, Q165R, Q165S, Q165T, Q165V, Q165W, Q165Y, E166D, E166K, E166L, E166N, E166P, E166R, E166S, E166T, E166Y, L167A, L167C, L167D, L167E, L167F, L167G, L167M, L167N, L167Q, L167R, L167S, L167V, L167Y, N168A, N168D, N168E, N168G, N168H, N168Q, N168R, N168T, N168Y, R169F, R169H, R169Q, R169S, R169T, E170D, E170F, E170I, E170M, E170V, P176E, P176F, P176H, P176K, P176L, P176M, P176Q, P176R, P176S, P176T, P176V, P176W, P176Y, D177A, D177E, D177F, D177H, D177L, D177M, D177Q, D177R, D177V, D177W, D177Y, D178C, D178K, D178N, D178Q, D178R, D178S, D178Y, R179A, R179C, R179G, R179I, R179K, R179S, R179T, R179V, R179W, Q194C, Q194E, Q194F, Q194G, Q194H, Q194K, Q194L, Q194M, Q194R, Q194T, Q194W, N196E, N196G, N196H, N196L, N196M, N196P, N196Q, N196R, N196T, S199G, S199N, S199T, S199V, Y204F, Y204M, N208K, N208R, T209C, T209D, T209E, T209G, T209H, T209I, T209K, T209L, T209M, T209Q, T209R, T209S, T209V, T209W, T209Y, E214A, E214C, E214D, E214G, E214H, E214K, E214L, E214M, E214N, E214P, E214Q, E214R, E214S, E214T, E214V, E214Y, D215A, D215C, D215E, D215F, D215G, D215H, D215L, D215M, D215N, D215Q, D215S, D215W, Q216A, Q216C, Q216F, Q216G, Q216H, Q216I, Q216K, Q216L, Q216M, Q216P, Q216S, Q216T, Q216W, Q216Y, K224H, K224R, K224V, D225A, D225E, D225F, D225G, D225H, D225I, D225L, D225M, D225Q, D225S, D225T, D225V, D225W, D225Y, Q226C, Q226D, Q226E, Q226F, Q226H, Q226I, Q226K, Q226L, Q226M, Q226N, Q226R, Q226S, Q226T, Q226V, Q226W, Q226Y, W237Y, N238A, N238C, N238D, N238E, N238F, N238G, N238M, N238P, N238S, N238T, N238W, T242A, T242C, T242E, T242F, T242G, T242H, T242I, T242K, T242L, T242M, T242N, T242Q, T242R, T242V, T242W, T242Y, N248F, N248G, N248L, N248T, N248W, N248Y, S249G, S249I, S249M, S249V, N263A, N263C, N263D, N263E, N263F, N263G, N263H, N263I, N263K, N263L, N263P, N263Q, N263R, N263S, N263T, N263V, N263Y, N264A, N264C, N264G, N264K, N264M, N264Q, N264T, R265E, R265F, R265I, R265K, R265L, R265M, R265N, R265P, R265Q, R265S, R265T, R265V, N276A, N276F, N276K, N276M, N276Q, S277C, S277E, S277F, S277G, S277H, S277I, S277M, S277N, S277P, S277Q, S277R, S277Y, N278C, N278F, N278G, N278H, N278I, N278L, N278M, N278Q, N278R, N278S, N278T, N278V, N278W, N278Y, Q279C, Q279D, Q279E, Q279G, Q279H, Q279I, Q279K, Q279N, Q279S, Q279T, Q279V, Q279Y, T282C, T282G, T282K, T282L, T282P, T282S, R284H, R284N, D287C, D287E, D287F, D287G, D287H, D287I, D287K, D287L, D287M, D287N, D287S, D287V, D287W, D287Y, Q301A, Q301E, Q301G, Q301L, Q301N, Q301R, Q301S, Q301T, Q301V, D302A, D302C, D302E, D302F, D302G, D302K, D302L, D302M, D302N, D302P, D302S, D302T, D302W, D302Y, Q303C, Q303D, Q303F, Q303G, Q303H, Q303I, Q303K, Q303L, Q303M, Q303P, Q303R, Q303S, Q303T, Q303V, Q303W, Q303Y, Y306C, Y306G, Y306I, Y306K, Y306M, Y306N, Y306P, Y306Q, Y306R, Y306S, Y306T, Y306V, S312C, S312K, S312L, S312M, S312Q, S312T, S312V, S312W, S312Y, R313A, R313C, R313G, R313K, R313L, R313N, R313S, R313V, R313W, Q316C, Q316E, Q316G, Q316H, Q316I, Q316K, Q316L, Q316M, Q316N, Q316P, Q316R, Q316S, Q316T, Q316V, Q316W, Q316Y, K320C, K320E, K320G, K320L, K320M, K320P, K320Q, K320R, K320T, R324C, R324D, R324E, R324F, R324H, R324I, R324K, R324L, R324M, R324Q, R324V, R324W, R324Y, R328C, R328E, R328G, R328I, R328K, R328L, R328M, R328Q, R328S, R328T, R328V, D329A, D329E, D329F, D329G, D329H, D329M, D329N, D329Q, D329S, D329T, D329Y, L334A, L334C, L334F, L334M, L334T, L334V, L334W, K335A, K335D, K335F, K335G, K335H, K335I, K335M, K335N, K335R, K335V, K335W, N336A, N336C, N336G, N336H, N336L, N336M, N336Q, N336R, N336S, N336T, N336V, N336Y, D337A, D337C, D337E, D337G, D337H, D337K, D337L, D337M, D337N, D337R, D337S, D337T, D337V, D337W, D337Y, A338C, A338F, A338H, A338I, A338K, A338L, A338M, A338P, A338Q, A338V, A338W, A338Y, N339D, N339E, N339G, N339H, N339I, N339K, N339L, N339P, N339Q, N339R, N339V, N339Y, K344D, K344E, K344F, K344G, K344I, K344L, K344M, K344N, K344P, K344Q, K344R, K344S, K344T, K344V, K345A, K345D, K345E, K345F, K345G, K345H, K345N, K345P, K345Q, K345R, K345S, K345T, K345V, K345W, K345Y, A347D, A347F, A347H, A347I, A347K, A347L, A347M, A347P, A347Q, A347R, A347S, H361A, H361C, H361D, H361E, H361G, H361K, H361L, H361M, H361N, H361P, H361S, H361T, H361Y, R363C, R363E, R363L, R363N, R363Q, R363S, R363T, R363W, R363Y, N369A, N369C, N369D, N369E, N369F, N369I, N369L, N369M, N369R, N369S, N369T, N369V, N369W, N369Y, D370F, D370G, D370S, D370W, D370Y, K371D, K371F, K371G, K371L, K371N, K371Q, K371R, K371S, K371T, K371V, K371W, G372A, G372C, G372D, G372E, G372L, G372M, G372N, G372S, G372T, G372V, G372Y, D374C, D374F, D374G, D374L, D374M, D374N, D374Q, D374S, D374T, D374V, D374Y, D375A, D375C, D375E, D375H, D375I, D375R, D375V, D375W, M380E, M380F, M380G, M380I, M380L, M380N, M380Q, M380S, M380T, M380V, M380Y, W382F, W382N, W382Y, Y396C, Y396D, Y396E, Y396F, Y396G, Y396H, Y396I, Y396K, Y396L, Y396M, Y396N, Y396Q, Y396R, Y396S, Y396T, Y396V, Y396W, D397A, D397C, D397E, D397F, D397H, D397I, D397K, D397L, D397M, D397P, D397Q, D397R, D397S, D397T, D397V, D397Y, A398C, A398D, A398E, A398F, A398G, A398H, A398I, A398K, A398L, A398M, A398N, A398P, A398Q, A398R, A398S, A398T, A398V, A398W, A398Y, I399A, I399C, I399D, I399E, I399F, I399G, I399M, I399Q, I399S, I399T, I399V, I399W, I399Y, R402A, R402C, R402E, R402F, R402G, R402I, R402L, R402P, R402Q, R402S, R402V, R402W, R402Y, Q409C, Q409D, Q409G, Q409H, Q409I, Q409V, V410A, V410C, V410F, V410G, V410H, V410I, V410L, V410N, V410S, V410T, V410W, V410Y, T411D, T411E, T411F, T411G, T411H, T411I, T411K, T411L, T411N, T411Q, T411R, T411S, T411V, T411Y, S420C, S420G, S420H, S420K, S420N, S420Q, S420T, S420Y, R426E, R426F, R426I, R426K, R426L, R426M, R426N, R426P, R426Q, R426S, R426T, R426W, R426Y, G427C, G427D, G427F, G427H, G427K, G427L, G427M, G427N, G427P, G427Q, G427R, G427T, G427V, G427W, G427Y, K428C, K428D, K428E, K428F, K428G, K428H, K428I, K428L, K428M, K428N, K428P, K428Q, K428R, K428S, K428T, K428V, K428W, K428Y, T445A, T445C, T445D, T445E, T445F, T445G, T445I, T445K, T445L, T445M, T445N, T445P, T445Q, T445R, T445S, T445V, T445Y, V446C, V446K, V446Q, V446R, E447K, E447L, E447S, E447V, E447W, E447Y, G448C, G448E, G448H, G448K, G448L, G448Q, G448V, N449A, N449C, N449E, N449F, N449G, N449H, N449K, N449L, N449M, N449P, N449R, N449T, N449V, N449W, D452N, R453A, R453L, R453M, N454F, N454G, N454K, N454L, N454M, N454R, N454S, N454T, N454V, N455A, N455C, N455D, N455E, N455F, N455G, N455H, N455I, N455L, N455M, N455S, N455T, N455V, N455W, N455Y, H460A, H460C, H460D, H460E, H460F, H460G, H460I, H460K, H460L, H460M, H460N, H460Q, H460R, H460S, H460W, H460Y, Q467A, Q467C, Q467D, Q467E, Q467H, Q467N, Q467S, Q467V, Q467W, Q467Y, N473A, N473C, N473E, N473F, N473G, N473H, N473K, N473L, N473M, N473P, N473Q, N473R, N473T, N473V, S474A, S474C, S474E, S474F, S474I, S474L, S474M, S474P, S474Q, N475I, N475L, N475M, N475P, N475Q, N475R, N475S, N475T, N475V, N475W, N475Y, E489N, Q490A, Q490C, Q490E, Q490F, Q490G, Q490H, Q490K, Q490L, Q490P, Q490R, Q490S, Q490T, Q490V, Q490W, Q490Y, L492A, L492D, L492F, L492H, L492I, L492M, L492N, L492Q, L492R, L492T, L492W, L492Y, Q496A, Q496G, Q496K, Q496N, Q496P, Q496S, Q496T, Q496V, V497A, V497C, V497I, V497M, V497N, V497T, K498A, K498C, K498E, K498F, K498G, K498H, K498I, K498L, K498M, K498N, K498Q, K498R, K498S, K498T, K498V, K498Y, D521C, D521E, D521F, D521G, D521H, D521I, D521K, D521L, D521M, D521P, D521R, D521S, D521T, D521V, D521W, D521Y, V522A, V522C, V522F, V522G, V522H, V522I, V522K, V522L, V522M, V522N, V522P, V522Q, V522R, V522S, V522T, V522W, V522Y, K534C, K534D, K534E, K534F, K534G, K534H, K534I, K534N, K534R, K534S, K534T, K534V, R542C, R542E, R542F, R542G, R542H, R542I, R542K, R542L, R542M, R542N, R542P, R542Q, R542S, R542T, R542V, R542W, R542Y, G547A, G547C, G547F, G547K, G547N, G547Q, G547R, G547T, G547V, G547Y, S548C, S548I, S548M, S548N, S548Q, S548R, S548T, S548V, S548W, S548Y, E553D, E553I, E553K, E553N, E553Q, E553W, E553Y, G554A, G554C, G554D, G554F, G554H, G554K, G554L, G554M, G554Q, G554R, G554S, G554T, G554V, G554W, L555A, L555C, L555D, L555E, L555F, L555G, L555H, L555I, L555K, L555M, L555N, L555P, L555Q, L555T, L555V, L555W, L555Y, K560A, K560C, K560E, K560G, K560I, K560L, K560M, K560N, K560P, K560Q, K560R, K560S, K560T, K560V, K560W, K560Y, H561A, H561C, H561D, H561E, H561F, H561G, H561I, H561M, H561N, H561Q, H561S, H561T, H561V, H561W, D563A, D563C, D563E, D563F, D563I, D563L, D563M, D563Q, D563R, D563S, D563T, D563V, D563W, D563Y, D564A, D564C, D564E, D564F, D564G, D564K, D564L, D564M, D564N, D564Q, D564R, D564S, D564T, D564V, D564Y, R570A, R570C, R570D, R570E, R570G, R570H, R570I, R570M, R570N, R570Q, R570S, R570T, R570V, Y571H, Y571M, Y571W, K581A, K581C, K581D, K581E, K581F, K581G, K581H, K581I, K581L, K581M, K581N, K581P, K581R, K581S, K581T, K581V, K581W, K581Y, N583A, N583C, N583E, N583F, N583G, N583H, N583I, N583K, N583L, N583M, N583P, N583R, N583S, N583T, N583V, N583W, N583Y, R586E, R586F, R586G, R586L, R586N, R586P, R586V, R586W, R586Y, S591C, S591D, S591F, S591G, S591H, S591I, S591K, S591M, S591P, S591Q, S591V, V603A, V603C, V603D, V603E, V603F, V603G, V603H, V603N, V603P, V603R, V603T, V603W, V603Y, F611A, F611C, F611D, F611G, F611I, F611K, F611L, F611M, F611N, F611R, F611S, F611T, F611V, F611W, F611Y, Q612C, Q612F, Q612H, Q612I, Q612L, Q612M, Q612R, Q612S, Q612W, A622D, A622E, A622F, A622G, A622H, A622I, A622K, A622M, A622N, A622P, A622R, A622S, A622T, A622V, Q626E, Q626F, Q626G, Q626M, V627D, V627K, V627P, V627Q, V627R, V627S, V627Y, T638A, T638E, T638F, T638G, T638I, T638K, T638L, T638M, T638P, T638Q, T638R, T638S, T638V, T638Y, S642A, S642C, S642E, S642F, S642G, S642H, S642I, S642K, S642L, S642M, S642N, S642P, S642Q, S642R, S642T, S642V, S642W, S642Y, A643C, A643E, A643F, A643G, A643H, A643K, A643L, A643N, A643Q, A643R, A643S, A643T, A643V, A643W, A643Y, R645A, R645D, R645E, R645F, R645G, R645H, R645I, R645K, R645L, R645M, R645P, R645Q, R645S, R645T, R645V, R645W, R645Y, K649C, K649F, K649I, K649L, K649M, K649Q, K649S, K649T, K649Y, Q650A, Q650C, Q650E, Q650F, Q650G, Q650H, Q650I, Q650K, Q650L, Q650M, Q650N, Q650R, Q650T, Q650V, Q650Y, K656R, T660C, T660D, T660E, T660F, T660G, T660H, T660I, T660K, T660M, T660N, T660P, T660Q, T660R, T660S, T660V, T660W, T660Y, P661A, P661C, P661D, P661E, P661F, P661G, P661H, P661I, P661K, P661L, P661M, P661Q, P661R, P661S, P661T, P661V, P661W, G662A, G662C, G662F, G662H, G662I, G662K, G662M, G662N, G662Q, G662R, G662W, G662Y, Q663A, Q663C, Q663E, Q663F, Q663H, Q663I, Q663K, Q663L, Q663M, Q663N, Q663R, Q663S, Q663V, Q663W, T666C, T666D, T666E, T666F, T666G, T666H, T666K, T666L, T666N, T666R, T666S, T666V, T666W, T666Y, R672C, R672E, R672F, R672G, R672H, R672I, R672K, R672L, R672M, R672N, R672T, R672V, R672W, R672Y, R673A, R673C, R673E, R673F, R673G, R673H, R673I, R673K, R673L, R673M, R673Q, R673S, R673T, R673V, R674K, R674L, R674M, R674Q, R674V, D675E, D675H, D675S, D675Y, D680A, D680C, D680E, D680F, D680H, D680I, D680K, D680L, D680M, D680N, D680Q, D680R, D680S, D680V, D680W, D680Y, T681A, T681G, T681H, T681K, T681L, T681M, T681N, T681P, T681Q, T681R, T681S, T681V, T681W, T681Y, A682C, A682E, A682I, A682L, A682M, A682N, A682P, A682S, A682W, A682Y, S683A, S683C, S683D, S683E, S683F, S683G, S683I, S683L, S683M, S683P, S683Q, S683R, S683V, S683W, Q684A, Q684C, Q684E, Q684G, Q684I, Q684N, Q684P, K685A, K685E, K685F, K685G, K685I, K685L, K685M, K685N, K685Q, K685R, K685S, K685T, K685V, K685W, K685Y, S692C, S692E, S692H, S692I, S692K, S692L, S692M, S692N, S692P, S692Q, S692T, S692V, S692W, R702C, R702D, R702F, R702G, R702H, R702I, R702K, R702L, R702M, R702N, R702Q, R702S, R702T, R702V, R702W, R705C, R705F, R705H, R705I, R705L, R705M, R705P, R705S, R705T, R705V, and R705W, wherein the substitution consists of no more than a single replacement at each of the positions, and wherein the positions are numbered by correspondence with the amino acid sequence of a reference beta-glucosidase 1 (BGL1) set forth as SEQ ID NO:3. As described in the experimental section, exemplary beta-glucosidase variants have reduced expression levels (PI greater than 0.1 but less than 1).

[0102] The present disclosure further provides a beta-glucosidase variant, wherein the substitution comprises one or more of the group consisting of: K022A, K022E, K022F, K022G, K022P, K022Q, K022S, K022V, K022W, K022Y, N024A, N024C, L025A, L025D, L025F, L025G, L025I, L025K, L025Q, L025R, L025S, L025T, L025V, L025W, L025Y, Q026C, Q026H, Q026I, Q026K, Q026L, Q026P, Q026R, Q026S, Q026T, Q026V, Q026W, D027A, D027C, D027E, D027L, D027M, D027Q, D027S, D027T, D027V, S033C, S033G, V035C, V035E, V035G, V035H, V035K, V035L, V035N, V035P, V035Q, V035R, V035S, V035T, V035Y, G036D, G036E, G036R, G036S, W037V, W037Y, S050A, K051C, K051D, K051E, K051G, K051H, K051M, K051Q, K051R, K051T, K051V, R067A, R067C, R067D, R067F, R067G, R067N, R067P, R067Q, R067S, R067W, R091A, R091D, R091F, R091L, R091Q, R091V, R091W, E092C, E092K, E092L, E092N, E100A, E100G, E100I, E100M, E100S, E100T, E164S, Q165C, Q165F, Q165H, Q165I, Q165L, Q165M, Q165R, Q165S, Q165T, Q165V, Q165W, Q165Y, E166D, E166K, E166L, E166P, E166R, E166T, L167A, L167C, L167D, L167E, L167G, L167Q, L167R, L167S, L167V, L167W, L167Y, N168A, N168D, N168E, N168G, N168Y, P176F, P176G, P176K, P176L, P176R, P176T, P176V, P176W, D177V, D177W, D178A, D178C, D178Q, D178R, R179W, Q194A, Q194K, Q194Y, N196E, Y204F, T209C, T209D, T209E, T209G, T209H, T209I, T209K, T209L, T209M, T209Q, T209R, T209S, T209V, T209W, T209Y, E214A, E214C, E214D, E214G, E214H, E214L, E214M, E214N, E214Q, E214R, E214S, E214T, E214Y, D215E, D215L, D215N, D215Q, D215S, Q216G, Q216I, Q216L, Q216N, Q216S, Q216Y, K224R, K224V, D225V, Q226A, Q226F, Q226L, Q226W, Q226Y, N238A, N238E, N238G, N238M, N238S, N238T, T242A, T242C, T242E, T242F, T242G, T242H, T242I, T242K, T242L, T242M, T242N, T242Q, T242R, T242V, T242W, T242Y, N248A, N248C, N248F, N248T, N248W, N263A, N263C, N263G, N263H, N263S, N263T, N264C, R265E, R265K, R265L, R265N, R265Q, S277W, N278F, Q279C, T282C, D287C, D287E, D287N, D287S, Q301A, Q301K, Q301L, Q301N, Q301R, Q301S, Q301T, Q301V, D302A, D302C, D302W, Q303A, Q303C, Q303E, Q303H, Q303I, Q303K, Q303L, Q303M, Q303N, Q303R, Q303S, Q303T, Q303V, Q303Y, Y306C, Y306G, Y306I, Y306K, Y306L, Y306M, Y306N, Y306P, Y306Q, Y306R, Y306S, Y306T, Y306V, S312C, S312T, S312V, S312W, S312Y, Q316C, Q316P, Q316T, K320C, R328C, R328E, R328G, R328K, R328L, R328M, R328Q, R328S, R328T, R328V, D329A, D329E, D329G, D329H, D329M, D329N, D329Q, D329S, D329T, K335A, K335D, K335F, K335H, K335I, K335L, K335M, K335N, K335R, K335S, K335T, K335V, K335W, D337A, D337C, D337E, D337G, D337H, D337K, D337L, D337M, D337N, D337R, D337S, D337T, D337V, D337Y, A338C, A338F, A338G, A338H, A338I, A338L, A338M, A338N, A338P, A338R, A338V, A338W, K344D, K344E, K344F, K344G, K344I, K344L, K344M, K344N, K344R, K344S, K344T, K344V, K345A, K345E, K345F, K345H, K345P, K345Q, K345R, K345S, K345T, K345V, K345Y, A347D, A347F, A347P, A347Y, H361G, R363C, R363K, R363L, R363Q, R363T, R363W, R363Y, N369C, N369D, N369E, N369F, N369L, N369M, N369S, N369T, N369V, N369W, N369Y, D370E, D370F, D370G, D370S, D370W, D370Y, K371A, K371F, K371G, K371L, K371N, K371Q, K371R, K371S, K371T, K371V, G372A, G372C, G372E, G372L, G372M, G372T, G372V, D374C, D374F, D374G, D374L, D374M, D374N, D374Q, D374S, D374V, D375A, D375C, D375E, D375I, D375V, M380I, M380L, M380Q, M380S, M380T, M380V, Y396A, Y396C, Y396D, Y396E, Y396F, Y396G, Y396I, Y396K, Y396T, D397C, D397E, D397H, D397I, D397K, D397L, D397M, D397N, D397P, D397Q, D397R, D397S, D397T, D397V, D397Y, A398C, A398D, A398E, A398F, A398G, A398H, A398I, A398K, A398L, A398M, A398N, A398P, A398Q, A398R, A398S, A398T, A398V, A398W, A398Y, I399L, I399V, R402A, V410C, T411D, T411E, T411F, T411G, T411H, T411I, T411K, T411L, T411N, T411Q, T411R, T411S, T411V, T411Y, S420C, S420D, S420G, S420K, S420N, S420Q, S420T, S420Y, R426A, R426T, G427C, G427F, G427H, G427L, G427M, G427S, G427Y, K428A, T445A, T445C, T445E, T445F, T445G, T445M, T445V, T445Y, G448A, G448C, G448D, G448E, G448H, G448T, N449A, N449C, N449E, N449F, N449M, N449P, N449T, N449V, N455C, N455D, N455W, N473S, S474C, S474F, S474I, S474L, S474M, S474N, S474P, S474R, S474T, S474Y, N475I, N475L, N475M, N475P, N475Q, N475R, N475S, N475T, N475V, N475W, N475Y, Q490A, Q490C, Q490E, Q490F, Q490G, Q490H, Q490K, Q490L, Q490R, Q490S, Q490T, Q490V, Q490Y, L492A, L492D, L492H, L492N, V497A, V497T, K498E, K498L, K498M, K498V, D521A, V522A, V522C, V522F, V522H, V522I, V522K, V522L, V522M, V522Q, V522R, V522S, V522T, V522W, V522Y, K534F, K534V, R542C, R542E, R542F, R542G, R542H, R542I, R542K, R542L, R542M, R542N, R542P, R542Q, R542S, R542T, R542V, R542W, R542Y, G547A, G547L, G547P, S548C, S548E, S548F, S548H, S548I, S548L, S548M, S548N, S548Q, S548R, S548T, S548V, S548W, S548Y, G554D, G554L, G554M, G554Q, G554W, L555C, L555I, L555V, H561M, H561N, D563A, D563M, D563Q, D564A, D564C, D564F, D564L, D564M, D564T, D564V, R570A, Y571W, K581A, K581D, K581E, K581F, K581G, K581H, K581I, K581L, K581M, K581N, K581R, K581S, K581T, K581V, K581W, K581Y, N583A, N583C, N583D, N583G, N583R, N583V, R586E, R586F, R586L, R586N, R586P, R586V, R586W, V603A, V603C, V603D, V603E, V603F, V603G, V603H, V603S, V603T, V603W, V603Y, F611A, Q612C, Q612G, Q612S, A622E, A622F, A622G, A622H, A622K, A622L, A622M, A622R, A622S, A622T, A622V, Q626E, Q626F, Q626G, Q626M, Q626T, T638A, T638D, T638E, T638G, T638I, T638K, T638L, T638M, T638Q, T638R, T638S, T638V, T638W, T638Y, S642C, S642E, S642F, S642H, S642I, S642L, S642M, S642N, S642P, S642Q, S642R, S642T, S642V, S642W, S642Y, A643L, A643M, R645G, R645K, K649A, K649C, K649F, K649I, K649L, K649M, K649Q, K649S, K649W, K649Y, T660C, T660D, T660I, T660W, P661C, P661D, P661F, P661I, P661L, P661S, P661V, P661W, G662A, G662C, G662F, G662H, G662T, G662W, G662Y, Q663A, Q663C, Q663D, Q663E, Q663G, Q663I, Q663K, Q663S, Q663W, T666A, T666C, T666N, R672K, R673K, R673N, R673S, R673T, D675E, D675H, D675S, D675Y, D680A, D680C, D680E, D680M, D680Q, D680R, D680V, D680Y, T681A, T681G, T681M, T681S, T681W, S683G, S683V, S683W, Q684A, Q684C, Q684G, Q684N, Q684P, K685A, K685E, K685F, K685G, K685I, K685L, K685M, K685N, K685Q, K685R, K685S, K685T, K685V, K685W, K685Y, S692C, S692E, S692H, S692I, S692K, S692L, S692M, S692N, S692P, S692Q, S692T, S692V, S692W, R702G, R705I, and R705V, wherein the substitution consists of no more than a single replacement at each of the positions, and wherein the positions are numbered by correspondence with the amino acid sequence of a reference beta-glucosidase 1 (BGL1) set forth as SEQ ID NO:3. As described in the experimental section, exemplary beta-glucosidase variants have improved CNPGase activity (PI greater than 1).

[0103] Also, the present disclosure provides a beta-glucosidase variant comprising a substitution, wherein the substitution comprises one or more of the group consisting of: K022A, K022E, K022S, N024D, N024E, N024F, N024G, N024K, N024L, N024M, N024P, N024T, L025T, Q026D, Q026P, Q026R, Q026W, Q026Y, K028L, K028M, K028N, W037F, W037I, S050C, S050F, S050G, S050K, S050P, S050R, S050T, S050Y, K051A, K051D, K051G, K051H, K051M, K051T, K051V, I052A, I052F, I052N, I052S, R067A, R067C, R067D, R067E, R067F, R067G, R067I, R067M, R067N, R067P, R067S, R067T, R067V, R067W, R067Y, R091A, R091D, R091E, R091F, R091G, R091H, R091I, R091K, R091L, R091N, R091Q, R091S, R091T, R091V, R091W, E092K, E092T, R093A, R093C, R093D, R093E, R093F, R093G, R093H, R093K, R093L, R093M, R093Q, R093S, R093T, R093V, R093W, E099A, E099F, E099I, E099M, E099W, E099Y, E100I, E100K, E100L, E100Y, H159E, H159G, Q165D, Q165I, Q165K, Q165L, Q165R, Q165V, Q165W, Q165Y, E166F, E166K, E166L, E166N, E166R, E166S, E166T, E166Y, R169A, R169C, R169E, R169F, R169H, R169Q, R169S, R169T, E170F, E170I, E170L, E170M, E170V, E170W, E170Y, P176A, P176D, P176R, 177A, D177G, D177L, D177M, D177N, D177Q, D177W, D178S, R179A, R179V, Q194A, N196E, N196G, N196M, N196P, S199A, Y204M, N208R, T209K, T209L, T209M, T209R, E214A, E214K, E214P, E214R, E214W, E214Y, D215A, D215C, D215F, D215G, D215H, D215M, D215N, D215Q, D215S, D215V, D215W, Q216A, Q216C, Q216D, Q216E, Q216F, Q216H, Q216I, Q216K, Q216L, Q216M, Q216P, Q216R, Q216T, Q216W, Q216Y, D225A, D225C, D225E, D225F, D225H, D225I, D225L, D225Q, D225T, D225V, D225W, D225Y, Q226A, Q226C, Q226D, Q226E, Q226I, Q226K, Q226W, W237Y, N238A, N238D, N238F, N238G, N238P, N238S, N238W, T242H, T242S, S249M, N263A, N263C, N263D, N263E, N263F, N263H, N263I, N263K, N263L, N263P, N263Q, N263S, N263T, N263V, N264A, N264C, N264D, N264E, N264G, N264H, N264K, N264L, N264M, N264Q, N264R, N264S, N264T, N264V, N264Y, R265A, R265E, R265F, R265G, R265I, R265M, R265P, R265Q, R265S, R265T, R265V, N276A, S277A, S277C, S277D, S277E, S277F, S277G, S277M, S277N, S277Q, S277R, S277W, N278C, N278D, N278F, N278G, 278Q, N278R, N278V, Q279C, Q279D, Q279V, T282D, T282H, T282K, R284N, D287C, D287F, D287H, D287I, D287K, D287L, D287M, D287V, D287W, D287Y, Q301E, Q301L, Q301T, Q301V, Y306C, S312A, S312C, S312D, S312G, S312I, S312K, S312L, S312M, S312N, S312Q, S312R, S312W, S312Y, R313E, R313G, R313L, R313S, R313V, R313W, Q316A, Q316C, Q316D, Q316E, Q316F, Q316G, Q316H, Q316I, Q316K, Q316N, Q316P, Q316R, Q316S, Q316T, Q316V, Q316W, Q316Y, K320E, K320G, K320M, K320N, K320T, R324C, R324D, R328C, R328E, R328I, R328L, R328Q, D329A, D329F, D329Q, D329T, D329Y, L334M, L334V, K335A, K335G, K335S, K335T, K335V, K335W, N336S, N336T, N336Y, D337A, D337C, D337W, A338F, A338P, A338Q, A338V, A338W, A338Y, N339D, N339I, N339L, N339P, N339Q, N339R, N339V, N339Y, K344D, K345A, K345D, K345E, K345G, K345H, K345Q, K345Y, A347D, A347F, A347I, A347K, A347L, A347M, A347P, A347Q, A347R, A347S, A347Y, H361A, H361C, H361E, H361G, H361K, H361L, H361M, H361P, H361S, H361Y, R363A, R363C, R363E, R363G, R363K, R363L, R363N, R363Q, R363S, R363T, R363V, R363W, N369A, N369C, N369D, N369E, N369I, N369L, N369M, N369R, N369T, N369V, N369W, N369Y, D370E, D370F, D370W, D370Y, K371A, K371D, K371F, K371G, K371L, K371S, K371T, K371W, G372A, G372C, G372D, G372N, G372S, D374A, D374I, D374R, D374W, M380E, M380F, M380G, M380L, M380Q, M380T, M380V, M380Y, W382N, W382Y, D397N, A398C, A398D, A398E, A398F, A398G, A398H, A398L, A398N, A398P, A398S, A398T, A398Y, I399L, R402C, R402E, R402G, R402I, R402L, R402P, R402Q, R402S, R402V, R402Y, Q409C, Q409D, Q409G, Q409H, Q409I, Q409V, V410G, V410L, R426A, R426E, R426F, R426I, R426K, R426L, R426M, R426N, R426Q, R426S, R426T, R426Y, G427D, G427E, G427F, G427L, G427N, G427Q, G427S, G427T, G427V, G427W, 428A, K428P, T445A, T445C, T445E, T445F, T445G, T445M, T445P, T445Q, T445S, T445V, T445Y, E447A, E447L, E447W, G448D, G448E, G448F, G448H, G448K, G448L, G448M, G448N, G448Q, G448S, G448T, G448V, G448Y, N449C, N449E, N449G, N449K, N449L, N449R, N449V, N449W, D452N, R453A, R453E, R453L, R453M, R453Q, R453S, N455A, N455C, N455D, N455I, Q467A, Q467C, Q467D, Q467E, Q467H, Q467K, Q467N, Q467S, Q467V, Q467W, Q467Y, N473F, N473P, N473Q, N473R, N473S, N473T, N473V, S474D, S474G, S474K, S474L, S474M, S474N, S474Q, S474R, S474V, N475I, N475K, N475L, N475P, N475T, N475V, N475W, N475Y, E489N, Q490C, Q490G, L492Y, Q496A, Q496G, Q496K, Q496N, Q496P, Q496T, Q496V, Q496W, V497C, V497N, K498A, K498C, K498E, K498F, K498G, K498H, K498I, K498Q, K498R, K498S, K498T, K498Y, D521E, D521F, D521P, D521T, D521V, V522G, V522K, V522N, K534D, K534E, K534G, K534H, K534I, K534N, K534Q, K534S, K534T, K534V, R542A, R542C, R542D, R542F, R542I, R542K, R542P, R542Q, R542S, R542W, R542Y, G547A, G547C, G547E, G547F, G547K, G547L, G547N, G547P, G547Q, G547R, G547T, G547V, G547Y, S548E, S548F, S548I, S548L, S548Q, S548T, S548V, S548W, E553D, E553I, E553K, E553Q, E553W, E553Y, G554A, G554C, G554D, G554F, G554H, G554K, G554L, G554M, G554Q, G554R, G554S, G554T, G554W, K560A, K560C, K560E, K560G, K560H, K560I, K560L, K560M, K560N, K560P, K560Q, K560R, K560S, K560T, K560V, K560Y, H561C, H561D, H561G, H561M, H561N, H561T, H561W, D563A, D563I, D563R, D563S, D563V, D563Y, D564A, D564C, D564E, D564F, D564G, D564N, D564Y, R570E, R570G, R570M, R570N, R570Q, R570T, R570V, Y571H, Y571M, Y571W, K581C, K581D, K581G, K581L, K581N, K581T, N583D, R586D, S591C, S591D, S591F, S591G, S591H, S591I, S591K, S591M, S591P, S591Q, S591V, F611G, F611I, F611L, F611N, F611R, F11S, F611T, F611V, F611Y, Q612C, Q612D, Q612G, Q612H, Q612I, Q612W, A622D, A622E, A622L, V627D, V627K, V627P, V627Q, V627R, V627S, V627Y, T638A, T638D, T638Q, T638R, S642D, S642E, S642F, S642G, S642I, S642L, S642M, S642N, A643E, A643F, A643G, A643H, A643K, A643M, A643N, A643Q, A643S, A643T, A643V, A643W, A643Y, R645A, R645D, R645E, R645F, R645H, R645I, R645L, R645M, R645P, R645Q, R645S, R645T, R645V, R645W, R645Y, K649A, K649Q, K649W, Q650D, K656R, T660C, T660E, T660F, T660G, T660I, T660K, T660M, T660N, T660P, T660Q, T660R, T660S, T660V, T660W, T660Y, P661A, P661C, P661D, P661F, P661G, P661I, G662E, G662F, G662I, G662K, G662L, G662M, G662N, G662Q, G662S, G662W, Q663V, Q663W, T666A, T666C, T666D, T666E, T666F, T666G, T666H, T666L, T666S, T666V, T666Y, R672M, R673F, R673N, R673W, R674K, R674L, R674Q, D675E, D675H, D675S, D675Y, T681G, T681L, A682C, A682E, A682I, A682L, A682M, A682N, A682P, A682S, A682W, A682Y, S683A, S683C, S683D, S683E, S683K, S683L, S683R, S683V, Q684C, Q684D, Q684E, Q684F, Q684G, Q684H, Q684I, Q684K, Q684L, Q684M, Q684N, Q684P, Q684R, Q684T, S692H, S692L, S692N, S692T, S692V, R702C, R702D, R702F, R702G, R702U, R702K, R702L, R702M, R702Q, R702S, R702V, R70W, and R705P, wherein the substitution consists of no more than a single replacement at each of the positions, and wherein the positions are numbered by correspondence with the amino acid sequence of a reference beta-glucosidase 1 (BGL1) set forth as SEQ ID NO:3. As described in the experimental section, exemplary beta-glucosidase variants have reduced glucose inhibition (PI greater than 1).

[0104] The present disclosure further provides a beta-glucosidase variant comprising a substitution, wherein the substitution comprises one or more of the group consisting of: K022A, K022E, K022F, K022G, K022I, K022P, K022Q, K022S, K022V, K022W, K022Y, N024A, N024C, N024E, N024M, L025I, L025T, Q026H, Q026K, Q026R, Q026S, V035L, V035S, V035T, W037E, W037F, W037H, W037M, W037S, W037Y, S050A, K051A, K051H, K051M, R091Y, E092K, E092L, E092M, L167R, L167W, E170L, P176A, P176G, D178A, D178T, Q194A, Q194K, Q194R, S199A, D215S, Q216E, Q216L, Q216N, D225C, D225G, D225L, D225Q, D225S, D225T, D225V, Q226A, N238A, T242H, T242S, N248A, N248C, N248L, N248T, S249I, N278D, T282C, T282D, T282G, T282N, T282R, T282V, Q303A, Q303D, Q303E, Q303F, Q303G, Q303I, Q303K, Q303L, Q303M, Q303N, Q303R, Q303S, Q303T, Q303V, Q303Y, Y306F, Y306I, Y306L, Y306R, Y306W, S312C, S312N, K320C, K320H, K320N, K320S, K320Y, D329A, K335A, K335L, K335R, K335S, K335T, K335W, A338C, A338D, A338G, A338I, A338N, A338V, A338W, K344S, A347D, A347F, A347Y, R363L, N369C, N369E, N369F, N369I, N369L, N369M, N369R, N369T, N369V, N369W, N369Y, G372A, I399L, R402A, V410L, T411F, T411H, T411L, T411Q, T411Y, S420A, S420D, R426F, R426N, G427E, G427S, K428A, K428S, T445D, T445G, N473S, S474D, S474G, S474I, S474K, S474M, S474N, S474R, S474T, S474V, S474Y, V497A, V497I, V497M, V497T, D521A, D521S, G547A, G547E, G547K, G547L, G547P, G547R, G547V, S548C, S548E, S548F, S548H, S548I, S548L, S548M, S548V, S548W, G554Q, H561N, D563A, D563E, N583D, N583R, R586D, V603A, V603E, V603H, V603M, V603N, V603Q, V603S, F611A, Q612D, Q612G, A622D, A622G, A622H, A622I, A622L, A622N, A622R, A622S, A622W, A622Y, Q626E, Q626L, Q626T, T638D, A643M, R645D, R645G, R645K, K649L, K649M, K649Q, K649W, T660C, T660D, T660E, T660F, T660H, T660I, T660K, T660M, T660Q, T660V, T660W, T660Y, P661C, P661D, P661S, P661V, G662C, G662D, G662E, G662F, G662H, G662K, G662L, G662M, G662N, G662Q, G662R, G662S, G662T, G662W, G662Y, T666A, R673W, S683V, Q684F, Q684H, Q684K, Q684L, Q684M, Q684S, Q684T, K685A, K685L, K685S, S692H, S692K, S692L, S692M, S692T, and R705L, wherein the substitution consists of no more than a single replacement at each of the positions, and wherein the positions are numbered by correspondence with the amino acid sequence of a reference beta-glucosidase 1 (BGL1) set forth as SEQ ID NO:3. As described in the experimental section, exemplary beta-glucosidase variants have improved thermostability (PI greater than 1).

[0105] Also, the present disclosure provides a beta-glucosidase variant comprising a substitution, wherein the substitution comprises one or more of the group consisting of: K022H, K022R, N024F, N024G, N024K, N024Q, N024R, N024S, N024T, N024V, L025A, L025D, L025F, L025G, L025K, L025N, L025Q, L025R, L025S, L025V, L025Y, Q026C, Q026D, Q026E, Q026G, Q026I, Q026L, Q026P, Q026T, Q026V, Q026W, Q026Y, D027A, D027C, D027E, D027L, D027M, D027Q, D027S, D027T, D027V, K028L, K028M, K028S, K028V, S033C, S033G, S033T, V035C, V035E, V035G, V035H, V035K, V035N, V035Q, V035R, G036R, G036S, W037V, S050C, S050F, S050G, S050I, S050K, S050L, S050M, S050N, S050P, S050R, S050T, S050V, S050Y, K051C, K051D, K051E, K051G, K051I, K051L, K051N, K051Q, K051R, K051S, K051T, K051V, I052V, R067Q, R091A, R091D, R091E, R091F, R091G, R091H, R091K, R091L, R091N, R091Q, R091S, R091V, R091W, E092A, E092C, E092D, E092F, E092H, E092I, E092N, E092Q, E092R, E092V, E099A, E099D, E099F, E099I, E099K, E099M, E099N, E099W, E099Y, E100M, E100Q, E100T, L167A, L167C, L167E, L167F, L167G, L167M, L167N, L167Q, L167S, L167V, L167Y, N168A, N168D, N168E, N168G, N168H, N168Q, N168R, N168T, N168Y, E170D, E170F, E170I, E170M, E170V, P176E, P176H, P176L, P176M, P176Q, P176R, P176S, P176T, P176V, P176Y, D177A, D177E, D177F, D177H, D177L, D177M, D177Q, D177R, D177V, D177Y, D178C, D178K, D178N, D178Q, D178R, D178S, R179A, R179C, R179G, R179I, R179K, R179S, R179T, R179V, R179W, Q194C, Q194E, Q194F, Q194G, Q194H, Q194L, Q194M, Q194T, Q194W, N196E, N196G, N196H, N196L, N196M, N196Q, N196R, N196T, S199G, S199T, S199V, Y204F, Y204M, N208K, T209C, T209D, T209E, T209G, T209H, T209I, T209K, T209L, T209M, T209Q, T209R, T209S, T209V, T209W, T209Y, E214D, E214Q, D215A, D215C, D215E, D215F, D215G, D215H, D215L, D215M, D215N, D215Q, D215W, Q216A, Q216C, Q216F, Q216G, Q216H, Q216I, Q216K, Q216M, Q216P, Q216S, Q216T, Q216W, Q216Y, K224R, K224V, D225A, D225E, D225F, D225H, D225I, D225M, D225W, D225Y, Q226C, Q226D, Q226E, Q226F, Q226H, Q226I, Q226K, Q226L, Q226M, Q226N, Q226R, Q226T, Q226V, Q226W, Q226Y, N238C, N238D, N238E, N238F, N238G, N238M, N238S, N238T, N238W, T242C, T242E, T242F, T242G, T242K, T242N, T242Q, T242R, T242W, T242Y, N248F, N248G, N248W, N248Y, S249G, S249M, S249V, N263A, N263C, N263D, N263E, N263F, N263G, N263H, N263I, N263K, N263L, N263P, N263Q, N263R, N263S, N263T, N263V, N263Y, N264C, N264G, N264K, N264M, N264Q, N264T, R265E, R265F, R265I, R265K, R265L, R265M, R265N, R265P, R265Q, R265S, R265T, R265V, N276A, N276F, N276M, N276Q, S277C, S277E, S277F, S277G, S277H, S277I, S277M, S277N, S277P, S277Q, S277R, S277Y, N278C, N278F, N278G, N278I, N278L, N278M, N278Q, N278R, N278S, N278T, N278V, N278W, N278Y, Q279C, Q279D, Q279E, Q279G, Q279H, Q279I, Q279K, Q279N, Q279S, Q279T, Q279V, Q279Y, T282K, T282L, T282P, T282S, D287C, D287E, D287G, D287H, D287I, D287K, D287L, D287M, D287N, D287S, D287V, Q301E, Q301N, D302A, D302C, D302E, D302F, D302G, D302K, D302M, D302N, D302P, D302S, D302T, D302W, D302Y, Q303C, Q303H, Q303P, Q303W, Y306C, Y306G, Y306K, Y306M, Y306N, Y306P, Y306Q, Y306S, Y306T, Y306V, S312K, S312L, S312M, S312Q, S312T, S312V, S312W, S312Y, R313A, R313C, R313G, R313K, R313L, R313N, R313S, R313V, R313W, Q316C, Q316E, Q316G, Q316K, Q316L, Q316M, Q316R, Q316S, Q316T, Q316V, Q316W, Q316Y, K320E, K320G, K320L, K320M, K320P, K320Q, K320R, K320T, R324C, R324D, R324E, R324F, R324H, R324I, R324K, R324L, R324M, R324Q, R324V, R324W, R324Y, R328C, R328E, R328G, R328K, R328L, R328M, R328Q, R328T, R328V, D329E, D329F, D329G, D329H, D329M, D329N, D329Q, D329S, D329T, D329Y, L334A, L334C, L334F, L334M, L334T, L334V, L334W, K335D, K335F, K335G, K335H, K335I, K335M, K335N, K335V, N336A, N336C, N336G, N336H, N336L, N336M, N336Q, N336R, N336S, N336T, N336V, N336Y, D337A, D337C, D337E, D337G, D337H, D337K, D337L, D337M, D337N, D337R, D337S, D337T, D337V, D337W, D337Y, A338F, A338H, A338K, A338L, A338M, A338P, A338Q, A338Y, N339D, N339E, N339G, N339H, N339I, N339K, N339L, N339P, N339Q, N339R, N339V, N339Y, K344D, K344E, K344F, K344G, K344I, K344L, K344M, K344N, K344P, K344Q, K344R, K344T, K344V, K345A, K345D, K345E, K345F, K345G, K345H, K345N, K345P, K345Q, K345R, K345S, K345T, K345V, K345W, K345Y, A347H, A347I, A347K, A347L, A347M, A347P, A347R, A347S, R363C, R363E, R363Q, R363T, N369A, N369D, N369S, G372C, G372N, G372V, D374C, D374N, D374Y, W382N, W382Y, Y396C, Y396D, Y396E, Y396F, Y396G, Y396H, Y396I, Y396K, Y396L, Y396M, Y396N, Y396Q, Y396S, Y396T, Y396V, Y396W, D397A, D397C, D397E, D397P, D397Q, D397R, D397S, D397T, D397V, A398C, A398D, A398E, A398F, A398G, A398H, A398I, A398K, A398L, A398M, A398N, A398P, A398Q, A398R, A398S, A398T, A398V, A398W, A398Y, I399A, I399C, I399D, I399E, I399F, I399G, I399M, I399Q, I399S, I399T, I399V, I399W, I399Y, R402C, R402E, R402F, R402G, R402I, R402L, R402P, R402Q, R402S, R402V, R402W, R402Y, Q409C, Q409D, Q409G, Q409H, Q409I, Q409V, V410A, V410C, V410F, V410G, V410H, V410I, V410N, V410S, V410T, V410W, V410Y, T411D, T411E, T411G, T411I, T411K, T411N, T411R, T411S, T411V, S420C, S420G, S420H, S420K, S420N, S420Q, S420T, S420Y, R426E, R426I, R426K, R426L, R426M, R426P, R426Q, R426S, R426T, R426W, R426Y, G427C, G427D, G427F, G427H, G427K, G427L, G427M, G427N, G427P, G427Q, G427R, G427T, G427V, G427W, G427Y, K428C, K428D, K428E, K428F, K428G, K428H, K428I, K428L, K428M, K428N, K428P, K428Q, K428R, K428T, K428V, K428W, K428Y, T445A, T445C, T445E, T445F, T445I, T445K, T445L, T445M, T445N, T445P, T445Q, T445R, T445S, T445V, T445Y, V446Q, N449C, N454F, N454K, N454L, N454M, N454R, N454S, N454T, N454V, N455A, N455C, N455D, N455E, N455F, N455G, N455H, N455L, N455M, N455S, N455T, N455V, N455W, N455Y, Q467A, Q467C, Q467D, Q467N, Q467S, N473A, N473C, N473E, N473G, N473H, N473K, N473L, N473M, N473P, N473Q, N473R, N473T, N473V, S474A, S474C, S474E, S474F, S474L, S474P, S474Q, N475I, N475L, N475M, N475P, N475Q, N475R, N475S, N475T, N475V, N475W, N475Y, E489N, Q490A, Q490C, Q490E, Q490F, Q490G, Q490H, Q490K, Q490L, Q490P, Q490R, Q490S, Q490T, Q490V, Q490W, Q490Y, L492A, L492D, L492F, L492H, L492I, L492M, L492N, L492Q, L492R, L492T, L492W, L492Y, Q496A, Q496G, Q496N, Q496P, Q496S, Q496T, V497C, V497N, K498A, K498C, K498E, K498F, K498G, K498H, K498I, K498L, K498M, K498N, K498Q, K498R, K498S, K498T, K498V, K498Y, D521C, D521E, D521F, D521G, D521H, D521I, D521K, D521L, D521M, D521P, D521R, D521T, D521V, D521W, V522A, V522C, V522F, V522G, V522H, V522I, V522K, V522L, V522M, V522N, V522P, V522Q, V522R, V522S, V522T, V522W, V522Y, K534C, K534D, K534E, K534F, K534H, K534I, K534N, K534R, K534S, K534T, K534V, R542C, R542E, R542F, R542G, R542H, R542K, R542L, R542M, R542N, R542P, R542Q, R542S, R542T, R542V, R542W, R542Y, G547C, G547F, G547N, G547Q, G547T, G547Y, S548N, S548Q, S548R, S548T, S548Y, E553D, E553K, E553N, E553W, G554A, G554C, G554D, G554F, G554H, G554K, G554L, G554M, G554R, G554S, G554T, G554V, G554W, L555A, L555C, L555D, L555E, L555F, L555G, L555H, L555I, L555K, L555M, L555N, L555P, L555Q, L555T, L555V, L555W, L555Y, K560A, K560C, K560G, K560I, K560L, K560M, K560N, K560P, K560R, K560S, K560T, K560V, K560Y, H561A, H561C, H561D, H561E, H561F, H561G, H561I, H561M, H561Q, H561S, H561T, H561V, H561W, D563C, D563F, D563I, D563L, D563M, D563Q, D563R, D563S, D563T, D563V, D563W, D563Y, D564A, D564C, D564E, D564F, D564G, D564K, D564L, D564M, D564N, D564Q, D564R, D564S, D564T, D564V, D564Y, R570A, R570C, R570D, R570E, R570G, R570H, R570I, R570M, R570Q, R570S, R570T, R570V, Y571H, Y571M, Y571W, K581A, K581C, K581D, K581E, K581F, K581G, K581H, K581I, K581L, K581M, K581N, K581P, K581R, K581S, K581T, K581V, K581W, K581Y, N583A, N583C, N583E, N583F, N583G, N583H, N583I, N583K, N583L, N583M, N583P, N583S, N583T, N583V, N583W, N583Y, R586E, R586F, R586G, R586L, R586N, R586P, R586V, R586W, R586Y, S591C, S591D, S591G, S591H, S591I, S591K, S591M, S591P, S591Q, S591V, V603C, V603D, V603F, V603G, V603P, V603R, V603T, V603W, V603Y, F611C, F611D, F611G, F611I, F611K, F611L, F611M, F611N, F611R, F611S, F611T, F611V, F611W, F611Y, Q612C, Q612F, Q612H, Q612I, Q612L, Q612M, Q612R, Q612S, Q612W, A622E, A622F, A622K, A622M, A622T, A622V, Q626F, Q626G, Q626M, V627K, V627P, V627Q, V627R, V627S, T638A, T638E, T638F, T638G, T638I, T638K, T638L, T638M, T638P, T638Q, T638R, T638S, T638V, T638Y, S642A, S642C, S642E, S642F, S642G, S642H, S642I, S642K, S642L, S642M, S642N, S642P, S642Q, S642R, S642T, S642V, S642W, S642Y, A643C, A643E, A643F, A643G, A643H, A643K, A643L, A643N, A643Q, A643R, A643S, A643T, A643V, A643W, A643Y, R645A, R645E, R645F, R645H, R645I, R645L, R645M, R645P, R645Q, R645S, R645T, R645V, R645W, R645Y, K649C, K649F, K649I, K649S, K649T, K649Y, Q650A, Q650C, Q650E, Q650F, Q650G, Q650H, Q650I, Q650K, Q650L, Q650M, Q650N, Q650R, Q650T, Q650V, Q650Y, K656R, T660G, T660N, T660P, T660R, T660S, P661A, P661E, P661F, P661G, P661H, P661I, P661K, P661L, P661M, P661Q, P661R, P661T, P661W, G662A, G662I, Q663A, Q663C, Q663E, Q663F, Q663H, Q663I, Q663K, Q663L, Q663M, Q663N, Q663R, Q663S, Q663V, Q663W, T666C, T666D, T666E, T666F, T666G, T666H, T666K, T666L, T666N, T666R, T666S, T666V, T666W, T666Y, R672C, R672F, R672G, R672H, R672I, R672K, R672L, R672N, R672T, R672V, R672W, R673A, R673C, R673E, R673G, R673H, R673I, R673K, R673L, R673M, R673Q, R673S, R673T, R673V, R674K, R674L, R674M, R674Q, R674V, D675E, D675H, D675S, D675Y, D680A, D680C, D680E, D680F, D680H, D680I, D680K, D680L, D680M, D680N, D680Q, D680R, D680S, D680V, D680W, D680Y, T681A, T681G, T681H, T681K, T681L, T681M, T681N, T681P, T681Q, T681R, T681S, T681V, T681W, T681Y, A682C, A682E, A682I, A682L, A682M, A682N, A682P, A682S, A682W, A682Y, S683A, S683C, S683D, S683E, S683F, S683G, S683I, S683L, S683M, S683P, S683Q, S683R, S683W, Q684A, Q684C, Q684E, Q684G, Q684I, Q684N, Q684P, K685E, K685F, K685G, K685I, K685M, K685N, K685Q, K685R, K685T, K685V, K685W, K685Y, S692C, S692E, S692I, S692N, S692P, S692Q, S692V, S692W, R702C, R702D, R702F, R702G, R702H, R702I, R702K, R702L, R702M, R702N, R702Q, R702S, R702T, R702V, R702W, R705C, R705F, R705H, R705I, R705M, R705P, R705S, R705T, R705V, and R705W, wherein the substitution consists of no more than a single replacement at each of the positions, and wherein the positions are numbered by correspondence with the amino acid sequence of a reference beta-glucosidase 1 (BGL1) set forth as SEQ ID NO:3. As described in the experimental section, exemplary beta-glucosidase variants have reduced thermostability (PI greater than 0.1 but less than 1).

[0106] The present disclosure further provides a beta-glucosidase variant comprising a substitution, wherein the substitution comprises one or more of the group consisting of: K022A, K022E, K022F, K022P, K022Q, N024C, N024F, N024Q, N024R, N024Y, L025D, Q026C, Q026E, Q026H, Q026I, Q026K, Q026L, Q026P, Q026R, Q026S, Q026T, Q026V, Q026W, D027A, D027C, D027E, S033C, V035C, V035P, V035T, G036D, G036E, G036K, G036R, G036S, G036Y, S050C, S050L, K051C, K051E, K051G, K051H, K051I, K051M, K051Q, K051T, K051V, I052D, I052T, R091D, R091G, R091K, R091Q, E092A, E092C, E092D, E099Y, E100A, E100G, E100N, E100Q, E100S, E100T, E100Y, K158H, K158T, Q165I, Q165K, Q165M, Q165N, Q165V, E166D, L167C, L167W, N168A, N168D, N168E, N168G, N168Y, E170D, E170F, P176K, P176R, P176W, P176Y, D177E, D177F, D177H, D177L, D177M, D177V, D177W, D177Y, 178C, D178Y, R179C, R179M, R179S, Q194C, N196E, N196L, N196Q, N196R, N196T, S199A, T209C, T209G, T209H, T209I, T209L, T209M, T209Q, T209S, T209V, T209Y, E214W, D215C, D215E, D215L, D215N, D215Q, D215S, Q216A, Q216E, Q216G, Q216H, Q216I, Q216L, Q216M, Q216N, Q216S, Q216W, Q216Y, K224H, K224R, K224V, D225G, D225H, D225I, D225L, D225M, D225T, D225V, D225W, D225Y, Q226F, Q226I, Q226L, Q226M, Q226R, Q226V, Q226W, Q226Y, N238A, N238C, N238E, N238G, T242A, T242C, T242E, T242F, T242G, T242H, T242K, T242L, T242M, T242N, T242Q, T242R, T242S, T242V, T242W, T242Y, N248A, N248F, N248G, N248T, N248W, S249M, S249V, N263C, N263D, N263G, N263S, N263T, N264C, N276A, N276C, N276F, S277C, S277F, S277W, S277Y, N278C, N278F, N278G, N278V, Q279C, T282C, D287C, D287S, Q301G, Q301K, Q301L, D302A, D302C, D302E, D302F, D302G, D302K, D302M, D302T, Q303A, Q303C, Q303K, Q303M, Q303P, Y306G, Y306K, Y306M, Y306Q, Y306R, Y306V, S312C, S312D, S312W, S312Y, Q316K, Q316P, Q316R, Q316S, Q316T, Q316Y, K320C, R328S, D329A, L334A, L334V, K335A, K335D, K335H, K335V, K335W, N336A, N336G, D337C, D337K, D337W, A338C, A338W, N339E, N339G, N339H, N339K, N339L, K344D, K344F, K344I, K344L, K344M, K344P, K344S, K344T, K344V, K345A, K345D, K345E, K345F, K345G, K345S, K345V, K345Y, A347S, A347Y, H361A, H361C, H361G, R363C, R363G, R363K, R363Q, R363S, R363W, R363Y, N369C, N369D, N369E, N369F, N369W, N369Y, K371A, K371G, K371L, K371T, G372A, G372K, G372W, D374C, D374L, D374M, D374Q, D374S, D374V, D375C, D375E, D375W, M380N, M380V, W382F, Y396A, Y396C, Y396E, Y396F, Y396K, Y396V, D397C, D397E, D397H, D397I, D397K, D397L, D397M, D397N, D397Q, D397R, D397S, D397T, D397V, D397Y, A398E, A398R, A398V, A398W, I399C, I399Y, R402A, R402E, R402G, R402L, R402Q, R402S, R402W, Q409G, T411D, T411E, T411F, T411G, T411H, T411I, T411K, T411L, T411N, T411Q, T411R, T411S, T411V, S420C, S420G, S420H, S420K, S420N, S420Q, S420T, S420V, S420Y, R426A, G427C, G427D, G427E, G427F, G427H, G427P, G427V, G427Y, K428A, K428N, T445A, T445C, T445E, T445F, T445G, T445M, T445P, T445V, T445Y, V446Q, V446R, E447V, G448C, G448D, G448E, G448F, G448N, N449A, N449C, N449E, N449G, N449K, 454F, N455C, N455D, N455S, N455V, N455W, H460E, H460G, H460M, H460Q, H460S, Q467P, Q467S, N473A, N473E, N473L, N473R, N473W, S474A, S474C, S474D, S474G, S474K, S474L, S474N, N475I, N475M, N475S, N475T, N475Y, Q490C, Q490H, Q490L, Q490R, Q490V, Q490W, Q490Y, L492A, L492D, L492F, L492W, L492Y, Q496G, Q496W, V497C, V497M, V497T, K498A, K498C, K498E, K498F, K498G, K498I, K498M, K498T, K498Y, D521A, D521C, D521W, V522A, V522C, V522K, V522L, V522M, V522Q, V522R, V522S, V522W, K534C, K534D, K534E, K534F, K534N, K534R, K534V, R542S, G547A, G547C, S548C, S548E, S548F, S548L, S548M, S548Q, S548T, S548W, G554C, G554D, G554F, G554H, G554M, G554Q, G554W, L555C, L555D, L555E, L555G, L555H, L555K, L555N, L555P, K560A, K560E, K560G, K560P, K560R, K560W, H561G, H561I, H561M, H561N, H561Q, H561S, H561V, H561W, D563A, D563Q, D563S, D563T, D563Y, D564A, D564C, D564F, D564G, D564K, D564L, D564M, D564N, D564Q, D564T, D564V, D564Y, R570A, R570C, R570D, R570E, R570G, R570I, R570Q, R570S, R570T, R570V, Y571H, Y571M, K581A, K581C, K581D, K581E, K581F, K581G, K581H, K581I, K581L, K581M, K581N, K581R, K581S, K581W, K581Y, N583A, N583C, N583D, N583G, N583H, R586N, R586P, R586V, R586W, V603G, V603H, V603Y, F611A, F611C, Q612C, Q612G, Q612S, A622E, Q626E, Q626H, T638A, T638D, T638G, T638W, S642E, S642F, S642G, S642H, 642I, S642L, S642Q, S642R, S642T, S642W, S642Y, A643K, A643V, R645A, R645G, R645I, R645K, R645L, R645M, R645W, R645Y, K649C, K649N, K649T, Q650A, Q650C, Q650D, Q650F, Q650G, Q650K, Q650N, Q650R, Q650T, Q650V, Q650Y, T660C, T660D, T660N, T660S, T660W, T660Y, P661A, P661C, P661D, P661E, P661F, P661H, P661I, P661K, P661L, P661M, P661Q, P661R, P661S, P661T, P661V, P661W, G662A, G662C, G662F, G662H, G662I, G662N, Q663E, T666C, T666D, T666N, R672C, R672D, R672G, R672L, R672M, R672N, R672T, R672V, R673G, R673K, R673L, R673N, R673S, R673T, R674T, R674Y, D680C, D680F, D680I, D680M, D680Q, D680V, D680Y, T681A, T681G, T681P, T681Q, T681S, T681V, T681W, S683F, S683V, S683W, Q684C, Q684G, Q684N, K685A, K685E, K685F, K685G, K685I, K685L, K685M, K685Q, K685S, K685T, K685W, K685Y, S692C, S692H, S692I, S692L, S692M, S692V, S692W, R702C, R702D, R702F, R702G, R702H, R702S, R702T, R702V, R705F, R705I, R705L, R705M, R705S, R705T, R705V, and R705W, wherein the substitution consists of no more than a single replacement at each of the positions, and wherein the positions are numbered by correspondence with the amino acid sequence of a reference beta-glucosidase 1 (BGL1) set forth as SEQ ID NO:3. As described in the experimental section, exemplary beta-glucosidase variants have improved PASC hydrolysis activity (PI greater than 1).

[0107] Also, the present disclosure provides a beta-glucosidase variant comprising a substitution, wherein the substitution comprises one or more of the group consisting of: K022E, K022F, K022G, K022W, N024A, N024C, N024D, N024E, N024F, N024G, N024L, N024P, N024Q, N024S, N024V, N024Y, L025K, L025N, L025T, L025V, L025Y, Q026C, Q026D, Q026E, Q026G, Q026I, Q026K, Q026L, Q026P, Q026R, Q026S, Q026T, Q026V, Q026W, Q026Y, S033C, S033T, V035C, V035E, V035N, G036S, S050C, S050F, S050G, S050I, S050L, S050M, S050N, S050P, S050R, S050T, S050V, K051A, K051C, K051D, K051H, K051I, K051L, K051Q, K051R, K051S, K051T, K051V, R091D, R091E, R091F, R091G, R091H, R091I, R091K, R091L, R091N, R091Q, R091T|R091V, R091W, R091Y, E092C, E092D, E092F, E092H, E092K, E092L, E092N, E092Q, E092R, E092T, E092V, E092Y, R093K, E099A, E099D, E099F, E099I, E099K, E099M, E099N, E099W, E099Y, L167C, L167D, L167E, L167F, L167G, L167M, L167V, L167W, N168A, N168D, N168E, N168G, N168Q, N168Y, E170D, E170F, P176E, P176F, P176G, P176H, P176L, P176M, P176Q, P176R, P176S, P176T, P176V, P176W, P176Y, D177F, D177K, D177L, D177M, D177N, D177Q, D177R, D177V, D178A, D178C, D178N, D178R, R179A, R179C, R179G, R179I, R179K, R179M, R179Q, R179S, R179T, R179V, Q194A, Q194C, Q194E, Q194F, Q194G, Q194H, Q194K, Q194L, Q194M, Q194R, Q194T, Q194W, Q194Y, N196E, N196H, N196L, N196R, N196T, S199G, S199N, S199T, S199V, T209C, T209D, T209E, T209G, T209H, T209I, T209K, T209L, T209M, T209Q, T209V, T209W, T209Y, E214D, D215C, D215L, D215M, D215S, D215W, Q216A, Q216C, Q216D, Q216E, Q216F, Q216G, Q216H, Q216I, Q216K, Q216N, Q216P, Q216R, Q216S, Q216T, Q216W, Q216Y, K224R, D225A, D225C, D225F, D225G, D225H, D225I, D225L, D225M, D225Q, D225S, D225T, D225V, D225W, D225Y, Q226C, Q226D, Q226E, Q226F, Q226H, Q226I, Q226K, Q226L, Q226M, Q226N, Q226R, Q226T, Q226V, Q226W, Q226Y, N238A, N238C, N238G, N238M, N238S, T242A, T242C, T242E, T242S, N248T, S249A, S249G, N263A, N263C, N263D, N263Q, N263S, N263T, N264C, R265A, R265E, R265I, R265L, R265M, R265Y, N276A, N276C, S277A, S277C, S277D, S277E, S277F, S277G, S277H, S277I, S277M, S277P, S277Q, S277W, S277Y, N278A, N278C, N278D, N278F, N278G, N278I, N278M, N278Q, N278R, N278S, N278T, N278V, N278W, N278Y, Q279C, Q279D, Q279E, Q279G, Q279H, Q279I, Q279K, Q279N, Q279S, Q279T, Q279V, Q279Y, T282K, D287C, D287G, D287H, D287I, D287K, D287M, D287N, D287S, Q301A, Q301E, Q301G, Q301K, Q301R, D302A, D302C, D302E, D302F, D302G, D302K, D302M, D302N, D302P, D302S, D302T, D302W, D302Y, Q303C, Q303D, Q303R, Q303W, Y306M, Y306R, Y306V, S312C, S312D, S312G, S312N, S312Q, S312R, S312T, S312V, S312Y, R313A, R313C, R313D, R313G, R313K, R313N, Q316K, Q316L, Q316M, Q316R, Q316T, Q316Y, K320C, K320G, K320N, K320P, K320S, K320Y, R324C, R324D, R324E, R324F, R324H, R324I, R324K, R324L, R324M, R324Q, R324V, R324W, R324Y, R328C, R328K, R328S, D329A, D329H, D329S, L334A, L334C, L334F, L334M, L334T, L334V, L334W, K335A, K335D, K335H, K335L, K335R, K335S, K335V, K335W, N336A, N336C, N336G, N336H, N336L, N336M, N336Q, N336R, N336T, N336V, N336Y, D337A, A338C, 338D, A338E, A338F, A338G, A338H, A338I, A338K, A338L, A338N, A338P, A338Q, A338R, A338V, A338W, A338Y, K344D, K344F, K344L, K344M, K344N, K344P, K344T, K344V, K345A, K345D, K345E, K345F, K345G, K345H, K345N, K345P, K345R, K345S, K345V, K345W, K345Y, A347D, A347F, A347H, A347I, A347K, A347L, A347M, A347P, A347Q, A347R, A347S, A347Y, H361A, H361G, H361N, R363C, R363K, R363M, R363Q, R363S, R363T, R363V, R363W, N369C, N369D, N369S, K371G, G372A, D374C, D374F, D374N, D374S, Y396D, Y396E, Y396F, Y396G, Y396H, Y396K, Y396L, Y396M, Y396N, Y396Q, Y396R, 397C, D397E, D397H, D397I, D397K, D397M, D397N, D397P, D397Q, D397R, D397S, D397T, D397V, D397Y, A398C, A398D, A398E, A398F, A398G, A398H, A398I, A398K, A398L, A398M, A398N, A398P, A398Q, A398R, A398S, A398T, A398V, A398W, A398Y, I399A, I399C, I399D, I399E, I399G, I399M, I399Q, I399S, I399T, I399W, I399Y, R402A, R402C, R402G, R402S, Q409D, V410A, V410C, V410I, V410L, V410N, V410R, V410S, V410T, V410W, T411D, T411E, T411G, T411N, T411Q, T411S, T411Y, S420C, R426A, R426E, R426F, R426I, R426K, R426N, R426P, R426Q, R426S, R426W, R426Y, G427C, G427E, G427F, G427H, G427K, G427N, G427Q, G427R, G427S, G427T, G427V, K428C, K428D, K428E, K428F, K428G, K428H, K428I, K428L, K428M, K428N, K428P, K428Q, K428R, K428S, K428T, K428V, K428W, K428Y, T445A, T445C, T445D, T445K, T445M, T445Q, T445S, V446C, G448A, G448C, G448D, G448E, G448F, G448N, G448S, G448T, G448Y, N449A, N449C, N454F, N454G, N454K, N454L, N454M, N454R, N454S, N454T, N454V, N455A, N455D, N455E, N455F, N455G, N455H, N455I, N455L, N455M, N455S, N455T, N455V, N455W, N455Y, Q467A, N473A, N473C, N473E, N473F, N473G, N473H, N473K, N473L, N473M, N473P, N473Q, N473R, N473S, N473T, N473V, N473W, S474A, S474C, S474D, S474F, S474G, S474I, S474K, S474M, S474N, S474Q, S474R, S474T, S474V, N475I, N475K, N475L, N475M, N475P, N475Q, N475R, N475S, N475T, N475V, N475W, N475Y, E489N, Q490P, Q490W, L492A, L492D, L492F, L492I, L492M, L492Q, L492T, L492W, Q496G, Q496S, Q496W, V497A, V497I, V497T, K498A, K498F, K498H, K498I, K498N, K498Q, D521A, D521C, D521E, D521F, D521G, D521H, D521I, D521K, D521L, D521M, D521P, D521R, D521S, D521T, D521V, D521W, D521Y, V522A, 522C, V522F, V522G, V522H, V522I, V522K, V522L, V522M, V522N, V522P, V522Q, V522R, V522S, V522T, V522W, V522Y, K534C, K534D, K534E, K534F, K534G, K534N, K534R, K534V, R542A, R542C, R542D, R542E, R542F, R542G, R542H, R542I, R542K, R542L, R542M, R542N, R542P, R542Q, R542S, R542T, R542V, R542W, R542Y, G547A, G547L, S548L, G554A, G554C, G554D, G554F, G554H, G554L, G554V, G554W, L555A, L555C, L555D, L555E, L555F, L555G, L555H, L555I, L555K, L555N, L555P, L555Q, L555V, L555W, L555Y, K560H, K560P, K560R, K560W, H561A, H561C, H561D, H561E, H561F, H561G, H561I, H561M, H561N, H561Q, H561S, H561V, H561W, D563A, D563C, D563F, D563I, D563L, D563Q, D563R, D563S, D563T, D563W, D563Y, D564A, D564C, D564F, D564K, D564L, D564R, D564T, D564V, R570A, R570D, R570G, R570H, R570I, R570S, R570V, Y571H, Y571M, K581W, N583A, N583C, N583D, N583E, N583F, N583G, N583H, N583I, N583K, N583L, N583M, N583P, N583R, N583S, N583T, N583W, N583Y, R586E, R586F, R586G, R586H, R586L, R586N, R586P, R586V, R586W, R586Y, S591D, V603A, V603D, V603G, V603H, V603N, V603Q, V603R, V603Y, F611A, F611C, F611D, F611K, F611L, F611M, F611N, F611R, F611S, F611W, Q612C, Q612F, Q612G, Q612H, Q612I, Q612K, Q612L, Q612M, Q612R, Q612S, Q612W, A622H, A622K, Q626H, Q626M, V627P, T638A, T638D, T638E, T638F, T638G, T638I, T638K, T638L, T638M, T638P, T638Q, T638R, T638S, T638V, T638W, T638Y, S642A, S642C, S642E, S642F, S642G, S642H, S642I, S642K, S642L, S642M, S642N, S642P, S642Q, S642R, S642T, S642V, S642W, S642Y, A643C, A643F, A643G, A643H, A643K, A643L, A643M, A643Q, A643R, A643S, A643T, A643V, A643Y, R645A, R645D, R645F, R645G, R645H, R645I, R645K, R645L, R645M, R645P, R645Q, R645T, R645V, R645W, R645Y, K649A, K649C, K649F, K649L, K649N, K649Q, K649S, K649T, K649Y, Q650C, Q650D, Q650E, Q650F, Q650G, Q650H, Q650I, Q650K, Q650L, Q650M, Q650N, Q650R, Q650V, Q650Y, T660C, T660W, T660Y, P661C, P661F, P661H, P661I, P661K, P661L, P661M, P661Q, P661R, P661T, P661V, P661W, G662A, G662C, G662F, G662H, G662I, G662K, G662R, G662Y, Q663A, Q663C, Q663D, Q663E, Q663F, Q663I, Q663L, Q663M, Q663N, Q663R, Q663S, Q663V, Q663W, T666A, T666C, T666H, T666K, T666N, T666R, T666W, R672C, R672D, R672G, R672I, R672K, R672V, R672W, R673A, R673C, R673G, R673H, R673I, R673K, R673L, R673Q, R673T, R673V, R673W, R674L, R674M, R674T, R674Y, D675C, D680A, D680C, D680E, D680F, D680H, D680I, D680K, D680L, D680M, D680N, D680Q, D680R, D680S, D680V, D680W, D680Y, T681A, T681G, T681H, T681K, T681L, T681M, T681N, T681Q, T681R, T681W, S683A, S683C, S683D, S683F, S683G, S683I, S683L, S683P, S683R, S683V, S683W, Q684A, Q684D, K685A, K685F, K685G, K685I, K685L, K685M, K685N, K685Q, K685R, K685S, K685T, K685V, K685W, K685Y, S692E, S692H, S692K, S692L, S692Q, S692T, S692V, S692W, R702C, R702D, R702F, R702G, R702H, R702I, R702K, R702L, R702M, R702N, R702Q, R702S, R702T, R702V, R702W, R705C, R705F, R705H, R705I, R705L, R705M, R705P, R705S, R705T, and R705W, wherein the substitution consists of no more than a single replacement at each of the positions, and wherein the positions are numbered by correspondence with the amino acid sequence of a reference beta-glucosidase 1 (BGL1) set forth as SEQ ID NO:3. As described in the experimental section, exemplary beta-glucosidase variants have improved PCS hydrolysis activity (PI greater than 1).

[0108] The present disclosure further provides a beta-glucosidase variant comprising a substitution, wherein the substitution comprises one or more of the group consisting of: K022A, K022E, K022F, K022P, K022Q, N024C, N024P, N024Q, L025A, L025D, Q026C, Q026I, Q026K, Q026L, Q026P, Q026R, Q026S, Q026T, Q026W, S033C, S033G, V035C, V035E, V035Q, V035R, V035S, V035T, V035Y, G036C, G036D, G036E, G036F, G036I, G036K, G036R, G036S, G036W, G036Y, S050C, S050P, K051A, K051C, K051D, K051G, K051H, K051M, K051Q, K051T, K051V, R091D, R091G, R091K, R091N, R091Q, E092A, E092C, E092D, E092F, E092H, E092I, E092L, E092N, E092R, E092V, E099Y, E100A, E100G, E100M, E100T, E100Y, E164G, E164S, Q165V, E166D, L167C, L167G, L167N, L167V, L167W, N168A, N168D, N168E, N168G, N168H, N168Q, N168R, N168T, N168Y, E170F, P176A, P176D, P176F, P176H, P176K, P176L, P176R, P176V, P176W, P176Y, D177E, D177F, D177H, D177L, D177M, D177R, D177W, D177Y, D178C, D178K, D178R, D178Y, R179K, R179M, R179S, R179W, R94A, Q194C, Q194E, Q194F, Q194G, Q194K, Q194L, N196E, N196L, N196Q, N196T, T209C, T209D, T209E, T209G, T209H, T209I, T209L, T209M, T209Q, T209S, T209V, T209Y, E214W, D215C, D215E, D215G, D215L, D215M, D215N, D215Q, D215S, Q216A, Q216C, Q216F, Q216G, Q216I, Q216K, Q216L, Q216M, Q216S, Q216T, Q216W, Q216Y, K224R, K224V, D225F, D225G, D225H, D225I, D225L, D225M, D225T, D225V, D225W, D225Y, Q226A, Q226C, Q226F, Q226I, Q226L, Q226M, Q226N, Q226R, Q226V, Q226W, Q226Y, N238A, N238C, N238E, T242A, T242C, T242E, T242F, T242H, T242K, T242L, T242M, T242Q, T242V, T242W, T242Y, N248G, N248W, N248Y, N263C, N263E, N263F, N263G, N263Q, N263S, N263T, N263V, N263Y, N264C, R265E, R265K, N276C, S277A, S277C, S277F, S277I, S277M, S277P, S277R, S277W, S277Y, N278A, N278C, N278F, N278G, N278H, N278I, N278L, N278M, N278R, N278S, N278T, N278V, N278Y, Q279C, Q279V, Q279Y, T282C, D287C, D287S, Q301G, Q301K, D302A, D302C, D302F, D302G, Q303A, Q303C, Q303D, Q303P, Y306K, Y306Q, Y306R, S312C, S312W, S312Y, Q316C, Q316P, Q316S, Q316T, Q316Y, K320C, K320N, K320S, K320T, K320Y, R324C, R324Y, R328M, R328S, D329A, D329G, D329N, D329S, L334A, L334C, L334F, L334M, L334T, L334V, K335D, K335R, K335V, K335W, N336R, D337T, D337V, A338C, A338D, A338G, A338I, A338V, A338W, N339E, K344D, K344F, K344I, K344L, K344P, K344Q, K344V, K345A, K345D, K345E, K345F, K345G, K345H, K345S, K345T, K345V, K345Y, A347D, A347Y, H361A, H361C, H361E, H361G, H361L, H361M, H361T, R363C, R363E, R363K, R363L, R363M, R363Q, R363W, R363Y, N369C, N369D, N369E, N369F, N369M, N369S, N369T, N369V, N369W, N369Y, K371T, G372A, G372C, G372D, G372K, G372M, G372N, G372V, G372W, G372Y, D374C, D374F, D374G, D374L, D374M, D374Q, D374S, D374V, D375C, D375E, D375V, D375W, M380N, Y396C, Y396G, Y396K, D397A, D397C, D397E, D397F, D397H, D397I, D397K, D397L, D397M, D397N, D397P, D397Q, D397R, D397S, D397T, D397V, D397Y, A398C, A398D, A398E, A398F, A398I, A398K, A398N, A398P, A398Q, A398R, A398S, A398T, A398V, A398W, A398Y, I399A, I399C, I399D, I399E, I399F, I399Q, I399S, I399T, I399V, I399Y, R402A, R402F, R402G, R402L, R402S, R402W, T411D, T411E, T411F, T411G, T411H, T411K, T411L, T411N, T411Q, T411R, T411S, T411V, S420C, S420G, S420N, S420Q, S420T, S420V, S420Y, R426A, R426F, R426N, R426Q, R426T, R426W, G427C, G427F, G427Y, K428A, T445A, T445C, T445E, T445F, T445G, T445I, T445K, T445L, T445M, T445N, T445P, T445Q, T445R, T445S, T445V, T445Y, V446K, V446Q, V446R, G448A, G448C, G448D, G448E, G448F, N449A, N449C, N449G, N449H, N449K, N449P, N454F, N455C, N455D, N455S, N455W, H460A, H460C, H460D, H460E, H460F, H460G, H460I, H460K, H460L, H460M, H460Q, H460R, H460S, H460W, H460Y, S474C, S474D, S474F, S474G, S474I, S474K, S474L, S474M, S474N, S474P, S474R, S474T, S474V, N475I, N475K, N475L, N475M, N475P, N475Q, N475R, N475S, N475T, N475V, N475W, N475Y, Q490C, Q490L, Q490V, Q490W, Q490Y, L492A, L492D, L492H, L492I, L492N, L492Q, L492T, L492Y, Q496S, Q496W, V497T, K498C, K498E, K498F, K498G, K498I, D521C, D521W, D521Y, V522A, V522C, V522F, V522G, V522K, V522L, V522M, V522N, V522P, V522Q, V522R, V522S, V522T, V522W, V522Y, K534C, K534D, K534E, K534F, K534G, K534V, R542A, R542D, R542I, R542L, R542N, R542T, R542W, G547A, S548C, S548E, S548F, S548L, S548N, S548Q, S548T, S548W, G554A, G554C, G554D, G554F, G554H, G554L, G554M, G554Q, G554W, L555C, L555E, L555G, L555H, L555K, L555M, L555P, L555Q, K560P, H561I, H561M, H561N, H561Q, H561S, H561V, H561W, D563A, D563I, D563L, D563Q, D563R, D563S, D563T, D563V, D563W, D563Y, D564A, D564C, D564F, D564G, D564K, D564L, D564M, D564N, D564R, D564T, D564V, D564Y, R570A, R570C, R570D, R570E, R570I, R570M, R570Q, R570T, R570V, Y571H, Y571M, Y571N, Y571R, K581A, K581C, K581D, K581E, K581F, K581G, K581M, K581W, N583A, N583C, N583D, N583G, N583V, R586D, R586F, R586N, R586P, R586V, R586W, R586Y, V603C, V603E, V603G, V603H, V603Y, F611A, F611C, F611D, F611K, F611M, F611R, F611W, Q612C, Q612D, Q612G, Q612S, A622E, A622H, Q626E, Q626F, Q626H, T638A, T638D, T638G, T638M, T638Q, T638R, T638S, T638V, T638W, T638Y, S642C, S642E, S642F, S642G, S642H, S642I, S642L, S642M, S642P, S642Q, S642T, S642V, S642W, S642Y, A643E, A643F, A643H, A643K, A643L, A643M, A643N, A643T, A643V, A643Y, R645G, R645K, R645M, R645W, R645Y, K649C, K649N, K649S, Q650C, Q650D, Q650T, Q650V, Q650Y, T660C, T660F, T660N, T660S, T660W, T660Y, P661C, P661D, P661E, P661F, P661H, P661I, P661K, P661L, P661M, P661Q, P661R, P661S, P661T, P661V, P661W, G662A, G662C, G662F, G662I, Q663D, Q663E, Q663G, Q663I, T666C, T666N, R672C, R672D, R672E, R672F, R672G, R672H, R672I, R672K, R672L, R672M, R672N, R672T, R672V, R672W, R673A, R673C, R673E, R673G, R673H, R673I, R673K, R673L, R673M, R673N, R673S, R673V, R674T, R674Y, D680A, D680C, D680E, D680F, D680H, D680I, D680M, D680Q, D680R, D680V, D680W, D680Y, T681G, T681H, T681K, T681L, T681M, T681P, T681Q, T681S, T681V, T681W, T681Y, S683C, S683D, S683E, S683F, S683G, S683I, S683M, S683P, S683Q, S683V, S683W, Q684C, Q684G, Q684N, K685A, K685E, K685G, K685I, K685L, K685M, K685N, K685Q, K685S, K685T, K685V, K685W, K685Y, S692C, S692H, S692I, S692L, S692M, S692W, R702C, R702D, R702F, R702G, R702H, R702I, R702K, R702L, R702N, R702Q, R702S, R702T, R702V, R702W, R705I, and R705V, wherein the substitution consists of no more than a single replacement at each of the positions, and wherein the positions are numbered by correspondence with the amino acid sequence of a reference beta-glucosidase 1 (BGL1) set forth as SEQ ID NO:3. As described in the experimental section, exemplary beta-glucosidase variants have improved cellobiose hydrolysis activity at pH 5 (PI greater than 1).

[0109] Also, the present disclosure provides a beta-glucosidase variant comprising a substitution, wherein the substitution comprises one or more of the group consisting of: K022E, K022F, K022H, K022P, K022Q, K022R, N024C, N024Q, L025A, L025D, L025N, Q026C, Q026H, Q026I, Q026K, Q026L, Q026P, Q026R, Q026S, Q026T, Q026W, D027C, K028S, K028V, S033C, S033G, V035C, V035E, V035G, V035H, V035K, V035L, V035N, V035Q, V035R, V035S, V035T, V035Y, G036C, G036D, G036E, G036F, G036I, G036K, G036N, G036R, G036S, G036W, G036Y, K051C, K051D, K051G, K051H, K051I, K051L, K051M, K051N, K051R, K051T, K051V, I052D, I052K, I052M, I052N, I052P, I052Q, I052T, I052V, R091C, R091Q, R091W, E092C, E092K, E099Y, E100A, E100G, E100I, E100M, E100N, E100Q, E100S, E100T, E100Y, Q165C, Q165G, Q165I, Q165K, Q165M, Q165N, Q165S, Q165V, E166D, L167C, L167V, L167W, L167Y, N168A, N168D, N168E, N168G, N168Y, E170F, E170Y, P176F, P176H, P176K, P176L, P176R, P176V, P176W, P176Y, D177E, D177F, D177H, D177L, D177M, D177Q, D177R, D177V, D177W, D177Y, D178Y, R179M, R179S, R179T, R179W, Q194L, N196L, N196T, N208K, T209C, T209D, T209E, T209G, T209H, T209I, T209K, T209L, T209M, T209Q, T209S, T209V, T209Y, E214A, E214D, E214G, E214R, E214S, E214W, D215E, D215H, D215L, D215N, D215S, D215W, Q216A, Q216D, Q216F, Q216G, Q216H, Q216I, Q216K, Q216L, Q216M, Q216N, Q216S, Q216T, Q216W, Q216Y, K224H, K224R, K224V, D225E, D225F, D225G, D225H, D225I, D225L, D225M, D225T, D225V, D225W, D225Y, Q226A, Q226C, Q226D, Q226F, Q226I, Q226L, Q226W, Q226Y, N238A, N238C, N238E, T242C, T242E, T242H, T242Q, T242W, T242Y, N248G, N248W, N248Y, N263C, N263G, N263S, N263T, N264C, N276A, N276C, N276F, N276M, N276Q, S277C, S277W, S277Y, N278C, N278F, N278V, N278W, Q279C, Q279D, Q279K, Q279V, Q279Y, T282C, T282D, T282P, T282S, R284H, R284M, D287C, D287S, Q301K, D302A, D302C, D302E, D302F, D302G, D302L, D302M, D302N, Q303C, Y306K, Y306M, Y306Q, Y306R, S312C, S312K, S312W, S312Y, Q316C, Q316D, Q316G, Q316H, Q316I, Q316K, Q316P, Q316R, Q316S, Q316T, Q316Y, K320C, K320E, K320H, K320L, K320M, K320P, K320Q, K320R, K320S, K320T, R324C, R324Y, R328C, R328E, R328F, R328G, R328I, R328K, R328L, R328M, R328Q, R328S, R328V, R328Y, D329A, D329M, D329S, D329T, D329Y, L334A, L334T, K335N, K335V, D337A, D337C, D337T, D337V, A338C, A338D, A338F, A338G, A338I, A338P, A338V, A338W, K344D, K344F, K344I, K344V, K345A, K345E, K345F, K345G, K345Q, K345S, K345T, K345V, K345W, K345Y, A347Y, H361C, H361G, H361M, R363C, R363E, R363G, R363K, R363Q, R363S, R363T, R363W, R363Y, N369C, N369D, N369E, N369F, N369W, N369Y, K371T, G372A, G372K, G372M, G372W, G372Y, D374C, D374F, D374G, D374I, D374L, D374M, D374Q, D374S, D374T, D374V, D374Y, D375C, D375E, D375H, D375I, D375R, D375V, D375W, M380I, M380N, M380T, M380V, M380Y, W382F, Y396C, Y396D, Y396E, Y396F, Y396G, Y396H, Y396I, Y396K, Y396L, Y396M, Y396N, Y396Q, Y396R, Y396S, Y396V, Y396W, D397A, D397C, D397E, D397H, D397I, D397K, D397M, D397N, D397P, D397Q, D397R, D397S, D397T, D397V, D397Y, A398K, A398Q, A398R, A398W, I399A, I399C, I399D, I399E, I399G, I399Q, I399S, I399T, I399V, I399Y, R402A, R402E, R402G, R402L, R402Q, R402S, R402W, R402Y, V410C, V410F, V410H, V410I, V410R, V410S, V410W, V410Y, T411D, T411E, T411F, T411G, T411H, T411I, T411N, T411Q, T411R, T411S, T411V, T411Y, S420C, S420G, S420N, S420Q, S420T, S420V, S420Y, R426A, R426L, R426T, R426Y, G427C, G427F, G427P, G427V, K428A, T445A, T445C, T445F, T445G, T445M, T445N, T445P, T445V, T445Y, V446K, V446Q, V446R, E447K, E447L, E447S, E447V, E447Y, G448C, G448Y, N449C, N449H, N449K, N454F, N454V, N455C, N455D, N455S, N455V, N455W, H460A, H460C, H460E, H460F, H460G, H460I, H460K, H460L, H460M, H460N, H460Q, H460R, H460S, H460W, H460Y, N473W, S474A, S474C, S474E, S474F, S474K, S474L, S474N, S474P, S474T, N475I, N475M, N475S, N475T, N475W, N475Y, E489D, E489N, Q490C, Q490V, Q490W, Q490Y, L492A, L492D, L492F, L492H, L492I, L492N, L492R, L492T, L492Y, Q496W, K498A, K498E, K498F, K498M, K498V, D521C, V522A, V522C, V522G, V522K, V522L, V522M, V522Q, V522R, V522S, V522T, V522W, V522Y, K534C, K534D, K534E, K534R, K534V, R542A, R542C, R542D, R542E, R542F, R542G, R542H, R542I, R542L, R542M, R542N, R542Q, R542S, R542T, R542V, R542W, R542Y, G547A, G547C, S548T, E553I, E553Y, G554C, G554D, G554F, G554H, G554Q, G554W, L555D, L555E, L555F, L555G, L555H, L555K, L555M, L555P, L555Q, L555T, L555V, L555W, L555Y, H561A, H561C, H561D, H561G, H561M, H561S, H561W, D563A, D563E, D563L, D563M, D563Q, D563S, D563T, D563V, D563W, D563Y, D564A, D564C, D564F, D564K, D564L, D564N, D564Q, D564R, D564T, D564V, D564Y, R570A, R570C, R570D, R570E, R570M, R570Q, R570S, R570T, R570V, Y571N, K581A, K581C, K581D, K581F, K581G, K581I, K581S, K581V, K581W, K581Y, N583A, N583C, N583D, N583E, N583F, N583G, N583H, N583I, N583K, N583L, N583M, N583P, N583R, N583S, N583T, N583V, N583W, N583Y, R586D, R586F, R586G, R586L, R586N, R586P, R586V, R586W, R586Y, S591D, V603C, V603D, V603F, V603G, V603H, V603M, V603N, V603P, V603Q, V603R, V603S, V603T, V603W, V603Y, F611A, F611C, F611K, F611R, F611V, F611W, F611Y, Q612C, Q612D, Q612G, Q612H, Q612S, A622D, A622G, A622H, A622I, A622K, A622L, A622M, A622P, A622S, A622T, A622V, A622Y, Q626G, Q626H, Q626L, Q626T, Q626V, T638A, T638D, T638G, T638M, T638Q, T638R, T638S, T638V, T638W, T638Y, S642C, S642E, S642F, S642H, S642L, S642P, S642Q, S642T, S642W, S642Y, A643H, A643K, A643L, A643Q, A643T, A643V, A643W, A643Y, R645A, R645D, R645F, R645G, R645I, R645K, R645L, R645M, R645T, R645V, R645W, R645Y, K649T, Q650E, Q650G, Q650H, Q650I, Q650K, Q650L, Q650N, Q650R, Q650T, Q650Y, T660C, T660D, T660N, T660S, T660W, T660Y, P661A, P661C, P661D, P661E, P661F, P661G, P661H, P661I, P661K, P661L, P661M, P661Q, P661R, P661S, P661T, P661V, P661W, G662A, G662C, G662F, G662I, Q663C, Q663D, Q663E, Q663F, Q663G, Q663H, Q663I, T666C, T666N, T666R, R672C, R672D, R672E, R672F, R672G, R672H, R672I, R672K, R672L, R672M, R672N, R672T, R672V, R672W, R672Y, R673E, R673G, R673I, R673L, R673M, R673Q, R673S, R674T, R674Y, D675L, D680A, D680C, D680E, D680F, D680H, D680I, D680L, D680M, D680Q, D680V, D680W, D680Y, T681A, T681G, T681H, T681K, T681L, T681M, T681N, T681P, T681Q, T681S, T681V, T681W, S683E, S683F, S683I, S683L, S683M, S683P, S683V, S683W, Q684A, Q684C, Q684G, K685I, K685L, K685M, K685N, K685S, K685T, K685V, K685W, K685Y, S692C, S692H, S692I, S692M, S692T, S692V, S692W, R702C, R702D, R702G, R702L, R702Q, R702S, R702T, R702V, R702W, R705F, R705H, R705I, R705L, R705S, R705T, R705V, and R705W, wherein the substitution consists of no more than a single replacement at each of the positions, and wherein the positions are numbered by correspondence with the amino acid sequence of a reference beta-glucosidase 1 (BGL1) set forth as SEQ ID NO:3. As described in the experimental section, exemplary beta-glucosidase variants have improved cellobiose hydrolysis activity at pH 6 (PI greater than 1).

[0110] The present disclosure further provides a beta-glucosidase variant comprising a substitution, wherein the substitution comprises one or more of the group consisting of: K022A, K022E, K022F, K022P, N024A, N024C, N024Q, L025A, L025D, L025S, Q026C, Q026D, Q026E, Q026K, Q026S, Q026T, Q026W, D027A, D027L, D027M, D027Q, D027S, S033C, V035C, V035E, V035G, G036D, G036E, G036S, G036Y, S050C, S050L, K051C, K051D, K051H, K051T, I052A, I052D, I052N, I052P, I052V, R091D, R091E, R091F, R091N, E092A, E092C, E100A, E100G, E100M, E100N, E100S, E100Y, L167C, L167W, N168Y, P176A, P176F, P176K, P176R, P176S, P176V, P176W, P176Y, D177E, D177F, D177M, D177N, D177V, D177W, D178A, Q194A, Q194C, N196E, N196L, T209C, T209D, T209E, T209G, T209L, T209S, T209V, T209W, T209Y, E214D, E214V, D215C, D215E, D215N, D215S, Q216A, Q216G, Q216H, Q216I, Q216L, Q216S, Q216W, Q216Y, D225E, D225G, D225H, D225I, D225L, D225T, D225W, D225Y, Q226C, Q226D, Q226F, Q226N, Q226R, Q226S, Q226W, Q226Y, N238A, T242C, T242E, T242H, T242L, T242S, T242W, T242Y, N248C, N248W, N263A, N263C, N263D, N263E, N263G, N263H, N263L, N263Q, N263R, N263S, N263T, N264C, N276A, N276C, N276F, N276K, S277C, S277W, N278F, N278W, Q279C, T282C, T282L, T282P, R284M, D287C, D287K, D302A, D302C, D302E, D302F, D302G, Q303C, Y306C, Y306G, Y306M, Y306Q, Y306V, S312C, S312D, S312W, S312Y, Q316C, Q316P, Q316S, Q316T, Q316Y, K320C, K320N, R324C, R328S, D329A, D329S, K335D, N336A, N336C, N336H, D337A, D337C, D337K, D337M, D337T, A338C, A338D, A338E, A338F, A338G, A338K, A338L, A338M, A338P, A338V, A338W, A338Y, K344D, K344F, K344G, K344L, K344Q, K344R, K344V, K345A, K345E, K345F, K345S, K345Y, A347Y, H361D, H361G, R363A, R363C, R363E, R363G, R363K, R363M, R363Q, R363T, R363V, R363W, R363Y, N369C, N369D, N369E, N369T, N369W, K371A, K371L, K371T, G372A, D374C, D374L, D374M, D374S, D375C, M380T, M380V, G381H, W382F, D397C, D397N, D397R, D397T, D397V, D397Y, A398R, A398W, I399L, I399V, R402A, R402I, V410F, V410R, V410S, T411E, T411H, T411N, S420C, S420D, S420G, S420N, S420T, S420V, R426A, G427C, G427E, G427F, G427H, G427L, G427Y, T445A, T445C, T445D, T445E, T445F, T445G, T445I, T445M, T445P, T445V, T445Y, V446A, V446C, G448C, G448F, G448H, N449A, N449C, N454F, N455C, N455D, N455W, H460D, H460F, H460G, H460K, H460Q, S474A, S474C, S474E, S474F, S474I, S474M, S474N, S474T, S474V, S474Y, N475S, Q490C, Q490E, Q490G, Q490H, Q490L, Q490V, Q490W, Q490Y, V497T, K498A, K498E, K498M, D521A, D521S, D521W, D521Y, V522C, V522S, V522W, V522Y, K534C, K534E, K534F, K534N, K534V, R542A, R542C, R542D, R542E, R542F, R542G, R542K, R542L, R542M, R542N, R542Q, R542T, R542V, R542W, G547A, S548C, S548E, S548F, S548W, E553N, G554C, G554F, G554Q, G554W, K560H, H561F, H561G, H561I, H561M, H561N, H561S, H561T, H561V, H561W, D563A, D563S, D564A, D564F, D564S, D564T, K581A, K581C, K581D, K581F, K581G, K581H, K581P, K581S, K581T, K581V, K581W, N583A, N583C, N583D, R586D, R586N, R586P, R586V, R586Y, V603A, V603C, V603D, V603E, V603F, V603G, V603H, V603Y, F611A, Q612C, A622D, A622E, A622F, A622G, A622H, A622M, A622N, A622R, A622S, A622T, A622V, Q626E, Q626F, Q626G, Q626H, Q626M, T638A, T638G, T638W, S642F, S642L, S642W, A643V, R645G, K649A, K649C, K649S, Q650E, Q650G, Q650H, Q650V, Q650Y, T660W, P661A, P661C, P661D, P661F, P661I, P661K, P661L, P661M, P661Q, P661R, P661S, P661T, P661V, P661W, G662A, G662C, G662D, G662F, Q663A, Q663C, Q663D, Q663E, Q663F, Q663G, Q663H, Q663L, Q663N, Q663R, Q663S, Q663V, T666A, T666C, T666N, R672C, R672K, R672T, R673A, R673C, R673G, R673H, R673I, R673K, R673L, R673M, R673S, R673T, R673V, R673W, R674M, R674T, R674V, D680A, D680C, D680M, D680Q, D680V, D680W, D680Y, T681A, T681G, T681K, T681L, T681M, T681P, T681Q, T681S, T681V, T681W, A682M, S683E, S683M, S683V, S683W, Q684A, Q684C, Q684G, Q684N, K685A, K685E, K685I, K685L, K685M, K685S, K685T, K685W, K685Y, S692C, S692H, S692I, S692L, S692M, S692P, S692V, S692W, R702C, R702G, R702S, R705C, R705I, R705T, and R705V, wherein the substitution consists of no more than a single replacement at each of the positions, and wherein the positions are numbered by correspondence with the amino acid sequence of a reference beta-glucosidase 1 (BGL1) set forth as SEQ ID NO:3. As described in the experimental section, exemplary beta-glucosidase variants have improved cellobiose hydrolysis in presence of ammonia pretreated corncob (PI greater than 1).

[0111] Also, the present disclosure provides a beta-glucosidase variant comprising a substitution, wherein the substitution comprises one or more of the group consisting of: K022A, K022E, K022F, K022G, K022H, K022P, K022Q, K022R, K022S, K022V, K022W, K022Y, N024A, N024C, N024D, N024E, N024F, N024G, N024L, N024P, N024Q, N024R, N024S, N024V, N024Y, L025A, L025D, L025F, L025G, L025I, L025K, L025N, L025Q, L025R, L025S, L025T, L025V, L025W, L025Y, Q026C, Q026D, Q026E, Q026G, Q026H, Q026I, Q026K, Q026L, Q026P, Q026R, Q026S, Q026T, Q026V, Q026W, Q026Y, D027A, D027C, D027E, D027L, D027M, D027Q, D027S, D027T, D027V, K028S, K028V, S033C, S033G, S033T, V035C, V035E, V035G, V035H, V035K, V035L, V035N, V035P, V035Q, V035R, V035S, V035T, V035Y, G036C, G036D, G036E, G036F, G036I, G036K, G036N, G036R, G036S, G036W, G036Y, W037V, W037Y, S050A, S050C, S050F, S050G, S050I, S050L, S050M, S050N, S050P, S050R, S050T, S050V, K051A, K051C, K051D, K051E, K051G, K051H, K051I, K051L, K051M, K051N, K051Q, K051R, K051S, K051T, K051V, I052A, I052D, I052K, I052M, I052N, I052P, I052Q, I052T, I052V, R067A, R067C, R067D, R067F, R067G, R067N, R067P, R067Q, R067S, R067W, R091A, R091C, R091D, R091E, R091F, R091G, R091H, R091I, R091K, R091L, R091N, R091Q, R091T, R091V, R091W, R091Y, E092A, E092C, E092D, E092F, E092H, E092I, E092K, E092L, E092N, E092Q, E092R, E092T, E092V, E092Y, R093K, E099A, E099D, E099F, E099I, E099K, E099M, E099N, E099W, E099Y, E100A, E100G, E100I, E100M, E100N, E100Q, E100S, E100T, E100Y, K158H, K158T, E164G, E164S, Q165C, Q165F, Q165G, Q165H, Q165I, Q165K, Q165L, Q165M, Q165N, Q165R, Q165S, Q165T, Q165V, Q165W, Q165Y, E166D, E166K, E166L, E166P, E166Q, E166R, E166T, L167A, L167C, L167D, L167E, L167F, L167G, L167M, L167N, L167Q, L167R, L167S, L167V, L167W, L167Y, N168A, N168D, N168E, N168G, N168H, N168Q, N168R, N168T, N168Y, E170D, E170F, E170Y, P176A, P176D, P176E, P176F, P176G, P176H, P176K, P176L, P176M, P176Q, P176R, P176S, P176T, P176V, P176W, P176Y, D177E, D177F, D177H, D177K, D177L, D177M, D177N, D177Q, D177R, D177V, D177W, D177Y, D178A, D178C, D178K, D178N, D178Q, D178R, D178Y, R179A, R179C, R179G, R179I, R179K, R179M, R179Q, R179S, R179T, R179V, R179W, Q194A, Q194C, Q194E, Q194F, Q194G, Q194H, Q194K, Q194L, Q194M, Q194R, Q194T, Q194W, Q194Y, N196E, N196H, N196L, N196Q, N196R, N196T, S199A, S199G, S199N, S199T, S199V, Y204F, N208K, T209C, T209D, T209E, T209G, T209H, T209I, T209K, T209L, T209M, T209Q, T209R, T209S, T209V, T209W, T209Y, E214A, E214C, E214D, E214G, E214H, E214L, E214M, E214N, E214Q, E214R, E214S, E214T, E214V, E214W, E214Y, D215C, D215E, D215G, D215H, D215L, D215M, D215N, D215Q, D215S, D215W, Q216A, Q216C, Q216D, Q216E, Q216F, Q216G, Q216H, Q216I, Q216K, Q216L, Q216M, Q216N, Q216P, Q216R, Q216S, Q216T, Q216W, Q216Y, K224H, K224R, K224V, D225A, D225C, D225E, D225F, D225G, D225H, D225I, D225L, D225M, D225Q, D225S, D225T, D225V, D225W, D225Y, Q226A, Q226C, Q226D, Q226E, Q226F, Q226H, Q226I, Q226K, Q226L, Q226M, Q226N, Q226R, Q226S, Q226T, Q226V, Q226W, Q226Y, N238A, N238C, N238E, N238G, N238M, N238S, N238T, T242A, T242C, T242E, T242F, T242G, T242H, T242I, T242K, T242L, T242M, T242N, T242Q, T242R, T242S, T242V, T242W, T242Y, N248A, N248C, N248F, N248G, N248T, N248W, N248Y, S249A, S249G, S249M, S249V, N263A, N263C, N263D, N263E, N263F, N263G, N263H, N263L, N263Q, N263R, N263S, N263T, N263V, N263Y, N264C, R265A, R265E, R265I, R265K, R265L, R265M, R265N, R265Q, R265Y, N276A, N276C, N276F, N276K, N276M, N276Q, S277A, S277C, S277D, S277E, S277F, S277G, S277H, S277I, S277M, S277P, S277Q, S277R, S277W, S277Y, N278A, N278C, N278D, N278F, N278G, N278H, N278I, N278L, N278M, N278Q, N278R, N278S, N278T, N278V, N278W, N278Y, Q279C, Q279D, Q279E, Q279G, Q279H, Q279I, Q279K, Q279N, Q279S, Q279T, Q279V, Q279Y, T282C, T282D, T282K, T282L, T282P, T282S, R284H, R284M, D287C, D287E, D287G, D287H, D287I, D287K, D287M, D287N, D287S, Q301A, Q301E, Q301G, Q301K, Q301L, Q301N, Q301R, Q301S, Q301T, Q301V, D302A, D302C, D302E, D302F, D302G, D302K, D302L, D302M, D302N, D302P, D302S, D302T, D302W, D302Y, Q303A, Q303C, Q303D, Q303E, Q303H, Q303I, Q303K, Q303L, Q303M, Q303N, Q303P, Q303R, Q303S, Q303T, Q303V, Q303W, Q303Y, Y306C, Y306G, Y306I, Y306K, Y306L, Y306M, Y306N, Y306P, Y306Q, Y306R, Y306S, Y306T, Y306V, S312C, S312D, S312G, S312K, S312N, S312Q, S312R, S312T, S312V, S312W, S312Y, R313A, R313C, R313D, R313G, R313K, R313N, Q316C, Q316D, Q316G, Q316H, Q316I, Q316K, Q316L, Q316M, Q316P, Q316R, Q316S, Q316T, Q316Y, K320C, K320E, K320G, K320H, K320L, K320M, K320N, K320P, K320Q, K320R, K320S, K320T, K320Y, R324C, R324D, R324E, R324F, R324H, R324I, R324K, R324L, R324M, R324Q, R324V, R324W, R324Y, R328C, R328E, R328F, R328G, R328I, R328K, R328L, R328M, R328Q, R328S, R328T, R328V, R328Y, D329A, D329E, D329G, D329H, D329M, D329N, D329Q, D329S, D329T, D329Y, L334A, L334C, L334F, L334M, L334T, L334V, L334W, K335A, K335D, K335F, K335H, K335I, K335L, K335M, K335N, K335R, K335S, K335T, K335V, K335W, N336A, N336C, N336G, N336H, N336L, N336M, N336Q, N336R, N336T, N336V, N336Y, D337A, D337C, D337E, D337G, D337H, D337K, D337L, D337M, D337N, D337R, D337S, D337T, D337V, D337W, D337Y, A338C, A338D, A338E, A338F, A338G, A338H, A338I, A338K, A338L, A338M, A338N, A338P, A338Q, A338R, A338V, A338W, A338Y, N339E, N339G, N339H, N339K, N339L, K344D, K344E, K344F, K344G, K344I, K344L, K344M, K344N, K344P, K344Q, K344R, K344S, K344T, K344V, K345A, K345D, K345E, K345F, K345G, K345H, K345N, K345P, K345Q, K345R, K345S, K345T, K345V, K345W, K345Y, A347D, A347F, A347H, A347I, A347K, A347L, A347M, A347P, A347Q, A347R, A347S, A347Y, H361A, H361C, H361D, H361E, H361G, H361L, H361M, H361N, H361T, R363A, R363C, R363E, R363G, R363K, R363L, R363M, R363Q, R363S, R363T, R363V, R363W, R363Y, N369C, N369D, N369E, N369F, N369L, N369M, N369S, N369T, N369V, N369W, N369Y, D370E, D370F, D370G, D370S, D370W, D370Y, K371A, K371F, K371G, K371L, K371N, K371Q, K371R, K371S, K371T, K371V, G372A, G372C, G372D, G372E, G372K, G372L, G372M, G372N, G372T, G372V, G372W, G372Y, D374C, D374F, D374G, D374I, D374L, D374M, D374N, D374Q, D374S, D374T, D374V, D374Y, D375A, D375C, D375E, D375H, D375I, D375R, D375V, D375W, M380I, M380L, M380N, M380Q, M380S, M380T, M380V, M380Y, G381H, W382F, Y396A, Y396C, Y396D, Y396E, Y396F, Y396G, Y396H, Y396I, Y396K, Y396L, Y396M, Y396N, Y396Q, Y396R, Y396S, Y396T, Y396V, Y396W, D397A, D397C, D397E, D397F, D397H, D397I, D397K, D397L, D397M, D397N, D397P, D397Q, D397R, D397S, D397T, D397V, D397Y, A398C, A398D, A398E, A398F, A398G, A398H, A398I, A398K, A398L, A398M, A398N, A398P, A398Q, A398R, A398S, A398T, A398V, A398W, A398Y, I399A, I399C, I399D, I399E, I399F, I399G, I399L, I399M, I399Q, I399S, I399T, I399V, I399W, I399Y, R402A, R402C, R402E, R402F, R402G, R402I, R402L, R402Q, R402S, R402W, R402Y, Q409D, Q409G, V410A, V410C, V410F, V410H, V410I, V410L, V410N, V410R, V410S, V410T, V410W, V410Y, T411D, T411E, T411F, T411G, T411H, T411I, T411K, T411L, T411N, T411Q, T411R, T411S, T411V, T411Y, S420C, S420D, S420G, S420H, S420K, S420N, S420Q, S420T, S420V, S420Y, R426A, R426E, R426F, R426I, R426K, R426L, R426N, R426P, R426Q, R426S, R426T, R426W, R426Y, G427C, G427D, G427E, G427F, G427H, G427K, G427L, G427M, G427N, G427P, G427Q, G427R, G427S, G427T, G427V, G427Y, K428A, K428C, K428D, K428E, K428F, K428G, K428H, K428I, K428L, K428M, K428N, K428P, K428Q, K428R, K428S, K428T, K428V, K428W, K428Y, T445A, T445C, T445D, T445E, T445F, T445G, T445I, T445K, T445L, T445M, T445N, T445P, T445Q, T445R, T445S, T445V, T445Y, V446A, V446C, V446K, V446Q, V446R, E447K, E447L, E447S, E447V, E447Y, G448A, G448C, G448D, G448E, G448F, G448H, G448N, G448S, G448T, G448Y, N449A, N449C, N449E, N449F, N449G, N449H, N449K, N449M, N449P, N449T, N449V, N454F, N454G, N454K, N454L, N454M, N454R, N454S, N454T, N454V, N455A, N455C, N455D, N455E, N455F, N455G, N455H, N455I, N455L, N455M, N455S, N455T, N455V, N455W, N455Y, H460A, H460C, H460D, H460E, H460F, H460G, H460I, H460K, H460L, H460M, H460N, H460Q, H460R, H460S, H460W, H460Y, Q467A, Q467P, Q467S, N473A, N473C, N473E, N473F, N473G, N473H, N473K, N473L, N473M, N473P, N473Q, N473R, N473S, N473T, N473V, N473W, S474A, S474C, S474D, S474E, S474F, S474G, S474I, S474K, S474L, S474M, S474N, S474P, S474Q, S474R, S474T, S474V, S474Y, N475I, N475K, N475L, N475M, N475P, N475Q, N475R, N475S, N475T, N475V, N475W, N475Y, E489D, E489N, Q490A, Q490C, Q490E, Q490F, Q490G, Q490H, Q490K, Q490L, Q490P, Q490R, Q490S, Q490T, Q490V, Q490W, Q490Y, L492A, L492D, L492F, L492H, L492I, L492M, L492N, L492Q, L492R, L492T, L492W, L492Y, Q496G, Q496S, Q496W, V497A, V497C, V497I, V497M, V497T, K498A, K498C, K498E, K498F, K498G, K498H, K498I, K498L, K498M, K498N, K498Q, K498T, K498V, K498Y, D521A, D521C, D521E, D521F, D521G, D521H, D521I, D521K, D521L, D521M, D521P, D521R, D521S, D521T, D521V, D521W, D521Y, V522A, V522C, V522F, V522G, V522H, V522I, V522K, V522L, V522M, V522N, V522P, V522Q, V522R, V522S, V522T, V522W, V522Y, K534C, K534D, K534E, K534F, K534G, K534N, K534R, K534V, R542A, R542C, R542D, R542E, R542F, R542G, R542H, R542I, R542K, R542L, R542M, R542N, R542P, R542Q, R542S, R542T, R542V, R542W, R542Y, G547A, G547C, G547L, G547P, S548C, S548E, S548F, S548H, S548I, S548L, S548M, S548N, S548Q, S548R, S548T, S548V, S548W, S548Y, E553I, E553N, E553Y, G554A, G554C, G554D, G554F, G554H, G554L, G554M, G554Q, G554V, G554W, L555A, L555C, L555D, L555E, L555F, L555G, L555H, L555I, L555K, L555M, L555N, L555P, L555Q, L555T, L555V, L555W, L555Y, K560A, K560E, K560G, K560H, K560P, K560R, K560W, H561A, H561C, H561D, H561E, H561F, H561G, H561I, H561M, H561N, H561Q, H561S, H561T, H561V, H561W, D563A, D563C, D563E, D563F, D563I, D563L, D563M, D563Q, D563R, D563S, D563T, D563V, D563W, D563Y, D564A, D564C, D564F, D564G, D564K, D564L, D564M, D564N, D564Q, D564R, D564S, D564T, D564V, D564Y, R570A, R570C, R570D, R570E, R570G, R570H, R570I, R570M, R570Q, R570S, R570T, R570V, Y571H, Y571M, Y571N, Y571R, Y571W, K581A, K581C, K581D, K581E, K581F, K581G, K581H, K581I, K581L, K581M, K581N, K581P, K581R, K581S, K581T, K581V, K581W, K581Y, N583A, N583C, N583D, N583E, N583F, N583G, N583H, N583I, N583K, N583L, N583M, N583P, N583R, N583S, N583T, N583V, N583W, N583Y, R586D, R586E, R586F, R586G, R586H, R586L, R586N, R586P, R586V, R586W, R586Y, S591D, V603A, V603C, V603D, V603E, V603F, V603G, V603H, V603M, V603N, V603P, V603Q, V603R, V603S, V603T, V603W, V603Y, F611A, F611C, F611D, F611K, F611L, F611M, F611N, F611R, F611S, F611V, F611W, F611Y, Q612C, Q612D, Q612F, Q612G, Q612H, Q612I, Q612K, Q612L, Q612M, Q612R, Q612S, Q612W, A622D, A622E, A622F, A622G, A622H, A622I, A622K, A622L, A622M, A622N, A622P, A622R, A622S, A622T, A622V, A622Y, Q626E, Q626F, Q626G, Q626H, Q626L, Q626M, Q626T, Q626V, V627P, T638A, T638D, T638E, T638F, T638G, T638I, T638K, T638L, T638M, T638P, T638Q, T638R, T638S, T638V, T638W, T638Y, S642A, S642C, S642E, S642F, S642G, S642H, S642I, S642K, S642L, S642M, S642N, S642P, S642Q, S642R, S642T, S642V, S642W, S642Y, A643C, A643E, A643F, A643G, A643H, A643K, A643L, A643M, A643N, A643Q, A643R, A643S, A643T, A643V, A643W, A643Y, R645A, R645D, R645F, R645G, R645H, R645I, R645K, R645L, R645M, R645P, R645Q, R645T, R645V, R645W, R645Y, K649A, K649C, K649F, K649I, K649L, K649M, K649N, K649Q, K649S, K649T, K649W, K649Y, Q650A, Q650C, Q650D, Q650E, Q650F, Q650G, Q650H, Q650I, Q650K, Q650L, Q650M, Q650N, Q650R, Q650T, Q650V, Q650Y, T660C, T660D, T660F, T660I, T660N, T660S, T660W, T660Y, P661A, P661C, P661D, P661E, P661F, P661G, P661H, P661I, P661K, P661L, P661M, P661Q, P661R, P661S, P661T, P661V, P661W, G662A, G662C, G662D, G662F, G662H, G662I, G662K, G662N, G662R, G662T, G662W, G662Y, Q663A, Q663C, Q663D, Q663E, Q663F, Q663G, Q663H, Q663I, Q663K, Q663L, Q663M, Q663N, Q663R, Q663S, Q663V, Q663W, T666A, T666C, T666D, T666H, T666K, T666N, T666R, T666W, R672C, R672D, R672E, R672F, R672G, R672H, R672I, R672K, R672L, R672M, R672N, R672T, R672V, R672W, R672Y, R673A, R673C, R673E, R673G, R673H, R673I, R673K, R673L, R673M, R673N, R673Q, R673S, R673T, R673V, R673W, R674L, R674M, R674T, R674V, R674Y, D675C, D675E, D675H, D675L, D675S, D675Y, D680A, D680C, D680E, D680F, D680H, D680I, D680K, D680L, D680M, D680N, D680Q, D680R, D680S, D680V, D680W, D680Y, T681A, T681G, T681H, T681K, T681L, T681M, T681N, T681P, T681Q, T681R, T681S, T681V, T681W, T681Y, A682M, S683A, S683C, S683D, S683E, S683F, S683G, S683I, S683L, S683M, S683P, S683Q, S683R, S683V, S683W, Q684A, Q684C, Q684D, Q684G, Q684N, Q684P, K685A, K685E, K685F, K685G, K685I, K685L, K685M, K685N, K685Q, K685R, K685S, K685T, K685V, K685W, K685Y, S692C, S692E, S692H, S692I, S692K, S692L, S692M, S692N, S692P, S692Q, S692T, S692V, S692W, R702C, R702D, R702F, R702G, R702H, R702I, R702K, R702L, R702M, R702N, R702Q, R702S, R702T, R702V, R702W, R705C, R705F, R705H, R705I, R705L, R705M, R705P, R705S, R705T, R705V, and R705W, wherein the substitution consists of no more than a single replacement at each of the positions, and wherein the positions are numbered by correspondence with the amino acid sequence of a reference beta-glucosidase 1 (BGL1) set forth as SEQ ID NO:3. As described in the experimental section, exemplary beta-glucosidase variants have improved beta-glucosidase activity (PI greater than 1 on either CNPG, PASC, PCS, G2 at pH 5, G2 at pH 6, or G2+CC).

[0112] In still further embodiments, the present disclosure provides a beta-glucosidase variant of any of the preceding paragraphs of the summary, wherein the variant is isolated. The present disclosure provides a composition comprising the beta-glucosidase variant. In a preferred embodiment, the composition is enriched in the beta-glucosidase variant.

[0113] In addition, the present disclosure provides an isolated nucleic acid encoding a beta-glucosidase variant of any of the preceding paragraphs of the summary. In a preferred embodiment, the disclosure provides an expression vector comprising the isolated nucleic acid operably linked to a regulatory sequence. In another embodiment, the disclosure provides a host cell comprising the expression vector. The present disclosure further provides a method for producing a beta-glucosidase variant, comprising culturing the host cell in a culture medium under suitable conditions to produce the beta-glucosidase variant. As such in another embodiment, the disclosure provides a composition comprising the host cell and culture medium. Similarly the disclosure also provides a composition comprising the beta-glucosidase variant in supernatant of the culture medium.

[0114] Moreover, the present disclosure provides a method of converting biomass to sugars comprising contacting the biomass with a beta-glucosidase variant of any of the preceding paragraphs of the summary. The present disclosure further provides a method of producing a fuel comprising contacting a biomass composition with a composition comprising a beta-glucosidase variant of any of the preceding paragraphs of the summary, to yield a sugar solution; and culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fuel.

BRIEF DESCRIPTION OF THE DRAWING

[0115] FIG. 1 provides an alignment of the amino acid sequences of the mature form of various cellulases: TrireBGL1, Hypocrea jecorina (also known as Trichoderma reesei) Q12715 beta-D-glucoside glucohydrolase 1 (SEQ ID NO:3); HananBglu, Hansenula anomala P06835 beta-glucosidase (SEQ ID NO:4); PirspBglu, Piromyces sp. E2 Q875K3 Beta-glucosidase (SEQ ID NO:5); CocimBglu, Coccidioides immitis 014424 Beta-glucosidase (SEQ ID NO:6); SacfiBglu2, Saccharomycopsis fibuligera beta-glucosidase 2 (SEQ ID NO:7); SacfiBglu1, Saccharomycopsis fibuligera P22506 beta-glucosidase 1 (SEQ ID NO:8); SeplyBglu, Septoria lycopersici Q99324 beta-1,2-D-glucosidase (SEQ ID NO:9); KurcaBglu, Kuraishia capsulata Q12653 beta-glucosidase (SEQ ID NO:10); TrireBGL7, Trichoderma reesei Q7Z9M0 beta-glucosidase 7 (SEQ ID NO:11); UrofaBglu, Uromyces fabae Q70KQ7 beta glucosidase (SEQ ID NO:12); AspteBglu, Aspergillus terreus (strain NIH 2624/FGSC A1156) Q0CEF3 beta-glucosidase (SEQ ID NO:13); ChaglBglu, Chaetomium globosum Q2GZ54 Putative beta-glucosidase (SEQ ID NO:14); TrireBGL3, Trichoderma reesei Q7Z9M5 beta-glucosidase 3 (SEQ ID NO:15); PenbrBGL, Penicillium brasilianum GH3 beta-glucosidase (SEQ ID NO:16); PerspBglu, Periconia sp. BCC 2871 A9UIG0 beta-glucosidase (SEQ ID NO:17); PhaavBglu, Phaeosphaeria avenaria Q9P879 beta-glucosidase (SEQ ID NO:18); AspfuBGL, Aspergillus fumigatus B0XPE1 beta-glucosidase (SEQ ID NO:19); AsporBGL1, Aspergillus oryzae Q2UUD6 beta-glucosidase (SEQ ID NO:20); AspacBGL1, Aspergillus aculeatus beta-glucosidase (SEQ ID NO:21); AspniBGL, Aspergillus niger Q9P8F4 beta-glucosidase (SEQ ID NO:22); TalemBglu, Talaromyces emersonii Q8TGI8 beta-glucosidase (SEQ ID NO:23); and TheauBGL, Thermoascus aurentiacus beta-glucosidase (SEQ ID NO:24). The full length sequences shown of the various cellulases correspond to SEQ ID NOS: 25-34, 2, 35-45, respectively.

[0116] FIG. 2 depicts a destination vector pTTT-pyrG13 and an expression vector pTTT-pyrG-bgl1 as described herein.

DETAILED DESCRIPTION

[0117] The present disclosure is generally directed to enzymes and in particular beta-glucosidase variants. Also described are nucleic acids encoding beta-glucosidase variants, compositions comprising beta-glucosidase variants, methods of using beta-glucosidase variants, and methods of identifying additional useful beta-glucosidase variants

[0118] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the compositions and methods described herein. Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In this application, the use of the singular includes the plural unless specifically stated otherwise. The use of "or" means "and/or" unless stated otherwise. Likewise, the terms "comprise," "comprising," "comprises," "include," "including" and "includes" are not intended to be limiting. All patents and publications, including all amino acid and nucleotide sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference. The headings provided herein are not limitations of the various aspects or embodiments of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms herein are more fully defined by reference to the specification as a whole.

[0119] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2nd Ed., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide one of skill with a general dictionary of many of the terms used in this disclosure. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxyl orientation, respectively. Practitioners are particularly directed to Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL (Second Edition), Cold Spring Harbor Press, Plainview, N.Y., 1989, and Ausubel F M et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1993, for definitions and terms of the art. It is to be understood that this disclosure is not limited to the particular methodology, protocols, and reagents described, as these may vary.

I. DEFINITIONS

[0120] The terms below are more fully defined by reference to the specification as a whole.

[0121] The term "polypeptide" as used herein refers to a compound made up of a single chain of amino acid residues linked by peptide bonds. The term "protein" as used herein may be synonymous with the term "polypeptide".

[0122] "Variant" means a protein which is derived from a precursor protein (e.g., the native protein) by one or more of: addition(s) of one or more amino acids to one or more of the C-terminal end, the N-terminal end, and site(s) within the amino acid sequence; substitution of one or more amino acids at site(s) within the amino acid sequence; and deletion of one or more amino acids at one or more of the C-terminal end, the N-terminal end, and sites within the amino acid sequence. The preparation of a beta-glucosidase variant may be performed by any means know in the art. In preferred embodiments, a beta-glucosidase variant is prepared by modifying a DNA sequence which encodes for the native protein, transformation of the modified DNA sequence into a suitable host, and expression of the modified DNA sequence to form the variant enzyme. The beta-glucosidase variant of the disclosure includes peptides comprising altered amino acid sequences in comparison with a precursor enzyme amino acid sequence wherein the variant beta-glucosidase retains the characteristic beta-glucosidase activity of the precursor enzyme but which may have altered properties in some specific aspect. For example, a variant beta-glucosidase may have an altered (increased or decreased) level of expression, activity and stability relative to a reference beta-glucosidase. It is contemplated that the variants according to the present disclosure may be derived from a DNA fragment encoding a cellulase variant wherein the functional activity of the expressed cellulase variant is retained. For example, a DNA fragment encoding a cellulase may further include a DNA sequence or portion thereof encoding a hinge or linker attached to the cellulase DNA sequence at either the 5' or 3' end wherein the functional activity of the encoded cellulase domain is retained. The terms variant and derivative may be used interchangeably herein. Moreover, "variant" as used herein can also refer to any polypeptide that has a different sequence from that of a wild type polypeptide. For example, a BGL1 variant polypeptide can be synthesized de novo, based on the variant sequence and one or more particular substitutions described herein. As such, the beta-glucosidase variants of the disclosure include polypeptides comprising altered amino acid sequences as compared to a wild-type BGL1.

[0123] For the purpose of the present disclosure, variants are often referred to by the substitutions at particular amino acid residues. For example, a variant can be referred to by the symbol "X(#)Y", which refers to a variant comprising a substitution at residue number "#," which is an X residue in the wild type polypeptide but is a Y residue at the same position in the variant. Accordingly a BGL1 variant X#Y refers to a BGL1 variant comprising a substitution at position or residue number #, where the X residue of the wild type BGL1 reference enzyme is replaced or substituted with a Y residue. Variants containing multiple substitutions are designated with "1" between different substitutions in the variant.

[0124] Equivalent residues which are functionally analogous to a specific residue of H. jecorina BGL1 are defined as those amino acids of a beta-glucosidase that may adopt a conformation such that they either alter, modify or contribute to protein structure, substrate binding or catalysis in a manner defined and attributed to a specific residue of the H. jecorina BGL1. In some preferred embodiments, "equivalent residues" are residues that align with the amino acid sequence of H. jecorina BGL1.

[0125] The term "nucleic acid molecule" includes RNA, DNA and cDNA molecules. It will be understood that, as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences encoding a given protein such as BGL1 and/or variants thereof may be produced. The present disclosure contemplates every possible variant nucleotide sequence, encoding variant cellulase such as BGL1, all of which are possible given the degeneracy of the genetic code.

[0126] A "heterologous" nucleic acid construct or sequence has a portion of the sequence which is not native to the cell in which it is expressed. Heterologous, with respect to a control sequence refers to a control sequence (i.e. promoter or enhancer) that does not function in nature to regulate the same gene the expression of which it is currently regulating. Generally, heterologous nucleic acid sequences are not endogenous to the cell or part of the genome in which they are present, and have been added to the cell, by infection, transfection, transformation, microinjection, electroporation, or the like. A "heterologous" nucleic acid construct may contain a control sequence/DNA coding sequence combination that is the same as, or different from a control sequence/DNA coding sequence combination found in the native cell.

[0127] As used herein, the term "vector" refers to a nucleic acid construct designed for transfer between different host cells. An "expression vector" refers to a vector that has the ability to incorporate and express heterologous DNA fragments in a foreign cell. Many prokaryotic and eukaryotic expression vectors are commercially available. Selection of appropriate expression vectors is within the knowledge of those having skill in the art.

[0128] Accordingly, an "expression cassette" or "expression vector" is a nucleic acid construct generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter.

[0129] As used herein, the term "plasmid" refers to a circular double-stranded (ds) DNA construct used as a cloning vector, and which forms an extrachromosomal self-replicating genetic element in many bacteria and some eukaryotes.

[0130] As used herein, the term "selectable marker-encoding nucleotide sequence" refers to a nucleotide sequence which is capable of expression in cells and where expression of the selectable marker confers to cells containing the expressed gene the ability to grow in the presence of a corresponding selective agent, or under corresponding selective growth conditions.

[0131] As used herein, the term "promoter" refers to a nucleic acid sequence that functions to direct transcription of a downstream gene. The promoter will generally be appropriate to the host cell in which the target gene is being expressed. The promoter together with other transcriptional and translational regulatory nucleic acid sequences (also termed "control sequences") are necessary to express a given gene. In general, the transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.

[0132] "Chimeric gene" or "heterologous nucleic acid construct", as defined herein refers to a non-native gene (i.e., one that has been introduced into a host) that may be composed of parts of different genes, including regulatory elements. A chimeric gene construct for transformation of a host cell is typically composed of a transcriptional regulatory region (promoter) operably linked to a heterologous protein coding sequence, or, in a selectable marker chimeric gene, to a selectable marker gene encoding a protein conferring, for example, antibiotic resistance to transformed cells. A typical chimeric gene of the present disclosure, for transformation into a host cell, includes a transcriptional regulatory region that is constitutive or inducible, a protein coding sequence, and a terminator sequence. A chimeric gene construct may also include a second DNA sequence encoding a signal peptide if secretion of the target protein is desired.

[0133] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA encoding a secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors, linkers or primers for PCR are used in accordance with conventional practice.

[0134] As used herein, the term "gene" means the segment of DNA involved in producing a polypeptide chain, that may or may not include regions preceding and following the coding region, e.g. 5' untranslated (5' UTR) or "leader" sequences and 3' UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0135] In general, nucleic acid molecules which encode the variant cellulase such as BGL1 will hybridize, under moderate to high stringency conditions to the wild type sequence such as provided herein as SEQ ID NO:1. However, in some cases a BGL1-encoding nucleotide sequence is employed that possesses a substantially different codon usage, while the protein encoded by the BGL1-encoding nucleotide sequence has the same or substantially the same amino acid sequence as the native protein. For example, the coding sequence may be modified to facilitate faster expression of BGL1 in a particular prokaryotic or eukaryotic expression system, in accordance with the frequency with which a particular codon is utilized by the host (Te'o et al., FEMS Microbiology Letters, 190: 13-19, 2000, for example, describes the optimization of genes for expression in filamentous fungi).

[0136] A nucleic acid sequence is considered to be "selectively hybridizable" to a reference nucleic acid sequence if the two sequences specifically hybridize to one another under moderate to high stringency hybridization and wash conditions. Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex or probe. For example, "maximum stringency" typically occurs at about Tm-5.degree. C. (5.degree. C. below the Tm of the probe); "high stringency" at about 5-10.degree. C. below the Tm; "moderate" or "intermediate stringency" at about 10-20.degree. C. below the Tm of the probe; and "low stringency" at about 20-25.degree. C. below the Tm of the probe. Functionally, maximum stringency conditions may be used to identify sequences having strict identity or near-strict identity with the hybridization probe; while high stringency conditions are used to identify sequences having about 80% or more sequence identity with the probe.

[0137] Moderate and high stringency hybridization conditions are well known in the art (see, for example, Sambrook, et al, 1989, Chapters 9 and 11, and in Ausubel et al., 1993, expressly incorporated by reference herein). An example of high stringency conditions includes hybridization at about 42.degree. C. in 50% formamide, 5.times.SSC, 5.times.Denhardt's solution, 0.5% SDS and 100 .mu.g/ml denatured carrier DNA followed by washing two times in 2.times.SSC and 0.5% SDS at room temperature and two additional times in 0.1.times.SSC and 0.5% SDS at 42.degree. C.

[0138] The term "recombinant" when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells can express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise over expressed, under expressed or not expressed at all.

[0139] As used herein, the terms "transformed", "stably transformed" or "transgenic" with reference to a cell means the cell has a non-native (heterologous) nucleic acid sequence integrated into its genome or as an episomal plasmid that is maintained through multiple generations.

[0140] As used herein, the term "expression" refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process includes both transcription and translation.

[0141] The term "introduced" in the context of inserting a nucleic acid sequence into a cell, means "transfection", or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid sequence into a eukaryotic or prokaryotic cell where the nucleic acid sequence may be incorporated into the genome of the cell (for example, chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (for example, transfected mRNA).

[0142] It follows that the term "BGL1 expression" refers to transcription and translation of the bgl1 gene or variants thereof, the products of which include precursor RNA, mRNA, polypeptide, post-translationally processed polypeptides, and derivatives thereof, including BGL1 from related species such as Trichoderma koningii, Hypocrea jecorina (also known as Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma viride) and Hypocrea schweinitzii. By way of example, assays for BGL1 expression include Western blot and HPLC for BGL1 protein, Northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR) assays for bgl1 mRNA.

[0143] The term "alternative splicing" refers to the process whereby multiple polypeptide isoforms are generated from a single gene, and involves the splicing together of nonconsecutive exons during the processing of some, but not all, transcripts of the gene. Thus a particular exon may be connected to any one of several alternative exons to form messenger RNAs. The alternatively-spliced mRNAs produce polypeptides ("splice variants") in which some parts are common while other parts are different.

[0144] The term "signal sequence" refers to a sequence of amino acids at the N-terminal portion of a protein that facilitates the secretion of the mature form of the protein outside the cell. The mature form of the extracellular protein lacks the signal sequence that is cleaved off during the secretion process.

[0145] Host cells for use in the present disclosure can be prokaryotic cells, such as E. coli, or eukaryotic cells such as yeast, plant, insect, amphibian, or mammalian cells.

[0146] The term "filamentous fungi" means any and all filamentous fungi recognized by those of skill in the art. A preferred fungus is selected from the group consisting of Aspergillus, Trichoderma, Fusarium, Chrysosporium, Penicillium, Humicola, Neurospora, or alternative sexual forms thereof such as Emericella, Hypocrea. It has now been demonstrated that the asexual industrial fungus Trichoderma reesei is a clonal derivative of the ascomycete Hypocrea jecorina (See, Kuhls et al., PNAS, 93:7755-7760, 1996).

[0147] The term "cellooligosaccharide" refers to oligosaccharide groups containing from 2-8 glucose units and having beta-1,4 linkages, e.g., cellobiose.

[0148] The terms "cellulase," "cellulolytic enzymes" or "cellulase enzymes" refer to a category of enzymes capable of hydrolyzing cellulose polymers to shorter cello-oligosaccharide oligomers, cellobiose and/or glucose. Numerous examples of cellulases, such as exoglucanases, exocellobiohydrolases, endoglucanases, and glucosidases have been obtained from cellulolytic organisms, particularly including fungi, plants and bacteria. The enzymes made by these microbes are mixtures of proteins with three types of actions useful in the conversion of cellulose to glucose: endoglucanases (EG), cellobiohydrolases (CBH), and beta-glucosidase. These three different types of cellulase enzymes act synergistically to convert cellulose and its derivatives to glucose.

[0149] The term "beta-glucosidase activity" as used herein refers to a polypeptide capable of catalyzing the hydrolysis of .beta.-D-glucoside substrates, such as cellobiose, laminaribiose, or para-nitrophenol-.beta.-D-glucose, resulting in the release of beta-D-glucose. For instance, beta-glucosidase and active variants thereof are capable of releasing a glucose monomer from cellooligosaccharides (e.g., cellobiose, cellotriose, and cellotetraose). Beta-glucosidase activity can be detected by the hydrolysis of synthetic glycoside substrates including but not limited to para-nitrophenyl-beta-D-glucopyranoside to produce glucose and para-nitrophenol, or by the hydrolysis of cellobiose to produce two glucose molecules. For instance, beta-glucosidase activity can be determined by measuring either a cellobiase activity in the presence of ammonia pretreated corncob (CC), or by a CC hydrolysis activity.

[0150] Many microbes make enzymes that hydrolyze cellulose, including the wood rotting fungus Trichoderma, the compost bacteria Thermomonospora, Bacillus, and Cellulomonas; Streptomyces; and the fungi Humicola, Aspergillus, Chrysosporium, and Fusarium.

[0151] The term "cellulose binding domain" as used herein refers to portion of the amino acid sequence of a cellulase or a region of the enzyme that is involved in the cellulose binding activity of a cellulase or derivative thereof. Cellulose binding domains generally function by non-covalently binding the cellulase to cellulose, a cellulose derivative or other polysaccharide equivalent thereof. Cellulose binding domains permit or facilitate hydrolysis of cellulose fibers by the structurally distinct catalytic core region, and typically function independent of the catalytic core. Thus, a cellulose binding domain will not possess the significant hydrolytic activity attributable to a catalytic core. In other words, a cellulose binding domain is a structural element of the cellulase enzyme protein tertiary structure that is distinct from the structural element which possesses catalytic activity. Cellulose binding domain and cellulose binding module may be used interchangeably herein.

[0152] As used herein, the term "surfactant" refers to any compound generally recognized in the art as having surface active qualities. Thus, for example, surfactants comprise anionic, cationic and nonionic surfactants such as those commonly found in detergents. Anionic surfactants include linear or branched alkylbenzenesulfonates; alkyl or alkenyl ether sulfates having linear or branched alkyl groups or alkenyl groups; alkyl or alkenyl sulfates; olefinsulfonates; and alkanesulfonates. Ampholytic surfactants include quaternary ammonium salt sulfonates, and betaine-type ampholytic surfactants. Such ampholytic surfactants have both the positive and negative charged groups in the same molecule. Nonionic surfactants may comprise polyoxyalkylene ethers, as well as higher fatty acid alkanolamides or alkylene oxide adduct thereof, fatty acid glycerine monoesters, and the like.

[0153] As used herein, the term "cellulose containing fabric" refers to any sewn or unsewn fabrics, yarns or fibers made of cotton or non-cotton containing cellulose or cotton or non-cotton containing cellulose blends including natural cellulosics and manmade cellulosics (such as jute, flax, ramie, rayon, and lyocell).

[0154] As used herein, the term "cotton-containing fabric" refers to sewn or unsewn fabrics, yarns or fibers made of pure cotton or cotton blends including cotton woven fabrics, cotton knits, cotton denims, cotton yarns, raw cotton and the like.

[0155] As used herein, the term "stonewashing composition" refers to a formulation for use in stonewashing cellulose containing fabrics. Stonewashing compositions are used to modify cellulose containing fabrics prior to sale, i.e., during the manufacturing process. In contrast, detergent compositions are intended for the cleaning of soiled garments and are not used during the manufacturing process.

[0156] As used herein, the term "detergent composition" refers to a mixture which is intended for use in a wash medium for the laundering of soiled cellulose containing fabrics. In the context of the present disclosure, such compositions may include, in addition to cellulases and surfactants, additional hydrolytic enzymes, builders, bleaching agents, bleach activators, bluing agents and fluorescent dyes, caking inhibitors, masking agents, cellulase activators, antioxidants, and solubilizers.

[0157] As used herein, the term "decrease or elimination in expression of the bgl1 gene" means that either that the bgl1 gene has been deleted from the genome and therefore cannot be expressed by the recombinant host microorganism; or that the bgl1 gene or transcript has been modified such that a functional BGL1 enzyme is not produced by the host microorganism or at levels that are significantly less than the unmodified bgl1 gene or transcript.

[0158] The term "variant bgl1 gene" means that the nucleic acid sequence of the bgl1 gene from H. jecorina has been altered by removing from, adding to, and/or manipulating the coding sequence.

[0159] As used herein, the terms "active" and "biologically active" refer to a biological activity associated with a particular protein and are used interchangeably herein. For example, the enzymatic activity associated with a protease is proteolysis and, thus, an active protease has proteolytic activity. It follows that the biological activity of a given protein refers to any biological activity typically attributed to that protein by those of skill in the art.

[0160] As used herein, the term "enriched" means that the beta-glucosidase such as BGL1 is found in a concentration that is greater relative to the BGL1 concentration found in a wild-type, or naturally occurring, fungal cellulase composition. The terms enriched, elevated and enhanced may be used interchangeably herein.

[0161] A wild type fungal cellulase composition is one produced by a naturally occurring fungal source and which comprises one or more BGL, CBH and EG components wherein each of these components is found at the ratio produced by the fungal source. Thus, an enriched BGL1 composition would have BGL1 at an altered ratio wherein the ratio of BGL1 to other cellulase components (i.e., EGs, CBHs and other endoglucanases) is elevated. This ratio may be increased by either increasing BGL1 or decreasing (or eliminating) at least one other component by any means known in the art.

[0162] The term "isolated" or "purified" as used herein refers to a nucleic acid or amino acid that is removed from at least one component with which it is naturally associated. For the purpose of this application, "isolated" refers to nucleic acids or amino acids that are not part of a library (e.g., screening library).

[0163] Thus, to illustrate, a naturally occurring cellulase system may be purified into substantially pure components by recognized separation techniques well published in the literature, including ion exchange chromatography at a suitable pH, affinity chromatography, size exclusion and the like. For example, in ion exchange chromatography (usually anion exchange chromatography), it is possible to separate the cellulase components by eluting with a pH gradient, or a salt gradient, or both a pH and a salt gradient. The purified BGL1 may then be added to the enzymatic solution resulting in an enriched BGL1 solution. It is also possible to elevate the amount of BGL1 produced by a microbe using molecular genetics methods to overexpress the gene encoding BGL1, possibly in conjunction with deletion of one or more genes encoding other cellulases.

[0164] Fungal cellulases may contain more than one beta-glucosidase component. The different components generally have different isoelectric points that allow for their separation via ion exchange chromatography and the like. Either a single BGL1 component or a combination of BGL1 components may be employed in an enzymatic solution.

[0165] When employed in enzymatic solutions, the variant BGL1 component is generally added in an amount sufficient to allow the highest rate of release of soluble sugars from the biomass. The amount of variant BGL1 component added depends upon the type of biomass to be saccharified, which can be readily determined by the skilled artisan. The weight percent of total protein of the variant BGL1 component present in the composition is from preferably between 0.1 and 100 with illustrative examples being about 0.1, preferably about 0.5, 1, preferably about 5, preferably about 10, preferably about 15, or preferably about 20 weight percent to preferably about 25, preferably about 30, preferably about 35, preferably about 40, preferably about 45 or preferably about 50 weight percent. Furthermore, preferred ranges may be about 0.5 to about 15 weight percent, about 0.5 to about 20 weight percent, from about 1 to about 10 weight percent, from about 1 to about 15 weight percent, from about 1 to about 20 weight percent, from about 1 to about 25 weight percent, from about 5 to about 20 weight percent, from about 5 to about 25 weight percent, from about 5 to about 30 weight percent, from about 5 to about 35 weight percent, from about 5 to about 40 weight percent, from about 5 to about 45 weight percent, from about 5 to about 50 weight percent, from about 10 to about 20 weight percent, from about 10 to about 25 weight percent, from about 10 to about 30 weight percent, from about 10 to about 35 weight percent, from about 10 to about 40 weight percent, from about 10 to about 45 weight percent, from about 10 to about 50 weight percent, from about 15 to about 60 weight percent, from about 15 to about 65 weight percent, from about 15 to about 70 weight percent, from about 15 to about 75 weight percent, from about 15 to about 80 weight percent, from about 15 to about 85 weight percent, from about 15 to about 95 weight percent. However, when employed, the weight percent of the variant BGL1 component relative to any (EG or CBH type) enzyme components present in the cellulase composition is from preferably about 1, preferably about 5, preferably about 10, preferably about 15, or preferably about 20 weight percent to preferably about 25, preferably about 30, preferably about 35, preferably about 40, preferably about 45 or preferably about 50 weight percent. Furthermore, preferred ranges may be about 0.5 to about 15 weight percent, about 0.5 to about 20 weight percent, from about 1 to about 10 weight percent, from about 1 to about 15 weight percent, from about 1 to about 20 weight percent, from about 1 to about 25 weight percent, from about 5 to about 20 weight percent, from about 5 to about 25 weight percent, from about 5 to about 30 weight percent, from about 5 to about 35 weight percent, from about 5 to about 40 weight percent, from about 5 to about 45 weight percent, from about 5 to about 50 weight percent, from about 10 to about 20 weight percent, from about 10 to about 25 weight percent, from about 10 to about 30 weight percent, from about 10 to about 35 weight percent, from about 10 to about 40 weight percent, from about 10 to about 45 weight percent, from about 10 to about 50 weight percent, from about 15 to about 20 weight percent, from about 15 to about 25 weight percent, from about 15 to about 30 weight percent, from about 15 to about 35 weight percent, from about 15 to about 40 weight percent, from about 15 to about 45 weight percent, from about 15 to about 50 weight percent.

[0166] As part of a composition, the weight percent (of total protein content) of the variant BGL1 component from preferably between 0.1 and 100, with illustrative examples being about 0.1, preferably about 0.5, 1, preferably about 5, preferably about 10, preferably about 15, or preferably about 20 weight percent to preferably about 25, preferably about 30, preferably about 35, preferably about 40, preferably about 45 or preferably about 50 weight percent. Furthermore, preferred ranges may be about 0.5 to about 15 weight percent, about 0.5 to about 20 weight percent, from about 1 to about 10 weight percent, from about 1 to about 15 weight percent, from about 1 to about 20 weight percent, from about 1 to about 25 weight percent, from about 5 to about 20 weight percent, from about 5 to about 25 weight percent, from about 5 to about 30 weight percent, from about 5 to about 35 weight percent, from about 5 to about 40 weight percent, from about 5 to about 45 weight percent, from about 5 to about 50 weight percent, from about 10 to about 20 weight percent, from about 10 to about 25 weight percent, from about 10 to about 30 weight percent, from about 10 to about 35 weight percent, from about 10 to about 40 weight percent, from about 10 to about 45 weight percent, from about 10 to about 50 weight percent, from about 15 to about 60 weight percent, from about 15 to about 65 weight percent, from about 15 to about 70 weight percent, from about 15 to about 75 weight percent, from about 15 to about 80 weight percent, from about 15 to about 85 weight percent, from about 15 to about 95 weight percent. However, when employed, the weight percent of the variant BGL1 component relative to any (EG or CBH type) enzyme components present in the cellulase composition is from preferably about 1, preferably about 5, preferably about 10, preferably about 15, or preferably about 20 weight percent to preferably about 25, preferably about 30, preferably about 35, preferably about 40, preferably about 45 or preferably about 50 weight percent. Furthermore, preferred ranges may be about 0.5 to about 15 weight percent, about 0.5 to about 20 weight percent, from about 1 to about 10 weight percent, from about 1 to about 15 weight percent, from about 1 to about 20 weight percent, from about 1 to about 25 weight percent, from about 5 to about 20 weight percent, from about 5 to about 25 weight percent, from about 5 to about 30 weight percent, from about 5 to about 35 weight percent, from about 5 to about 40 weight percent, from about 5 to about 45 weight percent, from about 5 to about 50 weight percent, from about 10 to about 20 weight percent, from about 10 to about 25 weight percent, from about 10 to about 30 weight percent, from about 10 to about 35 weight percent, from about 10 to about 40 weight percent, from about 10 to about 45 weight percent, from about 10 to about 50 weight percent, from about 15 to about 20 weight percent, from about 15 to about 25 weight percent, from about 15 to about 30 weight percent, from about 15 to about 35 weight percent, from about 15 to about 40 weight percent, from about 15 to about 45 weight percent, from about 15 to about 50 weight percent.

II. BGL1 VARIANTS

[0167] The invention provides, inter alia, H. jecorina beta-glucosidase 1 (BGL1) variants that have various improved activities over wild type BGL1. Exemplary improved activities include, but are not limited to, (a) pre-treated corn stover (PCS) hydrolysis activity, (b) cellobiase activity, (c) protein expression, (d) beta-glucosidase activity as measured by either a cellobiase activity in the presence of ammonia pretreated corncob (CC), or by a CC hydrolysis activity under the conditions described herein, (e) thermostability at 66 degrees Celsius, (f) phosphoric acid swollen cellulose (PASC) hydrolysis activity, and (g) hydrolytic activity in the presence of glucose.

[0168] In some aspects, the BGL1 variant has a single substitution. In other aspects, the BGL1 variant has two or more substitutions. In other aspects, the BGL1 variant has 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more substitutions. In any of these aspects, the BGL1 variant can have different activities and combinations of activities.

[0169] In some aspects, BGL1 variants with a single substitution have at least two improved activities (including two activities) over wild type BGL1, such as (a) pre-treated corn stover (PCS) hydrolysis activity, (b) cellobiase activity, (c) protein expression, (d) beta-glucosidase activity as measured by either a cellobiase activity in the presence of ammonia pretreated corncob (CC), or by a CC hydrolysis activity as described herein, (e) thermostability, (f) phosphoric acid swollen cellulose (PASC) hydrolysis activity, and (g) hydrolytic activity in the presence of glucose.

[0170] In some aspects, BGL1 variants with a single substitution have at least three improved activities over wild type BGL1, at least four improved activities over wild type BGL1, at least five improved activities over wild type BGL1, at least six improved activities over wild type BGL1, or at least seven improved activities over wild type BGL1.

[0171] In other aspects, BGL1 variants comprising two or more substitutions a have at least two improved activities (including two activities) over wild type BGL1, such as (a) pre-treated corn stover (PCS) hydrolysis activity, (b) cellobiase activity, (c) protein expression, (d) beta-glucosidase activity as measured by either a cellobiase activity in the presence of ammonia pretreated corncob (CC), or by a CC hydrolysis activity in accordance with the method described herein, (e) thermostability at 66 degrees Celsius, (f) phosphoric acid swollen cellulose (PASC) hydrolysis activity, and (g) hydrolytic activity in the presence of glucose.

[0172] In other aspects, BGL1 variants with a combination of substitutions have at least three improved activities over wild type BGL1, at least four improved activities over wild type BGL1, at least five improved activities over wild type BGL1, at least six improved activities over wild type BGL1, or at least seven improved activities over wild type BGL1.

[0173] Accordingly, in one aspect, the invention provides beta-glucosidase 1 (BGL1) variants having at least two improved activities over wild type BGL1 selected from the group consisting of: (a) pre-treated corn stover (PCS) hydrolysis activity, (b) cellobiase activity, (c) protein expression, (d) beta-glucosidase activity as measured by either a cellobiase activity in the presence of ammonia pretreated corncob (CC) or by a CC hydrolysis activity, (e) thermostability, (f) phosphoric acid swollen cellulose (PASC) hydrolysis activity, and (g) hydrolytic activity in the presence of glucose, wherein the BGL1 variant is any variant as shown in Tables 4-8, 3-2, 4-2, and 4-3.

[0174] Some BGL1 variants (e.g., variants comprising L266A, I567E, S283F, S283P, T258E, T258I, T258K, T258Q, P536T, P536W, I532Y, Y530T, P607D, Q406M, Q406S, V602T, G300M, A630S, A630T, T180H, T180M, A450M, I444E, I444F, I444N, I444W, I444Y, V500Q, A633I, S482P, A667V, A485L, A485W, Y678R, V603G, L266C, I567F, S624P, P607L, G606I, G606K, G606L, G606M, G606Q, G606V, G605E, I444V, A633V, A655W, Y678H, V522Y, G554F, L266N, F556L, S550I, S550T, S550V, T258L, P536I, P536V, F392R, S624G, S624N, S624Q, S624T, A601M, A630V, N463S, A450F, A450T, A450V, A450W, I486V, S482I, Y678I, G427F, D564T, Q684C, Q684G, Y530S, Q684N, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, F260D, F260G, F260Q, P607G, N400S, F260W, Y530F, Q406D, G605C, N263T, P607I, A450P, T242H, A630Y, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, F260L, L293F, A633C, S312C, or N455D) can have the combination of improved (b) and (d) activities over wild type BGL1.

[0175] Some BGL1 variants (e.g., variants comprising P607H, T011E, T011Y, N146E, I567V, N566G, A630K, Y639K, Q245N, K320Y, A347Y, P536Q, N369E, N369W, N369Y, N146A, N146Q, P607K, N369T, A655N, I671K, F260T, P607S, F260D, F260G, F260Q, P607G, N400S, P607F, P607I, A450P, T242H, T568E, A630Y, A655D, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, F260L, or L293F) can have improved (b) and (e) activities over wild type BGL1.

[0176] Some BGL1 variants (e.g., variants comprising N261C, T258C, F392Q, S624E, P607C, P604M, A377Q, N461A, N461F, N461P, T436A, T436C, T436F, T436I, T436M, T436Q, T436Y, Q220C, A655L, T646H, Y678F, A468I, D177M, P661E, L266N, F556L, S550I, S550T, S550V, T258L, P536I, P536V, F392R, S624G, S624N, S624Q, S624T, A601M, A630V, N463S, A450F, A450T, A450V, A450W, I486V, S482I, Y678I, G427F, D564T, Q684C, Q684G, N566H, F556V, P604Y, L293V, A630G, N461C, N463T, D457C, Q220M, T221A, T221G, T221I, A655R, A468F, A468S, Q216I, D564V, P536Q, N369E, N369W, N369Y, T436E, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, F260W, Y530F, N461V, I671C, K206A, A450P, T242H, E170F, S507G, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, L293F, A633C, S312C, or N455D) can have improved (b) and (f) activities over wild type BGL1.

[0177] Some BGL1 variants (e.g., variants comprising I567Q, A565F, A565K, A565Q, A565V, F556E, F260I, P607E, G605R, G300C, A377C, A377D, S308C, N146H, N146S, A655C, A655G, P176L, T209I, L266C, I567F, S624P, P607L, G606I, G606K, G606L, G606M, G606Q, G606V, G605E, I444V, A633V, A655W, Y678H, V522Y, G554F, N566H, F556V, P604Y, L293V, A630G, N461C, N463T, D457C, Q220M, T221A, T221G, T221I, A655R, A468F, A468S, Q216I, D564V, A565C, A655N, I671K, F260T, P607S, Y639V, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, F260D, F260G, F260Q, P607G, N400S, P607F, Q406D, G605C, N263T, N461V I671C, K206A, T568E, E170F, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, F260L, A633C, S312C, or N455D) can have improved (a) and (b) activities over wild type BGL1.

[0178] Some BGL1 variants (e.g., variants comprising I567K, I567R, A565E, A565S, A565Y, F392Y, Q406H, Q406T, P604C, N038F, T568A, N461G, Y639L, Y639M, T243A, T243C, Q245H, Q245M, Q245T, T646A, T646C, I671F, I671L, I567V, N566G, A630K, Y639K, Q245N, K320Y, A347Y, A565C, Y639G, Y530F, N461V, I671C, K206A, T568E, A630Y, A655D, S507G, F260E, T568K, or F260L) can have improved (b) and (c) activities over wild type BGL1.

[0179] Some BGL1 variants (e.g., variants comprising I567S, G606E, G606H, G606N, G606S, L293A, S308R, I444C, M201D, R542N, L266C, I567F, S624P, P607L, G606I, G606K, G606L, G606M, G606Q, G606V, G605E, I444V, A633V, A655W, Y678H, V522Y, G554F, N566F, L293M, Q220P, S692L, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, F260D, F260G, F260Q, P607G, N400S, Q406D, G605C, N263T, S308E, A338D, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, F260L, A633C, S312C, or N455D) can have improved (a) and (d) activities over wild type BGL1.

[0180] Some BGL1 variants (e.g., variants comprising L266F, I567Y, A270R, S384C, A630W, E128R, N146M, N146V, N146W, L181F, V043C, Y639P, S507F, Q245P, G662C, A630H, V466T, N146A, N146Q, P607K, N369T, S384E, L181M, V043A, V043G, V043N, Q060D, A655Y, T242S, S474D, P607F, A630Y, S308E, A655D, or L293F) can have improved (e) and (g) activities over wild type BGL1.

[0181] Some BGL1 variants (e.g., variants comprising N261E, N261K, N400A, V602K, L293I, N461S, D457A, V043Q, Q303N, K320S, G662D, F260A, S474R, I567V, N566G, A630K, Y639K, Q245N, K320Y, A347Y, A601D, S384E, L181M, V043A, V043G, V043N, Q060D, A655Y, T242S, S474D, D564T, T568E, A655D, A338D, F260E, T568K, or F260L) can have improved (c) and (e) activities over wild type BGL1.

[0182] BGL1 variants (e.g., variants comprising N566L, N566P, N566W, A270K, A270N, F556H, F556K, P604N, N461D, N463E, K206G, A468Q, A468Y, N566F, N566H, F556V, P604Y, L293V, A630G, N461C, N463T, D457C, Q220M, T221A, T221G, T221I, A655R, A468F, A468S, Q216I, D564V, A468T, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, N461V|I671C, K206A, E170F, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, A633C, S312C, or N455D) can have improved (a) and (f) activities over wild type BGL1.

[0183] Some BGL1 variants (e.g., variants comprising S283D, A270D, N146Y, F260A, S474R, A565C, K206S, D564T, N461V|I671C, K206A, T568E, A338D, F260E, T568K, or F260L) can have improved (a) and (c) activities over wild type BGL1. Other BGL1 variants (e.g., variants comprising F556G, F260S, P604E, P604V, N146D, Y639T, T221C, N473S, N583R, R645G, G662Y, F260A, S474R, A655N, I671K, F260T, P607S, S692L, D564T, F260D, F260G, F260Q, P607G, N400S, P607F, T568E, S308E, A338D, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, or F260L) can have improved (a) and (e) activities over wild type BGL1.

[0184] Some BGL1 variants (e.g., variants comprising D259S, T243V, Y530F, A338D, or F260L) can have improved (c) and (d) activities over wild type BGL1. Some BGL1 variants (e.g., variants comprising S550Q, P607R, N400Q, V602F, A601G, A601L, L293K, Y575C, Y575R, A450Q, I486C, I486Y, A655S, Q245F, D329A, P536G, P607Q, A655Q, Y575A, Y575K, A630H, V466T, S692L, F260D, F260G, F260Q, P607G, N400S, P607I, A450P, T242H, S308E, A630Y, A338D, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, F260L, or L293F) can have improved (d) and (e) activities over wild type BGL1.

[0185] Some BGL1 variants (e.g., variants comprising P536F, F392C, S624L, S624R, S624W, I486F, I486W, A667G, A667S, L266N, F556L, S550I, S550T, S550V, T258L, P536I, P536V, F392R, S624G, S624N, S624Q, S624T, A601M, A630V, N463S, A450F, A450T, A450V, A450W, I486V, S482I, Y678I, G427F, D564T, Q684C, Q684G, N566F, Y575A, Y575K, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, F260W, P607I, A450P, T242H, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, L293F, A633C, S312C, or N455D) can have improved (d) and (f) activities over wild type BGL1. Some BGL1 variants (e.g., variants comprising S384G, S384W, N038E, N038M, N038P, V043H, V043W, Y068E, Y068G, Y068M, L110C, L110G, L110Q, L110W, A655H, N264L, S384E, L181M, V043A, V043G, V043N, Q060D, A655Y, T242S, S474D, Y639G, K206S, A655D, or S507G) can have improved (c) and (g) activities over wild type BGL1.

[0186] Some BGL1 variants (e.g., variants comprising G606D, Y068V, L293M, Q220P, A630H, V466T, Y530S, Q684N, F260W, Q406D, G605C, N263T, S308E, A630Y, L293F, A633C, S312C, or N455D) can have (d) and (g) activities. Some BGL1 variants (e.g., variants comprising A377I, N461Y, N146A, N146Q, P607K, N369T, T436E, Y639G, Y530S, Q684N, Y639V, F260W, P607F, Q406D, G605C, N263T, A630Y, A655D, E170F, S507G, L293F, A633C, S312C, or N455D) can have improved (b) and (g) activities over wild type BGL1. Some BGL1 variants (e.g., variants comprising K206D, A601D, Y530F, N461V|I671C, K206A, S507G, F260E, or T568K) can have improved (c) and (f) activities over wild type BGL1. Other BGL1 variants (e.g., variants comprising A468G, P536Q, N369E, N369W, N369Y, A601D, Y575A, Y575K, P607I, A450P, T242H, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, or L293F) can have improved (e) and (f) activities over wild type BGL1.

[0187] Additionally, BGL1 variants can have at least three improved activities over wild type BGL1. For example, some BGL1 variants (e.g., variants comprising L266C, I567F, S624P, P607L, G606I, G606K, G606L, G606M, G606Q, G606V, G605E, I444V, A633V, A655W, Y678H, V522Y, G554F, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, F260D, F260G, F260Q, P607G, N400S, Q406D, G605C, N263T, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, F260L, A633C, S312C, or N455D) can have improved activities selected from any two or all three of (a), (b), and (d) over wild type BGL1, while others (e.g., variants comprising L266N, F556L, S550I, S550T, S550V, T258L, P536I, P536V, F392R, S624G, S624N, S624Q, S624T, A601M, A630V, N463S, A450F, A450T, A450V, A450W, I486V, S482I, Y678I, G427F, D564T, Q684C, Q684G, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, F260W, Y530F, P607I, A450P, T242H, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, A633C, S312C, N455D, or L293F) have improved activities selected from any two or all three of (b), (d), and (f) over wild type BGL1.

[0188] Some BGL1 variants (e.g., variants comprising F260A, S474R, D564T, T568E, A338D, F260E, T568K, or F260L) can have improved activities selected from any two or all three of (a), (c), and (e) over wild type BGL1, while other BGL1 variants (e.g., variants comprising I567V, N566G, A630K, Y639K, Q245N, K320Y, A347Y, T568E, A655D, F260E, T568K, or F260L) can have improved activities selected from any two or all three of (b), (c), and (e) over wild type BGL1. Some BGL1 variants (e.g., variants comprising N566F, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, A633C, S312C, or N455D) can have improved activities selected from any two or all three of (a), (d), and (f) over wild type BGL1. Other BGL1 variants (e.g., variants comprising N566H, F556V, P604Y, L293V, A630G, N461C, N463T, D457C, Q220M, T221A, T221G, T221I, A655R, A468F, A468S, Q216I, D564V, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, N461V, I671C, K206A, E170F, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, A633C, S312C, or N455D) can have improved activities selected from any two or all three of (a), (b), and (f) over wild type BGL1.

[0189] Some BGL1 variants (e.g., variants comprising A565C, N461V, I671C, K206A, T568E, F260E, T568K, or F260L) can have improved activities selected from any two or all three of (a), (b), and (c) over wild type BGL1. Other BGL1 variants (e.g., variants comprising P536G, P607Q, A655Q, F260D, F260G, F260Q, P607G, N400S, P607I, A450P, T242H, A630Y, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, F260L, or L293F) can have improved activities selected from any two or all three of (b), (d), and (e) over wild type BGL1. Yet other BGL1 variants (e.g., variants comprising P536Q, N369E, N369W, N369Y, P607I, A450P, T242H, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, or L293F) can have improved activities selected from any two or all three of (b), (e), and (f) over wild type BGL1.

[0190] Other BGL1 variants (e.g., variants comprising A601D, F260E or T568K) can have improved activities selected from any two or all three of (c), (e), and (f) over wild type BGL1. Some BGL1 variants (e.g., variants comprising L293M, Q220P, Q406D, G605C, N263T, S308E, A633C, S312C, or N455D) can have improved activities selected from any two or all three of (a), (d), and (g) over wild type BGL1. Other BGL1 variants (e.g., variants comprising Y575A, Y575K, P607I, A450P, T242H, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, or L293F) can have improved activities selected from any two or all three of (d), (e), and (f) over wild type BGL1. Some BGL1 variants (e.g., variants comprising A630H, V466T, S308E, A630Y, or L293F) can have improved activities selected from any two or all three of (d), (e), and (g) over wild type BGL1.

[0191] Some BGL1 variants (e.g., variants comprising N146A, N146Q, P607K, N369T, P607F, A630Y, A655D, or L293F) can have improved activities selected from any two or all three of (b), (e), and (g) over wild type BGL1. Other BGL1 variants (e.g., variants comprising S384E, L181M, V043A, V043G, V043N, Q060D, A655Y, T242S, S474D, or A655D) can have improved activities selected from any two or all three of (c), (e), and (g) over wild type BGL1. Some BGL1 variants (e.g., variants comprising T436E, F260W, E170F, S507G, L293F, A633C, S312C, or N455D) can have improved activities selected from any two or all three of (b), (f), and (g) over wild type BGL1. Other BGL1 variants (e.g., variants comprising Y639G, P607F, A655D, or S507G) can have improved activities selected from any two or all three of (b), (c), and (g) over wild type BGL1.

[0192] Some BGL1 variants (e.g., variants comprising A655N, I671K, F260T, P607S, F260D, F260G, F260Q, P607G, N400S, P607F, T568E, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, or F260L) can have improved activities selected from any two or all three of (a), (b), and (e) over wild type BGL1. Other BGL1 variants (e.g., variants comprising K206S or P607F) can have improved activities selected from any two or all three of (a), (c), and (g) over wild type BGL1. Other BGL1 variants (e.g., variants comprising Y530S, Q684N, F260W, Q406D, G605C, N263T, A630Y, L293F, A633C, S312C, or N455D) can have improved activities selected from any two or all three of (b), (d), and (g) over wild type BGL1. Some BGL1 variants (e.g., variants comprising A468T, E170F, A633C, S312C, or N455D) can have improved activities selected from any two or all three of (a), (f), and (g) over wild type BGL1.

[0193] Some BGL1 variants (e.g., variants comprising S692L, F260D, F260G, F260Q, P607G, N400S, S308E, A338D, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, or F260L) can have improved activities selected from any two or all three of (a), (d), and (e) over wild type BGL1 while other BGL1 variants (e.g., Y639V) can have improved activities selected from any two or all three of (a), (b), and (g) over wild type BGL1.

[0194] Other BGL1 variants (e.g., variants comprising A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, or S683W) can have improved activities selected from any two, any three, or all four of (a), (b), (d) and (f) over wild type BGL1.

[0195] Some BGL1 variants (e.g., variants comprising F260D, F260G, F260Q, P607G, or N400S) can have improved activities selected from any two, any three, or all four of (a), (b), (d) and (e) over wild type BGL1. Other BGL1 variants (e.g., variants comprising F260W, L293F, A633C, S312C, or N455D) can have improved activities selected from any two, any three, or all four of (b), (d), (f) and (g) over wild type BGL1. Other BGL1 variants (e.g., variants comprising Y530F) can have improved activities selected from any two, any three, or all four of (b), (c), (d) and (f) over wild type BGL1. Other BGL1 variants (e.g., variants comprising P607F) can have improved activities selected from any two, any three, or all four of (a), (b), (e) and (g) over wild type BGL1. Other BGL1 variants (e.g., variants comprising Q406D, G605C, N263T, A633C, S312C, or N455D) can have improved activities selected from any two, any three, or all four of (a), (b), (d) and (g) over wild type BGL1.

[0196] Other BGL1 variants (e.g., variants comprising N461V, I671C, K206A, F260E, or T568K) can have improved activities selected from any two, any three, or all four of (a), (b), (c) and (f) over wild type BGL1. Other BGL1 variants (e.g., variants comprising P607I, A450P, T242H, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, or L293F) can have improved activities selected from any two, any three, or all four of (b), (d), (e) and (f) over wild type BGL1. Some BGL1 variants (e.g., variants comprising T568E, F260E, T568K, or F260L) can have improved activities selected from any two, any three, or all four of (a), (b), (c) and (e) over wild type BGL1. Other BGL1 variants (e.g., variants comprising S308E) can have improved activities selected from any two, any three, or all four of (a), (d), (e) and (g) activities over wild type BGL1. Other BGL1 variants (e.g., variants comprising A630Y or L293F) can have improved (b), (d), (e) and (g) over wild type BGL1.

[0197] Other BGL1 variants (e.g., variants comprising A655D) can have improved activities selected from any two, any three, or all four of (b), (c), (e) and (g) over wild type BGL1. Other BGL1 variants (e.g., variants comprising E170F, A633C, S312C, or N455D) can have improved activities selected from any two, any three, or all four of (a), (b), (f) and (g) over wild type BGL1. Other BGL1 variants (e.g., variants comprising A338D, or F260L) can have improved activities selected from any two, any three, or all four of (a), (c), (d) and (e) over wild type BGL1. Other BGL1 variants (e.g., variants comprising S507G) can have improved activities selected from any two, any three, or all four of (b), (c), (f) and (g) over wild type BGL1.

[0198] Other BGL1 variants (e.g., variants comprising F260E or T568K) can have improved activities selected from any two, any three, any four, or all five of (a), (b), (c), (e) and (f) over wild type BGL1. Other BGL1 variants (e.g., variants comprising P536C, A630Q, D215S, G372A, G547A, F611A, G662C, or G662F) can have improved activities selected from any two, any three, any four, or all five of (a), (b), (d), (e) and (f) over wild type BGL1.

[0199] Other BGL1 variants (e.g., variants comprising F260L) can have improved (a), (b), (c), (d) and (e) activities over wild type BGL1. Other BGL1 variants (e.g., variants comprising L293F) can have improved activities selected from any two, any three, any four, or all five of (b), (d), (e), (f) and (g) over wild type BGL1. Other BGL1 variants (e.g., variants comprising A633C, S312C, or N455D) can have improved activities selected from any two, any three, any four, or all five of (a), (b), (d), (f) and (g) over wild type BGL1.

[0200] In one aspect, a suitable BGL1 variant can be any of the following: L266Y, I567S, A270D, S550D, T258S, P536D, P536V, F260D, F260G, Y530F, S624N, P607Q, G606M, Q406H, N400Q, G300M, N038L, N038M, A601Y, L293V, T568K, S308E, A630Y, N461D, N146D, A450E, V043L, Q220A, A655Q, S482A, A667L, A485T, K206A, or Y678Q.

[0201] The invention also provides for BGL1 variants that have at least two improved activities over wild type BGL1 selected from the group consisting of: (a) pre-treated corn stover (PCS) hydrolysis activity, (b) cellobiase activity, (c) protein expression, (d) beta-glycosidase activity as measured by an ammonia pretreated corncob (CC) hydrolysis activity, (e) thermostability, (f) phosphoric acid swollen cellulose (PASC) hydrolysis activity, and (g) hydrolytic activity in the presence of glucose, wherein the BGL1 variant comprises two or more substitutions from Table 5-1.

[0202] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are (a) and (c) and the substitutions are: L167W|D225Q, T242S|S312Y, D178K|A338K|S474D|G662L, K345E|N369T|G372A|K428N|P661L|S683W, D177M|D225Q|D564V|Q684G, and D178N|N264K|A338D|S474R|G662K, D177M|D564T|Q626F|Q684A, K428N|S683W, K345E|K428N|S683W, Q226Y|G372A|V603G|T666C, L167W|D177M|Q626F, L167W|D177M|D225Q|D564V|Q684G, D177M|D225Q|D564T|Q684N, D177M|Q626F|Q684R, N238W|R265P|K656R, N264M|R265P (optionally also G662F), N264L|A338I|S474R|G662D, L167W|D225Q|D564V|Q626F|Q684N, D177M|D225Q|D564T|Q684A, D177M|D225Q|D564V|Q626F|Q684N, K345E|N369E|G372A|P661E, N369T|P661L|S683W, R265M|K560S, N369T|G372A|P661L|S683W, P176L|Q226W|K320Y|R363E, K345E|N369T|G372A|P661E|S683W, K320S|R363E, E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C, T242S|T666C, Q226Y|T666C, Q216E|S312K|S692K, P176L|G662F, P176L|Q226W|Q316T|K320Y|R363E, P176L|Q226W|K320S|R363E|G662F, P176L|Q226W|Q316T|K320Y|V522Y, P176L|Q226W|K320Y|R363E|V522Y, Q226W|K320Y|R363E, Q316T|K320Y|R363E|G662F, Q226W|Q316T|R363E|V522Y|G662F, P176L|K320S|R363E|G662C, R363E|G547A|G662C, Q226W|K320S|G662C, P176L|Q226W|Q316T|K320Y|R363E|G662F, P176L|Q226W|Q316T|K320S|G662F, P176L|Q316T|K320S|R363E|V522Y|G662C, K320Y|R363E|G662C, K345E|N369E|P661L, E170F|V603G, K345E|N369E|G372A|S683W, N369E|S683W, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0203] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are (b) and (f) and the substitutions are: L167W|D177M|D225Q|Q626F|Q684G, L167W|D177M|D564V|Q684G, D215S|S312Y, E170F|S312Y|N369Y, L167W|D225Q|Q626F|Q684R, L167W|D564T|Q626F, P176L|Q226W|Q316T|K320S|V522Y|G662C, R363E|V522Y|G662F, Q316T|K320S|V522Y|G662F, Q226W|K320Y|V522Y, Q316T|K320S|V522Y, and Q226W|K320S|R363E|V522Y|G662F, L167W|D177M|D225Q|D564V, D177M|D225Q|D564T|Q684A, D177M|D225Q|D564V|Q626F|Q684N, L167W|D177M|Q626F|Q684G, L167W|D177M|D564V|Q626F|Q684A, P176L|K320S|V522Y|G662C, K345E|N369T|G372A|P661E|S683W, K320S|R363E, E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C, T242S|T666C, Q226Y|T666C, Q216E|S312K|S692K, P176L|G662F, P176L|Q226W|Q316T|K320Y|R363E, P176L|Q226W|K320S|R363E|G662F, P176L|Q226W|Q316T|K320Y|V522Y, P176L|Q226W|K320Y|R363E|V522Y, Q226W|K320Y|R363E, Q316T|K320Y|R363E|G662F, Q226W|Q316T|R363E|V522Y|G662F, P176L|K320S|R363E|G662C, R363E|G547A|G662C, Q226W|K320S|G662C, P176L|Q226W|Q316T|K320Y|R363E|G662F, P176L|Q226W|Q316T|K320S|G662F, P176L|Q316T|K320S|R363E|V522Y|G662C, K320Y|R363E|G662C, E170F|Q226Y|N369Y|G372A|P661F, and L167W|D177M|D564T|Q626F|Q684G, K345E|N369E|G372A|S683W, N369E|S683W, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0204] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are (e) and (g) and the substitutions are: L167W|D225Q|Q626F|Q684D, L167W|D225Q|Q684N, L167W|D225Q|D564T1, Q626F|Q684C, Q626F|Q684D, N264M|R265P|N369I|D370W, R179V|N238F|D370W, R179V|N238F|K656R, R179V|N264M|D370W, R179V|N238F|R265M, R179V|R265P|D370W|K656R, R179V|N238W|N264M|R265M|N369I, R179V|N369I|D370W|K656R, R179V|N264M|R265P|K656R, R179V|R265M|N369I, R179V|N264M|R265M|D370W|K656R, R179V|N264M|R265M|N369I, R179V|N238W|N264M, N238W|N264M|R265M|D370W, R179V|N238W|R265P|D370W, R179V|N238W|N264M|D370W|K656R, N264M|R265P, R265P|D370W (optionally also G662F), R179V|N264M|R265P|N369I|D370W, R265M|N369I, R179V|R265M|D370W, N238W|N264M|R265P, R179V|N238W|N264M|R265P, N264M|N369I, N238F|R265M|N369I, N263C|K345E|N369E|G372A|K428N|P661E|S683W, N263C|K345E|N369T|G372A|K428N|P661E|S683W, N263C|K345E|N369E|G372A, N263C|P661L|S683W, N263C|K345E|N369T|G372A|K428N, K345E|G372A|K428N|P661E, E170F|Q226Y|N369Y|G372A, Q226Y|T242S|G372A|P661F, Q216E|T282I|S312D|S692K, Q216I|T282K|S312K|A622K, P176L|Q316T|G662C, Q226W|Q316T|V522Y|G662F, P176L|Q316T, A347Y|R542N, N238F|N264M|R265M|N369I, L167W|D225Q|D564V|Q626F|Q684N, E170F|V603G, L167W|D177M|D564T|Q684N, K345E|N369E|G372A|S683W, N369E|S683W, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0205] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are (d) and (f) and the substitutions are: L167W|D177M|D564V|Q684R, L167W|D225Q|D564V, D177M|D225Q|D564T|Q626F|Q684N, L167W|Q626F, D225Q|D564V|Q626F|Q684R, D177M|D225Q|D564V|Q684R, Q226W|K320Y, P176L|V522Y, R363E|G662C, L167W|D225Q|Q626F|Q684R, L167W|D564T|Q626F, P176L Q226W|Q316T|K320S|V522Y|G662C, R363E|V522Y|G662F, Q316T|K320S|V522Y|G662F, Q226W|K320Y|V522Y, Q316T|K320S|V522Y, Q226W|K320S|R363E|V522Y|G662F, L167W|D177M|D564T|Q626F|Q684N, L167W|Q626F|Q684D, L167W|D177M|D564T|Q684R, L167W|D177M|D225Q|Q684D, R179V|R265P|N369I, Q316T|K320Y|R363E|V522Y|G662F, L167W|D177M|Q626F, L167W|D177M|D225Q|D564V|Q684G, D177M|D225Q|D564T|Q684N, D177M|Q626F|Q684R, L167W|D177M|Q626F|Q684G, L167W|D177M|D564V|Q626F|Q684A, P176L|K320S|V522Y|G662C, K345E|N369T|G372A|P661E|S683W, K320S|R363E, E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C, T242S|T666C, Q226Y|T666C, Q216E|S312K|S692K, P176L|G662F, P176L|Q226W|Q316T|K320Y|R363E, P176L|Q226W|K320S|R363E|G662F, P176L|Q226W|Q316T|K320Y|V522Y, P176L|Q226W|K320Y|R363E|V522Y, Q226W|K320Y|R363E, Q316T|K320Y|R363E|G662F, Q226W|Q316T|R363E|V522Y|G662F, P176L|K320S|R363E|G662C, R363E|G547A|G662C, Q226W|K320S|G662C, P176L|Q226W|Q316T|K320Y|R363E|G662F, P176L|Q226W|Q316T|K320S|G662F, P176L|Q316T|K320S|R363E|V522Y|G662C, K320Y|R363E|G662C, E170F|Q226Y|N369Y|G372A|P661F, L167W|D177M|D564T|Q626F|Q684G, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0206] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are (b) and (e) and the substitutions are: K345E|N369E|K428N|P661L, Q316T|K320Y|V522Y, N369T|G372A|P661L|S683W, P176L|Q226W|K320Y|R363E, P176L|K320S|V522Y|G662C, K345E|N369E|P661L, L167W|D177M|D564T|Q684N, K345E|N369E|G372A|S683W, N369E|S683W, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0207] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are (d) and (e) and the substitutions are: N263C|K345E|N369E|P661L, N238F|N264M|R265M|N369I, P176L|K320S|V522Y|G662C, K345E|N369E|P661L, E170F|V603G, L167W|D177M|D564T|Q684N, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0208] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are (b) and (g) and the substitutions are: E170F|T242S|N369Y|G372A|V603G|T666C, E170F|Q226Y|N369Y|V603G|T666C, E170F|Q226Y|S312Y, L167W|D177M|D225Q|D564V, L167W|D177M|Q626F|Q684G, L167W|D177M|D564V|Q626F|Q684A, E170F|Q226Y|N369Y|G372A|P661F, L167W|D177M|D564T|Q626F|Q684G, L167W|D177M|D564T|Q684N, K345E|N369E|G372A|S683W, N369E|S683W, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0209] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are (d) and (g) and the substitutions are: D178I|Q303E1A338I, Q316T|K320Y|G662F, L167W|D177M|D564T|Q626F|Q684N, L167W|Q626F|Q684D, L167W|D177M|D564T|Q684R, L167W|D177M|D225Q|Q684D, R179V|R265P|N369I, Q316T|K320Y|R363E|V522Y|G662F, N238F|N264M|R265M|N369I, N238W|R265P|K656R, N264M|R265P, (optionally also G662F), N264L A338I|S474R|G662D, L167W|D177M|Q626F|Q684G, and L167W|D177M|D564V|Q626F|Q684A, E170F|V603G, E170F|Q226Y|N369Y|G372A|P661F, L167W|D177M|D564T|Q626F|Q684G, L167W|D177M|D564T|Q684N, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0210] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two or all three of (b), (d), and (f) and the substitutions are: L167W|D225Q|Q626F|Q684R, L167W|D564T|Q626F, P176L|Q226W|Q316T|K320S|V522Y|G662C, R363E|V522Y|G662F, Q316T|K320S|V522Y|G662F, Q226W|K320Y|V522Y, Q316T|K320S|V522Y, Q226W|K320S|R363E|V522Y|G662F, L167W|D177M|Q626F|Q684G, L167W|D177M|D564V|Q626F|Q684A, P176L|K320S|V522Y|G662C, K345E|N369T|G372A|P661E|S683W, K320S|R363E, E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C, T242S|T666C, Q226Y|T666C, Q216E|S312K|S692K, P176L|G662F, P176L|Q226W|Q316T|K320Y|R363E, P176L|Q226W|K320S|R363E|G662F, P176L|Q226W|Q316T|K320Y|V522Y, P176L|Q226W|K320Y|R363E|V522Y, Q226W|K320Y|R363E, Q316T|K320Y|R363E|G662F, Q226W|Q316T|R363E|V522Y|G662F, P176L|K320S|R363E|G662C, R363E|G547A|G662C, Q226W|K320S|G662C, P176L|Q226W|Q316T|K320Y|R363E|G662F, P176L|Q226W|Q316T|K320S|G662F, P176L|Q316T|K320S|R363E|V522Y|G662C, K320Y|R363E|G662C, E170F|Q226Y|N369Y|G372A|P661F, L167W|D177M|D564T|Q626F|Q684G, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0211] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two or all three of (d), (f), and (g) and the substitutions are: L167W|D177M|D564T|Q626F|Q684N, L167W|Q626F|Q684D, L167W|D177M|D564T|Q684R, L167W|D177M|D225Q|Q684D, R179V|R265P|N369I, Q316T|K320Y|R363E|V522Y|G662F, L167W|D177M|Q626F|Q684G, L167W|D177M|D564V|Q626F|Q684A, E170F|Q226Y|N369Y|G372A|P661F, L167W|D177M|D564T|Q626F|Q684G, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0212] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two or all three of (a), (c), and (e) and the substitutions are: K345E|N369T|G372A|K428N|P661L|S683W, L167W|D225Q|D564V|Q626F|Q684N, N369T|G372A|P661L|S683W, P176L|Q226W|K320Y|R363E, K345E|N369E|P661L, E170F|V603G, K345E|N369E|G372A|S683W, N369E|S683W, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0213] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two or all three of (b), (f), and (g) and the substitutions are: L167W|D177M|D225Q|D564V, L167W|D177M|Q626F|Q684G, L167W|D177M|D564V|Q626F|Q684A, E170F|Q226Y|N369Y|G372A|P661F, L167W|D177M|D564T|Q626F|Q684G, K345E|N369E|G372A|S683W, N369E|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0214] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two or all three of (a), (c), and (d) and the substitutions are: D177M|D225Q|D564V|Q684G, D178N|N264K|A338D|S474R|G662K, L167W|D177M|Q626F, L167W|D177M|D225Q|D564V|Q684G, D177M|D225Q|D564T|Q684N, D177M|Q626F|Q684R, N238W|R265P|K656R, N264M|R265P (optionally also G662F), N264L|A338I|S474R|G662D, K345E|N369E|G372A|P661E, N369T|P661L|S683W, K345E|N369T|G372A|P661E|S683W, K320S|R363E, E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C, T242S|T666C, Q226Y|T666C, Q216E|S312K|S692K, P176L|G662F, P176L|Q226W|Q316T|K320Y|R363E, P176L|Q226W|K320S|R363E|G662F, P176L|Q226W|Q316T|K320Y|V522Y, P176L|Q226W|K320Y|R363E|V522Y, Q226W|K320Y|R363E, Q316T|K320Y|R363E|G662F, Q226W|Q316T|R363E|V522Y|G662F, P176L|K320S|R363E|G662C, R363E|G547A|G662C, Q226W|K320S|G662C, P176L|Q226W|Q316T|K320Y|R363E|G662F, P176L|Q226W|Q316T|K320S|G662F, P176L|Q316T|K320S|R363E|V522Y|G662C, K320Y|R363E|G662C, K345E|N369E|P661L, E170F|V603G, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0215] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two or all three of (a), (c), and (g) and the substitutions are: D177M|D564T|Q626F|Q684A, N238W|R265P|K656R, N264M|R265P (optionally also G662F), N264L|A338I|S474R|G662D, |D225Q|D564V|Q626F|Q684N, R265M|K560S, E170F|V603G, K345E|N369E|G372A|S683W, N369E|S683W, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C. In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two or all three of (d), (e), and (g) and the substitutions are: N238F|N264M|R265M|N369I, E170F|V603G, L167W|D177M|D564T|Q684N, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C. In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two or all three of (a), (b), and (c) and the substitutions are: K428N|S683W, K345E|K428N|S683W, Q226Y|G372A|V603G|T666C, D177M|D225Q|D564T|Q684A, D177M|D225Q|D564V|Q626F|Q684N, K345E|N369E|G372A|P661E, N369T|P661L|S683W, N369T|G372A|P661L|S683W, P176L|Q226W|K320Y|R363E, K345E|N369T|G372A|P661E|S683W, K320S|R363E, E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C, T242S|T666C, Q226Y|T666C, Q216E|S312K|S692K, P176L|G662F, P176L|Q226W|Q316T|K320Y|R363E, P176L|Q226W|K320S|R363E|G662F, P176L|Q226W|Q316T|K320Y|V522Y, P176L|Q226W|K320Y|R363E|V522Y, Q226W|K320Y|R363E, Q316T|K320Y|R363E|G662F, Q226W|Q316T|R363E|V522Y|G662F, P176L|K320S|R363E|G662C, R363E|G547A|G662C, Q226W|K320S|G662C, P176L|Q226W|Q316T|K320Y|R363E|G662F, P176L|Q226W|Q316T|K320S|G662F, P176L|Q316T|K320S|R363E|V522Y|G662C, K320Y|R363E|G662C, K345E|N369E|P661L, K345E|N369E|G372A|S683W, N369E|S683W, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0216] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, or all four of (a), (c), (d), and (f) and the substitutions are: L167W|D177M|Q626F, L167W|D177M|D225Q|D564V|Q684G, D177M|D225Q|D564T|Q684N, D177M|Q626F|Q684R, K345E|N369T|G372A|P661E|S683W, K320S|R363E, E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C, T242S|T666C, Q226Y|T666C, Q216E|S312K|S692K, P176L|G662F, P176L|Q226W|Q316T|K320Y|R363E, P176L|Q226W|K320S|R363E|G662F, P176L|Q226W|Q316T|K320Y|V522Y, P176L|Q226W|K320Y|R363E|V522Y, Q226W|K320Y|R363E, Q316T|K320Y|R363E|G662F, Q226W|Q316T|R363E|V522Y|G662F, P176L|K320S|R363E|G662C, R363E|G547A|G662C, Q226W|K320S|G662C, P176L|Q226W|Q316T|K320Y|R363E|G662F, P176L|Q226W|Q316T|K320S|G662F, P176L|Q316T|K320S|R363E|V522Y|G662C, K320Y|R363E|G662C, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0217] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, or all four of (a), (c), (d), and (g) and the substitutions are: N238W|R265P|K656R, N264M|R265P (optionally also G662F), N264L|A338I|S474R|G662DE170F|V603G, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C. In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, or all four of (a), (c), (e), and (g) and the substitutions are: L167W|D225Q|D564V|Q626F|Q684N, E170F|V603G, K345E|N369E|G372A|S683W, N369E|S683W, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L Q226W|G547A|G662C. In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, or all four of (a), (b), (c), and (f) and the substitutions are: D177M|D225Q|D564T|Q684A, D177M|D225Q|D564V|Q626F|Q684N, K345E|N369T|G372A|P661E|S683W, K320S|R363E, E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C, T242S|T666C, Q226Y|T666C, Q216E|S312K|S692K, P176L|G662F, P176L|Q226W|Q316T|K320Y|R363E, P176L|Q226W|K320S|R363E|G662F, P176L|Q226W|Q316T|K320Y|V522Y, P176L|Q226W|K320Y|R363E|V522Y, Q226W|K320Y|R363E, Q316T|K320Y|R363E|G662F, Q226W|Q316T|R363E|V522Y|G662F, P176L|K320S|R363E|G662C, R363E|G547A|G662C, Q226W|K320S|G662C, P176L|Q226W|Q316T|K320Y|R363E|G662F, P176L|Q226W|Q316T|K320S|G662F, P176L|Q316T|K320S|R363E|V522Y|G662C, K320Y|R363E|G662C, K345E|N369E|G372A|S683W, N369E|S683W, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0218] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, or all four of (a), (b), (c), and (d) and the substitutions are: K345E|N369E|G372A|P661E, N369T|P661L|S683W, K345E|N369T|G372A|P661E|S683W, K320S|R363E, E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C, T242S|T666C, Q226Y|T666C, Q216E|S312K|S692K, P176L|G662F, P176L|Q226W|Q316T|K320Y|R363E, P176L|Q226W|K320S|R363E|G662F, P176L|Q226W|Q316T|K320Y|V522Y, P176L|Q226W|K320Y|R363E|V522Y, Q226W|K320Y|R363E, Q316T|K320Y|R363E|G662F, Q226W|Q316T|R363E|V522Y|G662F, P176L|K320S|R363E|G662C, R363E|G547A|G662C, Q226W|K320S|G662C, P176L|Q226W|Q316T|K320Y|R363E|G662F, P176L|Q226W|Q316T|K320S|G662F, P176L|Q316T|K320S|R363E|V522Y|G662C, K320Y|R363E|G662C, K345E|N369E|P661L, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0219] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, or all four of (b), (d), (f), and (g) and the substitutions are: L167W|D177M|Q626F|Q684G, L167W|D177M|D564V|Q626F|Q684A, E170F|Q226Y|N369Y|G372A|P661F, L167W|D177M|D564T|Q626F|Q684G, K345E|N369E|G372A|S683W, N369E|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C. In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, or all four of (a), (c), (f), and (g) and the substitutions are: R265M|K560S, K345E|N369E|G372A|S683W, N369E|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0220] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, or all four of (a), (b), (c), and (e) and the substitutions are: N369T|G372A|P661L|S683W, P176L|Q226W|K320Y|R363E, K345E|N369E|P661L, K345E|N369E|G372A|S683W, N369E|S683W, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C. In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, or all four of (b), (d), (e), and (f) and the substitutions are: P176L|K320S|V522Y|G662C, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0221] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, any four, or all five of (a), (b), (c), (d), and (f) and the substitutions are: K345E|N369T|G372A|P661E|S683W, K320S|R363E, E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C, T242S|T666C, Q226Y|T666C, Q216E|S312K|S692K, P176L|G662F, P176L|Q226W|Q316T|K320Y|R363E, P176L|Q226W|K320S|R363E|G662F, P176L|Q226W|Q316T|K320Y|V522Y, P176L|Q226W|K320Y|R363E|V522Y, Q226W|K320Y|R363E, Q316T|K320Y|R363E|G662F, Q226W|Q316T|R363E|V522Y|G662F, P176L|K320S|R363E|G662C, R363E|G547A|G662C, Q226W|K320S|G662C, P176L|Q226W|Q316T|K320Y|R363E|G662F, P176L|Q226W|Q316T|K320S|G662F, P176L|Q316T|K320S|R363E|V522Y|G662C, K320Y|R363E|G662C, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0222] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, any four, or all five of (a), (b), (c), (d) and (e) and the substitutions are: K345E|N369E|P661L, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C. In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, any four, or all five of (a), (c), (d), (e) and (g) and the substitutions are: E170F|V603G, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, and P176L|Q226W|G547A|G662C. In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, any four, or all five of (a), (b), (d), (f) and (g) and the substitutions are: E170F|Q226Y|N369Y|G372A|P661F, L167W|D177M|D564T|Q626F|Q684G, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0223] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, any four, or all five of (a), (b), (d), (e) and (g) and the substitutions are: L167W|D177M|D564T|Q684N, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0224] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, any four, any five, or all six of (a), (b), (c), (e), (f) and (g) and the substitutions are: K345E|N369E|G372A|S683W, N369E|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C. In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, any four, any five, or all six of (a), (b), (c), (d), (e) and (g) and the substitutions are: G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C. In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, any four, any five, or all six of (a), (b), (c), (d), (e) and (f) and the substitutions are: K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

[0225] In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 are selected from any two, any three, any four, any five, any six, or all seven of (a), (b), (c), (d), (e), (f) and (g) and the substitutions are: N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G547A|G662C.

III. CELLULASES

[0226] Cellulases are known in the art as enzymes that hydrolyze cellulose (beta-1,4-glucan or beta D-glucosidic linkages) resulting in the formation of glucose, cellobiose, cellooligosaccharides, and the like. As set forth above, cellulases have been traditionally divided into three major classes: endoglucanases (EC 3.2.1.4) ("EG"), exoglucanases or cellobiohydrolases (EC 3.2.1.91) ("CBH") and beta-glucosidases (EC 3.2.1.21) ("BG").

[0227] Certain fungi produce complete cellulase systems which include exo-cellobiohydrolases or CBH-type cellulases, endoglucanases or EG-type cellulases and beta-glucosidases or BG-type cellulases. However, sometimes these systems lack CBH-type cellulases and bacterial cellulases also typically include little or no CBH-type cellulases. In addition, it has been shown that the EG components and CBH components synergistically interact to more efficiently degrade cellulose. The different components, i.e., the various endoglucanases and exocellobiohydrolases in a multi-component or complete cellulase system, generally have different properties, such as isoelectric point, molecular weight, degree of glycosylation, substrate specificity and enzymatic action patterns.

[0228] It is believed that endoglucanase-type cellulases hydrolyze internal beta-1,4-glucosidic bonds in regions of low crystallinity of the cellulose and exo-cellobiohydrolase-type cellulases hydrolyze cellobiose from the reducing or non-reducing end of cellulose. It follows that the action of endoglucanase components can greatly facilitate the action of exo-cellobiohydrolases by creating new chain ends which are recognized by exo-cellobiohydrolase components. Further, beta-glucosidase-type cellulases have been shown to catalyze the hydrolysis of alkyl and/or aryl beta-D-glucosides such as methyl beta-D-glucoside and p-nitrophenyl glucoside as well as glycosides containing only carbohydrate residues, such as cellobiose. This yields glucose as the sole product for the microorganism and reduces or eliminates cellobiose that inhibits cellobiohydrolases and endoglucanases.

[0229] Cellulases also find a number of uses in detergent compositions including to enhance cleaning ability, as a softening agent and to improve the feel of cotton fabrics (Hemmpel, ITB Dyeing/Printing/Finishing 3:5-14, 1991; Tyndall, Textile Chemist and Colorist 24:23-26, 1992; and Kumar et al., Textile Chemist and Colorist, 29:37-42, 1997). While the mechanism is not part of the disclosure, softening and color restoration properties of cellulase have been attributed to the alkaline endoglucanase components in cellulase compositions, as exemplified by U.S. Pat. Nos. 5,648,263, 5,691,178, and 5,776,757, which disclose that detergent compositions containing a cellulase composition enriched in a specified alkaline endoglucanase component impart color restoration and improved softening to treated garments as compared to cellulase compositions not enriched in such a component. In addition, the use of such alkaline endoglucanase components in detergent compositions has been shown to complement the pH requirements of the detergent composition (e.g., by exhibiting maximal activity at an alkaline pH of 7.5 to 10, as described in U.S. Pat. Nos. 5,648,263, 5,691,178, and 5,776,757).

[0230] Cellulase compositions have also been shown to degrade cotton-containing fabrics, resulting in reduced strength loss in the fabric (U.S. Pat. No. 4,822,516), contributing to reluctance to use cellulase compositions in commercial detergent applications. Cellulase compositions comprising endoglucanase components have been suggested to exhibit reduced strength loss for cotton-containing fabrics as compared to compositions comprising a complete cellulase system.

[0231] Cellulases have also been shown to be useful in degradation of cellulase biomass to ethanol (wherein the cellulase degrades cellulose to glucose and yeast or other microbes further ferment the glucose into ethanol), in the treatment of mechanical pulp (Pere et al., In Proc. Tappi Pulping Conf., Nashville, Tenn., 27-31, pp. 693-696, 1996), for use as a feed additive (WO 91/04673) and in grain wet milling.

[0232] Most CBHs and EGs have a multidomain structure consisting of a core domain separated from a cellulose binding domain (CBD) by a linker peptide (Suurnakki et al., 2000). The core domain contains the active site whereas the CBD interacts with cellulose by binding the enzyme to it (van Tilbeurgh et al., FEBS Lett. 204:223-227, 1986; Tomme et al., Eur. J. Biochem. 170:575-581, 1988). The CBDs are particularly important in the hydrolysis of crystalline cellulose. It has been shown that the ability of cellobiohydrolases to degrade crystalline cellulose clearly decreases when the CBD is absent (Linder and Teeri, J. Biotechnol. 57:15-28, 1997). However, the exact role and action mechanism of CBDs is still a matter of speculation. It has been suggested that the CBD enhances the enzymatic activity merely by increasing the effective enzyme concentration at the surface of cellulose (Stahlberg et al., Bio/Technol. 9:286-290, 1991), and/or by loosening single cellulose chains from the cellulose surface (Tormo et al., EMBO J. vol. 15, no. 21, pp. 5739-5751, 1996). Most studies concerning the effects of cellulase domains on different substrates have been carried out with core proteins of cellobiohydrolases, as their core proteins can easily be produced by limited proteolysis with papain (Tomme et al., 1988). Numerous cellulases have been described in the scientific literature, examples of which include: from Trichoderma reesei: Shoemaker et al., Bio/Technology, 1:691-696, 1983, which discloses CBH1; Teeri et al., Gene, 51:43-52, 1987, which discloses BGL1. Cellulases from species other than Trichoderma have also been described e.g., Ooi et al., Nucleic Acids Research, 18(19) 1990, which discloses the cDNA sequence coding for endoglucanase F1-CMC produced by Aspergillus aculeatus; Kawaguchi et al., Gene, 173:287-8, 1996, which discloses the cloning and sequencing of the cDNA encoding beta-glucosidase 1 from Aspergillus aculeatus; Sakamoto et al., Curr. Genet. 27:435-439, 1995, which discloses the cDNA sequence encoding the endoglucanase CMCase-1 from Aspergillus kawachii IFO 4308; Saarilahti et al., Gene, 90:9-14, 1990, which discloses an endoglucanase from Erwinia carotovara; Spilliaert et al., Eur J Biochem. 224:923-30, 1994, which discloses the cloning and sequencing of bglA, coding for a thermostable beta-glucanase from Rhodothermus marinus; and Halldorsdottir et al., Appl Microbiol Biotechnol., 49:277-84, 1998, which discloses the cloning, sequencing and overexpression of a Rhodothermus marinus gene encoding a thermostable cellulase of glycosyl hydrolase family 12. However, there remains a need for identification and characterization of novel cellulases, with improved properties, such as improved performance under conditions of thermal stress or in the presence of surfactants, increased specific activity, altered substrate cleavage pattern, and/or high level expression in vitro.

[0233] The development of new and improved cellulase compositions that comprise varying amounts CBH-type, EG-type and BG-type cellulases is of interest for use: (1) in detergent compositions that exhibit enhanced cleaning ability, function as a softening agent and/or improve the feel of cotton fabrics (e.g., "stone washing" or "biopolishing"); (2) in compositions for degrading wood pulp or other biomass into sugars (e.g., for bio-fuel production); and/or (3) in feed compositions.

[0234] Also provided are enzyme blends comprising one or more beta-glucosidase variants. In certain aspects, the enzyme blend comprises one or more beta-glucosidase variants and a whole cellulase. As used herein, a "whole cellulase" refers to both naturally occurring and non-naturally occurring cellulase containing compositions comprising at least two different enzyme types: (1) endoglucanase, which cleaves internal beta-1,4 linkages resulting in shorter glucooligosaccharides, (2) cellobiohydrolase, which acts in an "exo" manner releasing cellobiose units (beta-1,4 glucose-glucose disaccharide), and optionally (3) beta-glucosidase, releasing glucose monomer from short cellooligosaccharides (e.g., cellobiose).

[0235] A "naturally occurring" composition is one produced by a naturally occurring source and which comprises one or more cellobiohydrolase-type, one or more endoglucanase-type, and one or more beta-glucosidase components, wherein each of these components is found at the ratio produced by the source. A naturally occurring composition is one that is produced by an organism unmodified with respect to the cellulolytic enzymes such that the ratio of the component enzymes is unaltered from that produced by the native organism. A "non-naturally occurring" composition encompasses those compositions produced by: (1) combining component cellulolytic enzymes either in a naturally occurring ratio or non-naturally occurring, i.e., altered, ratio; or (2) modifying an organism to overexpress or underexpress one or more cellulolytic enzymes; or (3) modifying an organism such that at least one cellulolytic enzyme is deleted. Accordingly, in some embodiments, the whole cellulase preparation can have one or more of the various EGs and/or CBHs, and/or beta-glucosidase deleted or overexpressed.

[0236] In the present disclosure, the whole cellulase preparation can be from any microorganism that is useful for the hydrolysis of a cellulosic material. In some embodiments, the whole cellulase preparation is a filamentous fungal whole cellulase.

[0237] In some embodiments, the whole cellulase preparation is from an Acremonium, Aspergillus, Chrysosporium, Emericella, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia, Tolypocladium, or Trichoderma species.

[0238] In some embodiments, the whole cellulase preparation is an Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae whole cellulase. In another aspect, whole cellulase preparation is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum whole cellulase. In another aspect, the whole cellulase preparation is a Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Penicillium funiculosum, Scytalidium thermophilum, or Thielavia terrestris whole cellulase. In yet another aspect, the whole cellulase preparation is a Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei (e.g., RL-P37 (Sheir-Neiss et al., Appl. Microbiol. Biotechnology, 20:46-53, 1984; and Montenecourt, Can., 1-20, 1987), QM9414 (ATCC No. 26921), NRRL 15709, ATCC 13631, 56764, 56466, 56767), or Trichoderma viride, e.g., ATCC 32098 and 32086, whole cellulase.

[0239] In some embodiments, the whole cellulase preparation is a Trichoderma reesei RutC30 whole cellulase, which is available from the American Type Culture Collection as Trichoderma reesei ATCC 56765. In some embodiments, the whole cellulase is Penicillium funiculosum, which is available from the American Type Culture Collection as Penicillium funiculosum ATCC Number: 10446.

[0240] The whole cellulase preparation may also be obtained from commercial sources. Examples of commercial cellulase preparations suitable for use in the present disclosure include, for example, CELLUCLAST.TM. and CELLIC.TM. (available from Novozymes A/S) and LAMINEX.TM. BG, INDIAGE.TM. 44L, PRIMAFAST.TM. 100, PRIMAFAST.TM. 200, SPEZYME.TM. CP, ACCELLERASE.RTM. 1000 and ACCELLERASE.RTM. 1500 (Danisco US. Inc., Genencor).

[0241] In the present disclosure, the whole cellulase preparation can be from any microorganism cultivation method known in the art resulting in the expression of enzymes capable of hydrolyzing a cellulosic material. Fermentation can include shake flask cultivation, small- or large-scale fermentation, such as continuous, batch, fed-batch, or solid state fermentations in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the cellulase to be expressed or isolated.

[0242] Generally, the microorganism is cultivated in a cell culture medium suitable for production of enzymes capable of hydrolyzing a cellulosic material. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable culture media, temperature ranges and other conditions suitable for growth and cellulase production are known in the art. As a non-limiting example, the normal temperature range for the production of cellulases by Trichoderma reesei is 24.degree. C. to 28.degree. C.

[0243] Generally, the whole cellulase preparation is used as is produced by fermentation with no or minimal recovery and/or purification. For example, once cellulases are secreted by a cell into the cell culture medium, the cell culture medium containing the cellulases can be used. In some embodiments the whole cellulase preparation comprises the unfractionated contents of fermentation material, including cell culture medium, extracellular enzymes and cells. Alternatively, the whole cellulase preparation can be processed by any convenient method, e.g., by precipitation, centrifugation, affinity, filtration or any other method known in the art. In some embodiments, the whole cellulase preparation can be concentrated, for example, and then used without further purification. In some embodiments the whole cellulase preparation comprises chemical agents that decrease cell viability or kills the cells. In some embodiments, the cells are lysed or permeabilized using methods known in the art.

[0244] The endoglucanase activity of the whole cellulase preparation may be determined using carboxymethyl cellulose (CMC) as a substrate. Determination of whole cellulase activity, measured in terms of CMC activity. This method measures the production of reducing ends created by the enzyme mixture acting on CMC wherein 1 unit is the amount of enzyme that liberates 1 .mu.mol of product/minute (Ghose, Measurement of Cellulose Activities, Pure Appl. Chem., 59:257-268, 1987).

[0245] In certain aspects, the cellulase is a beta-glucosidase-enriched cellulase. Beta-glucosidase enhanced whole cellulases generally comprise beta-glucosidase and a whole cellulase preparation. However, it is to be understood that the beta-glucosidase enhanced whole cellulase compositions can be produced by recombinant means. For example, expressing beta-glucosidase in a microorganism capable of producing a whole cellulase. In some embodiments the beta-glucosidase enhanced whole cellulase composition comprises a whole cellulase preparation and beta-glucosidase. In specific embodiments, the beta-glucosidase enhanced whole cellulase composition comprises on a protein weight basis at least at least 5%, at least 7%, at least 10%, at least 15% or at least 20%, and up to 25%, 30%, 35%, up to 40%, or up to 50% beta-glucosidase.

IV. METHODS OF PRODUCING VARIANT BGL1 NUCLEIC ACID SEQUENCES

[0246] In one embodiment this disclosure provides for the expression of variant bgl1 genes under control of a promoter functional in a filamentous fungus. Therefore, this disclosure relies on routine techniques in the field of recombinant genetics (See, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., 1989; Kriegler, Gene Transfer and Expression: A Laboratory Manual, 1990; and Ausubel et al., eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing and Wiley-Interscience, New York, 1994). Any method known in the art that can introduce mutations is contemplated by the present disclosure.

[0247] The present disclosure relates to the expression, purification and/or isolation and use of variant BGL1. These enzymes are preferably prepared by recombinant methods utilizing the bgl1 gene from H. jecorina. The fermentation broth may be used with or without purification.

[0248] After the isolation and cloning of the bgl1 gene from H. jecorina, other methods known in the art, such as site directed mutagenesis, are used to make the substitutions, additions or deletions that correspond to substituted amino acids in the expressed bgl1 variant. Again, site directed mutagenesis and other methods of incorporating amino acid changes in expressed proteins at the DNA level are known in the art (Sambrook et al., supra; and Ausubel et al., supra).

[0249] DNA encoding an amino acid sequence variant of the H. jecorina BGL1 is prepared by a variety of methods known in the art. These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared DNA encoding the H. jecorina BGL1.

[0250] Site-directed mutagenesis is a preferred method for preparing substitution variants. This technique is well known in the art (see, e.g., Carter et al. Nucleic Acids Res. 13:4431-4443, 1985; and Kunkel et al., Proc. Natl. Acad. Sci. USA 82:488; 1987). Briefly, in carrying out site-directed mutagenesis of DNA, the starting DNA is altered by first hybridizing an oligonucleotide encoding the desired mutation to a single strand of such starting DNA. After hybridization, a DNA polymerase is used to synthesize an entire second strand, using the hybridized oligonucleotide as a primer, and using the single strand of the starting DNA as a template. Thus, the oligonucleotide encoding the desired mutation is incorporated in the resulting double-stranded DNA.

[0251] PCR mutagenesis is also suitable for making amino acid sequence variants of the starting polypeptide, i.e., H. jecorina BGL1 (See, e.g., Higuchi, in PCR Protocols, pp. 177-183, Academic Press, 1990; Vallette et al., Nuc. Acids Res. 17:723-733, 1989; and Cadwell et al., PCR Methods and Applications, 2:28-33, 1992). Briefly, when small amounts of template DNA are used as starting material in a PCR, primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence only at the positions where the primers differ from the template.

[0252] Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al., Gene 34:315-323, 1985. The starting material is the plasmid (or other vector) comprising the starting polypeptide DNA to be mutated. The codon(s) in the starting DNA to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist, they may be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate locations in the starting polypeptide DNA. The plasmid DNA is cut at these sites to linearize it. A double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures, wherein the two strands of the oligonucleotide are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is referred to as the cassette. This cassette is designed to have 5' and 3' ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid. This plasmid now contains the mutated DNA sequence.

[0253] Alternatively, or additionally, the desired amino acid sequence encoding a variant BGL1 can be determined, and a nucleic acid sequence encoding such amino acid sequence variant can be generated synthetically.

[0254] The variant BGL1(s) so prepared may be subjected to further modifications, oftentimes depending on the intended use of the cellulase. Such modifications may involve further alteration of the amino acid sequence, fusion to heterologous polypeptide(s) and/or covalent modifications.

V. BGL1 NUCLEIC ACIDS AND BGL1 POLYPEPTIDES

[0255] A. Variant Bgl1 Nucleic Acids

[0256] The nucleic acid sequence for the wild type bgl1 is shown in SEQ ID NO:1. The disclosure encompasses a nucleic acid molecule encoding the variant beta-glucosidase described herein. The nucleic acid may be a DNA molecule. The disclosure further provides isolated, synthetic or recombinant nucleic acids comprising a nucleic acid sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%; 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to a nucleic acid sequence encoding a variant beta-glucosidase described herein, over least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, or more residues.

[0257] The disclosure provides expression cassettes comprising a nucleic acid of the disclosure or a subsequence thereof. In one aspect, the expression cassette can comprise the nucleic acid operably linked to a promoter. The promoter can be a fungal, viral, bacterial, mammalian or plant promoter. The promoter can be a constitutive promoter or an inducible promoter. In one aspect, the promoter is expressible in filamentous fungi, e.g., Trichoderma reesei. In specific embodiments, the promoter is from a filamentous fungus, e.g., the Trichoderma reesei cellobiohydrolase I ("CBHI") gene promoter.

[0258] The disclosure provides a recombinant cell (e.g., host cell) engineered to express a nucleic acid of the disclosure or an expression cassette of the disclosure. In certain aspects, the recombinant cell is a bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insect cell or a plant cell. In a specific aspect, the recombinant cell is a filamentous fungal cell.

[0259] The disclosure provides transgenic plants comprising a nucleic acid of the disclosure or an expression cassette of the disclosure.

[0260] After DNA sequences that encode the BGL1 variants have been cloned into DNA constructs, the DNA is used to transform microorganisms. The microorganism to be transformed for the purpose of expressing a variant bgl1 according to the present disclosure may advantageously comprise a strain derived from Trichoderma sp. Thus, a preferred mode for preparing variant BGL1 cellulases according to the present disclosure comprises transforming a Trichoderma sp. host cell with a DNA construct comprising at least a fragment of DNA encoding a portion or all of the variant BGL1. The DNA construct will generally be functionally attached to a promoter. The transformed host cell is then grown under conditions so as to express the desired protein. Subsequently, the desired protein product may be purified to substantial homogeneity.

[0261] However, it may in fact be that the best expression vehicle for a given DNA encoding a variant BGL1 may differ from H. jecorina. Thus, it may be that it will be most advantageous to express a protein in a transformation host that bears phylogenetic similarity to the source organism for the variant BGL1. In an alternative embodiment, Aspergillus niger can be used as an expression vehicle. For a description of transformation techniques with A. niger, see WO 98/31821, the disclosure of which is incorporated by reference in its entirety.

[0262] Accordingly, the present description of an Aspergillus spp. expression system is provided for illustrative purposes only and as one option for expressing the variant BGL1 of the disclosure. One of skill in the art, however, may be inclined to express the DNA encoding variant BGL1 in a different host cell if appropriate and it should be understood that the source of the variant BGL1 should be considered in determining the optimal expression host. Additionally, the skilled worker in the field will be capable of selecting the best expression system for a particular gene through routine techniques utilizing the tools available in the art.

[0263] B. Variant BGL1 Polypeptides

[0264] The disclosure provides isolated, synthetic or recombinant polypeptides comprising an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to a polypeptide sequence of a variant beta-glucosidase over at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350 or more residues, or over the full length of the immature polypeptide or the full length mature polypeptide.

[0265] The variant beta-glucosidases of this disclosure have amino acid sequences that are derived from the amino acid sequence of a precursor BGL1. The amino acid sequence of the BGL1 variant differs from the precursor BGL1 amino acid sequence by the substitution, deletion or insertion of one or more amino acids of the precursor amino acid sequence. In a preferred embodiment, the precursor BGL1 is Hypocrea jecorina BGL1. The mature amino acid sequence of H. jecorina BGL1 is shown in Example 2 (SEQ ID NO:3). Thus, this disclosure is directed to BGL1 variants which contain amino acid residues at positions which are equivalent to the particular identified residue in H. jecorina BGL1. A residue (amino acid) of an BGL1 homolog is equivalent to a residue of Hypocrea jecorina BGL1 if it is either homologous (i.e., corresponding in position in either primary or tertiary structure) or is functionally analogous to a specific residue or portion of that residue in Hypocrea jecorina BGL1 (i.e., having the same or similar functional capacity to combine, react, or interact chemically or structurally). As used herein, numbering is intended to correspond to that of the mature BGL1 amino acid sequence (SEQ ID NO:3).

[0266] Alignment of amino acid sequences to determine homology is preferably determined by using a "sequence comparison algorithm." Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection, Visual inspection may utilize graphics packages such as, for example, MOE by Chemical Computing Group, Montreal Canada.

[0267] An example of an algorithm that is suitable for determining sequence similarity is the BLAST algorithm, which is described in Altschul, et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. These initial neighborhood word hits act as starting points to find longer HSPs containing them. The word hits are expanded in both directions along each of the two sequences being compared for as far as the cumulative alignment score can be increased. Extension of the word hits is stopped when: the cumulative alignment score falls off by the quantity X from a maximum achieved value; the cumulative score goes to zero or below; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989) alignments (B) of 50, expectation (E) of 10, M'S, N'-4, and a comparison of both strands.

[0268] The BLAST algorithm then performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, an amino acid sequence is considered similar to a protease if the smallest sum probability in a comparison of the test amino acid sequence to a protease amino acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0269] For purposes of the present disclosure, the degree of identity may be suitably determined by means of computer programs known in the art, such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman and Wunsch, Journal of Molecular Biology, 48, 443-45, 1970), using GAP with the following settings for polynucleotide sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3.

[0270] A structural alignment between a T. reesei BGL1 and other cellulases may be used to identify equivalent/corresponding positions in other cellulases having a moderate to high degree of homology, e.g., about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99%, with T. reesei BGL1 (SEQ ID NO: 3). One method of obtaining the structural alignment is to use the Pile Up program from the GCG package using default values of gap penalties, i.e., a gap creation penalty of 3.0 and gap extension penalty of 0.1. Other structural alignment methods include the hydrophobic cluster analysis (Gaboriaud et al., FEBS Letters, 224:149-155, 1987) and reverse threading (Huber and Torda, Protein Science, 7:142-149, 1998).

[0271] An exemplary alignment of the mature form of various reference beta-glucosidases is provided as FIG. 1. The reference cellulases include: TrireBGL1, Hypocrea jecorina (also known as Trichoderma reesei) Q12715 Beta-D-glucoside glucohydrolase 1 (SEQ ID NO:3); HananBglu, Hansenula anomala P06835 Beta-glucosidase (SEQ ID NO:4); PirspBglu, Piromyces sp. E2 Q875K3 Beta-glucosidase (SEQ ID NO:5); CocimBglu, Coccidioides immitis 014424 Beta-glucosidase (SEQ ID NO:6); SacfiBglu2, Saccharomycopsis fibuligera Beta-glucosidase 2 (SEQ ID NO:7); SacfiBglu1, Saccharomycopsis fibuligera P22506 Beta-glucosidase 1 (SEQ ID NO:8); SeplyBglu, Septoria lycopersici Q99324 Beta-1,2-D-glucosidase (SEQ ID NO:9); KurcaBglu, Kuraishia capsulata Q12653 Beta-glucosidase (SEQ ID NO:10); TrireBGL7, Trichoderma reesei Q7Z9M0 Beta-glucosidase 7 (SEQ ID NO:11); UrofaBglu, Uromyces fabae Q70KQ7 Beta glucosidase (SEQ ID NO:12); AspteBglu, Aspergillus terreus (strain NIH 2624/FGSC A1156) Q0CEF3 Beta-glucosidase (SEQ ID NO:13); ChaglBglu, Chaetomium globosum Q2GZ54 Putative beta-glucosidase (SEQ ID NO:14); TrireBGL3, Trichoderma reesei Q7Z9M5 Beta-glucosidase 3 (SEQ ID NO:15); PenbrBGL, Penicillium brasilianum GH3 Beta-glucosidase (SEQ ID NO:16); PerspBglu, Periconia sp. BCC 2871 A9UIG0 Beta-glucosidase (SEQ ID NO:17); PhaavBglu, Phaeosphaeria avenaria Q9P879 Beta-glucosidase (SEQ ID NO:18); AspfuBGL, Aspergillus fumigatus B0XPE1 Beta-glucosidase (SEQ ID NO:19); AsporBGL1, Aspergillus oryzae Q2UUD6 Beta-glucosidase (SEQ ID NO:20); AspacBGL1, Aspergillus aculeatus Beta-glucosidase (SEQ ID NO:21); AspniBGL, Aspergillus niger Q9P8F4 Beta-glucosidase (SEQ ID NO:22); TalemBglu, Talaromyces emersonii Q8TGI8 Beta-glucosidase (SEQ ID NO:23); and TheauBGL, Thermoascus aurentiacus Beta-glucosidase (SEQ ID NO:24). Sequences were aligned using the ClustalW and MUSCLE multiple sequence alignment algorithms. A matrix showing the percent identity of beta-glucosidases of the sequence alignment of FIG. 1 is provided in Table 1. Numbers shown in bold indicate percentage identity with T. reesei BGL1.

TABLE-US-00001 TABLE 1 Beta-Glucosidase Percent Identity Matrix* SEQ ID NO 04 05 06 07 08 09 10 11 12 13 04 14 15 16 17 18 19 20 21 22 23 24 04 + 21 29 37 37 30 31 28 29 30 30 30 32 35 33 36 34 36 36 35 34 35 HananBglu 05 + 22 25 25 25 24 26 26 32 31 31 26 26 27 26 26 26 27 27 26 27 PirspBglu 06 + 35 36 30 30 29 33 33 33 32 34 36 37 36 38 39 38 38 37 38 CocimBglu 07 + 82 32 34 33 33 35 34 33 38 39 39 39 39 39 41 40 38 38 SacfiBglu2 08 + 33 34 33 33 35 34 34 39 40 40 39 40 40 40 40 40 39 SacfiBglu1 09 + 37 38 34 39 39 38 35 37 37 36 37 38 37 38 38 37 SeplyBglu 10 + 47 32 37 35 36 35 36 38 38 36 37 37 37 38 38 KurcaBglu 11 + 31 38 38 37 33 35 36 36 36 34 37 36 37 36 TrireBGL7 12 + 38 35 34 34 35 35 36 36 36 36 36 37 36 UrofaBglu 13 + 58 58 41 40 41 41 40 40 41 39 41 41 AspteBglu 03 + 64 37 38 39 38 38 37 38 37 38 38 TrireBGL1 14 + 38 38 38 36 37 36 36 36 37 37 ChaglBglu 15 + 56 56 53 55 53 55 54 55 57 TrireBGL3 16 + 58 56 57 55 57 56 58 58 PenbrBGL 17 + 73 58 57 59 58 60 61 PerspBglu 18 + 56 57 59 58 58 59 PhaavBglu 19 + 76 76 75 68 70 AspfuBGL 20 + 79 77 68 69 AsporBGL1 21 + 82 67 68 AspacBGL1 22 + 66 68 AspniBGL 23 + 73 TalemBglu 24 + TheauBGL *Numbers in the top row and left column correspond to the SEQ ID NOS of the aligned sequences of FIG. 1. + indicates 100% amino acid sequence identity.

[0272] Sequence searches are typically carried out using the BLASTN program when evaluating a given nucleic acid sequence relative to nucleic acid sequences in the GenBank DNA Sequences and other public databases. The BLASTX program is preferred for searching nucleic acid sequences that have been translated in all reading frames against amino acid sequences in the GenBank Protein Sequences and other public databases. Both BLASTN and BLASTX are run using default parameters of an open gap penalty of 11.0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix. (See, e.g., Altschul, et al., 1997.)

VI. EXPRESSION OF RECOMBINANT BGL1 VARIANTS

[0273] The disclosure further provides methods of producing recombinant beta-glucosidase variants comprising the steps of: (a) culturing a host cell engineered to express a beta-glucosidase variant of the disclosure; and (b) recovering the beta-glucosidase variant. Step (b) can entail recovering fermentation broth comprising the beta-glucosidase variant, and optionally can include further purification step(s).

[0274] The methods of the disclosure rely on the use cells to express variant bgl1, with no particular method of bgl1 expression required. The variant BGL1 is preferably secreted from the cells. The disclosure provides host cells which have been transduced, transformed or transfected with an expression vector comprising a variant BGL1-encoding nucleic acid sequence. The culture conditions, such as temperature, pH and the like, are those previously used for the parental host cell prior to transduction, transformation or transfection and will be apparent to those skilled in the art.

[0275] In one approach, a filamentous fungal cell or yeast cell is transfected with an expression cassette having a promoter or biologically active promoter fragment or one or more (e.g., a series of) enhancers which functions in the host cell line, operably linked to a DNA segment encoding variant BGL1, such that variant bgl1 is expressed in the cell line.

[0276] A. Nucleic Acid Constructs/Expression Vectors

[0277] Natural or synthetic polynucleotide fragments encoding variant BGL1 ("BGL1-encoding nucleic acid sequences") may be incorporated into heterologous nucleic acid constructs or vectors, capable of introduction into, and replication in, a filamentous fungal or yeast cell. The vectors and methods disclosed herein are suitable for use in host cells for the expression of variant BGL1. Any vector may be used as long as it is replicable and viable in the cells into which it is introduced. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. Cloning and expression vectors are also described in Sambrook et al., 1989, Ausubel F M et al., 1989, and Strathern et al., The Molecular Biology of the Yeast Saccharomyces, 1981, each of which is expressly incorporated by reference herein. Appropriate expression vectors for fungi are described in van den Hondel et al. (1991) In: Bennett and Lasure (eds.) More Gene Manipulations in Fungi. Academic Press, pp. 396-428. The appropriate DNA sequence may be inserted into a plasmid or vector (collectively referred to herein as "vectors") by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by standard procedures. Such procedures and related sub-cloning procedures are deemed to be within the scope of knowledge of those skilled in the art.

[0278] Recombinant filamentous fungi comprising the coding sequence for variant bgl1 may be produced by introducing a heterologous nucleic acid construct comprising the variant bgl1 coding sequence into the cells of a selected strain of the filamentous fungi.

[0279] Once the desired form of a variant bgl1 nucleic acid sequence is obtained, it may be modified in a variety of ways. Where the sequence involves non-coding flanking regions, the flanking regions may be subjected to resection, mutagenesis, etc. Thus, transitions, transversions, deletions, and insertions may be performed on the naturally occurring sequence.

[0280] A selected variant bgl1 coding sequence may be inserted into a suitable vector according to well-known recombinant techniques and used to transform filamentous fungi capable of bgl1 expression. Due to the inherent degeneracy of the genetic code, other nucleic acid sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used to clone and express variant bgl1. Therefore it is appreciated that such substitutions in the coding region fall within the sequence variants covered by the present disclosure. Any and all of these sequence variants can be utilized in the same way as described herein for a parent BGL1-encoding nucleic acid sequence.

[0281] The present disclosure also includes recombinant nucleic acid constructs comprising one or more of the variant BGL1-encoding nucleic acid sequences as described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the disclosure has been inserted, in a forward or reverse orientation.

[0282] Heterologous nucleic acid constructs may include the coding sequence for variant bgl1. (i) in isolation; (ii) in combination with additional coding sequences; such as fusion protein or signal peptide coding sequences, where the bgl1 coding sequence is the dominant coding sequence; (iii) in combination with non-coding sequences, such as introns and control elements, such as promoter and terminator elements or 5' and/or 3' untranslated regions, effective for expression of the coding sequence in a suitable host; and/or (iv) in a vector or host environment in which the bgl1 coding sequence is a heterologous gene.

[0283] In one aspect of the present disclosure, a heterologous nucleic acid construct is employed to transfer a variant BGL1-encoding nucleic acid sequence into a cell in vitro, with established filamentous fungal and yeast lines preferred. For long-term, production of variant BGL1, stable expression is preferred. It follows that any method effective to generate stable transformants may be used in practicing the disclosure.

[0284] Appropriate vectors are typically equipped with a selectable marker-encoding nucleic acid sequence, insertion sites, and suitable control elements, such as promoter and termination sequences. The vector may comprise regulatory sequences, including, for example, non-coding sequences, such as introns and control elements, i.e., promoter and terminator elements or 5' and/or 3' untranslated regions, effective for expression of the coding sequence in host cells (and/or in a vector or host cell environment in which a modified soluble protein antigen coding sequence is not normally expressed), operably linked to the coding sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, many of which are commercially available and/or are described in Sambrook, et al., (supra).

[0285] Exemplary promoters include both constitutive promoters and inducible promoters, examples of which include a CMV promoter, an SV40 early promoter, an RSV promoter, an EF-1.alpha. promoter, a promoter containing the tet responsive element (TRE) in the tet-on or tet-off system as described (ClonTech and BASF), the beta actin promoter and the metallothionine promoter that can upregulated by addition of certain metal salts. A promoter sequence is a DNA sequence which is recognized by the particular filamentous fungus for expression purposes. It is operably linked to DNA sequence encoding a variant BGL1 polypeptide. Such linkage comprises positioning of the promoter with respect to the initiation codon of the DNA sequence encoding the variant BGL1 polypeptide in the disclosed expression vectors. The promoter sequence contains transcription and translation control sequence which mediate the expression of the variant BGL1 polypeptide. Examples include the promoters from the Aspergillus niger, A. awamori or A. oryzae glucoamylase, alpha-amylase, or alpha-glucosidase encoding genes; the A. nidulans gpdA or trpC Genes; the Neurospora crassa cbh1 or trp1 genes; the A. niger or Rhizomucor miehei aspartic proteinase encoding genes; the H. jecorina (T. reesei) bgl1, cbh1, cbh2, egl1, egl2, or other cellulase encoding genes.

[0286] The choice of the proper selectable marker will depend on the host cell, and appropriate markers for different hosts are well known in the art. Typical selectable marker genes include argB from A. nidulans or T. reesei, amdS from A. nidulans, pyr4 from Neurospora crassa or T. reesei, pyrG from Aspergillus niger or A. nidulans. Additional exemplary selectable markers include, but are not limited to trpc, trp1, oliC31, niaD or leu2, which are included in heterologous nucleic acid constructs used to transform a mutant strain such as trp.sup.-, pyr.sup.-, leu.sup.- and the like.

[0287] Such selectable markers confer to transformants the ability to utilize a metabolite that is usually not metabolized by the filamentous fungi. For example, the amdS gene from H. jecorina which encodes the enzyme acetamidase that allows transformant cells to grow on acetamide as a nitrogen source. The selectable marker (e.g. pyrG) may restore the ability of an auxotrophic mutant strain to grow on a selective minimal medium or the selectable marker (e.g. olic31) may confer to transformants the ability to grow in the presence of an inhibitory drug or antibiotic.

[0288] The selectable marker coding sequence is cloned into any suitable plasmid using methods generally employed in the art. Exemplary plasmids include pUC18, pBR322, pRAX and pUC100. The pRAX plasmid contains AMAL sequences from A. nidulans, which make it possible to replicate in A. niger.

[0289] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Sambrook et al., 1989; Freshney, Animal Cell Culture, 1987; Ausubel, et al., 1993; and Coligan et al., Current Protocols in Immunology, 1991.

[0290] B. Host Cells and Culture Conditions for BGL1 Production

[0291] (i) Filamentous Fungi

[0292] Thus, the present disclosure provides filamentous fungi comprising cells which have been modified, selected and cultured in a manner effective to result in variant BGL1 production or expression relative to the corresponding non-transformed parental fungi.

[0293] Examples of species of parental filamentous fungi that may be treated and/or modified for variant bgl1 expression include, but are not limited to Trichoderma, e.g., Trichoderma reesei, Trichoderma longibrachiatum, Trichoderma viride, Trichoderma koningii; Penicillium sp., Humicola sp., including Humicola insolens; Aspergillus sp., Chrysosporium sp., Fusarium sp., Hypocrea sp., and Emericella sp.

[0294] Cells expressing bgl1 are cultured under conditions typically employed to culture the parental fungal line. Generally, cells are cultured in a standard medium containing physiological salts and nutrients, such as described in Pourquie, J. et al., Biochemistry and Genetics of Cellulose Degradation, eds. Aubert et al., Academic Press, pp. 71-86, 1988 and Ilmen et al., Appl. Environ. Microbiol. 63:1298-1306, 1997. Culture conditions are also standard, e.g., cultures are incubated at 28.degree. C. in shaker cultures or fermenters until desired levels of bgl1 expression are achieved.

[0295] Preferred culture conditions for a given filamentous fungus may be found in the scientific literature and/or from the source of the fungi such as the American Type Culture Collection (ATCC; www.atcc.org/). After fungal growth has been established, the cells are exposed to conditions effective to cause or permit the expression of variant bgl1.

[0296] In cases where a BGL1 encoding sequence is under the control of an inducible promoter, the inducing agent, e.g., a sugar, metal salt or antibiotics, is added to the medium at a concentration effective to induce bgl1 expression.

[0297] In one embodiment, the strain comprises Aspergillus niger, which is a useful strain for obtaining overexpressed protein. For example A. niger var awamori dgr246 is known to secrete elevated amounts of secreted cellulases (Goedegebuur et al., Curr. Genet (2002) 41: 89-98). Other strains of Aspergillus niger var awamori such as GCDAP3, GCDAP4 and GAP3-4 are known (Ward et al., 1993, Appl. Microbiol. Biotechnol. 39:738-743).

[0298] In another embodiment, the strain comprises Trichoderma reesei, which is a useful strain for obtaining overexpressed protein. For example, RL-P37, described by Sheir-Neiss, et al., Appl. Microbiol. Biotechnol. 20:46-53 (1984) is known to secrete elevated amounts of cellulase enzymes. Functional equivalents of RL-P37 include Trichoderma reesei strain RUT-C30 (ATCC No. 56765) and strain QM9414 (ATCC No. 26921). It is contemplated that these strains would also be useful in overexpressing variant bgl1.

[0299] Where it is desired to obtain the variant BGL1 in the absence of potentially detrimental native cellulolytic activity, it is useful to obtain a Trichoderma host cell strain which has had one or more cellulase genes deleted prior to introduction of a DNA construct or plasmid containing the DNA fragment encoding the variant BGL1. Such strains may be prepared by the method disclosed in U.S. Pat. No. 5,246,853 and WO 92/06209, which disclosures are hereby incorporated by reference. By producing a variant BGL1 cellulase in a host microorganism that is missing one or more cellulase genes, the identification and subsequent purification procedures are simplified. Any gene from Trichoderma sp. which has been cloned can be deleted, for example, the bgl1, cbh1, cbh2, egl1, and egl2 genes as well as those encoding EG III and/or EGV protein (see e.g., U.S. Pat. No. 5,475,101 and WO 94/28117, respectively).

[0300] Gene deletion may be accomplished by inserting a form of the desired gene to be deleted or disrupted into a plasmid by methods known in the art. The deletion plasmid is then cut at an appropriate restriction enzyme site(s), internal to the desired gene coding region, and the gene coding sequence or part thereof replaced with a selectable marker. Flanking DNA sequences from the locus of the gene to be deleted or disrupted, preferably between about 0.5 to 2.0 kb, remain on either side of the selectable marker gene. An appropriate deletion plasmid will generally have unique restriction enzyme sites present therein to enable the fragment containing the deleted gene, including flanking DNA sequences, and the selectable marker gene to be removed as a single linear piece.

[0301] A selectable marker must be chosen so as to enable detection of the transformed microorganism. Any selectable marker gene that is expressed in the selected microorganism will be suitable. For example, with Aspergillus sp., the selectable marker is chosen so that the presence of the selectable marker in the transformants will not significantly affect the properties thereof. Such a selectable marker may be a gene that encodes an assayable product. For example, a functional copy of a Aspergillus sp. gene may be used which, if lacking in the host strain, results in the host strain displaying an auxotrophic phenotype. Similarly, selectable markers exist for Trichoderma sp.

[0302] In one embodiment, a pyrG.sup.- derivative strain of Aspergillus sp. is transformed with a functional pyrG gene, which thus provides a selectable marker for transformation. A pyrG.sup.- derivative strain may be obtained by selection of Aspergillus sp. strains that are resistant to fluoroorotic acid (FOA). The pyrG gene encodes orotidine-5'-monophosphate decarboxylase, an enzyme required for the biosynthesis of uridine. Strains with an intact pyrG gene grow in a medium lacking uridine but are sensitive to fluoroorotic acid. It is possible to select pyrG.sup.- derivative strains that lack a functional orotidine monophosphate decarboxylase enzyme and require uridine for growth by selecting for FOA resistance. Using the FOA selection technique it is also possible to obtain uridine-requiring strains which lack a functional orotate pyrophosphoribosyl transferase. It is possible to transform these cells with a functional copy of the gene encoding this enzyme (Berges and Barreau, Curr. Genet. 19:359-365, 1991; and van Hartingsveldt et al., Mol. Gen. Genet. 206:71-75, 1986). Selection of derivative strains is easily performed using the FOA resistance technique referred to above, and thus, the pyrG gene is preferably employed as a selectable marker.

[0303] In a second embodiment, a pyr4.sup.- derivative strain of Hypocrea sp. (Trichoderma sp.) is transformed with a functional pyr4 gene, which thus provides a selectable marker for transformation. A pyr4.sup.- derivative strain may be obtained by selection of Hyprocrea sp. (Trichoderma sp.) strains that are resistant to fluoroorotic acid (FOA). The pyr4 gene encodes orotidine-5'-monophosphate decarboxylase, an enzyme required for the biosynthesis of uridine. Strains with an intact pyr4 gene grow in a medium lacking uridine but are sensitive to fluoroorotic acid. It is possible to select pyr4.sup.- derivative strains that lack a functional orotidine monophosphate decarboxylase enzyme and require uridine for growth by selecting for FOA resistance. Using the FOA selection technique it is also possible to obtain uridine-requiring strains which lack a functional orotate pyrophosphoribosyl transferase. It is possible to transform these cells with a functional copy of the gene encoding this enzyme (Berges and Barreau, 1991). Selection of derivative strains is easily performed using the FOA resistance technique referred to above, and thus, the pyr4 gene is preferably employed as a selectable marker.

[0304] To transform pyrG.sup.- Aspergillus sp. or pyr4.sup.- Hyprocrea sp. (Trichoderma sp.) so as to be lacking in the ability to express one or more cellulase genes, a single DNA fragment comprising a disrupted or deleted cellulase gene is then isolated from the deletion plasmid and used to transform an appropriate pyr.sup.- Aspergillus or pyr.sup.- Trichoderma host. Transformants are then identified and selected based on their ability to express the pyrG or pyr4, respectively, gene product and thus compliment the uridine auxotrophy of the host strain. Southern blot analysis is then carried out on the resultant transformants to identify and confirm a double crossover integration event that replaces part or all of the coding region of the genomic copy of the gene to be deleted with the appropriate pyr selectable markers.

[0305] Although the specific plasmid vectors described above relate to preparation of pyr.sup.- transformants, the present disclosure is not limited to these vectors. Various genes can be deleted and replaced in the Aspergillus sp. or Hyprocrea sp. (Trichoderma sp.) strain using the above techniques. In addition, any available selectable markers can be used, as discussed above. In fact, any host, e.g., Aspergillus sp. or Hyprocrea sp., gene that has been cloned, and thus identified, can be deleted from the genome using the above described strategy.

[0306] As stated above, the host strains used may be derivatives of Hyprocrea sp. (Trichoderma sp.) that lack or have a nonfunctional gene or genes corresponding to the selectable marker chosen. For example, if the selectable marker of pyrG is chosen for Aspergillus sp., then a specific pyrG.sup.- derivative strain is used as a recipient in the transformation procedure. Also, for example, if the selectable marker of pyr4 is chosen for a Hyprocrea sp., then a specific pyr4.sup.- derivative strain is used as a recipient in the transformation procedure. Similarly, selectable markers comprising Hyprocrea sp.

[0307] (Trichoderma sp.) genes equivalent to the Aspergillus nidulans genes amdS, argB, trpC, niaD may be used. The corresponding recipient strain must therefore be a derivative strain such as argB.sup.-, trpC.sup.-, niaD.sup.-, respectively.

[0308] DNA encoding the BGL1 variant is then prepared for insertion into an appropriate microorganism. According to the present disclosure, DNA encoding a BGL1 variant comprises the DNA necessary to encode for a protein that has functional cellulolytic activity. The DNA fragment encoding the BGL1 variant may be functionally attached to a fungal promoter sequence, for example, the promoter of the glaA gene in Aspergillus or the promoter of the cbh1 or egl1 genes in Trichoderma.

[0309] It is also contemplated that more than one copy of DNA encoding a BGL1 variant may be recombined into the strain to facilitate overexpression. The DNA encoding the BGL1 variant may be prepared by the construction of an expression vector carrying the DNA encoding the variant. The expression vector carrying the inserted DNA fragment encoding the BGL1 variant may be any vector which is capable of replicating autonomously in a given host organism or of integrating into the DNA of the host, typically a plasmid. In preferred embodiments two types of expression vectors for obtaining expression of genes are contemplated. The first contains DNA sequences in which the promoter, gene-coding region, and terminator sequence all originate from the gene to be expressed. Gene truncation may be obtained where desired by deleting undesired DNA sequences (e.g., coding for unwanted domains) to leave the domain to be expressed under control of its own transcriptional and translational regulatory sequences. A selectable marker may also be contained on the vector allowing the selection for integration into the host of multiple copies of the novel gene sequences.

[0310] The second type of expression vector is preassembled and contains sequences required for high-level transcription and a selectable marker. It is contemplated that the coding region for a gene or part thereof can be inserted into this general-purpose expression vector such that it is under the transcriptional control of the expression cassette promoter and terminator sequences.

[0311] For example, in Aspergillus, pRAX is such a general-purpose expression vector. Genes or part thereof can be inserted downstream of the strong glaA promoter.

[0312] For example, in Hypocrea, pTREX is such a general-purpose expression vector. Genes or part thereof can be inserted downstream of the strong cbh1 promoter.

[0313] In the vector, the DNA sequence encoding the BGL1 variant of the present disclosure should be operably linked to transcriptional and translational sequences, i.e., a suitable promoter sequence and signal sequence in reading frame to the structural gene. The promoter may be any DNA sequence that shows transcriptional activity in the host cell and may be derived from genes encoding proteins either homologous or heterologous to the host cell. An optional signal peptide provides for extracellular production of the BGL1 variant. The DNA encoding the signal sequence is preferably that which is naturally associated with the gene to be expressed, however the signal sequence from any suitable source, for example an exo-cellobiohydrolase or endoglucanase from Trichoderma, is contemplated in the present disclosure. The procedures used to ligate the DNA sequences coding for the variant BGL1 of the present disclosure with the promoter, and insertion into suitable vectors are well known in the art.

[0314] The DNA vector or construct described above may be introduced in the host cell in accordance with known techniques such as transformation, transfection, microinjection, microporation, biolistic bombardment and the like.

[0315] In the preferred transformation technique, it must be taken into account that the permeability of the cell wall to DNA in Hyprocrea sp. (Trichoderma sp.) is very low. Accordingly, uptake of the desired DNA sequence, gene or gene fragment is at best minimal. There are a number of methods to increase the permeability of the Hyprocrea sp. (Trichoderma sp.) cell wall in the derivative strain (i.e., lacking a functional gene corresponding to the used selectable marker) prior to the transformation process.

[0316] It is understood that in certain circumstances higher or more efficient expression may be achieved by chromosomal integration, as compared to using expression using plasmids. Expression by chromosomal integration is also contemplated herein.

[0317] The preferred method in the present disclosure to prepare Aspergillus sp. or Hyprocrea sp. (Trichoderma sp.) for transformation involves the preparation of protoplasts from fungal mycelium (See Campbell et al., Curr. Genet. 16:53-56; 1989). The mycelium can be obtained from germinated vegetative spores. The mycelium is treated with an enzyme(s) that digests the cell wall resulting in protoplasts. The protoplasts are then protected by the presence of an osmotic stabilizer in the suspending medium. These stabilizers include sorbitol, mannitol, potassium chloride, magnesium sulfate and the like. Usually the concentration of these stabilizers varies between 0.8 M and 1.2 M. It is preferable to use about a 1.2 M solution of sorbitol in the suspension medium.

[0318] Uptake of the DNA into the host strain, (Aspergillus sp. or Hyprocrea sp. (Trichoderma sp.)), is dependent upon the calcium ion concentration. Generally between about 10 mM CaCl.sub.2 and 50 mM CaCl.sub.2 is used in an uptake solution. Besides the need for the calcium ion in the uptake solution, other items generally included are a buffering system such as TE buffer (10 Mm Tris, pH 7.4; 1 mM EDTA) or 10 mM MOPS, pH 6.0 buffer (morpholinepropanesulfonic acid) and polyethylene glycol (PEG). It is believed that the polyethylene glycol acts to fuse the cell membranes thus permitting the contents of the medium to be delivered into the cytoplasm of the host cell, by way of example either Aspergillus sp. or Hyprocrea sp. strain, and the plasmid DNA is transferred to the nucleus. This fusion frequently leaves multiple copies of the plasmid DNA integrated into the host chromosome.

[0319] Usually a suspension containing the Aspergillus sp. protoplasts or cells that have been subjected to a permeability treatment at a density of 10.sup.5 to 10.sup.6/mL, preferably 210.sup.5/mL are used in transformation. Similarly, a suspension containing the Hyprocrea sp. (Trichoderma sp.) protoplasts or cells that have been subjected to a permeability treatment at a density of 10.sup.8 to 10.sup.9/mL, preferably 2 times 10.sup.8/mL are used in transformation. A volume of 100 .mu.L of these protoplasts or cells in an appropriate solution (e.g., 1.2 M sorbitol; 50 mM CaCl.sub.2) are mixed with the desired DNA. Generally a high concentration of PEG is added to the uptake solution. From 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplast suspension. However, it is preferable to add about 0.25 volumes to the protoplast suspension. Additives such as dimethyl sulfoxide, heparin, spermidine, potassium chloride and the like may also be added to the uptake solution and aid in transformation.

[0320] Generally, the mixture is then incubated at approximately 0.degree. C. for a period of between 10 to 30 minutes. Additional PEG is then added to the mixture to further enhance the uptake of the desired gene or DNA sequence. The 25% PEG 4000 is generally added in volumes of 5 to 15 times the volume of the transformation mixture; however, greater and lesser volumes may be suitable. The 25% PEG 4000 is preferably about 10 times the volume of the transformation mixture. After the PEG is added, the transformation mixture is then incubated either at room temperature or on ice before the addition of a sorbitol and CaCl.sub.2 solution. The protoplast suspension is then further added to molten aliquots of a growth medium. This growth medium permits the growth of transformants only. Any growth medium can be used in the present disclosure that is suitable to grow the desired transformants. However, if pyr.sup.+ transformants are being selected it is preferable to use a growth medium that contains no uridine. The subsequent colonies are transferred and purified on a growth medium depleted of uridine.

[0321] At this stage, stable transformants may be distinguished from unstable transformants by their faster growth rate and, in Trichoderma, for example, the formation of circular colonies with a smooth, rather than ragged outline on solid culture medium lacking uridine. Additionally, in some cases a further test of stability may made by growing the transformants on solid non-selective medium (i.e. containing uridine), harvesting spores from this culture medium and determining the percentage of these spores which will subsequently germinate and grow on selective medium lacking uridine.

[0322] In a particular embodiment of the above method, the BGL1 variant(s) are recovered in active form from the host cell after growth in liquid media as a result of the appropriate post translational processing of the BGL1 variant.

[0323] (ii) Yeast

[0324] The present disclosure also contemplates the use of yeast as a host cell for BGL1 production. Several other genes encoding hydrolytic enzymes have been expressed in various strains of the yeast S. cerevisiae. These include sequences encoding for two endoglucanases (Penttila et al., Yeast, 3:175-185, 1987), two cellobiohydrolases (Penttila et al., Gene, 63: 103-112, 1988) and one beta-glucosidase from Trichoderma reesei (Cummings and Fowler, Curr. Genet. 29:227-233, 1996), a xylanase from Aureobasidlium pullulans (Li and Ljungdahl, Appl. Environ. Microbiol. 62:209-213, 1996), an alpha-amylase from wheat (Rothstein et al., Gene 55:353-356, 1987), etc. In addition, a cellulase gene cassette encoding the Butyrivibrio fibrisolvens endo-[beta]-1,4-glucanase (END1), Phanerochaete chrysosporium cellobiohydrolase (CBH1), the Ruminococcus flavefaciens cellodextrinase (CEL1) and the Endomyces fibrilizer cellobiase (BGL1) was successfully expressed in a laboratory strain of S. cerevisiae (Van Rensburg et al., Yeast, 14:67-76, 1998).

[0325] C. Introduction of a BGL1-Encoding Nucleic Acid Sequence into Host Cells.

[0326] The disclosure further provides cells and cell compositions which have been genetically modified to comprise an exogenously provided variant BGL1-encoding nucleic acid sequence. A parental cell or cell line may be genetically modified (i.e., transduced, transformed or transfected) with a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc, as further described above.

[0327] The methods of transformation of the present disclosure may result in the stable integration of all or part of the transformation vector into the genome of the filamentous fungus. However, transformation resulting in the maintenance of a self-replicating extra-chromosomal transformation vector is also contemplated.

[0328] Many standard transfection methods can be used to produce Trichoderma reesei cell lines that express large quantities of the heterologus protein. Some of the published methods for the introduction of DNA constructs into cellulase-producing strains of Trichoderma include Lorito, Hayes, DiPietro and Harman, 1993, Curr. Genet. 24: 349-356; Goldman, VanMontagu and Herrera-Estrella, 1990, Curr. Genet. 17:169-174; Penttila, Nevalainen, Ratto, Salminen and Knowles, 1987, Gene 6: 155-164, for Aspergillus Yelton, Hamer and Timberlake, 1984, Proc. Natl. Acad. Sci. USA 81: 1470-1474, for Fusarium Bajar, Podila and Kolattukudy, 1991, Proc. Natl. Acad. Sci. USA 88: 8202-8212, for Streptomyces Hopwood et al., 1985, The John Innes Foundation, Norwich, UK and for Bacillus Brigidi, DeRossi, Bertarini, Riccardi and Matteuzzi, 1990, FEMS Microbiol. Lett. 55: 135-138).

[0329] Other methods for introducing a heterologous nucleic acid construct (expression vector) into filamentous fungi (e.g., H. jecorina) include, but are not limited to the use of a particle or gene gun, permeabilization of filamentous fungi cells walls prior to the transformation process (e.g., by use of high concentrations of alkali, e.g., 0.05 M to 0.4 M CaCl.sub.2 or lithium acetate), protoplast fusion or Agrobacterium mediated transformation. An exemplary method for transformation of filamentous fungi by treatment of protoplasts or spheroplasts with polyethylene glycol and CaCl.sub.2 is described (Campbell et al., Curr. Genet. 16:53-56, 1989; and Penttila et al., Gene, 63:11-22, 1988).

[0330] Any of the well-known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, biolistics, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra). Also of use is the Agrobacterium-mediated transfection method described in U.S. Pat. No. 6,255,115. It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the heterologous gene.

[0331] In addition, heterologous nucleic acid constructs comprising a variant BGL1-encoding nucleic acid sequence can be transcribed in vitro, and the resulting RNA introduced into the host cell by well-known methods, e.g., by injection.

[0332] The disclosure further includes novel and useful transformants of filamentous fungi such as H. jecorina and A. niger for use in producing fungal cellulase compositions. The disclosure includes transformants of filamentous fungi especially fungi comprising the variant bgl1 coding sequence, or deletion of the endogenous bgl1 coding sequence.

[0333] Following introduction of a heterologous nucleic acid construct comprising the coding sequence for a variant bgl1, the genetically modified cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying expression of a variant BGL1-encoding nucleic acid sequence. The culture conditions, such as temperature, pH and the like, are those previously used for the host cell selected for expression, and will be apparent to those skilled in the art.

[0334] The progeny of cells into which such heterologous nucleic acid constructs have been introduced are generally considered to comprise the variant BGL1-encoding nucleic acid sequence found in the heterologous nucleic acid construct.

[0335] The disclosure further includes novel and useful transformants of filamentous fungi such as H. jecorina for use in producing fungal cellulase compositions. Aspergillus niger may also be used in producing the variant BGL1. The disclosure includes transformants of filamentous fungi especially fungi comprising the variant blg1 coding sequence, or deletion of the endogenous bgl1 coding sequence.

[0336] Stable transformants of filamentous fungi can generally be distinguished from unstable transformants by their faster growth rate and, in Trichoderma, for example, the formation of circular colonies with a smooth rather than ragged outline on solid culture medium. Additionally, in some cases, a further test of stability can be made by growing the transformants on solid non-selective medium, harvesting the spores from this culture medium and determining the percentage of these spores which will subsequently germinate and grow on selective medium.

VII. ISOLATION AND PURIFICATION OF RECOMBINANT BGL1 PROTEIN

[0337] In general, a variant BGL1 protein produced in cell culture is secreted into the medium and may be purified or isolated, e.g., by removing unwanted components from the cell culture medium. However, in some cases, a variant BGL1 protein may be produced in a cellular form necessitating recovery from a cell lysate. In such cases the variant BGL1 protein is purified from the cells in which it was produced using techniques routinely employed by those of skill in the art. Examples include, but are not limited to, affinity chromatography (Tilbeurgh et al., FEBS Lett. 16:215, 1984), ion-exchange chromatographic methods (Goyal et al., Bioresource Technol. 36:37-50, 1991; Fliess et al., Eur. J. Appl. Microbiol. Biotechnol. 17:314-318, 1983; Bhikhabhai et al., J. Appl. Biochem. 6:336-345, 1984; Ellouz et al., J. Chromatography 396:307-317, 1987), including ion-exchange using materials with high resolution power (Medve et al., J. Chromatography A 808:153-165, 1998), hydrophobic interaction chromatography (Tomaz and Queiroz, J. Chromatography A 865:123-128, 1999), and two-phase partitioning (Brumbauer, et al., Bioseparation 7:287-295, 1999).

[0338] Typically, the variant BGL1 protein is fractionated to segregate proteins having selected properties, such as binding affinity to particular binding agents, e.g., antibodies or receptors; or which have a selected molecular weight range, or range of isoelectric points.

[0339] Once expression of a given variant BGL1 protein is achieved, the BGL1 protein thereby produced is purified from the cells or cell culture. Exemplary procedures suitable for such purification include the following: antibody-affinity column chromatography, ion exchange chromatography; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; and gel filtration using, e.g., Sephadex G-75. Various methods of protein purification may be employed and such methods are known in the art and described e.g. in Deutscher, Methods in Enzymology, 182:779, 1990; Scopes, Methods Enzymol. 90:479-91, 1982. The purification step(s) selected will depend, e.g., on the nature of the production process used and the particular protein produced.

VIII. UTILITY OF BGL1 AND BGL1

[0340] It can be appreciated that the variant bgl1 nucleic acids, the variant BGL1 protein and compositions comprising variant BGL1 protein activity find utility in a wide variety applications, some of which are described below.

[0341] The present disclosure also provides variant beta-glucosidase and enzyme blends that break down lignocellulose material. Such enzyme combinations or mixtures include a multi-enzyme composition that contains at least one variant beta-glucosidase of the present disclosure. Synergistic enzyme combinations and related methods are contemplated.

[0342] Due to the complex nature of most biomass sources, which can contain cellulose, hemicellulose, pectin, lignin, protein, and ash, among other components, in certain aspects enzyme blends of the disclosure can contain enzymes with a range of substrate specificities that work together to degrade biomass into fermentable sugars in the most efficient manner.

[0343] One example of a multi-enzyme complex for lignocellulose saccharification is a mixture of cellobiohydrolase(s), xylanase(s), endoglucanase(s), beta-glucosidase(s), beta-xylosidase(s), and, optionally, accessory proteins.

[0344] Accordingly, the disclosure provides compositions (including products of manufacture, enzyme ensembles, or "blends") comprising a mixture (or "blend") of xylan-hydrolyzing, hemicellulose- and/or cellulose-hydrolyzing enzymes comprising at least one, several or all of a cellulase, a glucanase; a cellobiohydrolase; an L-alpha-arabinofuranosidase; a xylanase; optionally a beta-glucosidase; a beta-xylosidase, preferably including at least one a beta-glucosidase variant of the disclosure. The present disclosure provides enzyme blends that are non-naturally occurring. As used herein, the term "blend" refers to: (1) a composition made by combining component enzymes, whether in the form of fermentation broth or partially or completely isolated or purified; (2) a composition produced by an organism modified to express one or more component enzymes; optionally, the organism can be also modified to delete one or more genes, optionally encoding proteins affecting xylan hydrolysis, hemicellulose hydrolysis and/or cellulose hydrolysis; (3) a composition made by combining component enzymes simultaneously, separately or sequentially during a saccharification or fermentation reaction; (4) an enzyme mixture produced in situ, e.g., during a saccharification or fermentation reaction; and (5) a combination of any or all of the above (1)-(4).

[0345] The term "fermentation broth" as used herein refers to an enzyme preparation produced by fermentation that undergoes no or minimal recovery and/or purification. For example, microbial cultures are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of enzymes) and once the enzyme is secreted into the cell culture medium, the fermentation broth can be used. The fermentation broth can contain the unfractionated or fractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the fermentation broth is unfractionated and comprises the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells). In some embodiments, the fermentation broth contains the spent cell culture medium, extracellular enzymes, and live or killed microbial cells. In some embodiments, the fermentation broth is fractionated to remove the microbial cells, and comprises the spent cell culture medium and extracellular enzymes.

[0346] It is also to be understood that any of the enzymes described specifically herein can be combined with any one or more of the enzymes described herein or with any other available and suitable enzymes, to produce a multi-enzyme composition. The disclosure is not restricted or limited to the specific exemplary combinations listed below.

[0347] The disclosure provides methods and processes for biomass saccharification, using enzymes of the disclosure, including the enzyme mixtures or "blends" of the disclosure. The biomass can include any composition comprising cellulose and/or hemicellulose (lignocellulosic biomass also comprises lignin), e.g., seeds, grains, tubers, plant waste or byproducts of food processing or industrial processing (e.g., stalks), corn (including cobs, stover, and the like), grasses (e.g., Indian grass, such as Sorghastrum nutans; or, switchgrass, e.g., Panicum species, such as Panicum virgatum), wood (including wood chips, processing waste), paper, pulp, recycled paper (e.g., newspaper). Other biomass materials include, but are not limited to, potatoes, soybean (rapeseed), barley, rye, oats, wheat, beets or sugar cane bagasse.

[0348] The disclosure provides methods of saccharification comprising contacting a composition comprising a xylan, hemicellulose, cellulose or a fermentable sugar with a beta-glucosidase of the disclosure, or a polypeptide encoded by a nucleic acid of the disclosure, or any one of the mixtures or "blends" or products of manufacture of the disclosure.

[0349] The saccharified biomass (e.g., lignocellulosic material processed by enzymes of the disclosure) can be made into bio-based products by fermentation by a microorganism and/or by chemical synthesis. As used herein, a fermenting microorganism can be any microorganism suitable for use in a desired fermentation process for the production bio-based products. Suitable non-limiting examples of fermenting microorganisms include filamentous fungi, yeast, and bacteria. In some embodiments, the saccharified biomass can be made it into a fuel (e.g., a biofuel such as a bioethanol, biobutanol, biomethanol, a biopropanol, a biodiesel, jet fuel or the like) by fermentation and/or by chemical synthesis. In some embodiments, the saccharified biomass can be made into a commodity chemical (e.g., ascorbic acid, isoprene, 1,3-propanediol, lipids, amino acids, proteins and enzymes by fermentation and/or by chemical synthesis.

[0350] In addition to saccharification of biomass, the enzymes and enzyme blends of the disclosure can be used in industrial, agricultural, food and feed and food and feed supplement processing processes. Exemplary applications for the enzymes are described below.

[0351] The enzymes of the disclosure can be used in wood, wood product, wood waste or by-product, paper, paper product, paper or wood pulp, Kraft pulp, or wood or paper recycling treatment or industrial process, e.g., any wood, wood pulp, paper waste, paper or pulp treatment or wood or paper deinking process. In one aspect, enzymes of the disclosure can be used to treat/pretreat paper pulp, or recycled paper or paper pulp, and the like. In one aspect, enzyme(s) of the disclosure are used to increase the "brightness" of the paper via their use in treating/pretreating paper pulp, or recycled paper or paper pulp, and the like. The higher the grade of paper, the greater the brightness; paper brightness can impact the scan capability of optical scanning equipment; thus, the enzymes and processes of the disclosure can be used to make high grade, "bright" paper for, e.g., use in optical scanning equipment, including inkjet, laser and photo printing quality paper. The enzymes of the disclosure can be used to process or treat any cellulosic material, e.g., fibers from wood, cotton, hemp, flax or linen. In one aspect, the disclosure provides wood, wood pulp, paper, paper pulp, paper waste or wood or paper recycling treatment processes using an enzyme of the disclosure.

[0352] Enzymes of the disclosure can be used for deinking printed wastepaper, such as newspaper, or for deinking noncontact-printed wastepaper, e.g., xerographic and laser-printed paper, and mixtures of contact and noncontact-printed wastepaper (as described in U.S. Pat. Nos. 6,767,728 and 6,426,200; and Neo, J. Wood Chem. Tech. 6:147, 1986). Enzymes of the disclosure can be used in processes for the production of xylose from a paper-grade hardwood pulp by extracting xylan contained in pulp into a liquid phase, subjecting the xylan contained in the obtained liquid phase to conditions sufficient to hydrolyze xylan to xylose, and recovering the xylose, where the extracting step includes at least one treatment of an aqueous suspension of pulp or an alkali-soluble material an enzyme enzyme, as described in, e.g., U.S. Pat. No. 6,512,110. Enzymes of the disclosure can be used in processes for dissolving pulp from cellulosic fibers such as recycled paper products made from hardwood fiber, a mixture of hardwood fiber and softwood fiber, waste paper, e.g., from unprinted envelopes, de-inked envelopes, unprinted ledger paper, de-inked ledger paper, and the like, as described in, e.g., U.S. Pat. No. 6,254,722.

[0353] The disclosure provides methods of treating fibers and fabrics using one or more enzymes of the disclosure. The enzymes can be used in any fiber- or fabric-treating method, which are well known in the art, see, e.g., U.S. Pat. Nos. 6,261,828; 6,077,316; 6,024,766; 6,021,536; 6,017,751; 5,980,581; U.S. Patent Publication No. 20020142438 A1. For example, enzymes of the disclosure can be used in fiber and/or fabric desizing. In one aspect, the feel and appearance of a fabric is improved by a method comprising contacting the fabric with an enzyme of the disclosure in a solution. In one aspect, the fabric is treated with the solution under pressure. For example, enzymes of the disclosure can be used in the removal of stains.

[0354] The enzymes of the disclosure can be used to treat any cellulosic material, including fibers (e.g., fibers from cotton, hemp, flax or linen), sewn and unsewn fabrics, e.g., knits, wovens, denims, yarns, and toweling, made from cotton, cotton blends or natural or manmade cellulosics or blends thereof.

[0355] The textile treating processes of the disclosure (using enzymes of the disclosure) can be used in conjunction with other textile treatments, e.g., scouring and bleaching. Scouring is the removal of non-cellulosic material from the cotton fiber, e.g., the cuticle (mainly consisting of waxes) and primary cell wall (mainly consisting of pectin, protein and xyloglucan).

[0356] The enzymes of the disclosure have numerous applications in food processing industry. For example, in one aspect, the enzymes of the disclosure are used to improve the extraction of oil from oil-rich plant material, e.g., oil-rich seeds, for example, soybean oil from soybeans, olive oil from olives, rapeseed oil from rapeseed and/or sunflower oil from sunflower seeds.

[0357] The enzymes of the disclosure can be used for separation of components of plant cell materials. For example, enzymes of the disclosure can be used in the separation of plant cells into components. In one aspect, enzymes of the disclosure can be used to separate crops into valuable protein and oil and hull fractions. The separation process can be performed by use of methods known in the art.

[0358] The enzymes of the disclosure can be used in the preparation of fruit or vegetable juices, syrups, extracts and the like to increase yield. The enzymes of the disclosure can be used in the enzymatic treatment of various plant cell wall-derived materials or waste materials, e.g., from cereals, grains, wine or juice production, or agricultural residues such as vegetable hulls, bean hulls, sugar beet pulp, olive pulp, potato pulp, and the like. The enzymes of the disclosure can be used to modify the consistency and appearance of processed fruit or vegetables. The enzymes of the disclosure can be used to treat plant material to facilitate processing of plant material, including foods, facilitate purification or extraction of plant components. The enzymes of the disclosure can be used to improve feed value, decrease the water binding capacity, improve the degradability in waste water plants and/or improve the conversion of plant material to ensilage, and the like.

[0359] In one aspect, enzymes of the disclosure are used in baking applications, e.g., cookies and crackers. In one aspect, enzymes of the disclosure are used to create non-sticky doughs that are not difficult to machine and to reduce biscuit size. Enzymes of the disclosure can be used to hydrolyze arabinoxylans to prevent rapid rehydration of the baked product resulting in loss of crispiness and reduced shelf-life. In one aspect, enzymes of the disclosure are used as additives in dough processing.

[0360] The disclosure provides methods for treating animal feeds and foods and food or feed additives (supplements) using enzymes of the disclosure, animals including mammals (e.g., humans), birds, fish and the like. The disclosure provides animal feeds, foods, and additives (supplements) comprising enzymes of the disclosure. In one aspect, treating animal feeds, foods and additives using enzymes of the disclosure can help in the availability of nutrients, e.g., starch, protein, and the like, in the animal feed or additive (supplements). By breaking down difficult to digest proteins or indirectly or directly unmasking starch (or other nutrients), the enzymes make nutrients more accessible to other endogenous or exogenous enzymes. The enzymes can also simply cause the release of readily digestible and easily absorbed nutrients and sugars.

[0361] When added to animal feed, enzymes of the disclosure improve the in vivo break-down of plant cell wall material partly due to a reduction of the intestinal viscosity (see, e.g., Bedford et al., Proceedings of the 1st Symposium on Enzymes in Animal Nutrition, 1993, pp. 73-77), whereby a better utilization of the plant nutrients by the animal is achieved. Thus, by using enzymes of the disclosure in feeds the growth rate and/or feed conversion ratio (i.e. the weight of ingested feed relative to weight gain) of the animal is improved.

[0362] The animal feed additive of the disclosure may be a granulated enzyme product which may readily be mixed with feed components. Alternatively, feed additives of the disclosure can form a component of a pre-mix. The granulated enzyme product of the disclosure may be coated or uncoated. The particle size of the enzyme granulates can be compatible with that of feed and pre-mix components. This provides a safe and convenient mean of incorporating enzymes into feeds. Alternatively, the animal feed additive of the disclosure may be a stabilized liquid composition. This may be an aqueous or oil-based slurry. See, e.g., U.S. Pat. No. 6,245,546.

[0363] In another aspect, an enzyme of the disclosure can be supplied by expressing the enzymes directly in transgenic feed crops (as, e.g., transgenic plants, seeds and the like), such as grains, cereals, corn, soy bean, rape seed, lupin and the like. As discussed above, the disclosure provides transgenic plants, plant parts and plant cells comprising a nucleic acid sequence encoding a polypeptide of the disclosure. In one aspect, the nucleic acid is expressed such that the enzyme of the disclosure is produced in recoverable quantities. The xylanase can be recovered from any plant or plant part. Alternatively, the plant or plant part containing the recombinant polypeptide can be used as such for improving the quality of a food or feed, e.g., improving nutritional value, palatability, and rheological properties, or to destroy an antinutritive factor.

[0364] In one aspect, the disclosure provides methods for removing oligosaccharides from feed prior to consumption by an animal subject using an enzyme of the disclosure. In this process a feed is formed having an increased metabolizable energy value. In addition to enzymes of the disclosure, galactosidases, cellulases, xylanases, and combinations thereof can be used.

[0365] In another aspect, the disclosure provides methods for utilizing an enzyme of the disclosure as a nutritional supplement in the diets of animals by preparing a nutritional supplement containing a recombinant enzyme of the disclosure, and administering the nutritional supplement to an animal to increase the utilization of hemicellulase contained in food ingested by the animal.

[0366] The enzymes of the disclosure can be used in a variety of other industrial applications, e.g., in waste treatment. For example, in one aspect, the disclosure provides a solid waste digestion process using enzymes of the disclosure. The methods can comprise reducing the mass and volume of substantially untreated solid waste. Solid waste can be treated with an enzymatic digestive process in the presence of an enzymatic solution (including enzymes of the disclosure) at a controlled temperature. This results in a reaction without appreciable bacterial fermentation from added microorganisms. The solid waste is converted into a liquefied waste and any residual solid waste. The resulting liquefied waste can be separated from said any residual solidified waste. See e.g., U.S. Pat. No. 5,709,796.

[0367] The disclosure provides detergent, disinfectant or cleanser (cleaning or cleansing) compositions comprising one or more enzymes of the disclosure, and methods of making and using these compositions. The disclosure incorporates all methods of making and using detergent, disinfectant or cleanser compositions, see, e.g., U.S. Pat. Nos. 6,413,928; 6,399,561; 6,365,561; 6,380,147.

[0368] In specific embodiments, the detergent, disinfectant or cleanser compositions can be a one and two part aqueous composition, a non-aqueous liquid composition, a cast solid, a granular form, a particulate form, a compressed tablet, a gel and/or a paste and a slurry form. The enzymes of the disclosure can also be used as a detergent, disinfectant or cleanser additive product in a solid or a liquid form. Such additive products are intended to supplement or boost the performance of conventional detergent compositions and can be added at any stage of the cleaning process.

[0369] The present disclosure provides cleaning compositions including detergent compositions for cleaning hard surfaces, detergent compositions for cleaning fabrics, dishwashing compositions, oral cleaning compositions, denture cleaning compositions, and contact lens cleaning solutions.

[0370] When the enzymes of the disclosure are components of compositions suitable for use in a laundry machine washing method, the compositions can comprise in addition to an enzyme of the disclosure both a surfactant and a builder compound. They can additionally comprise one or more detergent components, e.g., organic polymeric compounds, bleaching agents, additional enzymes, suds suppressors, dispersants, lime-soap dispersants, soil suspension and anti-redeposition agents and corrosion inhibitors.

[0371] Laundry compositions of the disclosure can also contain softening agents, as additional detergent components. Such compositions containing carbohydrase can provide fabric cleaning, stain removal, whiteness maintenance, softening, color appearance, dye transfer inhibition and sanitization when formulated as laundry detergent compositions.

[0372] New and improved cellulase compositions that comprise varying amounts BG-type, EG-type and variant CBH-type cellulases find utility in detergent compositions that exhibit enhanced cleaning ability, function as a softening agent and/or improve the feel of cotton fabrics (e.g., "stone washing" or "biopolishing"), in compositions for degrading wood pulp into sugars (e.g., for bio-ethanol production), and/or in feed compositions. The isolation and characterization of cellulase of each type provides the ability to control the aspects of such compositions.

[0373] Since the rate of hydrolysis of cellulosic products may be increased by using a transformant having at least one additional copy of the bgl1 gene inserted into the genome, products that contain cellulose or heteroglycans can be degraded at a faster rate and to a greater extent. Products made from cellulose such as paper, cotton, cellulosic diapers and the like can be degraded more efficiently in a landfill. Thus, the fermentation product obtainable from the transformants or the transformants alone may be used in compositions to help degrade by liquefaction a variety of cellulose products that add to the overcrowded landfills.

[0374] Cellulose-based feedstocks are comprised of agricultural wastes, grasses and woods and other low-value biomass such as municipal waste (e.g., recycled paper, yard clippings, etc.). Ethanol may be produced from the fermentation of any of these cellulosic feedstocks. However, the cellulose must first be converted to sugars before there can be conversion to ethanol.

[0375] A large variety of feedstocks may be used with the inventive variant BGL1 and the one selected for use may depend on the region where the conversion is being done. For example, in the midwestern United States agricultural wastes such as wheat straw, corn stover and bagasse may predominate while in California rice straw may predominate. However, it should be understood that any available cellulosic biomass may be used in any region.

[0376] The methods of the present disclosure can be used in the production of monosaccharides, disaccharides, and polysaccharides as chemical or fermentation feedstocks for microorganism for the production of organic products, chemicals and fuels, plastics, and other products or intermediates. In particular, the value of processing residues (dried distillers grain, spent grains from brewing, sugarcane bagasse, etc.) can be increased by partial or complete solubilization of cellulose or hemicellulose. In addition to ethanol, some chemicals that can be produced from cellulose include acetone, acetate, glycine, lysine, organic acids (e.g., lactic acid), 1,3-propanediol, butanediol, glycerol, ethylene glycol, furfural, polyhydroxyalkanoates, cis, cis-muconic acid, animal feed and xylose. Moreover, proteins and cells can be produced from cellulose.

[0377] In addition the variant bgl1 nucleic acid sequence finds utility in the identification and characterization of related nucleic acid sequences. A number of techniques useful for determining (predicting or confirming) the function of related genes or gene products include, but are not limited to, (A) DNA/RNA analysis, such as (1) overexpression, ectopic expression, and expression in other species; (2) gene knock-out (reverse genetics, targeted knock-out, viral induced gene silencing (VIGS, see Baulcombe, 100 Years of Virology, Calisher and Horzinek eds., Springer-Verlag, New York, N.Y. 15:189-201, 1999); (3) analysis of the methylation status of the gene, especially flanking regulatory regions; and (4) in situ hybridization; (B) gene product analysis such as (1) recombinant protein expression; (2) antisera production, (3) immunolocalization; (4) biochemical assays for catalytic or other activity; (5) phosphorylation status; and (6) interaction with other proteins via yeast two-hybrid analysis; (C) pathway analysis, such as placing a gene or gene product within a particular biochemical or signaling pathway based on its overexpression phenotype or by sequence homology with related genes; and (D) other analyses which may also be performed to determine or confirm the participation of the isolated gene and its product in a particular metabolic or signaling pathway, and help determine gene function.

EXAMPLES

[0378] The present disclosure is described in further detail in the following examples, which are not in any way intended to limit the scope of the disclosure as claimed. The attached figures are meant to be considered as integral parts of the specification and description of the disclosure. The following examples are offered to illustrate, but not to limit the claimed disclosure

[0379] In the experimental disclosure which follows, the following abbreviations apply: M (molar); mM (millimolar); .mu.M (micromolar); nM (nanomolar); mol (moles); mmol (millimoles); .mu.mol (micromoles); nmol (nanomoles); g and gm (grams); mg (milligrams); .mu.g (micrograms); pg (picograms); L (liters); ml and mL (milliliters); .mu.l and .mu.L (microliters); cm (centimeters); mm (millimeters); .mu.m (micrometers); nm (nanometers); U (units); V (volts); MW (molecular weight); sec (seconds); min(s) (minute/minutes); h(s) and hr(s) (hour/hours); .degree. C. (degrees Centigrade); QS (quantity sufficient); ND (not done); NA (not applicable); rpm (revolutions per minute); H.sub.2O (water); dH.sub.2O (deionized water); HCl (hydrochloric acid); aa (amino acid); bp (base pair); kb (kilobase pair); kD (kilodaltons); cDNA (copy or complementary DNA); DNA (deoxyribonucleic acid); ssDNA (single stranded DNA); dsDNA (double stranded DNA); dNTP (deoxyribonucleotide triphosphate); RNA (ribonucleic acid); MgCl.sub.2 (magnesium chloride); NaCl (sodium chloride); w/v (weight to volume); v/v (volume to volume); g (gravity); OD (optical density); ABTS (2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt; APB (acid-pretreated bagasse); BGL (beta-glucosidase); CNP (2-chloro-4-nitrophenol); CNPG (chloro-nitro-phenyl-beta-D-glucoside); HPLC (high pressure liquid chromatography); PAGE (polyacrylamide gel electrophoresis); PASC (phosphoric acid swollen cellulose) PCR (polymerase chain reaction); PCS (acid-pretreated corn stover); Pi or PI (performance index); RT-PCR (reverse transcription PCR); and SEL (site evaluation library).

Example 1

Assays

[0380] The following assays were standard assays used in the examples described below. Occasionally specific protocols called for deviations from these standard assays. In those cases, deviations from these standard assay protocols below are identified in the examples. In these experiments, a spectrophotometer was used to measure the absorbance of the products formed after the completion of the reactions.

Measurement of Glucose

A. Hexokinase Assay for Measurement of Residual Glucose

[0381] Residual glucose from H. jecorina culture supernatants expressing BGL variants was measured using a hexokinase assay. Five (5) .mu.L of supernatant was added to 195 .mu.L of a glucose hexokinase assay mixture (Instrumentation Laboratory, Breda, Netherlands) in a 96-well microtiter plate (Costar Flat Bottom PS). The plates were incubated at room temperature for 15 min. Following incubation, absorbance of the supernatant was measured at 340 nm. Supernatants of cultures containing residual glucose were excluded from pooling for further studies.

B. ABTS Assay for Measurement of Glucose

[0382] Monomeric glucose generated in the beta-glucosidase activity assays was detected using the ABTS assay. The assay buffer contained 2.74 g/L 2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS, Sigma, catalog no. A1888), 0.1 U/mL horseradish peroxidase Type VI-A (Sigma, catalog no. P8375), and 1 Unit/mL food grade glucose oxidase (GENENCOR) in 50 mM sodium acetate buffer pH 5.0. Ten (10) .mu.L (diluted) sample was added to 100 .mu.L ABTS assay solution. The reaction was followed kinetically for 5 min at OD.sub.420, at ambient temperature of 22.degree. C. An appropriate calibration curve of glucose for each assay condition was always included.

HPLC Assay for Protein Content Determination

[0383] The concentration of BGL variant proteins from pooled culture supernatants was determined by an Agilent 1200 (Agilent Technologies) HPLC equipped with a Shodex HIC PH-814 PHM gel 75.times.8 mm column (Phenomenex). Fifty (50) .mu.L of sample was mixed with 50 .mu.L of 1.6 M (NH.sub.4).sub.2SO.sub.4 and after 5 min filtered under vacuum over a 0.22 .mu.m Millipore Multiscreen HTS 96 well filtration system. Forty (40) .mu.L of the filtered sample was injected on the column. Two elution buffers were employed to build an elution gradient: (1) Buffer A: 16 mM NaH.sub.2PO.sub.4, pH 6.75, 800 mM (NH.sub.4).sub.2SO.sub.4; and (2) Buffer B: 16 mM NaH.sub.2PO.sub.4, pH 6.75. Elution was carried out at a flow rate of 1.8 mL/min, using the following program: 0% to 50% Buffer B from 0.25 min to 1.5 min followed by a gradient of 50% to 100% Buffer B from 1.5 min to 4 min. 100% Buffer B was pumped over the column from 4 to 4.5 min. Protein concentrations of BGL variants were calculated from a calibration curve generated using purified wild-type BGL1 (15.625, 31.25, 62.5, 125, 250, 500 .mu.g/mL). To calculate performance index (PI), the concentration of a BGL variant was divided by that of the average wild-type BGL1 (e.g., a reference enzyme) in the same plate.

CNPGase Activity Assay

[0384] The activity of the BGL variants towards chloro-nitrophenol-.beta.-D-glucoside (CNPG) was determined. Culture supernatants expressing BGL variants were diluted 10-fold in a 50 mM sodium acetate buffer, pH 5.0. Twenty five (25) .mu.L aliquots of diluted supernatant were added to 75 .mu.L 1.33 mM CNPG in a 50 mM sodium acetate buffer, pH 5.0 (final concentration 1 mM CNPG) in quadruplicate. Kinetics of CNP release at 013405 was recorded in a microtiter plate reader (Spectramax, Molecular Devices) for 3 min. Average specific activities for the wild-type BGL1 and BGL variants were calculated by dividing the averaged CNPG hydrolyzing activity by the BGL concentration. A performance index (PI) was calculated by dividing the specific activity of a BGL variant by the average specific activity of the wild-type BGL1 (e.g., a reference enzyme) on the same plate.

Thermostability Assay

[0385] Residual activity of BGL1 polypeptides (including wild type and variants) after heat incubation was determined using the CNPG assay. Culture supernatants expressing BGL1 polypeptides (including wild type and variants) were diluted 10-fold in 50 mM sodium acetate buffer pH 5.0. Fifty (50) .mu.L aliquots were incubated in quadruplicate in a skirted 96-well PCR plate in a thermocycler at 66.degree. C. for 1 hr. After incubation the residual specific activity of BGL1 polypeptides was determined as described above. The residual activity of the variants and the wild-type enzyme was determined by the ratio of the averaged specific activity after incubation and the averaged specific activity before incubation. A performance index (PI) for the BGL variants was determined by dividing the residual activity of a BGL variant by the residual activity of the wild-type BGL1 (e.g., a reference enzyme).

Glucose Inhibition Assay

[0386] The effect of glucose on the hydrolytic activity of beta-glucosidase was determined by repeating the CNPGase activity assay as described above in the presence of 3.75 mM glucose. The residual activity of the variants and the wild-type protein was determined by the ratio of the averaged specific activity in the presence of glucose and the averaged specific activity in the absence of glucose. A performance index (PI) for the BGL variants was determined by dividing the residual activity of a BGL variant by the residual activity of the wild-type BGL1 (e.g., a reference enzyme).

Specific Activity in a Phosphoric Acid Swollen Cellulose (PASC) Hydrolysis Assay

[0387] Phosphoric acid swollen cellulose (PASC) was prepared from Avicel according to published methods (see, e.g., Walseth, Tappi, 35:228, 1971; and Wood, Biochem J., 121:353-362, 1971). This material was diluted with buffer and water to achieve a 1% w/v mixture wherein the final concentration of sodium acetate was 50 mM (pH 5.0). One hundred and fifty (150) .mu.L of a 1% suspension of PASC in a 50 mM sodium acetate buffer (pH 5.0) was dispensed into a 96-well microtiterplate (Costar Flat Bottom PS). Ten (10) .mu.L of a culture supernatant from a bgl1-deleted strain containing 0.75 mg/mL protein was added to the PASC suspension. Then 5, 10, 20, or 40 .mu.L of a 40.times. diluted (in 50 mM sodium acetate buffer pH 5.0) pooled culture supernatant from H. jecorina cells expressing either wild-type BGL1 or a BGL variant were added to the PASC/deletion mutant supernatant mixture. Compensating volumes of acetate buffer were added to make up for differences in total volume. The microtiter plate was sealed and incubated in a thermostatted incubator at 50.degree. C. with continuous shaking at 900 rpm. After 2 hr, the hydrolysis reaction was stopped by the addition of 100 .mu.L 100 mM glycine buffer, pH 10 to each well. The plates were sealed and centrifuged at 3,500 rpm at room temperature for 5 min. The hydrolysis reaction products in the supernatant were analyzed by the ABTS assay. A dose response curve was generated for the wild-type BGL1. To calculate performance index (PI), the (average) total sugar produced by a variant BGL was divided by the (average) total sugar produced by the wild-type BGL1 (e.g., a reference enzyme) at the same dose.

Specific Activity in a Dilute Acid Pretreated Corn Stover (PCS) Hydrolysis Assay

[0388] Corn stover was pretreated with 2% w/w H.sub.2SO.sub.4 (see, Schell et al., J. Appl. Biochem. Biotechnol., 105:69-86, 2003), followed by multiple washes with deinonized water to obtain a paste having a pH of 4.5. A sodium acetate buffer (pH 5.0) was then added (to a final concentration of 50 mM sodium acetate) and, if necessary, this mixture was titrated to pH 5.0 using 1 N NaOH. The cellulose concentration in the reaction mixture was about 7%. Sixty five (65) .mu.L of this cellulose suspension was added per well in a 96-well microtiter plate (Nunc Flat Bottom PS). Ten (10) .mu.L of a culture supernatant from a bgl1-deleted strain containing 10 mg/mL protein was added to the PCS. Then 5, 10, 15, or 20 .mu.L of a 5.times. diluted (in 50 mM sodium acetate buffer, pH 5.0) pooled culture supernatants from H. jecorina cells expressing either wild-type BGL1 or a BGL variant were added to the PCS/deletion mutant supernatant mixture. Compensating volumes of sodium acetate buffer were added to make up for the differences in total volume. After sealing, the plates were placed in a thermostatted incubator at 50.degree. C. with continuous shaking at 900 rpm. After 16 hr the plates were put on ice for 5 min and the hydrolysis reaction was stopped by the addition of 100 .mu.L 100 mM glycine buffer, pH 10, to each well. The plates were sealed and centrifuged at 3,000 rpm at room temperature for 5 min. The hydrolysis reaction products in the supernatant were analyzed by the ABTS assay. A dose response curve was generated for wild-type BGL1 protein. To calculate performance index (PI), the (average) total sugar produced by a variant BGL was divided by the (average) total sugar produced by the wild-type BGL1 (e.g., a reference enzyme) at the same dose.

Cellobiase Activity Assay (pH 5)

[0389] The cellobiose hydrolyzing ability at pH 5.0 of wild-type BGL1 and the BGL variants was tested. Varying amounts (e.g., 5, 10, 15, or 20 .mu.L) of 20.times. diluted (in 50 mM sodium acetate buffer, pH 5.0) pooled culture supernatants from H. jecorina cells expressing either wild-type BGL1 or BGL variants were added to 80 .mu.L of a 16.4 mM (5.63 mg/mL) cellobiose solution in a 50 mM sodium acetate buffer, pH 5.0. Compensating volumes of the sodium acetate buffer were added to make up for the differences in the total volume. The microtiter plate was sealed and incubated in a thermostatted incubator at 50.degree. C. under continuous shaking at 900 rpm. After 30 min, the hydrolysis reaction was stopped by the addition of 100 .mu.L 100 mM glycine buffer, pH 10 to each well. The hydrolysis reaction products were analyzed by the ABTS assay. A dose response curve was generated for the wild-type BGL1. To calculate performance index (PI), the (average) total sugar produced by a variant BGL was divided by the (average) total sugar produced by the wild-type BGL1 (e.g., a reference enzyme) at the same dose.

Cellobiase Activity Assay (pH 6)

[0390] The cellobiose hydrolyzing capability of wild-type BGL1 and the BGL1 variants at pH 6.0 was tested. Varying amounts (5, 10, 15, or 20 .mu.L) of 20.times. diluted (in 50 mM sodium citrate buffer, pH 6.0) pooled culture supernatants from H. jecorina cells expressing either wild-type BGL1 or a BGL variant were added to 80 .mu.L of a 16.4 mM (5.63 mg/mL) cellobiose solution in a 50 mM sodium citrate buffer, pH 6.0. Compensating volumes of citrate buffer were added to make up for the differences in total volume. The microtiter plate was sealed and incubated in a thermostatted incubator at 50.degree. C. with continuous shaking at 900 rpm. After 30 min, the hydrolysis reaction was stopped by the addition of 100 .mu.L of a 100 mM glycine buffer, pH 10, to each well. The hydrolysis reaction products were analyzed by the ABTS assay. A dose response curve was generated for wild-type BGL1 protein. To calculate performance index (PI), the (average) total sugar produced by a variant BGL was divided by the (average) total sugar produced by the wild-type BGL1 (e.g., a reference enzyme) at the same dose.

Determining Beta-Glucosidase Activity by Measuring Cellobiase Activity Assay in the Presence of Ammonia Pretreated Corncob (CC)

[0391] Corn cob was ground to pass a 0.9 mm screen and pretreated as described in PCT application publication WO 2006110901. Pretreated CC was used as a 7% cellulose suspension in a 50 mM sodium acetate buffer, pH 5.0. Sixty five (65) .mu.L of the suspension were added per well into a 96-well microtiter plate (Nunc Flat Bottom PS). Forty five (45) .mu.L of a 35.1 mM (12.0 mg/mL) cellobiose solution was added to the pretreated corncob, and varying amounts (5, 10, 15, or 20 .mu.L) of 20.times. diluted (in a 50 mM sodium acetate buffer, at pH 5.0) pooled culture supernatants from H. jecorina cells expressing either wild-type BGL1 or BGL variants were added. Compensating volumes of acetate buffer were added to make up for the differences in total volume. The microtiter plate was sealed and incubated in a thermostatted incubator at 50.degree. C. with continuous shaking at 900 rpm. After 30 min, the hydrolysis reaction was stopped by the addition of 100 .mu.L of a 100 mM glycine buffer, pH 10, to each well. After mixing, the plate was centrifuged for 5 min at 3,500 rpm. The hydrolysis reaction products were analyzed by the ABTS assay. A dose response curve was generated for wild-type BGL1 protein. To calculate performance index (PI), the (average) total sugar produced by a variant BGL was divided by the (average) total sugar produced by the wild-type BGL1 (e.g., a reference enzyme) at the same dose.

Example 2

[0392] Generation of Hypocrea jecorina BGL1 Site Evaluation Libraries ("SELs")

[0393] The pTTTpyrG-bgl1 plasmid containing the Hypocrea jecorina BGL1 protein encoding sequence (SEQ ID NO: 1) was sent to a number of vendors, for example, BASEClear (Leiden, The Netherlands), GeneArt AG (Regensburg, Germany), and Sloning BioTechnology GmbH (Puchheim, Germany) for the generation of Site Evaluation Libraries (SELs). The amino acid sequence of the full length BGL1 protein is shown in SEQ ID NO: 2. Vendors generated positional libraries at each of the sites in the BGL1 mature protein (SEQ ID NO: 3) shown in Table 2-1.

[0394] SEQ ID NO:1 sets forth the reference H. jecorina bgl1 coding DNA sequence:

TABLE-US-00002 atgcgctaccgcaccgctgccgctttagccttagccaccggccccttcgc cagagccgatagccacagcacctccggcgctagtgctgaagctgttgtcc ctcctgctggcaccccttggggcaccgcctacgacaaggccaaggccgcc ctcgccaagctcaacctccaggacaaggtcggcatcgtcagcggcgtcgg ctggaacggcggtccctgcgtcggcaacaccagccccgccagcaagatca gctaccccagcctctgcctccaggacggccccctcggcgtccgctacagc accggcagcaccgccttcacccctggcgtccaggccgccagcacctggga cgtcaacctcatccgcgagcgcggccagttcatcggcgaagaggtcaagg ccagcggcatccacgtcatcctcggtcccgttgctggtcccttaggcaag accccccagggcggtcgcaactgggagggcttcggcgtcgacccctacct caccggcattgccatgggccagaccatcaacggcatccagagcgtcggcg tccaggccaccgccaagcactacatcctcaacgagcaagagttaaaccgc gagactatcagcagcaaccccgacgaccgcaccctccacgagttatacac ctggcccttcgccgacgccgtccaggccaacgtcgccagcgtcatgtgca gctacaacaaggtcaacaccacctgggcctgcgaggaccagtacaccctc cagaccgtcctcaaggaccagctcggcttccccggctacgtcatgaccga ctggaacgcccagcacaccaccgtccagagcgccaacagcggcctcgaca tgagcatgcccggcaccgacttcaacggcaacaaccgcctctggggccct gccctcaccaacgccgtcaacagcaaccaggtccccacctcccgcgtcga cgacatggtcacccgcatcctcgccgcctggtacttaaccggccaagacc aggctggctatcccagcttcaacatcagccgcaacgtccagggcaaccac aagaccaacgtccgcgccattgcccgcgacggcatcgtcctcctcaagaa cgacgccaacatcctccccctcaagaagcccgcctctatcgccgtcgtcg gcagcgccgccatcatcggcaaccacgcccgcaacagccccagctgcaac gacaagggctgcgatgacggtgccctcggcatgggctggggctctggcgc cgtcaactacccctacttcgtcgccccctacgacgccatcaacacccgcg ccagcagccagggcacccaggtcaccctcagcaacaccgacaatacttct tctggcgcttctgctgctagaggcaaggacgtcgccatcgtttttatcac tgccgattctggcgaaggctacatcaccgtcgagggcaacgccggcgacc gcaacaacctcgacccctggcacaacggcaatgccctcgtccaggccgtt gctggtgctaacagcaacgtcatcgtcgtcgtccacagcgtcggcgccat catcctcgagcagatcctcgccctcccccaggtcaaggccgtcgtctggg ccggcttacccagccaggaaagcggcaacgccttagtcgacgtcctctgg ggtgacgtttccccctctggcaagctcgtctacaccattgccaagagccc caacgactacaacacccgcattgtcagcggcggcagcgacagcttcagcg agggcctcttcatcgactacaagcacttcgacgacgccaacattaccccc cgctacgagttcggctacggcctcagctacaccaagttcaactacagccg cctcagcgtcctcagcaccgccaagagcggccctgccactggtgctgtcg tccctggtggcccttctgacctcttccagaacgtcgccacggtcaccgtc gacattgccaactccggccaggtcactggcgccgaggtcgcccagctcta catcacctaccccagcagcgcccctcgcactcctcccaagcagctcagag gcttcgctaagttaaacttaacccctggccagagcggcaccgccaccttt aacatccgcagacgcgacctcagctactgggacaccgccagccagaagtg ggtcgtccccagcggcagcttcggcatctccgtcggcgccagctcccgcg acatccgcctcaccagcaccctcagcgtcgcctgatga*

[0395] SEQ ID NO:2 sets forth the sequence of the H. jecorina BGL1 full length protein:

TABLE-US-00003 MRYRTAAALALATGPFARADSHSTSGASAEAVVPPAGTPWGTAYDKAKAA LAKLNLQDKVGIVSGVGWNGGPCVGNTSPASKISYPSLCLQDGPLGVRYS TGSTAFTPGVQAASTWDVNLIRERGQFIGEEVKASGIHVILGPVAGPLGK TPQGGRNWEGFGVDPYLTGIAMGQTINGIQSVGVQATAKHYILNEQELNR ETISSNPDDRTLHELYTWPFADAVQANVASVMCSYNKVNTTWACEDQYTL QTVLKDQLGFPGYVMTDWNAQHTTVQSANSGLDMSMPGTDFNGNNRLWGP ALTNAVNSNQVPTSRVDDMVTRILAAWYLTGQDQAGYPSFNISRNVQGNH KTNVRAIARDGIVLLKNDANILPLKKPASIAVVGSAAIIGNHARNSPSCN DKGCDDGALGMGWGSGAVNYPYFVAPYDAINTRASSQGTQVTLSNTDNTS SGASAARGKDVAIVFITADSGEGYITVEGNAGDRNNLDPWHNGNALVQAV AGANSNVIVVVHSVGAIILEQILALPQVKAVVWAGLPSQESGNALVDVLW GDVSPSGKLVYTIAKSPNDYNTRIVSGGSDSFSEGLFIDYKHFDDANITP RYEFGYGLSYTKFNYSRLSVLSTAKSGPATGAVVPGGPSDLFQNVATVTV DIANSGQVTGAEVAQLYITYPSSAPRTPPKQLRGFAKLNLTPGQSGTATF NIRRRDLSYWDTASQKWVVPSGSFGISVGASSRDIRLTSTLSVA*

[0396] SEQ ID NO:3 sets forth the sequence of the H. jecorina BGL1 mature protein:

TABLE-US-00004 VVPPAGTPWGTAYDKAKAALAKLNLQDKVGIVSGVGWNGGPCVGNTSPAS KISYPSLCLQDGPLGVRYSTGSTAFTPGVQAASTWDVNLIRERGQFIGEE VKASGIHVILGPVAGPLGKTPQGGRNWEGFGVDPYLTGIAMGQTINGIQS VGVQATAKHYILNEQELNRETISSNPDDRTLHELYTWPFADAVQANVASV MCSYNKVNTTWACEDQYTLQTVLKDQLGFPGYVMTDWNAQHTTVQSANSG LDMSMPGTDFNGNNRLWGPALTNAVNSNQVPTSRVDDMVTRILAAWYLTG QDQAGYPSFNISRNVQGNHKTNVRAIARDGIVLLKNDANILPLKKPASIA VVGSAAIIGNHARNSPSCNDKGCDDGALGMGWGSGAVNYPYFVAPYDAIN TRASSQGTQVTLSNTDNTSSGASAARGKDVAIVFITADSGEGYITVEGNA GDRNNLDPWHNGNALVQAVAGANSNVIVVVHSVGAIILEQILALPQVKAV VWAGLPSQESGNALVDVLWGDVSPSGKLVYTIAKSPNDYNTRIVSGGSDS FSEGLFIDYKHFDDANITPRYEFGYGLSYTKFNYSRLSVLSTAKSGPATG AVVPGGPSDLFQNVATVTVDIANSGQVTGAEVAQLYITYPSSAPRTPPKQ LRGFAKLNLTPGQSGTATFNIRRRDLSYWDTASQKWVVPSGSFGISVGAS SRDIRLTSTLSVA*

TABLE-US-00005 TABLE 2-1 Positions In The Mature BGL1 Protein Selected For The Generation Of SELs 22 163 226 313 380 454 561 661 24 164 236 316 381 455 563 662 25 165 237 320 382 460 564 663 26 166 238 324 396 467 570 666 27 167 242 328 397 473 571 672 28 168 248 329 398 474 581 673 33 169 249 334 399 475 583 674 35 170 263 335 402 489 586 675 36 176 264 336 409 490 591 680 37 177 265 337 410 492 603 681 50 178 276 338 411 496 611 682 51 179 277 339 420 497 612 683 52 194 278 344 426 498 622 684 61 196 279 345 427 521 626 685 67 199 282 347 428 522 627 692 91 204 284 361 441 534 638 702 92 208 287 363 445 542 642 705 93 209 291 369 446 547 643 99 214 301 370 447 548 645 100 215 302 371 448 553 649 125 216 303 372 449 554 650 158 224 306 374 452 555 656 159 225 312 375 453 560 660

[0397] For each of the 178 sites listed in Table 2-1, typically 14-16 substitution variants were obtained. The SEL variants were received as individually purified plasmids each encoding a BGL1 variant sequence substituted at the indicated position.

Production of BGL1 Variants

[0398] To enable the expression of BGL1 and variant BGL proteins in Trichoderma reesei, the bgl1 coding sequence was cloned into the Gateway compatible destination vector pTTT-pyrG13 (Fig. X) via the Gateway.RTM. LR recombination reaction. This vector contained the T. reesei cbh1-derived promoter and terminator regions allowing for a strong inducible expression of a gene of interest, the Aspergillus nidulans amdS and pyrG selective markers conferring growth of transformants on acetamide as a sole nitrogen source, and the T. reesei telomere regions allowing for non-chromosomal plasmid maintenance in a fungal cell. In addition, this vector allowed for selecting transformants of T. reesei strains with uridine auxotrophy. The cbh1 promoter and terminator regions are separated by the chloramphenicol resistance gene, Cm.sup.R, and the lethal E. coli gene, ccdB, flanked by the bacteriophage lambda-based specific recombination sites attR1, attR2. Such configuration allowed for direct selection of recombinants containing the bgl1 gene under the control of the cbh1 regulatory elements in the right orientation via the Gateway.RTM. LR recombination reaction. The final expression vector pTTT-pyrG-bgl1 is shown in FIG. 2.

[0399] Purified pTTTpyrG-bgl1 plasmids (p.sub.cbh1, Amp.sup.R, acetamidase) expressing genes encoding BGL1 variant sequences were obtained from the vendors listed above. Protoplasts of H. jecorina strain (.DELTA.eg1, .DELTA.eg2, .DELTA.cbh1, .DELTA.cbh2, .DELTA.bgl1) were transformed with the individual pTTTpyrG constructs (a single BGL1 variant per transformation) and grown on selective agar containing acetamide at 28.degree. C. for 7 d as previously described in, for example, PCT Patent Application Publication WO 2009/048488. Protoplasts of H. jecorina were generated, harvested, plated on acetamide agar, and incubated at 28.degree. C. for 7 d. Spores were harvested in 15% glycerol and stored at -20.degree. C. For BGL1 variant production, a volume of 10 .mu.L spore suspension was added to 200 .mu.L of a glycine minimal medium supplemented with 2% glucose/sophorose mixture in a PVDF filter plate. Each BGL1 variant was grown in quadruplicate. After sealing the plate with an oxygen permeable membrane, the plates were incubated at 28.degree. C. for 6 d, with shaking at 220 rpm. Filtrates were harvested by transferring the culture medium to a microtiter plate under vacuum. Residual glucose was measured using the hexokinase assay as described in Example 1A.

Example 3

Expression, Activity and Performance of BGL 1 Variants

[0400] H. jecorina BGL1 SEL variant proteins were tested for various properties of interest. In particular, the beta-glucosidase variants were tested for protein expression using the HPLC assay (HPLC), CNPG hydrolyzing activity (CNPG), effect of glucose on activity (Gluc), thermostability (Heat), hydrolysis of PASC (PASC), hydrolysis of PCS (PCS), cellobiase activity at pH5.0 (G2 pH5), cellobiase activity at pH6.0 (G2 pH6), and beta-glucosidase activity measured by cellobiase activity in the presence of ammonia pretreated corncob (G2 CC) as described in Example 1. The performance indices for the BGL1 variants shown in Table 3-1 are rounded to the nearest hundredth. Performance index (PI) is the ratio of performance of the variant to wild-type BGL1. Performance indices less than or equal to 0.05 were generally fixed to 0.05. However, for HPLC protein values of 0.0, all values were fixed to 0.04. PI values for SEL enzymes with wild type residues were set at 1.00. PI values that were larger than 1 before rounding, are shown in bold, italic face in Table 3-1.

TABLE-US-00006 TABLE 3-1 Performance Index Data of BGL1 SEL Variants (3,153) variant HPLC CNPG Gluc Heat PASC PCS G2 pH 5 G2 pH 6 G2 CC K022A 0.95 0.99 K022C 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 K022E K022F 0.66 0.93 K022G 0.87 0.93 0.96 0.99 0.94 0.90 K022H 0.47 0.84 0.79 0.57 0.97 0.97 0.85 0.71 K022I 0.78 0.99 0.93 0.96 0.97 0.95 0.93 0.86 K022K 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 K022L 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 K022M 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 K022N 0.58 0.91 0.83 0.79 0.91 0.85 0.78 0.98 0.66 K022P 0.89 1.00 0.90 K022Q 0.82 0.96 0.84 0.98 K022R 0.61 0.98 0.84 0.90 0.97 0.89 0.93 0.74 K022S 0.95 0.87 0.99 0.90 0.89 K022T 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 K022V 0.75 0.92 0.93 1.00 0.95 0.98 0.66 K022W 0.90 0.89 0.88 0.83 0.78 K022Y 0.98 0.99 0.93 0.82 0.99 1.00 0.87 N024A 1.00 0.99 0.99 0.95 N024C 0.74 0.94 N024D 0.70 0.96 0.91 0.89 0.91 0.76 N024E 0.96 0.89 0.89 0.94 0.77 N024F 0.48 0.38 0.61 0.94 0.88 0.75 N024G 0.81 0.30 0.57 0.93 0.93 0.92 0.97 N024K 0.59 0.31 0.49 0.99 0.97 0.96 0.97 0.51 N024L 0.78 0.96 0.88 0.88 0.91 0.87 N024M 0.65 0.85 0.79 0.89 0.76 0.80 0.96 N024N 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 N024P 0.30 0.58 0.98 0.96 0.96 N024Q 0.34 0.28 0.05 0.68 N024R 0.67 0.05 0.05 0.22 0.92 0.90 0.73 0.77 N024S 0.96 0.30 0.05 0.79 0.88 0.85 0.88 0.88 N024T 0.96 0.35 0.67 0.92 0.82 0.84 0.86 0.82 N024V 0.62 0.30 0.98 0.54 0.93 0.86 0.78 0.60 N024Y 0.57 0.32 0.05 0.05 0.91 0.92 0.72 L025A 0.70 0.87 0.79 0.99 0.91 L025D 0.68 0.97 0.72 0.89 L025F 0.71 0.93 0.78 0.96 0.86 0.91 0.92 0.83 L025G 0.51 0.91 0.57 0.93 0.94 0.89 0.83 0.85 L025H 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 L025I 1.00 0.94 0.96 0.90 1.00 0.99 0.91 L025K 0.53 0.86 0.53 0.94 0.89 0.99 0.74 L025L 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 L025N 0.27 0.80 0.82 0.37 0.90 0.90 0.58 L025P 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 L025Q 0.64 0.93 0.92 0.92 0.94 0.98 0.92 0.84 L025R 0.60 0.89 0.63 0.93 0.99 0.91 0.85 0.84 L025S 0.85 0.96 0.89 0.91 0.82 0.89 0.88 L025T 0.96 0.92 0.95 0.91 0.82 L025V 0.68 0.95 0.83 0.92 0.92 0.89 0.92 L025W 0.99 0.98 0.98 0.77 0.95 0.92 0.97 L025Y 0.49 0.88 0.41 0.92 0.96 0.96 0.75 Q026A 0.94 0.90 0.80 0.74 0.89 0.89 0.85 0.89 0.88 Q026C 0.40 0.99 0.61 Q026D 0.82 0.99 0.75 1.00 0.99 0.98 Q026E 0.71 0.97 0.92 0.66 0.94 0.92 Q026F 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 Q026G 0.48 0.99 0.93 0.39 0.93 0.92 0.93 0.96 Q026H 0.55 0.97 1.00 0.99 0.85 Q026I 0.47 0.98 0.72 0.91 Q026K 0.43 0.96 Q026L 0.67 0.86 0.77 0.81 Q026M 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 Q026N 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 Q026P 0.42 0.51 0.89 Q026Q 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Q026R 0.47 0.88 Q026S 0.46 0.89 Q026T 0.17 0.89 0.67 Q026V 0.53 0.97 0.61 0.98 0.97 0.88 Q026W 0.39 0.56 Q026Y 0.70 0.86 0.60 0.84 0.80 0.82 0.73 D027A 0.81 1.00 0.74 0.70 0.93 1.00 D027C 0.28 0.99 0.34 0.22 0.92 0.92 D027E 0.71 0.99 0.88 0.86 0.92 0.99 0.82 D027F 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.55 D027G 0.20 0.90 0.93 0.21 0.22 0.08 0.76 0.79 1.00 D027I 0.17 0.87 0.92 0.17 0.47 0.06 0.71 0.73 0.83 D027K 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 D027L 0.34 0.93 0.44 0.60 0.24 0.84 0.92 D027M 0.29 0.89 0.39 0.22 0.21 0.83 0.88 D027P 0.06 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.48 D027Q 0.33 0.96 0.30 0.05 0.26 0.88 0.90 D027R 0.12 0.67 0.89 0.12 0.05 0.05 0.52 0.57 0.79 D027S 0.58 0.92 0.60 0.37 0.53 0.85 0.91 D027T 0.42 0.89 0.44 0.48 0.35 0.81 0.81 0.97 D027V 0.24 0.75 0.19 0.52 0.16 0.61 0.68 0.77 D027W 0.14 0.65 0.85 0.07 0.39 0.05 0.56 0.56 0.86 D027Y 0.23 0.78 0.94 0.17 0.37 0.12 0.58 0.71 0.91 K028C 0.44 0.60 0.90 0.57 0.91 0.76 0.73 0.93 0.73 K028E 0.44 0.56 0.95 0.50 0.84 0.79 0.70 0.90 0.64 K028F 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 K028G 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 K028I 0.20 0.29 0.89 0.20 0.75 0.75 0.54 0.95 0.46 K028K 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 K028L 0.19 0.29 0.20 0.79 0.74 0.59 0.98 0.58 K028M 0.70 0.82 0.95 0.88 0.92 0.80 0.92 0.75 K028N 0.09 0.13 0.08 0.46 0.50 0.43 0.88 0.39 K028P 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 K028Q 0.42 0.62 0.96 0.49 0.90 0.94 0.80 0.98 0.67 K028R 0.53 0.76 0.90 0.69 0.91 0.97 0.84 0.96 0.73 K028S 0.19 0.28 0.98 0.18 0.71 0.60 0.56 0.48 K028T 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 K028V 0.15 0.26 0.91 0.18 0.72 0.63 0.55 0.41 K028W 0.27 0.05 0.05 0.05 0.18 0.05 0.05 0.14 0.18 K028Y 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 S033A 0.11 0.35 0.82 0.05 0.62 0.89 0.64 0.64 0.36 S033C 0.42 0.88 0.49 S033D 0.21 0.58 0.75 0.05 0.75 0.94 0.73 0.81 0.61 S033E 0.11 0.05 0.05 0.05 0.05 0.33 0.05 0.05 0.37 S033F 0.08 0.21 0.71 0.05 0.49 0.46 0.40 0.54 0.34 S033G 0.46 0.89 0.39 1.00 0.97 1.00 S033H 0.20 0.60 0.82 0.09 0.84 0.70 0.80 0.82 0.64 S033I 0.09 0.19 0.75 0.05 0.38 0.43 0.26 0.30 0.29 S033K 0.12 0.43 0.76 0.05 0.76 0.24 0.71 0.80 0.40 S033L 0.09 0.21 0.67 0.05 0.36 0.38 0.22 0.28 0.31 S033M 0.21 0.70 0.75 0.05 0.85 0.72 0.71 0.88 0.64 S033N 0.18 0.62 0.81 0.05 0.89 0.90 0.78 0.91 0.60 S033P 0.09 0.05 0.05 0.05 0.05 0.11 0.05 0.05 0.11 S033Q 0.09 0.30 0.91 0.05 0.72 0.60 0.55 0.66 0.30 S033R 0.15 0.52 0.77 0.05 0.80 0.54 0.74 0.96 0.50 S033S 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 S033T 0.71 0.94 0.97 0.84 0.92 0.89 0.91 0.84 S033V 0.33 0.99 0.77 0.30 0.93 0.94 0.88 0.97 0.70 S033W 0.10 0.15 0.83 0.05 0.27 0.31 0.19 0.24 0.17 S033Y 0.10 0.15 0.05 0.05 0.24 0.21 0.17 0.26 0.26 V035C 0.77 0.90 0.85 V035D 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 V035E 0.77 0.92 0.77 0.95 V035F 0.26 0.05 0.05 0.07 0.20 0.09 0.12 0.10 0.10 V035G 0.99 0.90 0.36 0.86 0.66 0.98 V035H 0.81 0.86 0.31 0.96 0.72 0.98 0.83 V035K 0.59 0.87 0.74 0.99 0.89 0.97 0.91 V035L 0.78 0.95 0.98 0.87 0.98 0.79 V035N 0.69 0.88 0.64 0.90 0.97 0.85 V035P 0.44 0.88 0.05 0.33 0.92 0.92 0.79 V035Q 0.75 0.92 0.96 0.98 0.82 0.92 V035R 0.76 0.87 0.79 0.99 0.91 0.94 V035S 0.88 0.91 0.84 0.93 V035T 0.97 0.86 0.96 0.83 V035V 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 V035W 0.97 0.98 0.25 0.89 0.80 0.88 0.93 0.96 V035Y 0.83 0.99 0.09 0.96 0.71 0.90 G036A 0.33 0.53 0.74 0.05 0.74 0.70 0.73 0.65 0.70 G036C 0.18 0.60 0.77 0.05 1.00 0.70 0.83 G036D 0.52 0.86 0.05 0.67 G036E 0.46 0.88 0.05 0.63 G036F 0.09 0.41 0.71 0.05 0.70 0.17 0.65 G036H 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 G036I 0.10 0.43 0.75 0.05 0.70 0.28 0.68 G036K 0.27 0.89 0.73 0.05 0.85 0.82 G036N 0.10 0.43 0.70 0.05 0.86 0.55 0.78 0.64 G036P 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 G036Q 0.33 0.73 0.76 0.05 0.83 0.63 0.88 0.86 0.72 G036R 0.42 0.74 0.11 0.80 0.99 G036S 0.67 0.90 0.14 G036V 0.27 0.71 0.77 0.05 0.72 0.18 0.95 0.91 0.71 G036W 0.09 0.40 0.65 0.05 0.83 0.09 0.82 G036Y 0.07 0.83 0.71 0.05 0.15 W037A 0.85 0.88 0.66 0.79 0.73 0.63 0.78 W037C 0.46 0.70 0.64 0.35 0.62 0.85 0.84 0.75 0.82 W037E 0.91 0.98 0.60 0.82 0.67 0.66 0.68 W037F 1.00 0.59 0.68 0.68 0.63 0.65 W037G 0.93 0.72 0.70 0.45 0.53 0.67 0.66 0.55 0.62 W037H 0.85 0.83 0.61 0.75 0.69 0.71 0.71 W037I 0.07 0.31 0.05 0.05 0.27 0.14 0.05 0.31 W037K 0.80 0.68 0.65 0.63 0.45 0.68 0.56 0.42 0.49 W037L 0.11 0.32 0.84 0.08 0.44 0.90 0.56 0.39 0.57 W037M 0.99 0.91 0.97 0.56 0.69 0.72 0.61 0.69 W037P 0.88 0.90 0.72 0.84 0.59 0.71 0.73 0.62 0.71 W037R 0.69 0.87 0.88 0.73 0.53 0.78 0.63 0.53 0.59 W037S 0.93 0.87 0.63 0.87 0.75 0.67 0.82 W037T 0.64 0.91 0.80 0.63 0.54 0.69 0.66 0.55 0.75 W037V 0.65 0.98 0.90 0.56 0.76 0.65 0.64 0.70 W037W 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 W037Y 0.86 0.62 0.85 0.70 0.85 0.66 S050A 0.99 0.99 0.99 0.96 1.00 0.85 0.63 S050C 0.61 0.92 0.64 0.96 S050F 0.78 0.85 0.73 0.86 0.85 0.75 0.62 S050G 0.67 0.80 0.60 0.87 0.87 0.76 0.86 S050I 0.51 0.83 0.94 0.60 0.86 0.92 0.79 0.77 S050K 0.64 0.77 0.67 0.91 0.99 0.91 0.82 0.79 S050L 0.32 0.73 0.99 0.40 0.86 0.74 S050M 0.65 0.97 0.91 0.81 0.95 0.79 0.66 0.98 S050N 0.63 0.83 0.95 0.57 0.87 0.86 0.68 0.78 S050P 0.60 0.90 0.74 0.97 0.71 0.90 S050Q 0.65 0.86 0.98 0.73 0.89 0.98 0.93 0.71 0.64 S050R 0.77 0.91 0.81 0.92 0.95 0.80 0.82 S050S 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 S050T 0.55 0.75 0.63 0.91 0.89 0.80 0.83 S050V 0.73 0.99 0.96 0.79 0.88 0.95 0.86 0.86 S050W 0.74 0.91 0.98 0.75 0.93 0.97 0.91 0.90 0.92 S050Y 0.72 0.96 0.81 0.90 0.93 0.70 0.99 0.93 K051A 0.99 0.97 0.94 0.93 K051C 0.46 0.96 0.57 K051D 0.98 0.80 0.99 K051E 0.64 0.96 0.75 0.91 0.96 0.92 0.88 K051F 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 K051G 0.66 0.71 0.95 0.97 K051H 0.78 K051I 0.45 0.94 0.95 0.64 1.00 0.91 K051K 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 K051L 0.51 0.97 0.93 0.65 0.94 0.82 0.78 K051M 0.80 0.92 0.97 K051N 0.55 0.99 0.97 0.49 0.98 0.98 0.94 0.80 K051P 0.27 0.71 0.87 0.29 0.98 0.91 0.94 0.92 0.83 K051Q 0.55 0.94 0.77 0.99 0.81 K051R 0.64 0.96 0.78 0.99 0.98 0.81 K051S 0.34 0.84 0.91 0.49 0.94 0.91 0.90 0.74 K051T 0.75 0.93 K051V 0.50 0.70 0.90 K051W 0.44 0.77 0.95 0.23 0.89 0.85 0.86 0.95 0.82 I052A 0.27 0.21 0.05 0.62 0.44 0.44 0.83 I052C 0.32 0.12 0.05 0.11 0.24 0.07 0.09 0.52 0.33 I052D 0.10 0.19 0.84 0.10 0.69 0.62 I052F 0.26 0.17 0.05 0.45 0.25 0.28 0.82 0.54 I052I 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 I052K 0.10 0.22 0.57 0.06 0.74 0.37 0.31 0.64 I052L 0.27 0.28 0.87 0.12 0.61 0.43 0.50 0.92 0.80 I052M 0.17 0.18 0.87 0.05 0.61 0.34 0.34 0.86 I052N 0.09 0.17 0.05 0.77 0.29 0.40 I052P 0.10 0.13 0.05 0.05 0.88 0.26 0.41 I052Q 0.10 0.20 0.79 0.05 0.98 0.51 0.52 0.53 I052R 0.31 0.19 0.05 0.16 0.27 0.15 0.12 0.57 0.40 I052S 0.30 0.13 0.08 0.33 0.13 0.16 0.64 0.41 I052T 0.10 0.21 0.82 0.05 0.88 0.70 0.95 I052V 0.23 0.26 0.88 0.11 0.67 0.45 0.42 I052W 0.32 0.12 0.05 0.08 0.24 0.12 0.10 0.58 0.38 I052Y 0.32 0.18 0.65 0.05 0.32 0.16 0.15 0.61 0.56 D061A 0.92 0.06 0.12 0.05 0.05 0.05 0.05 0.05 0.07 D061C 0.95 0.07 0.05 0.05 0.05 0.05 0.05 0.05 0.07 D061D 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 D061E 0.10 0.60 0.05 0.19 0.54 0.11 0.07 0.12 D061F 0.81 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.10 D061G 0.05 0.05 0.05 0.05 0.07 0.05 0.05 0.05 D061H 0.78 0.22 0.49 0.12 0.23 0.30 0.18 0.08 0.15 D061I 0.33 0.10 0.05 0.07 0.05 0.05 0.07 0.05 0.19 D061K 0.38 0.11 0.05 0.13 0.05 0.05 0.06 0.05 0.11 D061L 0.97 0.07 0.05 0.08 0.05 0.07 0.05 0.05 0.07 D061M 0.96 0.05 0.05 0.05 0.05 0.06 0.05 0.05 0.05 D061N 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 D061P 0.06 0.05 0.36 0.05 0.05 0.05 0.05 0.05 D061R 0.49 0.09 0.05 0.11 0.05 0.05 0.05 0.05 0.06 D061T 0.52 0.07 0.05 0.16 0.05 0.05 0.05 0.05 0.09

D061V 0.19 0.10 0.05 0.31 0.05 0.19 0.16 0.05 0.18 D061W 0.24 0.22 0.49 0.19 0.28 0.24 0.22 0.10 0.29 D061Y 0.69 0.07 0.05 0.05 0.05 0.05 0.05 0.05 0.12 R067A 0.44 0.05 0.05 0.05 0.06 0.15 0.49 R067C 0.28 0.05 0.05 0.25 0.08 0.18 0.43 R067D 0.16 0.05 0.06 0.22 0.11 0.26 0.45 R067E 0.45 0.75 0.05 0.05 0.07 0.08 0.12 0.35 R067F 0.43 0.05 0.05 0.06 0.11 0.19 0.42 R067G 0.05 0.05 0.05 0.05 0.09 0.26 R067I 0.94 0.66 0.05 0.05 0.05 0.07 0.08 0.21 R067K 0.71 0.05 0.05 0.06 0.05 0.05 0.05 0.06 0.12 R067L 0.75 0.99 0.05 0.05 0.05 0.05 0.05 0.18 R067M 0.63 0.05 0.05 0.05 0.05 0.07 0.23 R067N 0.29 0.05 0.05 0.05 0.07 0.13 0.43 R067P 0.05 0.05 0.05 0.05 0.07 0.34 R067Q 0.19 0.05 0.10 0.05 0.05 0.08 0.23 0.98 R067R 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 R067S 0.85 0.05 0.05 0.14 0.07 0.12 0.24 R067T 0.77 0.11 0.05 0.05 0.05 0.08 0.22 R067V 0.78 0.05 0.05 0.05 0.05 0.10 0.25 R067W 0.31 0.07 0.05 0.05 0.14 0.21 0.25 R067Y 0.79 0.08 0.05 0.09 0.05 0.12 0.23 R091A 0.20 0.23 0.82 0.96 0.93 0.94 0.77 R091C 0.08 0.05 0.05 0.17 0.59 0.53 0.80 0.73 R091D 0.40 0.36 0.96 R091E 0.88 0.96 0.81 0.98 0.98 0.86 R091F 0.57 0.51 0.93 0.96 0.90 R091G 0.96 0.86 0.83 0.87 0.90 R091H 0.61 0.93 0.52 0.88 0.91 0.85 0.83 R091I 0.91 0.85 0.96 0.94 0.86 0.90 R091K 0.93 0.97 0.87 0.96 0.97 R091L 0.29 0.34 0.84 0.97 0.97 0.90 R091M 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 R091N 0.98 0.94 0.94 0.99 0.92 R091P 0.07 0.05 0.05 0.16 0.07 0.96 0.26 0.71 0.44 R091Q 0.63 0.61 0.80 R091R 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 R091S 0.73 0.88 0.62 0.86 0.89 0.89 0.88 0.72 R091T 0.83 0.95 0.93 0.93 0.87 0.81 R091V 0.34 0.35 0.94 0.94 0.98 0.78 R091W 0.57 0.57 0.96 0.96 0.82 R091Y 0.86 0.98 0.94 0.92 0.94 0.81 E092A 0.59 0.95 0.82 0.62 0.98 0.90 E092C 0.66 0.87 0.46 E092D 0.97 0.92 0.85 0.32 0.88 0.93 E092E 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 E092F 0.61 0.95 0.84 0.50 0.94 0.88 0.73 E092H 0.49 0.91 0.85 0.71 0.92 0.84 0.68 E092I 0.69 1.00 0.82 0.78 0.94 0.97 0.93 0.83 E092K 0.99 0.95 0.94 E092L 0.93 0.95 0.97 0.82 E092M 0.91 0.93 0.98 0.88 0.93 0.82 0.88 0.75 E092N 0.72 0.88 0.44 0.98 0.96 0.99 E092P 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E092Q 0.86 0.93 0.88 0.68 0.88 0.97 0.88 0.77 E092R 0.91 0.97 0.85 0.60 0.92 0.96 0.78 E092S 0.78 0.93 0.87 0.93 0.92 0.96 0.99 0.91 0.80 E092T 0.92 0.98 0.93 0.95 0.94 0.84 E092V 0.90 0.98 0.98 0.84 1.00 0.95 0.87 E092W 0.97 0.77 0.94 0.80 0.77 0.92 0.81 0.77 0.66 E092Y 0.77 0.91 0.95 0.05 0.90 0.96 0.92 0.76 R093A 0.40 0.43 0.05 0.48 0.12 0.32 0.47 0.28 R093C 0.38 0.37 0.05 0.45 0.18 0.29 0.50 0.33 R093D 0.14 0.10 0.08 0.31 0.66 0.20 0.32 0.21 R093E 0.37 0.40 0.05 0.44 0.12 0.31 0.43 0.20 R093F 0.20 0.34 0.05 0.30 0.12 0.31 0.49 0.15 R093G 0.08 0.15 0.05 0.29 0.43 0.22 0.50 0.24 R093H 0.23 0.49 0.05 0.78 0.70 0.62 0.76 0.47 R093I 0.11 0.05 0.05 0.15 0.05 0.05 0.05 0.18 0.11 R093K 0.18 0.31 0.05 0.56 0.44 0.62 0.26 R093L 0.51 0.51 0.08 0.37 0.10 0.20 0.41 0.22 R093M 0.63 0.54 0.05 0.43 0.17 0.21 0.54 0.19 R093P 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 R093Q 0.16 0.48 0.05 0.64 0.15 0.29 0.79 0.26 R093R 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 R093S 0.19 0.25 0.05 0.32 0.17 0.22 0.40 0.20 R093T 0.15 0.13 0.05 0.12 0.26 0.08 0.29 0.25 R093V 0.15 0.13 0.05 0.11 0.17 0.07 0.27 0.12 R093W 0.13 0.20 0.05 0.41 0.16 0.33 0.54 0.24 R093Y 0.11 0.05 0.05 0.05 0.05 0.05 0.05 0.16 0.09 E099A 0.74 0.75 0.71 0.91 0.83 0.80 0.80 E099C 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 E099D 0.16 0.26 0.98 0.17 0.81 0.66 0.76 0.46 E099E 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 E099F 0.48 0.72 0.56 0.96 0.97 0.86 0.90 E099G 0.11 0.05 0.05 0.05 0.11 0.22 0.11 0.27 0.10 E099I 0.53 0.67 0.62 0.90 0.79 0.83 0.77 E099K 0.59 0.75 0.97 0.72 0.89 0.79 0.84 0.69 E099L 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 E099M 0.70 0.78 0.78 0.85 0.75 0.80 0.70 E099N 0.70 0.90 0.94 0.86 0.87 0.84 0.83 0.88 E099P 0.10 0.05 0.05 0.05 0.10 0.16 0.06 0.29 0.24 E099Q 0.11 0.05 0.05 0.05 0.05 0.18 0.05 0.17 0.05 E099R 0.10 0.05 0.05 0.05 0.05 0.05 0.05 0.17 0.15 E099V 0.57 0.69 1.00 0.70 0.86 0.98 0.77 0.82 0.79 E099W 0.51 0.66 0.49 0.91 0.89 0.88 0.78 E099Y 0.58 0.85 0.79 0.85 E100A 0.60 0.99 0.05 0.64 E100C 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E100D 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E100E 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 E100G 0.26 0.82 0.05 0.69 E100H 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E100I 0.31 0.05 0.94 0.44 0.94 0.80 E100K 0.11 0.26 0.10 0.54 0.59 0.31 0.38 0.44 E100L 0.32 0.81 0.05 0.84 0.41 0.77 0.89 0.82 E100M 0.30 0.78 0.15 0.98 0.47 E100N 0.14 0.64 0.90 0.08 0.88 0.95 E100P 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E100Q 0.41 0.99 0.78 0.11 0.81 0.95 0.93 E100R 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E100S 0.52 0.81 0.06 0.78 0.97 E100T 0.47 0.86 0.10 0.76 0.96 E100V 0.30 0.82 0.77 0.08 0.89 0.40 0.86 0.96 0.75 E100W 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E100Y 0.27 0.91 0.06 0.34 R125A 0.05 0.05 0.05 0.38 0.13 0.05 0.05 0.05 R125C 0.51 0.05 0.05 0.05 0.71 0.07 0.05 0.05 0.14 R125D 0.05 0.05 0.05 0.31 0.19 0.05 0.05 0.06 R125E 0.65 0.05 0.05 0.05 0.85 0.11 0.05 0.05 0.14 R125F 0.07 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.82 R125G 0.83 0.05 0.05 0.05 0.32 0.38 0.05 0.05 0.07 R125I 0.17 0.81 0.95 0.15 0.24 0.05 0.73 0.76 0.97 R125L 0.54 0.05 0.05 0.05 0.72 0.07 0.05 0.05 0.13 R125M 0.58 0.05 0.05 0.05 0.28 0.07 0.05 0.05 0.10 R125P 0.15 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.23 R125R 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 R125S 0.84 0.05 0.05 0.05 0.66 0.08 0.05 0.05 0.05 R125T 0.93 0.05 0.05 0.05 0.71 0.18 0.05 0.05 0.08 R125V 0.68 0.05 0.05 0.05 0.05 0.06 0.05 0.05 0.08 R125W 0.21 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.21 R125Y 0.11 0.05 0.05 0.05 0.37 0.05 0.05 0.05 0.13 K158A 0.05 0.05 0.05 0.87 0.18 0.05 0.05 0.05 K158C 0.05 0.05 0.05 0.65 0.17 0.05 0.05 0.07 K158E 0.57 0.05 0.05 0.05 0.05 0.08 0.05 0.05 0.12 K158F 0.09 0.05 0.05 0.05 0.67 0.05 0.05 0.05 0.58 K158G 0.05 0.05 0.05 0.05 0.12 0.05 0.05 0.05 K158H 0.21 0.05 0.05 0.10 0.05 0.05 0.05 0.15 K158I 0.45 0.05 0.05 0.05 0.41 0.05 0.05 0.05 0.10 K158K 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 K158L 0.82 0.05 0.05 0.05 0.89 0.12 0.05 0.05 0.06 K158M 0.74 0.05 0.05 0.05 0.35 0.31 0.05 0.05 0.11 K158P 0.17 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.49 K158Q 0.64 0.05 0.05 0.05 0.05 0.10 0.05 0.05 0.09 K158R 0.75 0.05 0.05 0.05 0.05 0.13 0.05 0.05 0.09 K158S 0.67 0.05 0.05 0.05 0.51 0.09 0.05 0.05 0.08 K158T 0.69 0.05 0.05 0.05 0.10 0.05 0.05 0.06 K158V 0.57 0.05 0.05 0.05 0.53 0.10 0.05 0.05 0.10 K158Y 0.11 0.05 0.05 0.05 0.31 0.05 0.05 0.05 0.16 H159A 0.25 0.98 0.05 0.18 0.47 0.22 0.21 0.23 H159C 0.05 0.05 0.05 0.05 0.10 0.05 0.05 0.05 H159D 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 H159E 0.08 0.05 0.05 0.29 0.06 0.05 0.12 H159F 0.54 0.05 0.05 0.05 0.05 0.06 0.05 0.05 0.45 H159G 0.09 0.05 0.06 0.23 0.09 0.06 0.18 H159H 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 H159I 0.42 0.05 0.05 0.05 0.05 0.10 0.05 0.05 0.07 H159K 0.41 0.05 0.05 0.05 0.05 0.07 0.05 0.05 0.17 H159L 0.68 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 H159M 0.35 0.05 0.05 0.05 0.05 0.10 0.05 0.21 0.72 H159N 0.05 0.05 0.05 0.05 0.06 0.05 0.05 0.05 H159Q 0.40 0.05 0.05 0.05 0.05 0.15 0.05 0.05 0.14 H159R 0.67 0.05 0.05 0.05 0.05 0.09 0.05 0.05 0.08 H159S 0.12 0.62 0.05 0.05 0.14 0.06 0.05 0.11 H159T 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 H159V 0.61 0.05 0.05 0.05 0.05 0.07 0.05 0.38 0.08 H159W 0.05 0.05 0.05 0.05 0.06 0.05 0.05 0.05 H159Y 0.05 0.05 0.05 0.05 0.06 0.05 0.05 0.05 N163A 0.61 0.33 0.05 0.43 0.58 0.33 0.31 0.33 N163C 0.88 0.79 0.37 0.05 0.53 0.76 0.58 0.45 0.56 N163D 0.84 0.99 0.64 0.05 0.72 0.85 0.73 0.62 0.65 N163E 0.22 0.05 0.05 0.05 0.05 0.11 0.05 0.05 0.05 N163F 0.32 0.13 0.90 0.05 0.05 0.06 0.05 0.05 0.06 N163G 0.69 0.86 0.36 0.05 0.48 0.71 0.45 0.51 0.45 N163H 0.35 0.23 0.05 0.28 0.34 0.22 0.33 0.14 N163I 0.26 0.36 0.10 0.05 0.15 0.36 0.22 0.20 0.17 N163K 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 N163L 0.94 0.41 0.35 0.05 0.19 0.30 0.14 0.26 0.21 N163M 0.74 0.17 0.43 0.05 0.15 0.25 0.08 0.12 0.09 N163N 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 N163P 0.90 0.45 0.31 0.05 0.28 0.37 0.19 0.23 0.21 N163Q 0.93 0.60 0.56 0.05 0.54 0.61 0.44 0.81 0.29 N163R 0.45 0.05 0.05 0.05 0.05 0.08 0.05 0.05 0.07 N163S 0.71 0.38 0.05 0.43 0.54 0.39 0.35 0.41 N163T 0.63 0.52 0.11 0.05 0.21 0.38 0.25 0.23 0.33 N163V 0.27 0.48 0.08 0.05 0.13 0.38 0.39 0.25 0.29 N163W 0.16 0.05 0.05 0.05 0.05 0.10 0.05 0.05 0.15 N163Y 0.86 0.56 0.38 0.58 0.40 0.69 0.46 0.32 0.45 E164A 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E164C 0.65 0.95 0.91 0.05 0.75 0.84 0.93 0.80 0.96 E164E 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 E164F 0.12 0.34 0.70 0.05 0.19 0.30 0.57 0.09 0.22 E164G 0.20 0.67 0.83 0.05 0.74 0.74 0.53 0.61 E164H 0.06 0.05 0.05 0.05 0.05 0.17 0.13 0.05 0.15 E164I 0.08 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.07 E164K 0.10 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.17 E164L 0.09 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.17 E164M 0.10 0.26 0.68 0.06 0.14 0.21 0.44 0.05 0.17 E164N 0.11 0.45 0.28 0.05 0.18 0.05 0.70 0.05 0.37 E164P 0.11 0.32 0.92 0.05 0.14 0.05 0.59 0.05 0.16 E164Q 0.15 0.50 0.83 0.05 0.55 0.23 0.96 0.42 0.58 E164R 0.10 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.12 E164S 0.69 0.93 0.05 0.80 0.65 0.99 0.87 E164T 0.43 0.98 0.86 0.67 0.78 0.72 0.90 0.80 0.96 E164V 0.10 0.45 0.65 0.05 0.18 0.05 0.68 0.10 0.39 E164W 0.07 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.12 E164Y 0.08 0.05 0.05 0.05 0.07 0.05 0.05 0.05 0.26 Q165C 0.53 0.98 0.05 0.94 0.75 0.90 0.88 Q165D 0.20 0.05 0.05 0.85 0.63 0.75 0.82 0.85 Q165F 0.66 0.96 0.09 0.82 0.69 0.75 0.95 0.63 Q165G 0.22 0.05 0.88 0.05 0.82 0.61 0.76 0.65 Q165H 0.28 0.92 0.05 0.81 0.59 0.68 0.94 0.63 Q165I 0.25 0.05 0.79 0.98 0.54 Q165K 0.16 0.05 0.05 0.82 0.75 0.45 Q165L 0.23 0.05 0.82 0.58 0.53 0.81 0.77 Q165M 0.37 0.91 0.05 0.87 0.96 0.69 Q165N 0.20 0.05 0.95 0.05 0.73 0.80 0.51 Q165P 0.31 0.78 0.05 0.05 0.16 0.26 0.08 0.07 0.27 Q165Q 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Q165R 0.26 0.05 0.89 0.57 0.64 0.95 0.77 Q165S 0.49 0.91 0.05 0.91 0.64 0.81 0.98 Q165T 0.25 0.05 0.07 0.19 0.24 0.07 0.10 0.27 Q165V 0.25 0.05 0.84 0.93 Q165W 0.51 0.05 0.80 0.70 0.71 0.94 0.79 Q165Y 0.87 0.09 0.87 0.91 0.85 0.98 0.62 E166A 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E166C 0.07 0.05 0.83 0.05 0.60 0.75 0.40 0.40 0.45 E166D 0.39 0.84 0.05 0.70 0.87 E166E 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 E166F 0.07 0.05 0.05 0.46 0.53 0.25 0.37 0.48 E166G 0.18 0.05 0.73 0.05 0.22 0.33 0.15 0.12 0.45 E166H 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E166I 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E166K 0.23 0.05 0.16 0.23 0.07 0.10 0.26 E166L 0.23 0.05 0.20 0.25 0.07 0.09 0.35 E166M 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E166N 0.17 0.05 0.05 0.22 0.29 0.17 0.17 0.28 E166P 0.24 0.85 0.05 0.18 0.12 0.08 0.09 0.22 E166Q 0.51 0.47 0.05 0.12 0.14 0.12 0.10 0.10 E166R 0.28 0.05 0.21 0.11 0.05 0.08 0.26 E166S 0.14 0.05 0.05 0.28 0.38 0.15 0.21 0.40 E166T 0.23 0.05 0.21 0.18 0.12 0.13 0.38 E166V 0.20 0.05 0.05 0.05 0.21 0.22 0.11 0.11 0.46 E166W 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E166Y 0.32 0.74 0.05 0.17 0.12 0.06 0.06 0.34 L167A 0.97 0.77 0.94 0.93 0.83 0.91 0.84 0.89 L167C 0.74 0.72 0.82 L167D 0.76 0.68 0.05 0.99 0.99 0.97 0.91 L167E 0.72 0.72 0.65 0.97 0.99 0.94 0.85 L167F 0.59 0.91 0.73 0.55 0.98 0.95 0.94 0.77 L167G 0.68 0.68 0.33 0.98 0.99 0.85

L167I 0.72 0.94 0.69 0.88 0.90 0.93 0.87 0.82 0.75 L167K 0.62 0.97 0.63 0.87 0.88 0.91 0.94 0.87 0.90 L167L 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 L167M 0.55 0.94 0.69 0.70 0.83 0.95 0.79 0.81 L167N 0.48 0.88 0.69 0.41 0.94 1.00 0.88 0.87 L167P 0.12 0.07 0.05 0.05 0.13 0.05 0.05 0.17 0.10 L167Q 0.74 0.68 0.75 0.83 0.80 0.98 0.82 0.87 L167R 0.80 0.68 0.83 0.99 0.97 0.86 0.89 L167S 0.94 0.76 0.31 0.91 1.00 0.99 0.88 0.94 L167T 0.70 0.99 0.73 0.47 0.94 0.97 0.96 0.92 0.91 L167V 0.60 0.70 0.74 0.98 0.90 L167W 0.91 L167Y 0.94 0.75 0.66 0.96 0.90 0.97 0.88 N168A 0.72 0.69 0.67 0.97 N168D 0.66 0.67 0.70 0.98 N168E 0.54 0.65 0.66 1.00 N168G 0.66 0.70 0.76 0.91 N168H 0.54 0.90 0.62 0.58 0.94 0.95 0.93 0.99 N168I 0.56 0.83 0.64 0.24 0.93 0.99 0.89 0.86 0.77 N168K 0.61 0.85 0.65 0.38 0.90 0.95 0.96 0.87 0.74 N168L 0.66 0.94 0.70 0.55 0.92 0.90 0.98 0.82 0.82 N168M 0.52 0.77 0.66 0.47 0.80 0.92 0.87 0.75 0.76 N168N 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 N168P 0.15 0.05 0.05 0.05 0.05 0.16 0.05 0.05 0.05 N168Q 0.54 0.90 0.63 0.45 0.84 0.87 0.85 N168R 0.51 0.87 0.58 0.35 0.84 0.89 0.92 0.77 N168S 0.58 0.93 0.66 0.49 0.87 0.94 0.99 0.86 0.83 N168T 0.58 0.92 0.66 0.55 0.88 0.96 0.99 0.97 N168V 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 N168W 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 N168Y 0.76 0.70 0.60 R169A 0.05 0.05 0.13 0.19 0.06 0.05 0.09 R169C 0.07 0.05 0.12 0.18 0.08 0.06 0.09 R169D 0.05 0.05 0.05 0.15 0.23 0.08 0.05 0.08 R169E 0.18 0.05 0.23 0.32 0.10 0.10 0.11 R169F 0.42 0.11 0.05 0.05 0.16 0.05 0.05 0.06 R169G 0.55 0.05 0.05 0.05 0.05 0.13 0.05 0.05 0.12 R169H 0.77 0.07 0.05 0.15 0.27 0.07 0.05 0.15 R169I 0.69 0.05 0.05 0.05 0.05 0.10 0.05 0.05 0.10 R169K 0.05 0.05 0.05 0.19 0.37 0.08 0.06 0.10 R169L 0.72 0.05 0.05 0.05 0.08 0.20 0.05 0.05 0.05 R169M 0.40 0.09 0.05 0.05 0.05 0.20 0.05 0.05 0.08 R169P 0.18 0.05 0.05 0.05 0.05 0.31 0.05 0.05 0.18 R169Q 0.56 0.13 0.05 0.39 0.37 0.21 0.20 0.23 R169R 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 R169S 0.96 0.05 0.05 0.10 0.18 0.07 0.05 0.08 R169T 0.67 0.10 0.05 0.07 0.12 0.05 0.05 0.14 R169V 0.56 0.08 0.05 0.05 0.05 0.15 0.05 0.05 0.06 R169W 0.34 0.05 0.05 0.05 0.05 0.12 0.05 0.05 0.13 R169Y 0.29 0.12 0.05 0.05 0.05 0.15 0.05 0.05 0.11 E170A 0.77 0.93 0.96 0.74 0.95 0.77 0.51 0.62 E170D 0.93 0.95 0.97 0.71 0.90 0.78 0.77 E170E 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 E170F 0.51 0.98 0.84 0.84 E170G 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 E170H 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E170I 0.70 0.63 0.58 0.87 0.85 0.82 0.72 0.70 E170K 0.41 0.31 0.59 0.52 0.67 0.58 0.56 0.58 E170L 0.82 0.78 0.84 0.68 0.60 0.55 E170M 0.75 0.52 0.64 0.72 0.79 0.66 0.50 0.61 E170N 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 E170P 0.79 0.90 0.61 0.75 0.82 0.78 0.56 0.62 E170Q 0.71 0.71 0.87 0.70 0.74 0.95 0.72 0.60 0.62 E170R 0.74 0.56 0.58 0.47 0.53 0.68 0.58 0.47 0.56 E170S 0.90 0.88 0.82 0.83 0.81 0.94 0.86 0.67 0.86 E170T 0.10 0.05 0.05 0.05 0.05 0.87 0.05 0.05 0.55 E170V 0.75 0.66 0.64 0.85 0.86 0.78 0.67 0.61 E170W 0.63 0.51 0.74 0.61 0.69 0.58 0.60 E170Y 0.53 0.69 0.82 0.93 0.86 0.67 P176A 1.00 0.84 0.89 0.97 P176C 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 P176D 0.95 0.48 0.82 0.92 0.93 0.94 P176E 0.86 0.98 0.90 0.30 0.81 0.99 0.96 0.78 P176F 0.73 0.85 0.06 0.87 P176G 0.94 0.86 1.00 0.93 0.78 P176H 0.61 0.91 0.89 0.20 0.96 0.92 P176I 0.26 0.05 0.05 0.12 0.18 0.47 0.14 0.08 0.78 P176K 0.27 0.82 0.05 0.90 P176L 0.64 0.91 0.73 0.99 0.92 P176M 0.79 0.99 0.99 0.27 0.84 0.93 0.97 0.63 P176N 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 P176P 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 P176Q 0.72 0.93 0.94 0.35 0.85 0.90 0.92 0.83 P176R 0.35 0.23 P176S 0.54 0.98 0.85 0.53 0.98 0.98 0.96 P176T 0.88 0.90 0.89 0.84 0.95 0.97 0.91 P176V 0.68 0.91 0.71 0.96 P176W 0.20 0.87 0.05 P176Y 0.64 0.98 0.92 0.20 D177A 0.99 0.89 0.52 0.91 0.90 0.84 0.86 0.85 D177C 0.91 0.98 0.45 0.94 0.91 0.88 0.89 0.95 D177D 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 D177E 0.53 0.91 0.98 0.71 0.87 D177F 0.72 0.89 0.98 0.15 D177G 0.94 0.80 0.96 0.99 0.88 0.92 0.99 D177H 0.44 0.84 0.99 0.54 0.98 0.84 D177I 0.67 0.87 0.91 0.35 0.97 0.85 0.96 0.98 0.94 D177K 0.98 0.98 0.52 0.97 0.95 0.97 0.94 D177L 0.58 0.81 0.54 0.87 D177M 0.60 0.94 0.73 D177N 0.98 0.87 0.96 0.92 0.98 D177Q 0.72 0.89 0.61 0.97 0.98 0.80 D177R 0.73 0.91 0.96 0.40 0.98 0.92 D177S 0.13 0.05 0.05 0.05 0.05 0.05 0.05 0.14 0.23 D177V 0.95 0.99 0.59 0.97 D177W 0.34 0.09 0.05 D177Y 0.60 0.87 0.98 0.28 0.96 0.94 D178A 0.93 0.98 0.99 0.79 D178C 0.83 0.90 0.89 0.81 0.96 D178D 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 D178E 0.93 0.94 0.97 0.87 0.85 0.84 0.65 0.81 D178G 0.81 0.99 0.66 0.84 0.94 0.78 0.65 0.74 D178I 0.79 0.82 0.90 0.66 0.84 0.87 0.85 0.67 0.78 D178K 0.75 0.99 0.91 0.83 0.95 0.97 0.82 0.84 D178L 0.92 0.87 0.99 0.78 0.89 0.92 0.88 0.66 0.76 D178M 0.91 0.84 0.96 0.73 0.85 0.81 0.85 0.68 0.78 D178N 0.84 0.91 0.97 0.78 0.86 0.85 0.71 0.82 D178P 0.88 0.93 0.90 0.85 0.92 0.81 0.68 0.71 D178Q 0.92 0.98 0.94 0.95 0.90 0.97 0.84 0.86 D178R 0.73 0.88 0.84 0.97 0.92 0.87 D178S 0.89 0.81 0.71 0.88 0.92 0.82 0.71 0.76 D178T 1.00 0.94 0.88 0.83 0.88 0.77 0.77 D178V 0.65 0.82 0.94 0.57 0.84 0.81 0.80 0.73 0.76 D178W 0.90 0.90 0.75 0.86 0.99 0.85 0.70 0.72 D178Y 0.22 0.80 0.97 0.05 0.25 0.91 R179A 0.24 0.56 0.19 0.99 0.94 0.84 0.55 R179C 0.12 0.34 0.96 0.10 0.86 0.77 0.38 R179D 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 R179F 0.09 0.05 0.05 0.05 0.10 0.18 0.05 0.05 0.19 R179G 0.23 0.56 0.90 0.19 0.95 0.85 0.80 0.53 R179H 0.09 0.12 0.05 0.07 0.05 0.23 0.05 0.05 0.15 R179I 0.12 0.42 0.86 0.14 0.84 0.84 0.78 0.28 R179K 0.45 0.79 0.99 0.43 0.94 0.88 0.72 R179L 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 R179M 0.07 0.40 0.88 0.13 0.34 R179N 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 R179P 0.11 0.05 0.05 0.06 0.05 0.66 0.05 0.05 0.08 R179Q 0.09 0.29 0.89 0.08 0.77 0.69 0.71 0.28 R179R 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 R179S 0.30 0.86 0.97 0.48 0.73 R179T 0.16 0.52 0.96 0.28 0.95 0.96 0.46 R179V 0.19 0.38 0.18 0.95 0.99 0.91 0.57 R179W 0.67 0.91 0.71 0.91 0.93 0.88 R179Y 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 Q194A 0.99 0.89 Q194C 0.67 0.95 0.98 0.89 0.94 Q194E 0.62 0.87 0.98 0.81 0.97 0.87 0.95 Q194F 0.72 0.95 0.98 0.92 0.96 0.91 0.89 Q194G 0.54 0.80 0.94 0.73 0.96 0.88 0.81 Q194H 0.73 0.84 0.95 0.86 0.89 0.94 0.79 0.90 Q194I 0.54 0.73 0.89 0.15 0.89 0.99 0.95 0.83 0.78 Q194K 0.90 0.92 0.90 0.91 0.87 Q194L 0.12 0.32 0.89 0.24 1.00 0.56 Q194M 0.66 0.91 0.89 0.91 0.92 0.92 0.87 0.98 Q194N 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 Q194P 0.13 0.07 0.05 0.12 0.05 0.20 0.05 0.05 0.10 Q194Q 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Q194R 0.84 0.93 0.93 0.86 0.90 0.91 0.75 Q194S 0.77 0.91 0.95 0.86 0.87 0.94 0.88 0.85 0.80 Q194T 0.56 0.79 0.98 0.72 0.88 0.93 0.93 0.84 Q194W 0.83 0.89 0.98 0.70 0.89 0.88 0.89 0.83 Q194Y 0.92 0.99 0.92 0.96 0.98 0.94 N196A 0.25 0.43 0.93 0.22 0.81 0.95 0.85 0.69 0.61 N196E 0.59 0.69 0.88 N196F 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 N196G 0.70 0.89 0.66 0.91 0.89 0.89 0.81 0.80 N196H 0.65 0.95 0.98 0.69 0.97 0.99 0.88 0.88 N196I 0.15 0.05 0.05 0.05 0.05 0.07 0.07 0.05 0.13 N196K 0.14 0.05 0.05 0.05 0.05 0.05 0.06 0.05 0.14 N196L 0.33 0.95 0.97 0.55 N196M 0.48 0.76 0.45 0.89 0.99 0.92 0.76 0.77 N196N 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 N196P 0.17 0.19 0.07 0.41 0.69 0.50 0.39 0.29 N196Q 0.57 0.98 0.98 0.66 0.96 0.97 0.98 N196R 0.42 0.79 0.97 0.46 0.99 1.00 0.96 N196T 0.15 0.49 0.97 0.22 0.75 N196V 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 N196Y 0.40 0.50 0.99 0.25 0.73 0.89 0.71 0.74 0.58 S199A 0.88 0.92 0.88 0.91 0.94 S199C 0.92 0.96 0.95 0.80 0.90 0.90 0.85 0.95 0.99 S199F 0.06 0.05 0.05 0.05 0.05 0.38 0.05 0.13 0.26 S199G 0.56 1.00 0.89 0.52 0.98 0.94 0.96 0.99 S199H 0.10 0.05 0.05 0.05 0.08 0.19 0.05 0.05 0.14 S199I 0.15 0.42 0.63 0.05 0.47 0.55 0.52 0.75 0.57 S199K 0.12 0.05 0.05 0.05 0.05 0.17 0.05 0.05 0.13 S199L 0.07 0.10 0.57 0.05 0.14 0.19 0.17 0.18 0.19 S199M 0.07 0.05 0.05 0.05 0.06 0.38 0.11 0.11 0.36 S199N 0.17 0.40 0.97 0.07 0.61 0.58 0.88 0.68 S199Q 0.15 0.10 0.05 0.05 0.05 0.34 0.15 0.17 0.22 S199R 0.09 0.05 0.05 0.05 0.05 0.08 0.05 0.05 0.15 S199T 0.47 0.86 0.93 0.56 0.96 0.90 0.99 0.86 S199V 0.30 0.69 0.87 0.26 0.68 0.78 0.80 0.53 S199W 0.08 0.05 0.05 0.05 0.05 0.11 0.05 0.05 0.05 S199Y 0.16 0.05 0.05 0.05 0.05 0.48 0.05 0.05 0.09 Y204A 0.05 0.05 0.05 0.05 0.12 0.10 0.05 0.06 Y204C 0.27 0.05 0.05 0.08 0.05 0.19 0.32 0.05 0.42 Y204E 0.05 0.05 0.21 0.06 0.21 0.15 0.05 0.23 Y204F 0.59 0.56 0.40 0.65 0.77 0.62 0.50 0.53 Y204G 0.05 0.05 0.05 0.05 0.13 0.09 0.05 0.20 Y204H 0.05 0.05 0.17 0.05 0.08 0.08 0.05 0.06 Y204I 0.08 0.05 0.11 0.05 0.13 0.13 0.05 0.09 Y204K 0.05 0.05 0.08 0.05 0.14 0.10 0.05 0.10 Y204L 0.74 0.60 0.55 0.46 0.31 0.53 0.44 0.27 0.32 Y204M 0.80 0.16 0.15 0.21 0.33 0.31 0.14 0.23 Y204P 0.05 0.05 0.06 0.05 0.09 0.08 0.05 0.05 Y204Q 0.05 0.05 0.05 0.05 0.14 0.13 0.05 0.22 Y204R 0.05 0.05 0.05 0.05 0.07 0.07 0.05 0.05 Y204S 0.06 0.05 0.17 0.05 0.06 0.10 0.05 0.17 Y204T 0.05 0.05 0.05 0.07 0.22 0.15 0.16 0.20 Y204V 0.07 0.05 0.05 0.13 0.25 0.16 0.20 0.17 Y204W 0.06 0.50 0.07 0.05 0.14 0.11 0.05 0.13 Y204Y 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 N208A 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 N208C 0.33 0.11 0.05 0.05 0.24 0.19 0.08 0.54 0.34 N208D 0.30 0.25 0.96 0.17 0.61 0.42 0.39 0.89 0.87 N208E 0.27 0.11 0.05 0.11 0.29 0.14 0.08 0.60 0.33 N208F 0.29 0.25 0.95 0.11 0.51 0.34 0.30 0.82 0.53 N208G 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 N208H 0.31 0.33 0.05 0.29 0.67 0.53 0.44 0.92 0.64 N208K 0.25 0.34 0.86 0.15 0.84 0.63 0.53 0.68 N208N 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 N208R 0.30 0.25 0.07 0.56 0.52 0.36 0.83 0.86 N208T 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 N208V 0.25 0.27 0.90 0.11 0.65 0.47 0.41 0.98 0.74 N208W 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 T209A 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 T209C 0.65 0.92 0.86 T209D 0.73 0.87 0.73 0.98 T209E 0.63 0.91 0.79 0.99 T209G 0.70 0.97 0.73 T209H 0.42 0.97 0.48 0.81 T209I 0.45 0.92 0.48 0.97 T209K 0.35 0.33 0.97 0.98 0.39 T209L 0.44 0.45 T209M 0.47 0.58 0.98 T209P 0.81 0.94 0.95 0.05 0.84 0.75 0.79 0.82 0.73 T209Q 0.63 0.95 0.63 0.86 T209R 0.69 0.61 0.99 1.00 1.00 0.98 0.92 T209S 0.72 0.97 0.82 0.97 T209T 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 T209V 0.73 0.96 0.77 T209W 0.60 0.95 0.41 0.95 0.95 0.99 T209Y 0.25 0.99 0.63 E214A 0.58 0.05 0.86 0.56 0.90 0.92 E214C 0.24 0.82 0.05 0.70 0.64 0.69 0.84 0.92 E214D 0.58 0.83 0.36 0.99 0.99 E214E 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 E214F 0.20 0.05 0.84 0.05 0.58 0.31 0.44 0.49 0.46 E214G 0.35 0.89 0.05 0.88 0.72 0.87 0.94 E214H 0.36 0.85 0.05 0.78 0.42 0.80 0.96 0.88 E214I 0.20 0.05 0.85 0.05 0.68 0.43 0.73 0.84 0.61

E214K 0.21 0.05 0.05 0.76 0.41 0.62 0.74 0.62 E214L 0.31 0.79 0.05 0.85 0.31 0.87 0.95 0.74 E214M 0.40 0.69 0.05 0.82 0.63 0.87 0.97 0.63 E214N 0.34 0.74 0.05 0.78 0.66 0.79 0.92 0.66 E214P 0.17 0.05 0.05 0.35 0.18 0.31 0.38 0.44 E214Q 0.52 0.89 0.30 0.91 0.88 0.83 0.97 0.57 E214R 0.26 0.05 0.74 0.32 0.76 0.61 E214S 0.40 0.87 0.05 0.87 0.62 0.86 0.82 E214T 0.31 0.63 0.05 0.80 0.65 0.78 0.93 0.64 E214V 0.21 0.05 0.81 0.05 0.75 0.31 0.73 0.86 E214W 0.08 0.05 0.05 0.69 0.59 E214Y 0.31 0.05 0.47 0.36 0.40 0.46 0.66 D215A 0.48 0.98 0.51 0.99 0.97 0.96 1.00 0.97 D215C 0.50 0.96 0.59 0.98 D215D 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 D215E 0.39 0.97 0.65 0.86 D215F 0.16 0.49 0.19 0.63 0.67 0.46 0.63 0.45 D215G 0.44 0.96 0.60 0.99 0.93 0.95 0.93 D215H 0.34 0.94 0.50 0.98 0.90 0.97 0.65 D215I 0.10 0.18 0.05 0.17 0.12 0.26 0.05 0.27 0.05 D215K 0.25 0.79 0.90 0.34 0.82 0.81 0.67 0.85 0.46 D215L 0.14 0.87 0.40 0.60 D215M 0.25 0.98 0.47 0.90 0.98 0.66 D215N 0.46 0.97 0.99 D215Q 0.36 0.61 0.91 0.93 0.72 D215R 0.18 0.66 0.99 0.28 0.55 0.81 0.58 0.62 0.27 D215S 0.42 D215V 0.06 0.34 0.12 0.38 0.64 0.05 0.76 0.38 D215W 0.16 0.65 0.28 0.83 0.61 0.36 Q216A 0.93 0.97 0.73 Q216C 0.53 0.83 0.31 1.00 0.91 0.81 Q216D 0.79 0.76 0.99 0.94 0.92 Q216E 1.00 0.99 0.99 0.95 Q216F 0.72 0.97 0.66 0.98 0.98 Q216G 0.83 0.99 0.85 Q216H 0.81 0.97 0.62 0.99 Q216I 0.61 0.76 Q216K 0.95 0.92 0.89 0.97 0.96 Q216L 0.77 0.99 Q216M 0.51 0.94 0.92 0.98 0.93 Q216N 1.13 0.99 0.96 0.96 Q216P 0.53 0.82 0.52 0.94 1.00 0.96 0.85 Q216Q 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Q216R 0.90 0.65 0.96 0.96 0.94 0.94 Q216S 0.46 0.96 0.87 Q216T 0.77 0.99 0.92 0.99 0.89 Q216W 0.58 0.97 0.79 Q216Y 0.73 0.86 1.27 K224A 0.08 0.05 0.05 0.05 0.14 0.06 0.05 0.05 0.37 K224C 0.11 0.22 0.05 0.05 0.18 0.29 0.08 0.05 0.30 K224D 0.09 0.05 0.05 0.05 0.05 0.26 0.05 0.05 0.41 K224E 0.08 0.19 0.05 0.05 0.36 0.37 0.37 0.16 0.39 K224F 0.10 0.20 0.05 0.05 0.53 0.18 0.37 0.20 0.46 K224G 0.09 0.22 0.05 0.05 0.63 0.49 0.49 0.23 0.61 K224H 0.21 0.66 0.81 0.07 0.98 0.97 0.60 K224I 0.10 0.27 0.90 0.05 0.54 0.44 0.47 0.22 0.45 K224K 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 K224L 0.11 0.23 0.69 0.11 0.53 0.29 0.42 0.23 0.36 K224M 0.07 0.05 0.05 0.05 0.25 0.18 0.20 0.05 0.33 K224N 0.10 0.26 0.68 0.05 0.60 0.70 0.53 0.24 0.52 K224P 0.09 0.05 0.05 0.05 0.05 0.07 0.05 0.05 0.23 K224R 0.52 0.73 0.33 0.93 K224S 0.08 0.05 0.05 0.05 0.29 0.18 0.26 0.05 0.47 K224T 0.07 0.05 0.05 0.05 0.28 0.23 0.31 0.05 0.30 K224V 0.43 0.82 0.55 0.98 0.86 K224W 0.10 0.05 0.05 0.05 0.14 0.13 0.18 0.05 0.29 K224Y 0.12 0.34 0.72 0.05 0.64 0.70 0.54 0.35 0.53 D225A 0.94 0.76 0.94 0.89 0.93 0.90 0.96 D225C 0.85 0.91 0.96 0.99 0.90 D225D 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 D225E 0.25 0.49 0.46 0.91 0.81 0.91 D225F 0.53 0.81 0.86 0.95 0.94 D225G 0.63 0.95 1.00 D225H 0.40 0.73 0.76 D225I 0.47 0.77 0.62 D225L 0.67 0.88 D225M 0.47 0.81 0.96 0.84 0.99 D225P 0.05 0.05 0.05 0.05 0.10 0.50 0.05 0.05 0.18 D225Q 0.64 0.81 0.92 0.98 0.99 0.90 D225R 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 D225S 0.72 0.83 0.98 0.91 0.90 0.92 0.92 D225T 0.57 0.94 D225V 0.71 0.97 D225W 0.48 0.82 0.73 D225Y 0.52 0.88 0.92 Q226A 0.94 0.95 0.98 Q226C 0.63 0.96 0.63 0.99 1.15 Q226D 0.52 0.89 0.45 1.00 0.98 Q226E 0.69 0.92 0.58 0.88 0.95 1.00 0.88 Q226F 0.53 0.92 0.51 Q226H 0.65 0.86 0.95 0.41 0.89 0.93 0.87 0.69 Q226I 0.41 0.89 0.45 0.79 Q226K 0.48 0.78 0.42 0.93 0.97 0.90 0.65 Q226L 0.52 0.91 0.48 0.88 Q226M 0.47 0.90 0.99 0.45 0.99 0.85 Q226N 0.33 0.85 0.97 0.29 0.98 0.91 Q226Q 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Q226R 0.44 0.86 0.90 0.53 0.89 Q226S 0.22 0.05 0.05 0.09 0.26 0.50 0.15 0.13 Q226T 0.59 0.98 0.96 0.55 0.99 0.98 0.98 0.99 Q226V 0.31 0.85 0.96 0.37 0.94 0.98 Q226W 0.37 0.54 Q226Y 0.42 0.99 0.51 D236A 0.05 0.05 0.05 0.05 0.07 0.05 0.05 0.07 D236C 0.57 0.05 0.05 0.05 0.05 0.06 0.05 0.05 0.21 D236D 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 D236E 0.85 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.13 D236F 0.19 0.05 0.05 0.05 0.05 0.14 0.05 0.05 0.26 D236G 0.05 0.05 0.05 0.05 0.08 0.05 0.05 0.10 D236H 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 D236I 0.26 0.05 0.05 0.05 0.05 0.14 0.05 0.05 0.18 D236K 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 D236L 0.15 0.05 0.05 0.12 0.05 0.23 0.05 0.24 0.23 D236M 0.86 0.05 0.05 0.05 0.05 0.12 0.05 0.05 0.10 D236P 0.05 0.05 0.05 0.05 0.07 0.05 0.05 0.14 D236Q 0.05 0.05 0.05 0.05 0.07 0.05 0.05 0.05 D236R 0.06 0.05 0.05 0.05 0.05 0.64 0.05 0.07 0.33 D236S 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.08 D236T 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.06 D236V 0.21 0.05 0.05 0.05 0.05 0.20 0.05 0.06 0.45 D236W 0.21 0.05 0.05 0.05 0.05 0.09 0.05 0.05 0.30 D236Y 0.16 0.05 0.05 0.05 0.05 0.19 0.05 0.06 0.23 W237A 1.00 0.06 0.05 0.05 0.06 0.13 0.05 0.05 0.14 W237C 0.43 0.05 0.05 0.05 0.05 0.06 0.05 0.05 0.12 W237D 0.58 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.16 W237E 0.78 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.07 W237G 0.46 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.30 W237H 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.10 W237I 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 W237K 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.21 W237M 0.05 0.05 0.05 0.05 0.06 0.05 0.05 0.05 W237N 0.93 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.09 W237P 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 W237Q 0.93 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.10 W237R 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.10 W237S 0.05 0.05 0.05 0.05 0.08 0.05 0.05 0.15 W237T 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.08 W237V 0.98 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.09 W237W 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 W237Y 0.74 0.39 0.05 0.22 0.17 0.21 0.28 0.30 N238A 0.78 N238C 0.38 0.92 1.00 0.48 0.96 N238D 0.89 0.88 0.69 0.95 0.95 0.92 0.93 0.79 N238E 0.44 0.91 0.45 0.92 0.83 N238F 0.49 0.20 0.27 0.49 0.50 0.33 0.36 0.31 N238G 0.43 0.71 0.93 0.89 0.61 N238H 0.49 0.80 0.78 0.20 0.66 0.70 0.80 0.69 0.59 N238I 0.39 0.86 0.67 0.06 0.46 0.51 0.68 0.61 0.45 N238K 0.33 0.79 0.73 0.05 0.34 0.40 0.68 0.51 0.32 N238L 0.60 0.82 0.90 0.42 0.45 0.78 0.76 0.74 0.54 N238M 0.32 0.77 0.54 0.58 0.92 0.87 0.60 N238P 0.11 0.42 0.05 0.20 0.25 0.16 0.32 0.24 N238R 0.39 0.52 0.57 0.10 0.27 0.27 0.57 0.42 0.24 N238S 0.56 0.73 0.85 0.88 0.89 0.76 N238T 0.55 0.87 0.47 0.65 0.96 0.94 0.84 0.72 N238V 0.37 0.82 0.97 0.33 0.58 0.85 0.84 0.93 0.61 N238W 0.44 0.30 0.16 0.94 0.63 0.59 0.75 0.44 N238Y 0.53 0.83 0.87 0.48 0.75 0.77 0.77 0.89 0.59 T242A 0.75 0.82 0.09 0.95 0.92 T242C 0.62 0.94 0.47 T242E 0.96 0.89 0.46 T242F 0.50 0.84 0.17 0.70 0.92 0.84 T242G 0.75 0.86 0.27 0.89 1.00 0.89 0.87 T242H 0.94 0.93 T242I 0.38 0.66 0.05 0.96 0.05 0.94 0.81 0.73 T242K 0.48 0.81 0.36 0.72 0.99 0.82 T242L 0.80 0.87 0.05 0.46 0.99 T242M 0.52 0.85 0.07 0.58 0.98 0.94 T242N 0.82 0.90 0.93 0.86 0.95 0.91 0.90 T242P 0.09 0.05 0.05 0.05 0.09 0.05 0.05 0.05 0.39 T242Q 0.50 0.77 0.49 0.86 0.88 T242R 0.72 0.80 0.38 0.65 0.93 0.94 0.79 T242S 0.93 1.00 0.95 T242T 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 T242V 0.40 0.86 0.05 0.27 0.97 0.88 T242W 0.54 0.86 0.19 0.84 T242Y 0.52 0.82 0.21 0.95 N248A 0.98 0.80 0.95 0.95 0.99 N248C 0.99 0.95 0.89 0.95 0.93 N248F 0.58 0.88 0.79 0.72 0.94 0.99 0.96 N248G 0.25 0.90 0.84 0.45 0.92 0.99 N248H 0.70 0.88 0.89 0.76 0.94 0.68 0.86 0.82 0.77 N248K 0.51 0.84 0.87 0.59 0.96 0.71 0.87 0.83 0.78 N248L 0.93 0.98 0.93 0.96 0.75 0.86 0.84 0.82 N248M 0.61 0.89 0.83 0.70 0.85 0.69 0.77 0.79 0.75 N248P 0.13 0.34 0.92 0.08 0.72 0.30 0.70 0.74 0.58 N248Q 0.56 0.88 0.93 0.70 0.94 0.80 0.89 0.88 0.78 N248R 0.44 0.89 0.88 0.54 0.99 0.81 0.94 0.95 0.82 N248S 0.75 0.85 0.87 0.73 0.86 0.77 0.76 0.76 0.75 N248T 0.84 0.87 0.98 1.00 0.96 N248V 0.58 0.91 0.89 0.74 0.92 0.85 0.87 0.92 0.76 N248W 0.64 0.91 1.00 0.96 N248Y 0.51 0.98 0.87 0.74 1.00 0.93 0.89 S249A 0.05 0.05 0.32 0.89 0.91 0.97 0.75 S249C 0.93 0.43 0.90 0.50 0.84 0.96 0.88 0.93 0.81 S249D 0.51 0.29 0.05 0.67 0.73 0.80 0.63 0.64 0.55 S249E 0.24 0.05 0.05 0.36 0.17 0.33 0.09 0.05 0.26 S249G 0.58 0.05 0.05 0.57 0.84 0.79 0.85 0.67 S249H 0.69 0.05 0.05 0.48 0.88 0.83 0.79 0.75 0.53 S249I 0.73 0.70 0.88 0.90 0.88 0.87 0.78 0.67 S249K 0.43 0.27 0.05 0.68 0.88 0.94 0.74 0.70 0.40 S249L 0.48 0.28 0.90 0.97 0.98 0.85 0.87 0.87 0.72 S249M 0.66 0.69 0.84 0.92 0.84 0.71 0.61 S249N 0.65 0.45 0.90 0.73 0.83 0.80 0.75 0.61 0.50 S249P 0.27 0.05 0.05 0.57 0.17 0.16 0.10 0.05 0.20 S249Q 0.81 0.05 0.05 0.44 0.92 0.97 0.90 0.72 0.62 S249R 0.74 0.40 0.87 0.63 0.79 0.81 0.77 0.57 0.56 S249S 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 S249T 0.81 0.28 0.05 0.66 0.87 0.99 0.83 0.73 0.82 S249V 0.55 0.53 0.76 0.36 0.76 0.97 0.71 0.74 S249W 0.52 0.05 0.05 0.05 0.86 0.85 0.79 0.81 0.62 S249Y 0.63 0.72 0.97 0.05 0.95 0.86 0.86 0.84 0.57 N263A 0.96 0.96 0.99 0.96 0.96 N263C 0.61 0.91 N263D 0.93 0.96 0.93 0.95 0.97 N263E 0.92 0.93 0.87 0.94 0.97 0.89 N263F 0.59 0.97 0.71 0.98 0.68 0.99 0.97 N263G 0.76 1.00 0.93 0.99 N263H 0.84 0.94 0.97 0.82 0.89 0.91 N263I 0.50 0.78 0.50 0.91 0.85 0.98 0.87 0.91 N263K 0.57 0.83 0.59 0.89 0.80 0.94 0.92 0.93 N263L 0.68 0.93 0.78 0.96 0.99 0.98 0.92 N263M 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 N263N 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 N263P 0.62 0.77 0.53 0.66 0.42 0.77 0.73 0.75 N263Q 0.61 0.95 0.67 0.96 0.97 N263R 0.68 0.93 0.97 0.68 0.92 0.82 0.92 0.87 N263S 0.47 0.86 N263T 0.51 0.71 N263V 0.47 0.83 0.52 0.94 0.85 0.94 0.88 N263W 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 N263Y 0.60 0.91 0.95 0.65 0.88 0.86 0.89 0.90 N264A 0.37 0.91 0.06 0.93 0.96 0.96 0.71 0.95 N264C 0.71 0.90 N264D 0.86 0.71 0.90 0.89 0.87 0.71 0.75 N264E 0.77 0.70 0.84 0.86 0.82 0.62 0.70 N264G 0.97 0.88 0.68 0.86 0.78 0.88 0.66 0.70 N264H 0.94 0.94 0.90 0.87 0.91 0.71 0.71 N264K 0.78 0.85 0.68 0.80 0.79 0.87 0.67 0.70 N264L 0.66 0.58 0.79 0.83 0.75 0.61 0.65 N264M 0.81 0.74 0.61 0.80 0.85 0.81 0.71 0.66 N264N 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 N264P 0.85 0.81 0.76 0.05 0.57 0.44 0.74 0.51 0.58 N264Q 0.78 0.91 0.66 0.91 1.00 0.96 0.80 0.91 N264R 0.85 0.91 0.77 0.78 0.83 0.72 0.76 N264S 0.87 0.89 0.90 0.87 0.88 0.75 0.82 N264T 0.86 0.81 0.61 0.81 0.95 0.81 0.71 0.70 N264V 0.84 0.70 0.83 0.76 0.84 0.69 0.73 N264W 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 N264Y 0.76 0.73 0.79 0.75 0.76 0.70 0.71 R265A 0.89 0.72 0.92 0.87 0.73 0.85

R265E 0.87 0.61 0.97 0.83 0.89 R265F 0.98 0.90 0.41 0.89 0.90 0.95 0.78 0.82 R265G 0.96 0.16 0.86 0.83 0.88 0.73 0.79 R265I 0.84 0.96 0.29 0.99 0.96 0.92 0.92 R265K 0.92 0.96 0.95 0.98 0.99 0.90 0.86 R265L 0.92 0.92 0.52 0.92 0.92 0.80 0.82 R265M 0.89 0.92 0.61 0.95 0.94 0.84 0.82 R265N 0.90 0.95 0.35 0.96 0.94 0.91 0.84 0.91 R265P 0.75 0.77 0.36 0.78 0.80 0.79 0.70 0.68 R265Q 0.98 0.87 0.93 0.99 0.93 0.86 0.95 R265R 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 R265S 0.84 0.94 0.26 0.82 0.90 0.86 0.85 0.64 R265T 0.89 0.76 0.20 0.82 0.84 0.86 0.85 0.77 R265V 0.74 0.84 0.27 0.91 0.87 0.86 0.95 0.76 R265W 0.87 0.97 0.95 0.37 0.83 0.97 0.90 0.90 0.88 R265Y 0.91 0.96 0.50 0.85 0.90 0.91 0.72 N276A 0.28 0.51 0.27 0.95 N276C 0.09 0.34 0.98 0.17 N276E 0.27 0.26 0.90 0.14 0.62 0.44 0.39 0.87 0.45 N276F 0.19 0.39 0.98 0.21 0.87 0.95 N276H 0.30 0.28 0.85 0.11 0.49 0.40 0.27 0.77 0.72 N276K 0.25 0.35 0.82 0.05 0.68 0.52 0.48 0.98 N276M 0.24 0.29 0.86 0.15 0.70 0.49 0.48 0.50 N276N 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 N276P 0.20 0.19 0.73 0.12 0.41 0.21 0.13 0.74 0.28 N276Q 0.30 0.39 0.90 0.24 0.80 0.66 0.52 1.00 N276R 0.32 0.31 0.94 0.15 0.59 0.48 0.41 0.85 0.80 N276T 0.34 0.35 0.87 0.16 0.68 0.63 0.46 0.91 0.86 N276V 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 N276W 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 S277A 0.96 0.91 0.95 0.86 0.81 S277C 0.53 0.94 0.60 S277D 0.88 0.89 0.94 0.99 0.84 0.76 S277E 0.86 0.84 0.86 0.87 0.90 0.79 0.76 S277F 0.56 0.82 0.60 0.93 0.73 S277G 0.74 0.83 0.70 0.90 0.89 0.86 0.73 S277H 0.62 0.75 0.99 0.61 0.90 0.92 0.87 0.80 S277I 0.54 0.77 1.00 0.54 0.92 0.90 0.82 S277K 0.74 0.77 0.96 0.51 0.79 0.95 0.84 0.61 0.68 S277L 0.56 0.83 0.94 0.70 0.92 0.91 0.98 0.56 0.76 S277M 0.54 0.90 0.63 0.98 0.65 0.82 S277N 0.70 0.78 0.65 0.89 1.00 0.89 0.80 0.71 S277P 0.26 0.59 0.92 0.18 0.94 0.72 0.93 S277Q 0.69 0.87 0.73 0.95 0.99 0.88 0.87 S277R 0.73 0.89 0.68 0.92 0.97 0.71 0.74 S277S 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 S277T 0.49 0.76 0.93 0.05 0.89 0.96 0.96 0.92 0.91 S277V 0.62 0.64 0.49 0.05 0.41 0.55 0.46 0.40 0.47 S277W 0.06 0.60 S277Y 0.56 0.97 0.94 0.79 0.93 N278A 0.97 1.00 0.79 1.00 0.93 0.90 N278C 0.90 1.00 0.72 0.98 N278D 0.88 0.97 0.94 0.88 0.80 N278F 0.50 0.69 N278G 0.80 0.92 0.79 0.92 0.88 N278H 0.15 0.41 0.73 0.05 0.61 0.12 0.64 0.59 N278I 0.59 0.84 0.98 0.57 0.94 0.95 0.88 N278L 0.67 0.89 0.97 0.74 0.91 0.99 0.78 0.87 N278M 0.72 0.93 0.92 0.65 0.91 0.90 0.76 N278N 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 N278P 0.22 0.34 0.90 0.05 0.70 0.44 0.85 0.69 0.52 N278Q 0.74 0.79 0.67 0.88 0.99 0.87 0.78 N278R 0.94 0.89 0.82 0.92 0.98 0.78 N278S 0.60 0.90 0.99 0.66 0.97 0.86 0.89 N278T 0.79 0.98 0.95 0.65 0.92 0.98 0.88 N278V 0.80 0.98 0.81 0.94 N278W 0.70 0.93 0.95 0.55 0.91 0.87 N278Y 0.60 0.82 0.99 0.74 0.98 0.92 0.95 Q279A 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Q279C 0.63 0.81 Q279D 0.87 0.97 0.85 0.99 0.96 1.00 Q279E 0.68 0.78 0.95 0.60 0.81 0.78 0.81 0.82 Q279G 0.73 1.00 0.94 0.84 0.95 0.95 0.96 0.92 Q279H 0.52 0.89 0.93 0.58 0.91 0.85 0.95 0.75 Q279I 0.39 0.76 0.99 0.37 0.86 0.87 0.97 0.83 Q279K 0.28 0.79 0.89 0.37 0.88 0.99 0.70 Q279L 0.77 0.74 0.99 0.65 0.78 0.79 0.67 0.72 0.60 Q279M 0.73 0.74 0.97 0.59 0.73 0.94 0.63 0.69 0.70 Q279N 0.27 0.41 0.95 0.20 0.58 0.55 0.66 0.51 Q279P 0.12 0.18 0.96 0.05 0.35 0.47 0.51 0.46 0.32 Q279S 0.56 0.85 0.94 0.56 0.82 0.86 0.92 0.83 Q279T 0.61 0.87 0.93 0.64 0.85 0.82 0.86 0.80 Q279V 0.49 0.90 0.58 0.95 0.83 Q279Y 0.45 0.90 0.98 0.55 0.95 0.86 T282C 0.92 0.99 0.95 T282D 0.94 0.82 0.96 0.92 0.99 T282E 0.72 0.96 0.97 0.84 0.92 0.81 0.87 0.94 0.92 T282F 0.79 0.99 0.91 0.75 0.83 0.84 0.85 0.93 0.79 T282G 0.99 0.99 0.98 0.97 0.97 0.87 0.97 0.87 T282H 0.10 0.13 0.05 0.05 0.05 0.05 0.05 0.65 T282I 0.87 0.83 0.97 0.82 0.81 0.84 0.79 0.84 0.73 T282K 0.91 0.86 0.97 0.87 0.83 0.93 0.79 T282L 0.61 0.94 0.92 0.78 0.87 0.88 0.84 0.93 T282M 0.75 1.00 0.93 0.85 0.89 0.92 0.90 0.99 0.96 T282N 0.96 0.97 0.86 0.94 0.82 0.87 0.79 T282P 0.58 0.91 0.98 0.78 0.94 0.84 0.94 T282Q 0.88 0.87 0.98 0.84 0.82 0.79 0.80 0.85 0.78 T282R 0.96 0.97 0.89 0.96 0.91 0.96 0.84 T282S 0.47 0.98 0.91 0.71 0.98 0.94 0.97 0.99 T282T 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 T282V 0.99 0.97 0.90 0.95 0.90 0.97 0.87 T282Y 0.70 0.82 0.97 0.74 0.85 0.87 0.89 0.93 0.85 R284A 0.19 0.13 0.68 0.05 0.35 0.64 0.14 0.92 0.52 R284C 0.39 0.16 0.05 0.05 0.26 0.20 0.14 0.51 0.30 R284E 0.31 0.15 0.05 0.08 0.27 0.16 0.11 0.57 0.76 R284F 0.35 0.13 0.05 0.05 0.23 0.21 0.11 0.49 0.28 R284G 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 R284H 0.14 0.15 0.05 0.06 0.53 0.36 0.23 0.74 R284I 0.36 0.15 0.05 0.08 0.23 0.20 0.10 0.50 0.50 R284K 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 R284M 0.10 0.20 0.05 0.12 0.83 0.73 0.45 R284N 0.36 0.12 0.05 0.29 0.22 0.17 0.54 0.41 R284P 0.29 0.14 0.92 0.05 0.26 0.15 0.10 0.54 0.43 R284R 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 R284S 0.24 0.14 0.05 0.15 0.29 0.15 0.11 0.68 0.59 R284T 0.36 0.19 0.89 0.14 0.33 0.27 0.20 0.59 0.86 R284V 0.31 0.64 0.25 0.05 0.47 0.43 0.27 0.73 0.43 R284W 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 R284Y 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 D287C 0.96 0.76 D287D 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 D287E 0.72 0.94 0.61 0.99 0.99 0.99 0.98 0.91 D287F 0.31 0.29 0.07 0.33 0.41 0.27 0.19 0.66 D287G 0.71 0.98 0.92 0.60 0.97 0.96 0.94 0.93 D287H 0.22 0.78 0.36 0.85 0.80 0.74 0.73 D287I 0.31 0.63 0.18 0.62 0.61 0.57 0.55 D287K 0.35 0.92 0.21 0.94 0.93 0.94 D287L 0.28 0.47 0.31 0.48 0.59 0.44 0.37 0.47 D287M 0.30 0.85 0.22 0.83 0.83 0.82 0.63 D287N 0.65 0.89 0.62 0.91 0.92 0.93 0.90 D287P 0.14 0.05 0.05 0.08 0.39 0.44 0.28 0.17 0.27 D287R 0.34 0.20 0.05 0.05 0.23 0.33 0.16 0.14 0.47 D287S 0.46 0.91 0.52 0.97 D287V 0.36 0.56 0.12 0.66 0.80 0.59 0.54 0.93 D287W 0.23 0.52 0.08 0.61 0.75 0.52 0.47 0.62 D287Y 0.29 0.32 0.05 0.37 0.44 0.29 0.22 0.08 R291A 0.08 0.30 0.91 0.05 0.29 0.83 0.35 0.41 0.30 R291C 0.08 0.05 0.05 0.05 0.05 0.48 0.05 0.11 0.23 R291D 0.07 0.05 0.05 0.05 0.05 0.34 0.05 0.05 0.26 R291E 0.07 0.05 0.05 0.05 0.05 0.50 0.05 0.05 0.21 R291G 0.06 0.05 0.05 0.05 0.05 0.87 0.05 0.18 0.25 R291H 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 R291I 0.05 0.05 0.05 0.05 0.05 0.25 0.05 0.05 0.31 R291K 0.08 0.27 0.05 0.05 0.20 0.98 0.31 0.47 0.37 R291L 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 R291M 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 R291N 0.06 0.05 0.05 0.05 0.05 0.33 0.05 0.09 0.31 R291Q 0.06 0.05 0.05 0.05 0.05 0.32 0.05 0.09 0.29 R291S 0.07 0.31 0.98 0.05 0.28 0.75 0.28 0.39 0.22 R291T 0.05 0.05 0.05 0.05 0.05 0.40 0.05 0.18 0.43 R291V 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 R291W 0.06 0.05 0.05 0.05 0.05 0.68 0.05 0.05 0.09 R291Y 0.06 0.05 0.05 0.05 0.05 0.32 0.05 0.13 0.22 Q301A 0.67 0.88 0.06 0.93 1.00 0.84 0.93 Q301E 0.42 0.84 0.30 0.89 0.91 0.76 0.78 Q301F 0.22 0.75 0.95 0.05 0.61 0.68 0.64 0.53 0.34 Q301G 0.12 0.99 0.97 0.05 0.85 0.47 Q301H 0.20 0.96 0.96 0.05 0.82 0.90 0.86 0.75 0.47 Q301K 0.08 0.91 0.08 0.48 Q301L 0.33 0.05 0.98 0.95 0.95 0.79 Q301N 0.22 0.95 0.14 0.98 0.98 0.98 0.98 0.60 Q301Q 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Q301R 0.22 0.85 0.05 0.88 0.88 0.97 0.57 Q301S 0.35 0.96 0.05 0.87 0.76 0.90 0.88 0.78 Q301T 0.21 0.05 0.89 0.83 0.95 0.90 0.58 Q301V 0.26 0.05 0.84 0.85 0.88 0.95 0.77 Q301Y 0.24 0.95 0.94 0.05 0.78 0.67 0.86 0.90 0.59 D302A 0.38 0.93 0.55 D302C 0.23 0.99 0.36 D302D 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 D302E 0.35 0.83 0.89 0.51 0.97 D302F 0.33 0.88 0.81 0.48 D302G 0.39 0.98 0.86 0.49 D302I 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 D302K 0.29 0.74 0.94 0.38 0.88 0.98 0.93 D302L 0.29 0.72 0.91 0.09 0.90 0.86 0.83 0.81 D302M 0.27 0.77 0.90 0.27 0.95 0.79 D302N 0.51 0.87 0.86 0.71 0.93 0.94 0.65 D302P 0.24 0.75 0.88 0.31 1.00 0.85 0.95 0.67 D302S 0.48 0.94 0.86 0.68 0.96 0.93 0.99 0.87 D302T 0.41 0.83 0.93 0.39 0.90 0.93 0.64 D302V 0.23 0.30 0.82 0.19 0.55 0.84 0.34 0.29 0.16 D302W 0.59 0.99 0.58 0.87 0.85 0.92 0.85 D302Y 0.50 0.93 0.85 0.48 0.97 0.83 0.94 0.83 Q303A 0.85 0.92 0.82 0.90 Q303C 0.56 0.91 0.95 Q303D 0.95 0.98 0.93 0.98 0.86 0.97 Q303E 0.96 0.98 0.91 0.94 0.79 0.89 Q303F 0.88 0.91 0.91 0.92 0.95 0.86 0.71 0.85 Q303G 0.80 0.99 0.92 0.98 0.90 0.95 0.80 0.84 Q303H 0.74 0.94 0.93 0.98 0.92 0.99 0.85 0.86 Q303I 0.82 0.96 0.95 0.89 0.95 0.86 0.83 Q303K 0.74 0.88 0.93 0.99 0.88 0.94 Q303L 0.79 0.90 0.98 0.90 0.92 0.88 0.93 Q303M 0.87 0.91 0.95 0.87 0.80 0.87 Q303N 0.97 0.99 0.89 0.81 0.88 0.84 Q303P 0.37 0.97 0.85 0.66 0.85 0.87 0.91 Q303Q 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Q303R 0.94 0.91 0.91 0.93 0.86 0.80 Q303S 0.92 0.91 0.93 0.92 0.90 0.90 0.80 Q303T 0.87 0.94 0.94 0.98 0.90 0.88 0.91 Q303V 0.85 0.91 0.86 0.84 0.95 0.94 0.86 Q303W 0.71 0.95 0.92 0.85 0.94 0.99 0.89 0.81 Q303Y 0.69 0.90 0.94 0.88 0.94 0.92 0.93 Y306A 0.84 0.88 0.44 0.81 0.83 0.77 0.77 0.86 Y306C 0.65 0.24 0.89 0.87 0.85 0.89 Y306D 0.11 0.05 0.05 0.05 0.36 0.44 0.30 0.22 0.81 Y306E 0.96 0.84 0.35 0.85 0.79 0.84 0.86 0.93 Y306F 0.95 0.90 0.92 0.79 0.86 0.88 0.91 Y306G 0.85 0.83 0.62 0.93 0.94 0.94 Y306I 1.00 0.87 0.88 0.97 0.84 0.86 0.90 Y306K 0.44 0.99 0.12 0.99 0.94 Y306L 0.89 0.92 0.83 0.94 0.94 0.78 Y306M 0.61 0.78 0.78 0.98 Y306N 0.57 0.86 0.48 0.98 0.94 0.93 0.92 1.00 Y306P 0.85 0.79 0.56 0.87 0.86 0.86 0.87 0.84 Y306Q 0.17 0.87 0.24 0.10 Y306R 0.44 0.05 0.98 Y306S 0.89 0.88 0.39 0.88 0.91 0.84 0.87 0.84 Y306T 0.79 0.81 0.52 0.86 0.91 0.83 0.85 0.94 Y306V 0.42 0.83 0.90 0.95 0.98 Y306W 0.95 0.92 0.88 0.89 0.84 0.87 0.88 Y306Y 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 S312A 0.87 0.90 0.92 1.00 0.84 0.87 0.68 S312C 0.97 S312D 0.89 0.88 0.99 0.67 S312F 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 S312G 0.89 0.84 0.96 0.91 0.88 0.89 S312I 0.87 0.56 0.88 0.92 0.85 0.87 0.71 S312K 0.77 1.00 0.73 0.98 0.94 0.97 0.75 S312L 0.73 0.88 0.56 0.76 0.81 0.82 0.83 0.55 S312M 0.64 0.98 0.60 0.88 0.97 0.90 0.93 0.69 S312N 0.85 0.93 0.88 0.71 0.70 S312P 0.26 0.85 0.87 0.05 0.51 0.15 0.67 0.97 0.20 S312Q 0.94 0.96 0.90 0.92 0.96 0.99 0.67 S312R 0.81 0.68 0.85 0.83 0.91 0.70 S312S 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 S312T 0.86 0.98 0.94 0.92 0.96 0.97 0.72 S312V 0.85 0.99 0.68 0.98 0.97 0.99 0.86 S312W 0.13 0.88 0.06 S312Y 0.51 0.78 R313A 0.78 0.86 0.95 0.47 0.92 0.88 0.76 0.93 R313C 0.67 0.83 0.97 0.35 0.92 0.97 0.78 0.91 R313D 0.94 0.97 0.05 0.91 0.89 0.78 0.90 R313E 0.95 0.46 0.88 0.85 0.69 0.71 0.83 R313F 0.51 0.68 0.99 0.26 0.80 0.93 0.75 0.66 0.71 R313G 0.83 0.92 0.52 0.86 0.84 0.73 0.79 R313H 0.93 0.90 0.91 0.73 0.84 0.88 0.82 0.72 0.76 R313I 0.85 0.84 0.98 0.62 0.84 0.99 0.80 0.67 0.69 R313K 0.67 0.88 0.92 0.75 0.95 0.95 0.82 0.75 R313L 0.97 0.98 0.83 0.86 0.97 0.77 0.74 0.78

R313M 0.89 0.87 0.99 0.63 0.85 0.89 0.75 0.70 0.68 R313N 0.84 0.86 0.97 0.57 0.83 0.78 0.71 0.70 R313P 0.23 0.49 0.87 0.05 0.76 0.42 0.80 0.72 0.55 R313R 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 R313S 1.00 0.89 0.54 0.83 0.93 0.73 0.77 0.72 R313V 0.63 0.80 0.50 0.85 0.88 0.81 0.82 0.75 R313W 0.70 0.83 0.34 0.86 0.92 0.85 0.83 0.73 Q316A 0.97 0.58 0.86 0.99 0.94 0.99 0.92 Q316C 0.89 0.33 0.95 0.97 Q316D 0.93 0.27 0.88 0.86 0.95 0.98 Q316E 0.87 0.93 0.75 0.89 0.92 0.88 0.89 0.79 Q316F 0.91 0.24 0.91 0.93 0.87 0.95 0.90 Q316G 0.66 0.78 0.24 0.92 0.99 0.71 0.82 Q316H 0.27 0.42 0.06 0.90 0.88 0.82 0.69 Q316I 0.74 0.91 0.05 0.95 0.91 0.92 0.92 Q316K 0.53 0.81 0.20 0.99 0.93 Q316L 0.84 0.92 0.97 0.15 0.87 0.77 0.83 0.79 Q316M 0.23 0.42 0.89 0.25 0.97 0.76 0.97 0.63 Q316N 0.49 0.66 0.06 0.92 0.94 0.80 0.95 0.73 Q316P 0.47 0.05 0.78 Q316Q 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Q316R 0.37 0.61 0.16 0.90 0.79 Q316S 0.38 0.82 0.13 0.85 Q316T 0.50 0.52 Q316V 0.72 0.94 0.20 0.97 0.92 0.92 0.92 0.89 Q316W 0.46 0.54 0.14 0.84 0.91 0.68 0.85 0.62 Q316Y 0.78 1.00 0.14 K320A 0.73 0.97 0.77 0.77 0.89 0.82 0.70 0.69 K320C 0.86 1.00 K320E 0.94 0.93 0.30 0.81 0.91 0.96 0.95 K320G 0.98 0.82 0.80 0.84 0.87 0.78 0.93 K320H 0.80 0.98 0.78 0.93 0.87 0.66 K320K 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 K320L 0.52 0.74 0.99 0.40 0.57 0.86 0.66 0.77 K320M 0.87 0.84 0.80 0.75 0.98 0.79 0.91 K320N 0.95 0.84 0.89 K320P 0.82 0.93 0.94 0.65 0.84 1.02 0.90 0.87 K320Q 1.00 0.77 0.93 0.80 0.73 0.95 0.77 0.63 K320R 0.98 0.86 0.92 0.49 0.78 0.99 0.90 0.80 K320S 0.98 0.97 0.90 0.78 K320T 0.81 0.92 0.60 0.89 0.91 0.90 K320V 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 K320W 0.85 0.88 0.95 0.71 0.59 0.79 0.92 0.64 0.90 K320Y 0.88 0.97 0.82 0.94 0.91 R324C 0.65 0.94 0.57 0.98 R324D 0.61 0.80 0.26 0.90 0.94 0.98 0.90 R324E 0.56 0.96 0.95 0.63 0.87 0.95 0.98 0.77 R324F 0.68 0.94 0.93 0.68 0.89 0.94 0.96 0.90 R324H 0.56 0.87 0.97 0.60 0.84 0.87 0.92 0.89 R324I 0.26 0.49 0.92 0.21 0.72 0.85 0.78 0.64 R324K 0.38 0.68 0.99 0.40 0.75 0.95 0.94 0.81 R324L 0.57 0.87 0.92 0.57 0.83 0.88 0.89 0.77 R324M 0.55 0.88 0.95 0.54 0.87 0.96 0.98 0.83 R324N 0.25 0.05 0.05 0.05 0.17 0.13 0.05 0.05 0.15 R324P 0.07 0.05 0.05 0.05 0.05 0.11 0.08 0.09 0.36 R324Q 0.46 0.77 0.91 0.41 0.79 0.91 0.90 0.75 R324S 0.14 0.05 0.05 0.05 0.05 0.16 0.05 0.06 0.11 R324V 0.28 0.53 0.92 0.22 0.73 0.89 0.89 0.63 R324W 0.36 0.67 0.97 0.35 0.76 0.91 0.92 0.79 R324Y 0.77 0.98 0.99 0.84 0.95 0.93 R328C 0.39 0.36 0.97 0.94 0.54 R328D 0.14 0.05 0.05 0.05 0.08 0.06 0.05 0.49 0.05 R328E 0.35 0.44 0.89 0.82 0.93 0.67 R328F 0.06 0.05 0.05 0.05 0.09 0.53 0.05 0.15 R328G 0.36 1.00 0.45 0.90 0.97 0.89 0.42 R328I 0.14 0.62 0.05 0.48 0.59 0.54 0.05 R328K 0.50 0.94 0.51 0.85 0.96 0.70 R328L 0.13 0.13 0.70 0.73 0.89 0.32 R328M 0.13 0.99 0.16 0.86 0.75 0.40 R328N 0.23 0.89 0.88 0.12 0.52 0.77 0.73 0.99 0.32 R328P 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 R328Q 0.39 0.43 0.82 0.99 0.97 0.57 R328R 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 R328S 0.12 0.97 0.05 R328T 0.83 0.94 0.91 0.93 0.88 0.98 0.97 0.79 R328V 0.41 0.91 0.30 0.99 0.97 0.99 0.61 R328W 0.12 0.05 0.05 0.05 0.06 0.21 0.05 0.51 0.39 R328Y 0.06 0.05 0.05 0.05 0.20 0.44 0.09 0.16 D329A 0.93 D329D 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 D329E 0.69 0.97 0.91 0.94 0.85 0.92 0.91 0.72 D329F 0.12 0.60 0.18 0.40 0.44 0.37 0.85 0.53 D329G 0.67 0.99 0.80 0.94 0.98 0.94 0.73 D329H 0.24 0.95 0.26 0.77 0.93 0.97 0.41 D329I 0.07 0.05 0.05 0.05 0.05 0.05 0.05 0.73 0.05 D329K 0.09 0.48 0.05 0.05 0.22 0.28 0.27 0.90 0.34 D329L 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 D329M 0.21 1.00 0.29 0.69 0.87 0.92 0.38 D329N 0.60 0.95 0.85 0.77 0.89 0.99 0.72 D329P 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 D329Q 0.45 0.59 0.74 0.98 0.93 0.95 0.62 D329R 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 D329S 0.66 0.96 0.97 0.97 D329T 0.15 0.24 0.85 0.94 0.90 0.46 D329V 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 D329W 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 D329Y 0.22 0.95 0.20 0.76 1.00 0.72 0.39 L334A 0.12 0.33 0.90 0.19 0.70 L334C 0.49 0.67 0.97 0.47 0.99 0.89 0.89 L334E 0.09 0.07 0.05 0.05 0.05 0.10 0.05 0.05 0.14 L334F 0.13 0.31 0.87 0.15 0.93 0.95 0.97 L334G 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 L334H 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 L334I 0.20 0.38 0.98 0.23 0.91 0.99 0.98 0.89 0.44 L334K 0.09 0.06 0.05 0.08 0.07 0.43 0.05 0.05 0.16 L334L 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 L334M 0.44 0.72 0.53 0.93 0.95 0.66 L334N 0.12 0.08 0.05 0.09 0.05 0.18 0.06 0.06 0.10 L334P 0.10 0.13 0.05 0.11 0.17 0.37 0.24 0.31 0.22 L334R 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 L334S 0.11 0.07 0.05 0.10 0.12 0.31 0.14 0.15 0.07 L334T 0.16 0.35 0.93 0.24 0.96 0.70 L334V 0.53 0.70 0.67 0.98 0.88 L334W 0.90 0.92 0.93 0.84 0.86 0.94 0.96 0.86 L334Y 0.14 0.09 0.05 0.09 0.15 0.28 0.15 0.16 0.18 K335A 0.99 0.88 0.19 0.98 K335D 0.47 0.98 0.77 0.49 K335F 0.59 1.00 0.87 0.99 1.00 0.94 0.63 0.83 K335G 0.12 0.75 0.15 0.56 0.62 0.47 0.94 0.37 K335H 0.77 0.98 1.00 0.96 0.83 0.80 K335I 0.41 0.95 0.54 0.87 0.91 0.85 0.89 0.79 K335K 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 K335L 0.95 0.95 0.89 0.80 0.89 K335M 0.68 0.93 0.91 0.99 0.91 0.92 0.89 0.84 K335N 0.20 0.90 0.31 0.79 0.87 0.85 0.59 K335P 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 K335R 0.73 0.98 0.96 0.96 0.87 K335S 0.93 0.86 0.81 0.88 K335T 0.97 0.88 0.94 0.87 0.85 K335V 0.50 0.87 0.90 K335W 0.69 0.82 0.96 N336A 0.48 0.72 0.99 0.64 0.95 0.77 N336C 0.71 0.85 0.97 0.79 0.95 0.94 0.77 N336F 0.14 0.16 0.83 0.15 0.42 0.64 0.42 0.33 0.51 N336G 0.21 0.47 0.94 0.36 0.95 0.79 0.66 N336H 0.46 0.74 1.00 0.59 0.95 0.93 0.77 N336I 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 N336K 0.12 0.17 0.76 0.11 0.40 0.81 0.45 0.34 0.41 N336L 0.24 0.51 0.97 0.31 0.94 0.94 0.79 0.82 N336M 0.30 0.55 0.98 0.45 0.92 0.69 0.76 0.78 N336N 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 N336P 0.13 0.07 0.05 0.05 0.06 0.07 0.05 0.05 0.33 N336Q 0.21 0.43 0.96 0.30 0.89 0.93 0.82 0.77 N336R 0.10 0.30 0.86 0.19 0.97 0.92 0.40 N336S 0.56 0.72 0.59 0.93 0.90 0.86 0.78 0.88 N336T 0.27 0.51 0.37 0.90 0.90 0.82 0.74 N336V 0.12 0.32 0.97 0.17 0.96 0.92 0.92 0.63 N336W 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 N336Y 0.32 0.55 0.42 0.91 0.87 0.81 0.84 D337A 0.83 0.90 0.53 0.91 D337C 0.71 0.87 0.83 0.97 D337E 0.57 0.97 0.66 0.21 0.52 0.93 0.93 0.90 D337F 0.48 0.99 0.98 0.51 0.05 0.46 0.83 0.89 0.94 D337G 0.92 0.95 0.93 0.91 0.97 0.93 0.98 0.81 D337H 0.36 0.96 0.45 0.57 0.37 0.92 0.93 0.98 D337K 0.53 0.95 0.54 0.56 0.81 0.88 D337L 0.35 0.89 0.40 0.30 0.33 0.87 0.94 0.93 D337M 0.37 0.99 0.47 0.61 0.38 0.96 0.92 D337N 0.68 0.95 0.75 0.84 0.66 0.94 0.98 0.97 D337P 0.24 0.67 0.96 0.20 0.53 0.15 0.53 0.65 0.66 D337Q 0.56 0.99 0.99 0.62 0.44 0.66 0.88 0.86 0.94 D337R 0.57 0.96 0.67 0.89 0.61 0.87 0.97 0.97 D337S 0.56 0.99 0.65 0.05 0.59 0.92 0.88 0.81 D337T 0.46 1.00 0.67 0.77 0.45 D337V 0.84 0.93 0.84 0.40 0.95 0.98 D337W 0.42 0.96 0.46 0.42 0.91 0.90 0.90 D337Y 0.54 0.96 0.56 0.57 0.46 0.88 0.88 0.97 A338A 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 A338C 0.92 0.97 A338D 0.93 1.00 0.95 A338E 0.97 0.98 0.95 0.83 0.94 0.96 A338F 0.81 0.85 0.87 0.95 A338G 1.00 0.95 A338H 0.69 0.99 0.65 0.84 0.90 0.97 0.92 A338I 0.89 0.99 0.92 0.98 A338K 0.84 0.96 0.99 0.86 0.81 0.90 0.94 A338L 0.85 0.96 0.91 0.87 1.00 1.00 A338M 0.88 0.91 0.74 0.84 0.94 0.97 1.00 A338N 0.93 0.81 0.91 0.93 0.97 A338P 0.56 0.57 0.91 0.98 A338Q 0.81 0.96 0.73 0.87 0.92 0.95 0.92 A338R 1.00 0.95 0.86 0.93 0.95 0.98 A338S 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 A338T 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 A338V 0.86 0.98 A338W 0.66 A338Y 0.96 0.92 0.79 0.84 0.89 0.92 1.04 N339A 0.31 0.63 0.97 0.32 0.75 0.92 0.73 0.63 0.76 N339C 0.26 0.67 0.98 0.27 0.86 0.65 0.85 0.82 0.90 N339D 0.22 0.59 0.22 0.78 0.71 0.75 0.66 0.66 N339E 0.16 0.72 0.95 0.25 0.91 0.90 0.89 N339F 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 N339G 0.15 0.48 1.00 0.15 0.67 0.67 0.52 0.63 N339H 0.21 0.56 0.99 0.24 0.83 0.70 0.64 0.67 N339I 0.16 0.36 0.15 0.91 0.41 0.49 0.45 0.68 N339K 0.14 0.42 0.05 0.16 0.68 0.66 0.45 0.14 N339L 0.23 0.52 0.23 0.66 0.60 0.59 0.72 N339N 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 N339P 0.20 0.38 0.13 0.53 0.58 0.51 0.41 0.39 N339Q 0.14 0.39 0.14 0.64 0.56 0.56 0.50 0.42 N339R 0.18 0.46 0.16 0.65 0.81 0.64 0.46 0.75 N339S 0.26 0.65 0.89 0.25 0.80 0.73 0.71 0.68 0.63 N339T 0.20 0.48 0.95 0.19 0.71 0.67 0.64 0.56 0.49 N339V 0.19 0.41 0.14 0.57 0.53 0.58 0.43 0.61 N339W 0.29 0.46 0.97 0.18 0.58 0.62 0.48 0.44 0.73 N339Y 0.21 0.59 0.22 0.75 0.68 0.71 0.65 0.58 K344D 0.85 0.88 K344E 0.58 0.94 0.75 0.99 0.98 0.99 0.86 0.98 K344F 0.51 1.00 0.65 K344G 0.60 0.93 0.68 0.95 1.00 0.94 0.87 K344I 0.42 0.93 0.59 0.97 0.95 K344K 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 K344L 0.47 0.92 0.76 0.89 K344M 0.52 0.98 0.78 0.99 0.98 0.80 K344N 0.56 0.93 0.74 0.95 0.95 0.91 0.84 K344P 0.26 0.93 0.94 0.36 0.89 0.78 K344Q 0.37 0.97 0.93 0.51 0.98 0.95 0.92 K344R 0.69 0.94 0.79 0.97 0.99 0.92 0.94 K344S 0.80 0.95 1.00 0.99 0.98 0.98 K344T 0.45 0.98 0.67 0.98 0.95 0.81 K344V 0.48 0.96 0.72 K344W 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 K345A 0.94 0.96 K345D 0.51 0.86 0.49 0.94 0.95 K345E 0.43 0.80 K345F 0.12 0.99 0.89 K345G 0.23 0.91 0.33 0.76 K345H 0.68 0.76 0.99 0.94 0.94 K345I 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 K345K 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 K345L 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 K345M 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 K345N 0.25 0.78 0.99 0.34 0.94 0.92 0.80 0.41 K345P 0.48 0.98 0.62 0.98 1.00 0.91 0.93 K345Q 0.66 0.81 0.98 0.93 1.00 0.73 K345R 0.70 0.96 0.72 0.95 0.78 0.97 0.98 K345S 0.41 0.89 0.58 K345T 0.51 0.91 0.71 0.99 0.97 0.98 K345V 0.37 0.94 0.54 0.91 K345W 0.44 0.93 0.99 0.44 0.97 0.99 0.94 K345Y 0.11 0.58 A347D 1.00 0.96 0.96 0.98 A347F 0.99 0.96 0.97 0.92 0.96 A347H 0.81 0.94 0.99 0.85 0.91 0.92 0.84 0.95 A347I 0.66 0.93 0.74 0.92 0.95 0.90 0.79 A347K 0.68 0.88 0.77 0.91 0.97 0.88 0.81 A347L 0.61 0.91 0.76 0.99 0.93 0.93 0.94 A347M 0.77 0.98 0.94 0.98 0.95 0.87 0.84 A347P 0.72 0.96 0.99 0.99 0.86 0.96 A347Q 0.61 0.88 0.09 0.97 0.93 0.86 0.75 A347R 0.73 0.99 0.98 0.96 0.96 0.92 0.73

A347S 0.65 0.94 0.73 0.94 0.87 0.84 A347T 0.21 0.40 0.91 0.13 0.60 0.83 0.24 0.37 0.50 A347V 0.20 0.05 0.05 0.05 0.32 0.34 0.10 0.16 0.60 A347Y H361A 0.89 0.92 0.05 0.99 0.97 H361C 0.83 0.93 0.05 0.45 0.93 H361D 0.94 0.88 1.00 0.05 0.96 0.61 0.90 0.74 H361E 0.56 0.88 0.05 0.89 0.73 0.77 0.78 H361F 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 H361G 0.92 0.05 H361H 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 H361I 0.15 0.12 0.92 0.05 0.05 0.12 0.13 0.05 0.21 H361K 0.16 0.47 0.05 0.15 0.32 0.89 0.15 0.39 H361L 0.17 0.63 0.05 0.77 0.07 0.73 0.48 H361M 0.40 0.82 0.05 0.95 0.18 0.68 H361N 0.98 0.95 0.97 0.05 0.92 0.99 0.99 0.92 H361P 0.43 0.66 0.05 0.83 0.25 0.97 0.85 0.58 H361R 0.11 0.42 0.97 0.05 0.21 0.41 0.95 0.22 0.36 H361S 0.93 0.84 0.05 0.89 0.95 0.94 0.96 0.82 H361T 0.28 0.76 0.95 0.05 0.72 0.17 0.73 0.40 H361V 0.18 0.11 0.05 0.05 0.05 0.20 0.11 0.05 0.19 H361Y 0.19 0.13 0.05 0.05 0.15 0.19 0.05 0.07 R363A 0.92 0.62 0.99 0.96 0.99 0.99 R363C 0.45 0.16 R363E 0.38 0.90 0.86 0.92 0.05 R363F 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 R363G 0.97 0.05 0.93 0.97 R363K 0.80 R363L 0.81 0.99 0.91 0.98 0.80 R363M 0.98 0.98 0.98 0.96 0.98 R363N 0.22 0.51 0.10 0.60 0.93 0.41 0.72 0.45 R363P 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 R363Q 0.80 0.28 R363R 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 R363S 0.21 0.81 0.06 0.83 0.78 R363T 0.68 0.30 1.00 0.83 R363V 0.77 0.09 0.97 0.91 0.93 R363W 0.14 0.05 R363Y 0.13 0.93 0.05 0.10 N369A 0.11 0.38 0.24 0.62 0.80 0.30 0.72 0.27 N369C 0.30 N369D 0.96 0.32 N369E 0.74 0.86 N369F 0.50 0.98 0.74 0.95 N369I 0.46 0.90 0.85 0.69 0.83 0.76 0.54 N369K 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 N369L 0.63 0.91 0.84 0.97 0.94 0.77 N369M 0.70 0.97 0.85 0.96 0.84 N369N 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 N369R 0.94 0.88 0.76 0.80 0.82 0.69 0.72 N369S 0.47 0.96 0.93 0.97 0.93 0.94 N369T 0.76 0.94 0.96 0.93 N369V 0.91 0.89 0.68 0.91 0.83 N369W 0.26 0.98 N369Y 0.34 0.96 0.91 D370D 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 D370E 0.05 0.41 0.43 0.47 0.50 0.70 D370F 0.72 0.05 0.42 0.17 0.57 0.37 0.66 D370G 0.54 0.87 0.05 0.59 0.31 0.64 0.52 0.80 D370I 0.39 0.81 0.82 0.05 0.05 0.05 0.11 0.07 0.38 D370K 0.59 0.28 0.57 0.05 0.05 0.08 0.05 0.05 0.27 D370L 0.58 0.64 0.84 0.05 0.05 0.09 0.05 0.05 0.46 D370M 0.66 0.77 0.92 0.05 0.14 0.17 0.25 0.11 0.43 D370N 0.99 0.99 0.83 0.05 0.30 0.43 0.40 0.27 0.48 D370P 0.18 0.38 0.98 0.05 0.05 0.18 0.13 0.05 0.55 D370Q 0.78 0.78 0.05 0.23 0.37 0.32 0.21 0.48 D370R 0.36 0.19 0.64 0.05 0.05 0.09 0.05 0.05 0.21 D370S 0.75 0.82 0.05 0.49 0.55 0.56 0.45 0.70 D370T 0.36 0.94 0.83 0.05 0.13 0.06 0.29 0.13 0.66 D370V 0.62 0.99 0.85 0.05 0.12 0.05 0.20 0.12 0.42 D370W 0.75 0.05 0.25 0.07 0.32 0.21 0.52 D370Y 0.75 0.05 0.33 0.18 0.43 0.33 0.57 K371A 0.05 0.94 0.93 0.95 K371C 0.47 0.92 0.97 0.05 0.90 0.94 0.89 0.93 0.98 K371D 0.94 0.95 0.05 0.85 0.73 0.89 0.85 0.86 K371F 0.38 0.05 0.75 0.05 0.82 0.80 0.87 K371G 0.65 0.05 0.67 0.86 0.92 K371H 0.97 0.95 0.30 0.85 0.86 0.85 0.77 1.00 K371K 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 K371L 0.88 0.05 0.90 0.95 0.88 K371N 0.41 0.91 0.05 0.81 0.56 0.84 0.73 0.98 K371P 0.37 0.84 0.72 0.05 0.14 0.07 0.21 0.10 0.36 K371Q 0.49 0.98 0.05 0.78 0.46 0.81 0.76 0.60 K371R 0.58 0.85 0.05 0.89 0.87 0.98 0.88 0.85 K371S 0.72 0.05 0.97 0.48 0.92 0.89 0.98 K371T 0.19 0.05 0.10 K371V 0.27 0.94 0.05 0.74 0.12 0.78 0.79 0.61 K371W 0.25 0.62 0.05 0.36 0.08 0.42 0.15 0.47 K371Y 0.32 0.58 0.98 0.05 0.27 0.06 0.31 0.16 0.32 G372A 0.65 G372C 0.51 0.11 0.98 0.98 0.89 0.77 G372D 0.66 0.97 0.05 0.93 0.94 0.92 0.86 G372E 0.59 0.91 0.05 0.70 0.48 0.86 0.79 0.73 G372F 0.14 0.69 0.61 0.05 0.42 0.05 0.74 0.48 0.38 G372G 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 G372H 0.20 0.88 0.68 0.05 0.55 0.12 0.94 0.73 0.48 G372I 0.10 0.14 0.05 0.06 0.05 0.28 0.05 0.05 0.26 G372K 0.09 0.67 0.96 0.07 0.65 0.73 G372L 0.14 0.75 0.05 0.39 0.08 0.63 0.52 0.43 G372M 0.28 0.85 0.05 0.63 0.41 0.73 G372N 0.35 0.96 0.12 0.61 0.53 0.98 0.71 G372Q 0.39 0.90 0.99 0.06 0.62 0.71 0.98 0.96 0.73 G372R 0.42 0.93 0.95 0.05 0.53 0.54 0.84 0.76 0.66 G372S 0.41 0.81 0.05 0.73 0.95 0.87 0.78 0.71 G372T 0.46 0.82 0.05 0.54 0.54 0.93 0.77 0.74 G372V 0.18 0.74 0.11 0.54 0.12 0.96 0.57 G372W 0.06 0.74 0.84 0.05 0.33 0.57 G372Y 0.17 0.97 0.74 0.05 0.84 0.20 0.54 D374A 0.76 0.05 0.89 0.82 0.82 0.74 0.79 D374C 0.70 0.97 0.15 D374F 0.16 0.95 0.09 0.90 0.78 D374G 0.21 0.93 0.05 0.98 0.35 0.93 D374I 0.08 0.81 0.05 0.98 0.76 0.96 0.62 D374K 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 D374L 0.32 0.90 0.05 0.80 D374M 0.19 0.93 0.05 0.28 D374N 0.59 0.90 0.90 0.86 0.83 0.93 0.84 D374P 0.11 0.05 0.05 0.05 0.05 0.45 0.05 0.05 0.13 D374Q 0.20 0.92 0.05 0.44 0.68 D374R 0.06 0.36 0.05 0.09 0.78 0.21 0.37 0.43 D374S 0.50 0.93 0.05 D374T 0.21 0.88 0.92 0.05 0.76 0.24 0.82 0.65 D374V 0.21 0.93 0.05 0.56 0.78 D374W 0.07 0.39 0.05 0.31 0.59 0.36 0.67 0.44 D374Y 0.26 0.98 0.93 0.22 0.75 0.90 0.99 0.61 D375A 0.78 0.87 0.05 0.98 0.90 0.98 0.88 0.95 D375C 0.66 0.89 0.05 0.99 D375D 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 D375E 0.65 0.98 0.05 0.99 0.89 D375F 0.56 0.85 0.89 0.05 0.91 0.55 0.92 0.90 0.81 D375G 0.54 0.85 0.93 0.05 0.91 0.61 0.93 0.96 0.85 D375H 0.56 0.89 0.88 0.05 0.93 0.86 0.95 0.87 D375I 0.73 0.95 0.05 0.90 0.82 0.93 0.85 D375K 0.47 0.81 0.87 0.05 0.89 0.68 0.91 0.98 0.85 D375L 0.54 0.89 0.93 0.05 0.92 0.74 0.87 0.93 0.76 D375M 0.57 0.89 0.93 0.05 0.89 0.77 0.92 0.97 0.76 D375N 0.65 0.94 0.93 0.05 0.92 0.79 0.92 0.92 0.85 D375P 0.09 0.24 0.86 0.05 0.07 0.26 0.05 0.26 0.35 D375Q 0.31 0.62 0.90 0.05 0.88 0.96 0.86 0.91 0.56 D375R 0.45 0.80 0.85 0.05 0.88 0.76 0.85 0.76 D375S 0.58 0.90 0.91 0.05 0.88 0.92 0.86 0.90 0.69 D375T 0.55 0.91 0.96 0.05 0.96 0.80 0.95 0.97 0.80 D375V 0.60 0.92 0.05 0.97 0.68 0.83 D375W 0.41 0.93 0.91 0.05 0.66 0.96 D375Y 0.62 0.90 0.88 0.05 0.89 0.56 0.87 0.93 0.73 M380A 0.18 0.05 0.05 0.05 0.05 0.07 0.05 0.05 0.09 M380E 0.79 0.98 0.05 0.89 0.84 0.95 0.85 0.84 M380F 0.52 0.97 0.05 0.97 0.77 0.95 0.99 0.94 M380G 0.14 0.47 0.05 0.79 0.22 0.84 0.95 0.62 M380I 0.50 0.94 0.05 1.00 0.69 0.97 1.12 0.98 M380K 0.06 0.05 0.05 0.05 0.15 0.36 0.13 0.40 0.35 M380L 0.56 0.05 1.00 0.77 0.91 0.98 0.83 M380M 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 M380N 0.12 0.75 0.95 0.05 0.13 0.53 M380Q 0.59 0.05 0.92 0.81 0.92 0.99 1.00 M380R 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 M380S 0.60 0.99 0.05 0.97 0.60 0.90 0.94 0.85 M380T 0.59 0.05 1.00 0.93 0.90 M380V 0.59 0.05 0.69 0.93 M380W 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 M380Y 0.12 0.55 0.05 0.79 0.40 0.96 0.42 G381A 0.21 0.05 0.05 0.05 0.05 0.08 0.05 0.05 0.16 G381C 0.18 0.05 0.05 0.05 0.05 0.07 0.05 0.05 0.07 G381D 0.11 0.05 0.05 0.05 0.05 0.07 0.05 0.06 0.16 G381E 0.18 0.05 0.05 0.05 0.05 0.11 0.05 0.05 0.07 G381F 0.17 0.05 0.05 0.05 0.05 0.05 0.05 0.08 0.08 G381G 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 G381H 0.06 0.05 0.05 0.05 0.05 0.19 0.05 0.05 G381I 0.05 0.05 0.05 0.05 0.05 0.24 0.05 0.06 0.13 G381K 0.07 0.05 0.05 0.05 0.05 0.17 0.05 0.05 0.22 G381L 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 G381N 0.13 0.05 0.05 0.06 0.05 0.10 0.05 0.05 0.05 G381P 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 G381Q 0.06 0.05 0.05 0.05 0.05 0.07 0.05 0.05 0.05 G381R 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 G381S 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 G381T 0.05 0.05 0.05 0.05 0.05 0.26 0.05 0.05 0.18 G381V 0.06 0.05 0.05 0.05 0.05 0.42 0.05 0.05 0.32 G381W 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 G381Y 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 W382A 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 W382C 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 W382D 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 W382E 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 W382F 0.25 0.74 0.76 0.06 0.45 0.96 W382G 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 W382H 0.18 0.16 0.05 0.23 0.29 0.26 0.08 0.05 0.31 W382I 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 W382K 0.17 0.22 0.05 0.19 0.24 0.20 0.11 0.09 0.44 W382L 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 W382M 0.18 0.17 0.05 0.25 0.25 0.35 0.14 0.08 0.83 W382N 0.18 0.25 0.17 0.58 0.92 0.37 0.34 0.77 W382P 0.19 0.38 0.05 0.15 0.28 0.30 0.09 0.06 0.72 W382R 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 W382S 0.26 0.19 0.05 0.24 0.24 0.23 0.14 0.09 0.63 W382T 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 W382V 0.28 0.15 0.05 0.25 0.18 0.16 0.09 0.05 0.58 W382Y 0.49 0.91 0.14 1.00 0.26 0.95 0.99 0.87 Y396A 0.92 0.48 0.97 1.00 0.99 1.00 Y396C 0.86 0.98 0.41 0.97 0.92 Y396D 0.78 0.90 0.33 0.99 0.99 0.94 Y396E 0.79 0.91 0.39 0.99 0.86 Y396F 0.67 0.97 0.88 0.97 0.91 Y396G 0.74 0.94 0.12 1.00 0.92 Y396H 0.85 0.92 0.94 0.80 0.96 0.97 0.85 Y396I 0.76 1.02 0.94 0.75 0.92 0.95 0.95 0.84 Y396K 0.62 0.92 0.25 0.91 Y396L 0.67 0.98 0.93 0.66 0.93 0.96 0.75 Y396M 0.51 0.94 0.94 0.56 0.90 0.92 0.64 Y396N 0.56 0.94 0.95 0.48 0.99 0.98 0.82 Y396P 0.15 0.05 0.05 0.05 0.05 0.10 0.05 0.10 0.05 Y396Q 0.60 0.97 0.95 0.22 0.94 1.00 0.76 Y396R 0.61 1.00 0.96 0.06 0.97 0.97 0.67 Y396S 0.86 1.00 0.90 0.38 0.92 0.93 0.95 0.79 Y396T 0.77 1.00 0.10 0.94 0.93 0.92 0.99 0.72 Y396V 0.54 0.93 0.96 0.38 0.97 0.93 0.71 Y396W 0.82 0.93 0.92 0.81 0.96 0.99 0.91 0.80 Y396Y 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 D397A 0.67 0.97 0.94 0.11 0.97 0.99 0.99 D397C 0.61 1.00 0.24 D397D 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 D397E 0.70 0.89 0.44 0.93 D397F 0.18 0.05 0.05 0.05 0.05 0.05 0.05 0.25 D397H 0.50 0.77 0.09 0.93 D397I 0.60 0.74 0.05 0.90 D397K 0.87 0.88 0.05 1.00 D397L 0.56 0.79 0.05 0.98 0.94 0.92 D397M 0.57 0.84 0.07 0.91 D397N 0.77 D397P 0.73 0.72 0.33 0.93 0.87 D397Q 0.64 0.95 0.29 0.98 D397R 0.69 0.90 0.14 D397S 0.61 0.83 0.23 0.90 D397T 0.83 0.94 0.17 D397V 0.61 0.95 0.24 D397Y 0.58 0.91 0.05 A398C 0.87 0.72 0.98 0.74 0.72 A398D 0.59 0.46 0.99 0.99 0.77 A398E 0.65 0.77 0.97 0.74 A398F 0.46 0.36 0.89 0.84 0.82 A398G 0.49 0.28 0.84 0.91 0.80 0.76 A398H 0.63 0.58 1.00 0.99 0.93 0.93 A398I 0.55 0.97 0.63 0.86 0.80 0.82 A398K 0.39 0.97 0.45 0.99 0.90 A398L 0.58 0.61 0.97 0.97 0.90 0.91 A398M 0.39 0.98 0.41 0.72 0.96 0.75 0.62 A398N 0.33 0.42 0.97 0.93 0.84 A398P 0.69 0.66 0.98 0.92 0.94 A398Q 0.48 0.98 0.58 0.94 0.90 A398R 0.36 0.91 0.51 A398S 0.42 0.47 0.90 0.82 0.77

A398T 0.64 0.86 0.97 0.91 0.93 A398V 0.54 0.99 0.76 0.93 0.92 A398W 0.30 0.97 0.43 A398Y 0.44 0.37 0.89 0.94 0.81 I399A 0.41 0.78 0.94 0.66 0.99 1.00 I399C 0.19 0.46 0.85 0.33 0.78 I399D 0.14 0.36 0.86 0.24 0.90 0.68 I399E 0.30 0.62 0.88 0.41 0.97 0.87 I399F 0.60 0.97 0.94 0.68 0.95 0.91 0.96 0.95 I399G 0.11 0.31 0.76 0.18 0.89 0.69 0.55 I399H 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 I399I 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 I399K 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 I399L 0.97 0.98 0.95 0.92 I399M 0.55 0.89 0.89 0.66 0.93 0.90 0.93 0.86 I399N 0.13 0.05 0.05 0.05 0.06 0.15 0.06 0.22 0.26 I399P 0.17 0.05 0.05 0.05 0.05 0.14 0.06 0.18 0.09 I399Q 0.14 0.39 0.80 0.18 0.94 0.49 I399R 0.14 0.05 0.05 0.05 0.05 0.10 0.08 0.07 0.17 I399S 0.22 0.52 0.86 0.33 0.90 0.74 I399T 0.24 0.63 0.83 0.39 1.00 0.74 I399V 0.58 0.90 0.78 0.99 0.94 I399W 0.43 0.73 0.89 0.34 0.93 0.96 0.85 0.74 I399Y 0.22 0.53 0.84 0.26 0.65 R402A 0.62 0.91 R402C 0.57 0.81 0.64 0.95 0.96 0.93 0.78 R402D 0.22 0.33 0.89 0.15 0.74 0.87 0.70 0.79 0.50 R402E 0.31 0.61 0.55 0.89 1.00 0.66 R402F 0.59 0.84 0.96 0.92 1.00 0.90 0.99 0.81 R402G 0.25 0.63 0.41 0.78 R402I 0.54 0.89 0.80 0.98 0.83 0.98 0.98 R402K 0.07 0.15 0.05 0.13 0.32 0.46 0.28 0.62 0.50 R402L 0.41 0.85 0.92 0.99 0.77 R402N 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 R402P 0.16 0.36 0.27 0.82 0.97 0.77 0.96 0.49 R402Q 0.32 0.65 0.49 0.90 0.96 0.46 R402R 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 R402S 0.18 0.76 0.34 0.87 R402T 0.12 0.05 0.05 0.11 0.05 0.05 0.05 0.07 0.26 R402V 0.52 0.80 0.77 0.95 0.83 0.95 0.95 0.89 R402W 0.47 0.94 0.98 0.85 0.98 0.94 R402Y 0.60 0.86 0.80 0.95 1.00 0.94 0.80 Q409C 0.18 0.48 0.14 0.76 0.79 0.68 0.63 0.52 Q409D 0.18 0.63 0.18 0.97 0.98 0.75 0.24 Q409E 0.13 0.43 0.86 0.31 0.92 0.84 0.63 0.59 0.35 Q409G 0.15 0.57 0.19 0.91 0.83 0.71 0.65 Q409H 0.15 0.51 0.16 0.74 0.72 0.75 0.66 0.53 Q409I 0.20 0.43 0.16 0.59 0.51 0.54 0.41 0.22 Q409L 0.20 0.35 0.96 0.15 0.52 0.55 0.33 0.33 0.48 Q409M 0.15 0.39 0.71 0.26 0.69 0.66 0.47 0.48 0.43 Q409Q 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Q409R 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 Q409S 0.18 0.47 0.84 0.20 0.67 0.54 0.66 0.50 0.82 Q409T 0.23 0.58 0.89 0.26 0.81 0.74 0.77 0.70 0.52 Q409V 0.17 0.51 0.12 0.73 0.75 0.74 0.58 0.44 Q409W 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 V410A 0.96 0.97 0.99 0.65 0.97 0.98 1.00 0.96 V410C 0.95 0.97 0.93 0.93 0.95 0.96 V410D 0.14 0.14 0.97 0.05 0.42 0.61 0.29 0.66 0.38 V410F 0.67 0.86 1.00 0.53 0.98 0.92 0.88 V410G 0.22 0.25 0.16 0.60 0.73 0.49 0.82 0.49 V410H 0.22 0.39 0.96 0.18 0.91 0.95 0.72 0.64 V410I 0.63 0.82 0.99 0.79 0.92 0.95 0.87 V410L 0.95 0.97 0.92 0.86 0.91 0.89 V410M 0.68 0.87 0.98 0.89 0.96 0.98 0.90 0.86 0.84 V410N 0.22 0.46 0.91 0.32 0.99 0.87 0.96 0.75 V410P 0.27 0.41 0.98 0.24 0.85 0.97 0.68 0.89 0.61 V410Q 0.17 0.30 0.92 0.11 0.81 0.92 0.62 0.93 0.47 V410R 0.08 0.20 0.92 0.05 1.00 0.78 V410S 0.44 0.69 0.98 0.50 0.98 0.96 V410T 0.83 0.95 0.97 0.88 0.90 0.87 0.89 0.81 V410V 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 V410W 0.35 0.53 0.99 0.28 0.91 0.82 0.64 V410Y 0.51 0.71 0.97 0.33 0.95 0.92 0.89 0.82 T411D 0.76 0.88 0.91 0.96 T411E 0.55 0.92 0.73 T411F 0.76 0.92 1.00 0.94 T411G 0.47 0.84 0.68 0.91 T411H 0.68 0.85 0.87 T411I 0.68 0.80 0.87 0.94 0.97 0.80 T411K 0.55 0.79 0.70 0.90 0.97 0.84 T411L 0.84 0.91 0.90 0.93 0.86 T411M 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.26 T411N 0.57 0.90 0.90 T411P 0.17 0.70 0.75 0.25 0.92 0.74 0.83 0.68 0.47 T411Q 0.74 0.92 0.95 T411R 0.77 0.85 0.86 0.98 0.89 T411S 0.60 0.92 0.86 0.92 T411T 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 T411V 0.82 0.90 0.98 0.99 0.97 T411W 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 T411Y 0.98 0.97 0.98 0.95 0.91 S420A 0.95 0.92 0.96 0.86 0.88 0.90 0.86 S420C 0.41 0.94 0.85 S420D 0.88 0.99 0.94 0.93 0.95 S420F 0.71 1.00 0.92 0.80 0.95 0.85 0.87 0.91 0.96 S420G 0.51 0.91 0.88 0.92 S420H 0.58 0.95 0.94 0.71 0.79 0.93 0.92 0.84 S420I 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 S420K 0.66 0.86 0.74 0.82 0.94 0.97 0.98 S420L 0.56 0.93 0.86 0.69 0.90 0.75 0.82 0.87 0.81 S420M 0.15 0.35 0.90 0.16 0.59 0.70 0.54 0.67 0.58 S420N 0.46 0.95 0.73 0.83 S420Q 0.41 0.84 0.68 0.96 0.97 S420S 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 S420T 0.48 0.93 0.74 0.84 S420V 0.05 0.90 0.97 0.29 0.19 S420W 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 S420Y 0.70 0.86 0.92 0.96 0.96 R426A 0.06 0.84 R426E 0.90 0.95 0.89 0.95 0.99 0.98 0.89 R426F 0.87 0.96 0.98 0.99 0.93 R426G 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 R426I 0.89 0.80 0.76 0.88 0.96 0.90 0.91 R426K 0.90 0.85 0.97 0.92 0.95 0.92 0.80 R426L 0.77 0.99 0.97 0.93 0.93 1.00 0.85 R426M 0.68 0.80 0.61 0.88 0.98 0.92 0.95 0.78 R426N 0.97 0.94 0.95 0.97 0.86 R426P 0.24 0.52 0.92 0.19 0.98 0.99 0.93 0.48 R426Q 0.81 0.86 0.78 0.96 0.99 0.79 R426R 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 R426S 0.84 0.82 0.80 0.90 0.68 0.87 0.72 R426T 0.93 0.94 0.98 0.98 0.99 R426V 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 R426W 0.73 0.95 0.97 0.78 0.96 0.99 0.95 R426Y 0.92 0.90 0.92 0.96 1.00 0.74 G427C 0.38 0.99 0.68 G427D 0.95 0.97 0.88 0.90 0.97 0.98 0.95 G427E 1.00 0.95 0.94 G427F 0.53 0.94 G427G 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 G427H 0.93 0.95 0.98 0.93 0.84 G427I 0.42 0.90 0.93 0.48 1.00 1.00 0.87 0.82 0.78 G427K 0.61 0.93 0.96 0.69 0.88 0.89 0.86 0.82 G427L 0.89 0.98 0.99 0.93 0.94 0.91 G427M 0.59 0.98 0.74 0.97 0.88 0.90 0.93 0.92 G427N 0.57 0.94 0.65 0.89 0.77 0.91 0.81 G427P 0.19 0.65 0.97 0.13 0.97 0.94 0.73 G427Q 0.72 0.98 0.87 0.94 0.90 0.93 0.85 G427R 0.58 0.95 0.98 0.64 0.84 0.89 0.89 0.86 G427S 0.99 0.80 0.83 0.97 G427T 0.60 0.96 0.70 0.96 0.85 0.91 0.76 G427V 0.32 0.94 0.45 0.96 0.76 G427W 0.94 0.89 0.89 0.93 0.88 0.80 0.68 0.82 G427Y 0.62 0.98 0.82 0.94 0.98 K428A 0.83 0.91 K428C 0.95 0.97 0.98 0.99 0.94 0.88 0.91 0.92 K428D 0.31 0.47 0.90 0.30 0.77 0.63 0.75 0.36 K428E 0.62 0.78 0.98 0.67 0.91 0.78 0.85 0.62 K428F 0.42 0.52 0.89 0.31 0.71 0.57 0.62 0.48 K428G 0.50 0.79 0.88 0.60 0.93 0.80 0.85 0.65 K428H 0.81 0.84 0.94 0.82 0.88 0.77 0.80 0.77 K428I 0.43 0.70 0.97 0.55 0.92 0.79 0.85 0.77 K428K 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 K428L 0.63 0.76 0.94 0.60 0.85 0.73 0.77 0.70 K428M 0.57 0.90 0.91 0.67 0.91 0.85 0.88 0.84 K428N 0.27 0.67 0.89 0.40 0.91 0.99 0.82 K428P 0.70 0.78 0.77 0.86 0.79 0.74 0.76 K428Q 0.47 0.74 0.93 0.56 0.87 0.76 0.83 0.83 K428R 0.52 0.86 0.92 0.62 0.92 0.91 0.91 0.85 K428S 0.96 0.90 0.92 0.90 0.77 0.78 0.80 K428T 0.66 0.82 0.93 0.73 0.87 0.76 0.80 0.77 K428V 0.52 0.81 0.96 0.62 0.91 0.84 0.85 0.78 K428W 0.43 0.50 0.97 0.31 0.70 0.58 0.68 0.53 K428Y 0.55 0.78 0.94 0.53 0.87 0.82 0.84 0.75 E441A 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 E441C 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.08 E441D 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 E441E 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 E441F 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E441G 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 E441H 0.17 0.05 0.05 0.05 0.05 0.10 0.05 0.05 0.08 E441I 0.70 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 E441K 0.35 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.07 E441L 0.31 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.08 E441M 0.54 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 E441N 0.87 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.12 E441P 0.28 0.05 0.05 0.05 0.05 0.09 0.05 0.05 0.05 E441Q 0.69 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.08 E441R 0.08 0.05 0.05 0.10 0.05 0.20 0.05 0.05 0.05 E441S 0.98 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 E441T 0.67 0.05 0.05 0.05 0.05 0.07 0.05 0.05 0.15 E441V 0.74 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 E441Y 0.27 0.08 0.05 0.05 0.05 0.05 0.05 0.05 0.05 T445A 0.88 0.91 T445C 0.32 0.60 T445D 0.74 0.96 0.98 0.97 0.95 0.97 T445E 0.61 0.74 0.91 0.94 T445F 0.55 0.78 0.99 T445G 0.61 0.89 T445H 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 T445I 0.57 1.00 1.00 0.68 0.95 0.95 0.98 T445K 0.48 0.85 0.96 0.53 0.86 0.97 0.99 T445L 0.57 0.98 0.97 0.65 0.94 0.85 0.89 0.88 T445M 0.46 0.72 T445N 0.27 0.75 0.93 0.36 0.97 1.00 0.96 T445P 0.43 0.99 0.42 0.60 T445Q 0.42 0.81 0.47 0.88 0.92 0.94 T445R 0.65 0.92 0.92 0.67 0.88 0.91 0.95 0.86 T445S 0.43 0.83 0.52 0.96 0.99 0.97 T445T 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 T445V 0.49 0.70 0.89 T445W 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 T445Y 0.61 0.75 0.78 V446A 0.99 0.94 0.05 0.92 0.90 0.96 0.96 V446C 0.82 0.87 0.89 0.05 0.95 0.96 0.90 V446E 0.26 0.54 0.81 0.05 0.85 0.18 0.88 0.94 0.60 V446F 0.17 0.29 0.87 0.05 0.67 0.33 0.72 0.79 0.70 V446G 0.23 0.39 0.91 0.05 0.84 0.18 0.84 0.78 0.79 V446I 0.70 0.83 0.85 0.05 0.86 0.86 0.89 0.84 0.86 V446K 0.11 0.34 0.72 0.05 0.92 0.36 0.48 V446L 0.23 0.52 0.89 0.05 0.92 0.54 0.79 0.84 0.70 V446M 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 V446N 0.29 0.55 0.90 0.09 0.81 0.32 0.82 0.81 0.71 V446P 0.12 0.17 0.71 0.05 0.39 0.13 0.39 0.50 0.34 V446Q 0.26 0.73 0.86 0.11 0.92 0.82 V446R 0.12 0.43 0.78 0.05 0.54 0.50 V446S 0.35 0.71 0.91 0.05 0.96 0.76 1.00 0.94 0.86 V446V 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 V446W 0.12 0.29 0.88 0.05 0.75 0.23 0.79 0.94 0.36 E447A 0.96 0.05 0.87 0.82 0.83 0.87 0.81 E447C 0.08 0.08 0.05 0.05 0.09 0.05 0.05 0.05 0.14 E447E 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 E447F 0.40 0.60 0.92 0.05 0.92 0.25 0.86 0.96 0.52 E447G 0.07 0.05 0.05 0.05 0.05 0.39 0.05 0.05 0.28 E447I 0.48 0.56 0.96 0.05 0.81 0.20 0.78 0.91 0.69 E447K 0.42 0.62 0.96 0.05 0.87 0.34 0.85 0.66 E447L 0.42 0.66 0.05 0.92 0.56 0.89 0.80 E447M 0.05 0.05 0.05 0.05 0.05 0.11 0.05 0.05 0.05 E447N 0.86 0.95 0.05 0.82 0.70 0.75 0.88 0.74 E447P 0.07 0.11 0.98 0.05 0.13 0.15 0.06 0.05 0.25 E447R 0.56 0.56 0.95 0.06 0.69 0.28 0.59 0.68 0.59 E447S 0.53 0.76 0.93 0.05 0.92 0.60 0.89 0.92 E447T 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.17 E447V 0.35 0.63 0.93 0.05 0.23 0.99 0.85 E447W 0.45 0.48 0.10 0.75 0.20 0.61 0.72 0.59 E447Y 0.25 0.44 0.87 0.05 0.96 0.30 0.90 0.56 G448A 0.97 0.05 0.98 0.90 0.95 G448C 0.82 0.92 0.07 G448D 0.06 0.90 0.92 G448E 0.85 0.05 0.90 0.98 G448F 0.99 0.05 0.90 G448G 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 G448H 0.87 0.05 0.97 1.00 0.96 0.99 G448K 0.85 0.94 0.05 0.93 0.93 0.92 0.90 0.88 G448L 0.69 0.99 0.05 0.97 0.89 0.92 0.88 0.74 G448M 1.00 0.05 0.95 0.98 0.94 0.88 0.85 G448N 0.97 0.21 0.96 0.95 0.98 G448P 0.18 0.87 0.99 0.05 0.61 0.11 0.97 0.76 0.44 G448Q 0.82 0.94 0.05 0.97 0.98 1.00 0.98 0.82 G448R 0.94 0.97 0.05 0.95 0.90 0.92 0.99 0.88

G448S 0.99 0.05 0.93 0.93 0.99 0.91 G448T 0.05 0.98 0.99 0.98 0.89 G448V 0.96 0.85 0.05 0.93 0.63 0.83 0.85 0.81 G448W 0.98 0.92 0.99 0.05 0.88 0.81 0.86 0.91 0.73 G448Y 0.89 0.05 0.94 0.94 0.93 N449A 0.83 0.99 0.05 1.00 N449C 0.66 0.23 N449E 0.73 0.05 0.88 0.95 0.94 0.93 N449F 0.47 0.97 0.05 0.92 0.47 0.97 0.93 0.93 N449G 0.22 0.64 0.05 0.11 0.87 0.69 N449H 0.41 0.88 0.95 0.05 0.98 0.94 0.73 N449K 0.16 0.46 0.05 0.14 0.79 N449L 0.34 0.85 0.05 0.87 0.27 0.81 0.89 0.66 N449M 0.73 0.94 0.05 0.94 0.78 0.82 0.85 0.91 N449N 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 N449P 0.12 0.60 0.05 0.69 0.10 0.54 0.36 N449Q 0.68 0.98 0.98 0.05 0.86 0.93 0.81 0.80 0.79 N449R 0.74 0.87 0.05 0.91 0.76 0.84 0.81 0.85 N449S 0.66 1.00 0.92 0.05 0.93 0.92 0.84 0.87 0.77 N449T 0.76 0.99 0.05 0.94 0.90 0.87 0.86 0.66 N449V 0.79 0.05 0.92 0.81 0.85 0.86 0.75 N449W 0.43 0.92 0.05 0.87 0.26 0.82 0.85 0.72 N449Y 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 D452A 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 D452C 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 D452D 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 D452E 0.14 0.05 0.05 0.05 0.05 0.17 0.05 0.05 0.29 D452F 0.18 0.05 0.05 0.05 0.05 0.05 0.07 0.05 0.39 D452G 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 D452H 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 D452I 0.13 0.05 0.05 0.05 0.07 0.17 0.13 0.05 0.30 D452K 0.14 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.12 D452L 0.14 0.05 0.05 0.05 0.05 0.05 0.07 0.05 0.20 D452M 0.14 0.05 0.05 0.05 0.05 0.05 0.11 0.05 0.26 D452N 0.26 0.20 0.05 0.20 0.13 0.24 0.23 0.29 D452P 0.13 0.05 0.05 0.05 0.05 0.06 0.05 0.05 0.25 D452Q 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 D452R 0.14 0.05 0.05 0.05 0.11 0.10 0.05 0.05 0.16 D452S 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 D452T 0.14 0.08 0.05 0.05 0.05 0.05 0.14 0.05 0.24 D452V 0.15 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.28 D452W 0.13 0.05 0.05 0.05 0.05 0.07 0.17 0.05 0.24 D452Y 0.14 0.05 0.05 0.05 0.05 0.05 0.17 0.05 0.28 R453A 0.19 0.08 0.05 0.05 0.10 0.18 0.05 0.24 R453C 0.20 0.05 0.05 0.05 0.05 0.11 0.08 0.05 0.54 R453D 0.20 0.05 0.05 0.05 0.05 0.05 0.10 0.05 0.14 R453E 0.08 0.12 0.05 0.18 0.25 0.74 0.16 0.38 R453F 0.08 0.05 0.05 0.08 0.05 0.15 0.09 0.05 0.14 R453I 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 R453K 0.09 0.17 1.00 0.07 0.25 0.14 0.70 0.41 0.34 R453L 0.11 0.12 0.05 0.22 0.25 0.52 0.37 0.49 R453M 0.17 0.30 0.05 0.59 0.25 0.70 0.68 0.52 R453N 0.09 0.09 0.05 0.05 0.09 0.09 0.42 0.20 0.23 R453P 0.11 0.05 0.05 0.05 0.05 0.05 0.08 0.05 0.31 R453Q 0.10 0.12 0.05 0.25 0.06 0.62 0.40 0.63 R453S 0.05 0.10 0.05 0.06 0.28 0.73 0.26 0.18 R453T 0.17 0.05 0.05 0.05 0.05 0.05 0.11 0.05 0.09 R453V 0.15 0.05 0.05 0.05 0.05 0.05 0.12 0.05 0.16 R453W 0.19 0.05 0.05 0.05 0.05 0.08 0.05 0.05 0.25 R453Y 0.18 0.05 0.05 0.05 0.05 0.06 0.05 0.05 0.12 N454A 0.98 0.92 0.48 0.94 0.91 0.91 0.85 0.94 N454C 0.80 1.00 0.90 0.47 0.98 0.70 0.94 0.95 0.97 N454D 0.77 0.88 0.96 0.05 0.93 0.90 0.87 0.88 0.83 N454F 0.27 0.95 0.92 0.30 N454G 0.58 0.81 0.87 0.09 0.86 0.74 0.83 0.80 N454K 0.61 0.92 0.84 0.52 0.92 0.82 0.91 0.86 N454L 0.52 0.81 0.86 0.32 0.88 0.79 0.85 0.69 N454M 0.49 0.83 0.78 0.33 0.88 0.80 0.85 0.72 N454N 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 N454R 0.62 0.77 0.88 0.35 0.79 0.79 0.84 0.65 N454S 0.62 0.88 0.86 0.39 0.88 0.81 0.85 0.82 N454T 0.62 0.82 0.86 0.30 0.85 0.78 0.83 0.72 N454V 0.49 0.96 0.86 0.43 0.99 0.93 0.84 N455A 1.00 0.94 0.54 0.93 0.90 0.93 0.96 N455C 0.81 0.27 0.93 N455D 0.43 0.94 N455E 0.65 0.81 0.98 0.21 0.89 0.78 0.81 0.91 N455F 0.70 0.84 0.96 0.40 0.89 0.83 0.83 0.83 N455G 0.73 0.81 0.91 0.26 0.82 0.77 0.82 0.75 N455H 0.59 0.93 0.95 0.56 0.98 0.95 0.99 0.89 N455I 0.49 0.64 0.05 0.84 0.77 0.86 0.66 N455L 0.60 0.78 0.87 0.16 0.78 0.76 0.82 0.59 N455M 0.48 0.78 0.93 0.25 0.86 0.79 0.91 0.78 N455N 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 N455R 0.79 0.79 0.96 0.21 0.77 0.73 0.75 0.78 0.57 N455S 0.54 0.95 0.94 0.39 0.82 N455T 0.41 0.70 0.92 0.22 0.88 0.82 0.89 0.71 N455V 0.37 0.76 0.91 0.11 0.94 0.58 N455W 0.38 0.91 0.45 N455Y 0.52 0.82 0.94 0.38 0.90 0.90 0.98 0.83 H460A 0.21 0.45 0.84 0.05 0.93 0.36 0.76 H460C 0.24 0.56 0.82 0.05 0.98 0.49 0.82 H460D 0.60 0.89 0.90 0.05 0.97 0.56 0.96 H460E 0.33 0.72 0.94 0.05 0.67 0.93 H460F 0.51 0.94 0.81 0.05 0.99 0.87 H460G 0.39 0.84 0.92 0.05 0.26 H460H 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 H460I 0.43 0.83 0.78 0.05 0.94 0.68 0.92 H460K 0.52 0.91 0.88 0.05 0.96 0.90 H460L 0.24 0.64 0.82 0.05 0.98 0.42 0.71 H460M 0.34 0.85 0.77 0.05 0.53 0.96 H460N 0.61 1.00 0.94 0.05 0.96 0.79 0.97 0.95 H460Q 0.41 0.91 0.87 0.05 0.81 H460R 0.27 0.65 0.80 0.05 1.00 0.44 0.97 H460S 0.25 0.65 0.93 0.05 0.48 0.81 H460V 0.11 0.05 0.05 0.05 0.05 0.17 0.05 0.12 0.05 H460W 0.45 0.83 0.86 0.05 0.97 0.61 0.96 H460Y 0.53 0.92 0.89 0.05 0.98 0.95 0.93 Q467A 0.12 0.47 0.10 0.83 0.81 0.73 0.54 Q467C 0.18 0.54 0.14 0.82 0.91 0.77 0.62 0.58 Q467D 0.18 0.50 0.11 0.76 0.77 0.77 0.56 0.30 Q467E 0.13 0.42 0.10 0.71 0.66 0.72 0.57 0.67 Q467H 0.14 0.45 0.09 0.90 0.59 0.69 0.56 0.33 Q467I 0.10 0.39 0.83 0.06 0.85 0.78 0.76 0.49 0.42 Q467K 0.07 0.30 0.05 0.62 0.80 0.64 0.38 0.10 Q467L 0.18 0.41 0.85 0.11 0.61 0.58 0.54 0.44 0.40 Q467N 0.13 0.49 0.23 0.77 0.95 0.78 0.61 0.43 Q467P 0.08 0.29 0.05 0.11 0.86 0.59 0.34 0.63 Q467Q 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Q467S 0.15 0.57 0.17 0.90 0.94 0.80 0.65 Q467T 0.09 0.34 0.05 0.05 0.68 0.52 0.53 0.28 0.81 Q467V 0.13 0.40 0.09 0.81 0.90 0.78 0.51 0.46 Q467W 0.16 0.44 0.07 0.72 0.63 0.73 0.50 0.60 Q467Y 0.15 0.32 0.05 0.53 0.55 0.55 0.40 0.38 N473A 0.74 0.90 0.99 0.81 0.88 0.81 0.92 N473C 0.94 0.92 0.91 0.80 0.92 0.83 0.78 0.83 N473E 0.67 0.98 0.99 0.87 0.91 0.84 0.92 N473F 0.14 0.22 0.06 0.60 0.56 0.60 0.25 N473G 0.61 0.96 0.97 0.94 0.97 0.94 0.83 0.89 N473H 0.83 0.90 0.95 0.83 0.89 0.87 0.77 0.75 N473K 0.33 0.59 0.94 0.52 0.88 0.80 0.77 0.75 N473L 0.56 0.93 0.98 0.84 0.94 0.83 0.84 N473M 0.58 0.86 0.96 0.84 0.93 0.84 0.80 0.69 N473N 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 N473P 0.29 0.42 0.30 0.76 0.63 0.61 0.44 N473Q 0.67 0.77 0.79 0.81 0.78 0.71 0.63 N473R 0.30 0.68 0.54 0.90 0.98 0.76 N473S 0.94 0.91 0.84 0.99 N473T 0.64 0.96 0.92 0.98 0.98 0.90 0.79 N473V 0.42 0.77 0.60 0.94 0.83 0.84 0.67 N473W 0.05 0.19 0.97 0.08 0.98 0.20 S474A 0.89 0.99 1.00 0.89 0.96 S474C 0.76 0.94 0.82 S474D 0.76 0.93 0.91 S474E 0.86 0.96 0.98 0.76 0.95 0.86 0.98 S474F 0.91 0.96 0.94 0.98 S474G 0.98 0.94 0.96 S474I 0.98 0.97 0.96 0.99 S474K 0.94 0.99 S474L 0.56 0.74 0.98 0.97 S474M 0.91 0.95 0.99 S474N S474P 0.87 0.97 1.00 0.96 0.98 0.96 S474Q 0.95 0.96 0.94 0.92 1.00 0.90 0.90 S474R 0.97 0.91 0.99 S474S 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 S474T 0.97 0.97 S474V 0.98 0.99 0.99 S474W 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 S474Y 0.93 0.93 0.91 0.99 0.99 N475I 0.27 0.37 0.87 N475K 0.08 0.05 0.05 0.30 0.43 0.19 N475L 0.33 0.45 0.89 0.90 0.79 N475M 0.42 0.98 0.61 0.98 N475N 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 N475P 0.31 0.44 0.97 0.96 0.79 N475Q 0.59 0.95 0.72 1.00 0.91 0.91 N475R 0.62 0.94 0.70 0.98 0.94 0.84 N475S 0.23 0.99 0.53 N475T 0.21 0.32 0.80 N475V 0.34 0.46 0.98 0.93 0.78 N475W 0.40 0.54 0.99 0.88 N475Y 0.41 0.50 0.76 E489A 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E489D 0.94 0.97 0.99 0.99 0.94 0.94 0.96 E489E 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 E489F 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E489G 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E489H 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E489I 0.13 0.05 0.05 0.05 0.05 0.05 0.05 0.11 0.33 E489K 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E489L 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E489M 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E489N 0.14 0.23 0.13 0.82 0.62 0.56 E489P 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E489Q 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E489R 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E489S 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 E489T 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E489V 0.19 0.05 0.05 0.05 0.05 0.10 0.05 0.14 0.26 E489W 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 E489Y 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 Q490A 0.81 0.99 0.90 0.96 0.92 0.89 0.95 0.91 Q490C 0.61 0.79 0.95 Q490E 0.65 0.97 0.68 0.91 0.98 0.91 0.93 Q490F 0.51 0.92 0.61 0.99 0.91 0.90 0.86 1.00 Q490G 0.54 0.58 0.97 0.91 0.95 0.98 Q490H 0.47 0.90 0.62 0.86 0.96 0.98 Q490K 0.64 0.94 0.79 0.95 0.79 0.83 0.91 0.83 Q490L 0.54 0.96 0.84 0.89 1.00 Q490P 0.50 0.43 0.92 0.67 0.99 0.92 0.89 0.95 Q490Q 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Q490R 0.57 0.99 0.75 0.86 0.91 0.95 0.96 Q490S 0.59 0.95 0.74 0.99 0.89 0.90 0.92 0.95 Q490T 0.74 0.96 0.96 1.00 0.85 0.93 1.00 0.88 Q490V 0.44 0.99 0.67 0.85 Q490W 0.40 0.48 0.99 0.74 Q490Y 0.57 0.97 0.76 0.97 L492A 0.56 0.91 0.57 0.95 L492D 0.46 0.97 0.61 0.89 L492F 0.27 0.74 0.91 0.30 0.80 0.69 L492G 0.21 0.43 0.96 0.18 0.76 0.68 0.63 0.78 0.38 L492H 0.46 0.90 0.45 0.94 0.93 0.87 L492I 0.59 0.97 0.98 0.58 0.97 0.84 L492K 0.13 0.34 0.88 0.09 0.55 0.78 0.55 0.75 0.27 L492L 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 L492M 0.45 0.91 0.98 0.52 0.69 0.97 0.91 0.52 L492N 0.37 0.90 0.56 0.91 0.97 0.55 L492P 0.17 0.43 0.96 0.05 0.42 0.69 0.63 0.80 0.56 L492Q 0.48 0.97 0.93 0.46 0.76 1.00 0.79 L492R 0.17 0.50 0.95 0.17 0.62 0.82 0.80 0.30 L492S 0.12 0.05 0.05 0.05 0.08 0.05 0.05 0.12 0.17 L492T 0.39 0.99 0.95 0.48 0.86 0.54 L492V 0.13 0.05 0.05 0.05 0.05 0.16 0.05 0.13 0.05 L492W 0.37 0.85 0.86 0.35 0.97 0.97 0.54 L492Y 0.53 0.92 0.55 0.96 0.74 Q496A 0.17 0.61 0.15 0.90 0.90 0.92 0.76 0.67 Q496C 0.16 0.40 0.81 0.09 0.61 0.80 0.60 0.49 0.53 Q496D 0.16 0.43 0.96 0.05 0.67 0.71 0.67 0.43 0.51 Q496F 0.09 0.05 0.05 0.05 0.42 0.50 0.27 0.10 0.36 Q496G 0.17 0.53 0.16 0.80 0.60 0.53 Q496K 0.18 0.29 0.05 0.38 0.52 0.40 0.30 0.64 Q496L 0.13 0.05 0.05 0.05 0.35 0.27 0.29 0.10 0.56 Q496N 0.17 0.49 0.14 0.65 0.74 0.66 0.55 0.59 Q496P 0.13 0.36 0.16 0.56 0.60 0.83 0.44 0.16 Q496Q 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Q496S 0.25 0.78 0.99 0.28 1.00 0.93 0.74 Q496T 0.15 0.44 0.10 0.74 0.52 0.69 0.45 0.64 Q496V 0.21 0.34 0.09 0.48 0.50 0.45 0.33 0.48 Q496W 0.07 0.56 0.09 0.47 V497A 0.78 0.98 0.96 0.90 0.84 0.90 V497C 0.74 0.96 0.77 0.98 0.97 0.95 0.95 V497D 0.05 0.05 0.05 0.05 0.07 0.46 0.05 0.05 0.17 V497E 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 V497F 0.05 0.05 0.05 0.05 0.05 0.10 0.05 0.05 0.07 V497G 0.07 0.05 0.05 0.10 0.17 0.28 0.19 0.05 0.46 V497H 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 V497I 0.59 0.89 0.94 0.97 0.89 0.80 0.88 V497K 0.08 0.05 0.05 0.05 0.05 0.11 0.05 0.05 0.13 V497M 0.51 0.99 0.96 0.90 0.97 0.83 0.96 V497N 0.17 0.46 0.35 0.78 0.81 0.78 0.42 0.48

V497Q 0.05 0.05 0.05 0.05 0.05 0.33 0.05 0.05 0.13 V497R 0.06 0.05 0.05 0.05 0.09 0.47 0.19 0.05 0.17 V497S 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 V497T 0.45 0.94 0.90 V497V 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 V497W 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 V497Y 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 K498A 0.87 0.99 0.76 0.98 K498C 0.63 0.91 0.56 0.98 0.97 0.96 K498E 0.45 0.60 0.94 K498F 0.29 0.81 0.38 0.83 K498G 0.37 0.81 0.40 0.87 0.88 0.81 K498H 0.66 0.92 0.68 0.99 0.99 0.97 0.94 K498I 0.43 0.91 0.53 0.95 0.72 K498K 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 K498L 0.69 0.98 0.79 0.89 0.89 0.89 0.88 0.59 K498M 0.44 0.95 0.62 0.94 0.97 K498N 0.59 0.93 0.98 0.68 0.96 0.95 0.95 0.78 K498P 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 K498Q 0.67 0.93 0.81 0.97 0.94 0.94 0.87 K498R 0.60 0.85 0.66 0.99 0.89 0.99 0.96 0.82 K498S 0.54 0.88 0.63 0.97 0.88 0.76 0.81 0.85 K498T 0.48 0.90 0.60 0.97 0.92 0.93 0.98 K498V 0.55 0.99 0.63 0.98 0.92 0.95 0.97 K498W 0.06 0.23 0.05 0.05 0.21 0.09 0.10 0.05 0.20 K498Y 0.44 0.90 0.52 0.90 0.98 0.92 0.77 D521A 0.97 0.89 0.78 D521C 0.15 0.55 0.93 0.25 0.76 D521D 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 D521E 0.85 0.88 0.88 0.92 0.88 0.70 0.94 D521F 0.82 0.87 0.90 0.87 0.81 0.69 0.91 D521G 0.44 0.70 0.99 0.56 0.93 0.92 0.67 0.85 D521H 0.89 0.94 0.97 0.90 0.93 0.85 0.75 0.91 D521I 0.47 0.71 0.97 0.49 0.86 0.86 0.69 0.79 D521K 0.47 0.78 0.98 0.57 0.92 0.94 0.75 0.72 D521L 0.72 0.89 1.00 0.82 0.92 0.89 0.72 0.95 D521M 0.62 0.87 0.90 0.75 0.94 0.90 0.75 0.99 D521P 0.24 0.40 0.26 0.83 0.77 0.61 0.80 D521R 0.43 0.73 0.98 0.50 0.95 0.91 0.73 0.81 D521S 0.92 0.98 0.94 0.97 0.87 0.79 D521T 0.66 0.88 0.79 0.91 0.89 0.83 1.00 D521V 0.56 0.89 0.76 0.95 0.92 0.66 0.90 D521W 0.63 0.95 1.00 0.76 0.76 D521Y 0.22 0.58 0.87 0.05 0.87 0.88 V522A 0.86 1.00 0.73 0.94 V522C 0.90 1.00 0.89 V522F 0.51 0.99 0.51 0.92 0.97 0.77 V522G 0.11 0.05 0.13 0.91 0.54 V522H 0.73 0.90 0.81 0.99 1.00 0.93 0.84 V522I 0.56 0.92 0.57 0.97 0.98 0.92 0.70 V522K 0.36 0.43 0.81 V522L 0.46 0.99 0.57 0.92 V522M 0.60 0.95 0.70 0.83 V522N 0.19 0.05 0.21 0.68 0.86 0.48 V522P 0.18 0.05 0.84 0.14 0.71 0.87 0.59 V522Q 0.37 0.94 0.42 0.76 V522R 0.30 0.94 0.39 0.92 V522S 0.46 0.96 0.55 V522T 0.51 0.98 0.59 0.97 0.98 V522V 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 V522W 0.24 0.98 0.49 V522Y 0.56 0.96 0.56 1.00 K534A 0.69 0.89 0.90 0.49 0.95 0.95 0.92 0.85 0.90 K534C 0.63 0.99 0.92 0.62 K534D 0.19 0.50 0.18 0.87 K534E 0.48 0.97 0.64 K534F 0.68 0.99 0.65 0.97 K534G 0.22 0.46 0.05 0.86 0.87 0.77 K534H 0.55 0.76 0.43 0.90 0.99 0.85 0.90 0.81 K534I 0.66 0.84 0.57 0.93 0.96 0.87 0.89 0.83 K534K 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 K534M 0.53 0.79 0.94 0.41 0.90 0.88 0.86 0.88 0.76 K534N 0.65 0.91 0.56 0.97 0.95 K534P 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 K534Q 0.97 0.83 0.94 0.91 0.89 0.98 0.97 K534R 0.84 0.98 0.98 0.81 0.98 0.98 K534S 0.53 0.78 0.48 0.88 0.93 0.85 0.88 0.84 K534T 0.62 0.78 0.52 0.88 0.95 0.82 0.89 0.80 K534V 0.75 0.70 K534W 0.16 0.05 0.05 0.05 0.05 0.17 0.11 0.05 0.26 R542A 0.98 0.91 0.95 R542C 0.76 0.47 0.93 0.98 R542D 0.95 0.44 0.89 R542E 0.51 0.90 0.37 0.90 0.92 R542F 0.55 0.38 0.92 0.95 R542G 0.83 0.99 0.47 0.93 0.97 R542H 0.60 0.92 0.49 0.89 0.94 0.86 R542I 0.54 0.05 0.91 0.98 R542K 0.81 0.60 0.81 0.96 0.99 R542L 0.54 0.86 0.41 0.93 R542M 0.55 0.89 0.42 0.96 0.97 R542N 0.90 0.88 0.70 0.90 R542P 0.54 0.18 0.89 0.69 0.91 0.64 R542Q 0.62 0.39 0.87 0.87 R542R 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 R542S 0.68 0.55 0.98 0.85 R542T 0.75 0.99 0.53 0.97 R542V 0.74 0.82 0.57 0.90 0.97 R542W 0.60 0.45 0.99 R542Y 0.62 0.44 0.94 0.96 0.90 G547A 0.38 G547C 0.56 0.95 0.88 0.96 0.93 0.88 G547E 0.96 0.85 0.97 0.71 0.57 0.74 G547F 0.54 0.76 0.83 0.95 0.74 0.80 0.79 0.75 G547G 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 G547I 0.63 0.80 0.99 0.86 0.90 0.76 0.78 0.82 0.79 G547K 0.81 0.87 0.90 0.87 0.80 0.87 0.77 G547L 0.93 0.79 0.86 0.91 G547N 0.37 0.59 0.61 0.95 0.76 0.73 0.82 0.80 G547P 0.97 0.89 0.91 0.90 0.92 G547Q 0.45 0.67 0.74 0.95 0.80 0.76 0.80 0.73 G547R 0.77 0.96 0.96 0.91 0.86 0.94 0.84 G547T 0.63 0.82 0.96 0.96 0.81 0.83 0.86 0.83 G547V 0.75 0.93 0.97 0.91 0.82 0.83 0.91 G547W 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 G547Y 0.61 0.80 0.87 0.96 0.78 0.82 0.86 0.92 S548A 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 S548C 0.87 0.94 0.92 0.96 S548E 0.89 0.91 S548F 0.93 0.92 S548H 0.99 0.97 0.86 0.90 0.84 0.79 S548I 0.91 0.98 0.82 1.00 0.88 0.97 S548K 0.50 0.98 0.97 0.60 0.88 0.78 0.84 0.84 0.69 S548L 0.91 0.95 S548M 0.74 0.97 0.94 0.96 0.88 0.88 S548N 0.50 0.98 0.83 0.96 0.92 0.96 0.79 S548Q 0.46 0.73 0.96 0.97 0.73 S548R 0.53 0.93 0.73 0.84 0.89 0.86 0.86 0.61 S548S 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 S548T 0.56 0.97 0.94 0.89 S548V 0.65 0.91 0.92 0.98 0.90 0.74 S548W 0.77 0.94 0.96 S548Y 0.55 0.95 0.81 0.98 0.89 0.94 0.96 0.72 E553A 0.42 0.50 0.87 0.13 0.70 0.71 0.75 0.85 0.67 E553C 0.33 0.35 0.95 0.12 0.54 0.59 0.56 0.68 0.27 E553D 0.38 0.30 0.14 0.59 0.56 0.56 0.51 0.47 E553E 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 E553H 0.41 0.33 0.95 0.05 0.53 0.49 0.42 0.47 0.44 E553I 0.11 0.22 0.08 0.75 0.83 0.99 0.94 E553K 0.25 0.22 0.13 0.40 0.42 0.46 0.61 0.39 E553M 0.24 0.26 0.94 0.10 0.47 0.48 0.52 0.61 0.55 E553N 0.28 0.26 0.88 0.25 0.46 0.66 0.48 0.63 E553Q 0.19 0.14 0.09 0.35 0.47 0.28 0.44 0.90 E553R 0.25 0.20 0.87 0.07 0.33 0.36 0.40 0.51 0.22 E553V 0.19 0.16 0.93 0.08 0.36 0.38 0.29 0.45 0.79 E553W 0.36 0.28 0.12 0.47 0.49 0.44 0.41 0.56 E553Y 0.10 0.23 0.05 0.88 0.51 0.75 0.65 G554A 0.87 1.00 0.69 0.98 1.00 0.98 G554C 0.62 0.95 0.59 G554D 0.99 0.75 0.96 G554F 0.43 0.95 0.44 G554G 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 G554H 0.59 0.94 0.60 0.94 G554K 0.80 0.84 0.74 0.82 0.95 0.86 0.83 0.75 G554L 0.74 0.56 0.97 0.97 0.89 G554M 0.62 0.74 0.97 0.96 0.77 G554P 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 G554Q 0.86 0.87 G554R 0.84 0.85 0.86 0.83 0.97 0.82 0.77 0.89 G554S 0.31 0.71 0.40 0.92 0.96 0.94 0.76 0.61 G554T 0.62 0.95 0.71 0.93 1.00 0.98 0.90 0.78 G554V 0.55 0.88 0.95 0.47 0.94 0.94 1.00 0.66 G554W 0.47 0.54 L555A 0.69 0.97 0.63 0.51 0.97 0.95 0.92 0.82 L555C 0.88 0.77 0.80 0.98 0.90 L555D 0.69 0.86 0.77 0.59 0.98 0.77 L555E 0.36 0.71 0.60 0.44 0.75 L555F 0.49 0.84 0.60 0.54 0.98 0.96 0.78 L555G 0.50 0.95 0.64 0.53 0.87 L555H 0.45 0.86 0.64 0.42 0.97 L555I 0.69 0.66 0.73 0.95 0.93 0.96 0.82 L555K 0.47 0.89 0.68 0.45 0.89 L555L 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 L555M 0.60 0.97 0.66 0.66 1.00 0.93 0.87 L555N 0.63 0.94 0.68 0.63 0.89 0.96 0.76 L555P 0.43 0.92 0.59 0.53 0.84 L555Q 0.61 0.90 0.64 0.53 0.97 0.77 L555R 0.16 0.05 0.05 0.05 0.05 0.21 0.05 0.05 0.05 L555S 0.58 0.91 0.66 0.51 0.93 0.85 0.99 0.93 0.75 L555T 0.62 0.97 0.67 0.65 0.99 0.99 0.97 0.77 L555V 0.90 0.76 0.82 0.99 1.00 0.91 L555W 0.39 0.73 0.59 0.25 0.89 0.94 0.87 L555Y 0.58 0.91 0.63 0.43 0.97 0.98 0.78 K560A 0.63 0.80 0.44 0.96 0.84 0.81 1.00 K560C 0.59 0.73 0.32 0.93 0.93 0.78 0.83 0.93 K560D 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 K560E 0.43 0.69 0.05 0.82 0.80 0.74 0.97 K560G 0.40 0.74 0.29 0.94 0.81 0.74 0.95 K560H 0.90 0.42 0.98 0.87 0.83 K560I 0.19 0.33 0.11 0.76 0.60 0.55 0.42 0.49 K560K 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 K560L 0.63 0.83 0.49 1.00 0.88 0.82 0.82 0.96 K560M 0.67 0.84 0.52 0.97 0.87 0.83 0.86 0.94 K560N 0.25 0.51 0.15 0.90 0.72 0.66 0.59 0.50 K560P 0.12 0.55 0.10 0.94 0.89 K560Q 0.65 0.80 0.09 0.97 0.79 0.82 0.82 0.79 K560R 0.72 0.98 0.90 0.92 0.94 0.78 K560S 0.37 0.57 0.27 0.98 0.92 0.76 0.69 0.96 K560T 0.39 0.66 0.26 0.94 0.74 0.73 0.68 0.74 K560V 0.19 0.39 0.10 0.83 0.68 0.59 0.36 0.58 K560W 0.64 0.90 0.99 0.07 0.91 0.76 0.94 K560Y 0.50 0.78 0.45 0.98 0.87 0.85 0.90 0.66 H561A 0.68 0.05 0.05 0.37 0.94 0.91 1.00 H561C 0.63 0.80 0.75 0.97 0.96 0.91 H561D 0.54 0.50 0.53 0.93 0.90 0.85 H561E 0.36 0.05 0.05 0.47 1.00 0.88 0.92 0.88 H561F 0.43 0.05 0.05 0.54 0.89 0.84 0.85 H561G 0.57 0.94 0.87 0.99 H561H 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 H561I 0.51 0.56 1.00 0.53 0.97 H561M 0.61 0.82 H561N 0.36 0.94 H561P 0.30 0.05 0.05 0.48 0.22 0.20 0.12 0.05 0.25 H561Q 0.55 0.49 0.93 0.61 0.92 0.87 H561R 0.89 0.42 0.05 0.27 0.84 0.75 0.88 0.74 0.91 H561S 0.40 0.41 0.97 0.49 H561T 0.65 0.34 0.56 0.90 0.95 0.90 0.79 H561V 0.52 0.27 0.99 0.35 0.87 H561W 0.64 0.48 0.61 D563A 0.89 D563C 0.28 0.66 0.94 0.16 0.87 0.89 0.67 0.68 D563D 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 D563E 0.82 0.98 0.97 0.98 0.91 0.98 0.89 D563F 0.39 0.81 0.95 0.47 0.92 0.96 0.93 0.89 D563I 0.39 0.79 0.42 0.99 0.89 0.84 D563L 0.56 0.98 0.98 0.65 0.98 0.90 D563M 0.79 0.93 0.99 0.94 0.94 0.93 0.96 D563Q 0.53 0.99 0.96 0.90 D563R 0.24 0.63 0.38 0.99 0.83 0.74 D563S 0.45 0.97 0.78 D563T 0.45 0.94 0.96 0.73 0.85 D563V 0.43 0.79 0.54 0.95 1.00 0.88 D563W 0.42 0.82 0.94 0.30 0.95 0.80 D563Y 0.47 0.87 0.51 0.96 D564A 0.62 0.70 D564C 0.77 0.67 0.78 D564D 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 D564E 0.49 0.74 0.50 0.95 0.95 0.92 0.85 0.76 D564F 0.65 0.74 D564G 0.52 0.89 0.56 0.99 0.99 0.79 D564I 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 D564K 0.49 0.85 0.97 0.48 0.73 D564L 0.56 0.94 0.69 0.89 D564M 0.60 0.90 0.66 0.96 0.97 0.83 D564N 0.64 0.98 0.74 0.91 0.86 D564P 0.16 0.47 0.91 0.13 0.98 0.78 0.71 0.79 0.36 D564Q 0.56 0.90 0.93 0.55 0.97 0.98 0.77 D564R 0.63 0.94 0.97 0.65 0.97 0.90 D564S 0.67 0.99 0.98 0.76 0.99 0.97 0.76 0.98 D564T 0.59 0.98 0.78 D564V 0.40 0.96 0.72 0.88

D564Y 0.58 0.99 0.63 0.91 0.92 R570A 0.58 0.92 0.71 0.96 R570C 0.31 0.78 0.93 0.29 0.96 0.87 R570D 0.20 0.61 1.00 0.17 0.62 R570E 0.48 0.86 0.57 0.98 0.77 R570F 0.12 0.22 0.95 0.09 0.65 0.56 0.46 0.56 0.45 R570G 0.18 0.45 0.17 0.81 0.94 0.65 R570H 0.19 0.47 0.95 0.17 0.82 0.73 0.92 0.58 R570I 0.22 0.69 0.96 0.28 0.97 0.68 R570M 0.27 0.67 0.34 0.99 0.99 0.51 R570N 0.11 0.29 0.07 0.91 0.76 0.65 0.93 0.31 R570P 0.15 0.05 0.05 0.05 0.10 0.20 0.05 0.14 0.05 R570Q 0.52 0.91 0.67 0.97 0.57 R570R 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 R570S 0.22 0.63 0.96 0.23 0.93 0.42 R570T 0.36 0.85 0.44 0.90 0.68 R570V 0.23 0.62 0.27 0.59 R570W 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 R570Y 0.18 0.40 0.88 0.14 0.82 0.90 0.68 0.80 0.42 Y571A 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Y571D 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Y571E 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Y571H 0.14 0.05 0.28 0.88 0.98 Y571K 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Y571L 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Y571M 0.15 0.05 0.29 0.78 0.91 Y571N 0.05 0.05 0.05 0.17 0.21 0.66 0.75 Y571P 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Y571Q 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 Y571R 0.05 0.05 0.05 0.12 0.05 0.76 0.05 0.87 Y571S 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Y571T 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 Y571V 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Y571W 0.65 0.59 0.94 0.98 0.97 0.97 0.98 Y571Y 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 K581A 0.69 0.96 0.80 0.85 K581C 0.39 0.05 0.51 0.97 K581D 0.53 0.64 0.94 K581E 0.49 0.79 0.61 0.71 0.99 0.88 K581F 0.47 0.94 0.64 0.78 K581G 0.51 0.70 0.88 K581H 0.54 0.97 0.77 0.91 0.88 0.93 K581I 0.55 0.98 0.68 0.97 0.91 0.90 K581K 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 K581L 0.66 0.85 0.93 0.96 0.98 0.99 K581M 0.63 0.99 0.73 0.92 0.90 1.00 K581N 0.55 0.69 0.89 0.93 0.89 0.81 K581P 0.40 0.05 0.91 0.44 0.97 0.86 0.91 0.79 K581R 0.57 0.92 0.73 0.96 1.00 0.99 0.90 K581S 0.67 1.77 1.00 0.80 0.89 0.92 K581T 0.60 0.64 1.00 0.79 0.91 0.99 K581V 0.69 0.98 0.80 0.98 0.88 0.92 K581W 0.62 0.80 0.90 K581Y 0.81 0.96 0.90 0.95 0.96 0.97 N583A 0.82 0.92 0.95 N583C 0.57 0.88 0.97 N583D N583E 0.60 0.86 0.91 0.66 0.99 0.94 0.92 N583F 0.62 0.85 0.92 0.65 0.95 0.91 0.90 N583G 0.66 0.92 0.91 0.96 N583H 0.56 0.92 0.83 0.75 0.96 0.89 N583I 0.48 0.83 0.89 0.67 0.99 0.93 0.87 N583K 0.60 0.88 0.89 0.67 0.93 0.93 0.84 N583L 0.53 0.81 0.92 0.75 0.97 0.94 0.63 N583M 0.51 0.82 0.84 0.71 0.91 0.91 0.68 N583N 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 N583P 0.21 0.43 0.77 0.29 0.79 0.74 0.32 N583R 0.85 0.97 0.96 0.92 0.90 N583S 0.53 0.80 0.90 0.71 0.94 0.93 0.67 N583T 0.53 0.81 0.85 0.71 0.94 0.90 0.67 N583V 0.75 0.88 0.98 0.99 0.80 0.84 N583W 0.70 0.92 0.89 0.82 0.89 0.91 0.78 N583Y 0.66 0.88 0.83 0.57 0.92 0.91 0.81 R586C 0.13 0.05 0.05 0.05 0.05 0.18 0.05 0.05 0.26 R586D 0.98 1.00 0.94 R586E 0.73 0.90 0.72 0.94 0.97 0.94 0.94 R586F 0.64 0.98 0.67 0.99 0.96 R586G 0.38 0.84 0.93 0.48 0.92 0.90 0.87 R586H 0.08 0.14 0.05 0.05 0.36 0.16 0.38 0.34 R586I 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 R586K 0.61 0.94 0.97 0.60 0.93 0.99 0.90 0.98 0.87 R586L 0.45 0.96 0.56 0.98 0.96 0.90 R586N 0.66 0.98 0.82 R586P 0.18 0.90 0.37 R586Q 0.10 0.05 0.05 0.05 0.05 0.29 0.05 0.05 0.09 R586R 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 R586S 0.10 0.05 0.05 0.05 0.05 0.16 0.05 0.05 0.06 R586V 0.53 0.96 0.99 R586W 0.49 0.89 0.55 0.96 R586Y 0.51 0.98 0.94 0.52 0.99 S591C 0.29 0.26 0.16 0.44 0.66 0.45 0.61 0.32 S591D 0.25 0.42 0.22 0.82 0.87 0.48 S591F 0.23 0.18 0.09 0.33 0.53 0.38 0.47 0.25 S591G 0.20 0.19 0.14 0.44 0.80 0.49 0.63 0.44 S591H 0.23 0.27 0.36 0.61 0.79 0.56 0.72 0.27 S591I 0.27 0.20 0.20 0.39 0.55 0.37 0.47 0.14 S591K 0.17 0.14 0.17 0.36 0.55 0.33 0.41 0.26 S591M 0.23 0.15 0.18 0.32 0.33 0.32 0.45 0.30 S591N 0.26 0.28 0.97 0.35 0.53 0.81 0.51 0.52 0.31 S591P 0.13 0.32 0.36 0.90 0.11 0.64 0.67 0.38 S591Q 0.22 0.22 0.29 0.44 0.77 0.52 0.56 0.43 S591R 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 S591S 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 S591V 0.29 0.29 0.39 0.50 0.73 0.53 0.65 0.36 V603A 0.93 0.99 0.89 0.97 0.98 V603C 0.95 0.94 0.75 0.97 1.00 V603D 0.54 0.96 0.82 0.76 0.82 V603E 0.94 0.96 0.94 0.96 0.75 V603F 0.71 0.91 0.81 0.77 1.00 0.84 V603G 0.40 0.94 0.72 V603H 0.59 0.91 V603L 0.59 0.97 0.93 0.64 0.83 0.60 0.42 0.59 V603M 0.72 0.92 0.71 0.87 0.69 0.59 V603N 0.82 0.95 0.94 0.88 0.87 0.87 V603P 0.21 0.82 0.90 0.28 0.54 0.83 0.65 0.56 V603Q 0.90 0.88 0.84 0.83 0.73 V603R 0.62 0.85 0.86 0.86 0.77 0.70 0.59 V603S 0.91 0.87 0.86 0.86 0.67 V603T 0.48 0.95 0.57 0.70 0.91 0.69 0.97 V603V 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 V603W 0.16 0.05 0.13 0.16 0.26 0.25 0.72 V603Y 0.33 0.96 0.87 F611A 0.67 0.93 F611C 0.38 0.65 0.94 0.41 0.86 F611D 0.21 0.33 0.92 0.19 0.79 0.93 0.65 F611G 0.67 0.74 0.58 0.84 0.96 0.92 0.83 0.77 F611I 0.16 0.29 0.17 0.77 0.98 0.95 0.99 0.52 F611K 0.32 0.57 0.95 0.42 0.91 0.77 F611L 0.70 0.92 0.77 0.89 0.97 0.90 0.82 F611M 0.58 0.82 0.99 0.70 0.90 0.97 0.83 F611N 0.65 0.80 0.66 0.86 0.93 0.86 0.81 F611P 0.54 0.70 0.98 0.53 0.86 0.86 0.91 0.95 0.66 F611Q 0.45 0.61 0.93 0.46 0.82 0.79 0.92 0.95 0.74 F611R 0.23 0.41 0.30 0.91 0.75 F611S 0.60 0.76 0.63 0.86 0.92 0.97 0.89 F611T 0.52 0.66 0.50 0.80 0.87 0.92 0.99 0.69 F611V 0.53 0.69 0.51 0.81 0.92 0.96 0.87 F611W 0.61 0.78 0.96 0.58 0.94 0.86 F611Y 0.70 0.80 0.70 0.96 0.93 0.96 0.94 Q612C 0.79 0.84 Q612D 0.94 1.00 0.77 0.97 Q612F 0.66 0.92 0.95 0.70 0.93 0.91 1.00 0.72 Q612G 0.84 Q612H 0.42 0.77 0.50 0.97 2.04 0.94 0.84 Q612I 0.75 0.76 0.67 0.81 0.80 0.83 0.68 Q612K 0.96 0.95 0.73 0.87 0.87 0.92 0.80 Q612L 0.53 0.78 0.95 0.56 0.82 0.80 0.84 0.76 Q612M 0.56 0.81 0.94 0.57 0.87 0.81 0.90 0.65 Q612P 0.42 0.64 0.97 0.45 0.83 0.94 0.83 0.92 0.66 Q612Q 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Q612R 0.98 0.91 0.99 0.84 0.85 0.88 0.90 0.80 Q612S 0.47 0.92 0.39 0.89 Q612T 0.66 0.74 0.95 0.28 0.79 0.80 0.73 0.79 0.60 Q612V 0.96 0.99 0.97 0.92 0.94 0.90 0.94 0.91 Q612W 0.72 0.78 0.66 0.81 0.83 0.89 0.76 Q612Y 0.88 0.75 0.99 0.66 0.81 0.93 0.75 0.76 0.72 A622A 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 A622D 0.88 0.99 0.90 0.85 0.98 A622E 0.87 0.91 0.95 0.66 A622F 0.68 0.95 0.93 0.80 0.79 0.84 0.54 A622G 0.56 0.92 0.95 0.95 0.99 A622H 0.71 0.91 0.98 A622I 0.83 0.95 0.91 0.78 0.88 0.82 0.91 A622K 0.69 0.95 0.93 0.78 0.82 0.98 A622L 0.91 0.97 0.90 0.91 A622M 0.48 0.90 0.68 0.60 0.83 0.67 A622N 0.90 0.98 0.95 0.84 0.84 0.79 0.63 A622P 0.11 0.05 0.05 0.05 0.05 0.19 0.10 0.74 A622R 0.99 0.95 0.92 0.95 0.93 0.62 A622S 0.85 0.99 0.90 0.98 0.98 A622T 0.52 0.97 0.57 0.73 0.84 0.82 A622V 0.47 1.00 0.59 0.78 0.90 0.82 A622W 0.93 0.96 0.84 0.86 0.83 0.38 0.87 A622Y 0.94 0.90 0.78 0.89 0.89 0.83 Q626D 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 Q626E 0.82 0.99 0.89 0.73 Q626F 0.53 0.96 0.87 0.94 0.93 0.69 Q626G 0.47 0.88 0.51 0.70 0.80 0.77 Q626H 0.09 0.91 0.93 0.24 Q626I 0.05 0.05 0.05 0.05 0.12 0.05 0.05 0.07 Q626K 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Q626L 0.95 0.96 0.81 0.88 0.75 0.85 Q626M 0.31 0.87 0.36 0.66 0.58 0.81 Q626P 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 Q626Q 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Q626R 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 Q626S 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 Q626T 0.96 0.89 0.92 0.88 0.88 Q626V 0.95 0.92 0.96 0.80 0.89 0.79 0.80 Q626W 0.40 0.66 0.88 0.54 0.37 0.60 0.43 0.93 0.47 Q626Y 0.12 0.05 0.05 0.05 0.09 0.79 0.08 0.41 0.64 V627D 0.29 0.17 0.06 0.36 0.47 0.32 0.44 0.35 V627K 0.26 0.15 0.30 0.28 0.65 0.31 0.37 0.12 V627P 0.11 0.20 0.13 0.76 0.88 0.86 0.57 V627Q 0.28 0.21 0.21 0.37 0.67 0.40 0.30 0.78 V627R 0.26 0.21 0.24 0.39 0.59 0.43 0.36 0.52 V627S 0.31 0.25 0.31 0.38 0.53 0.42 0.52 0.72 V627V 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 V627Y 0.27 0.21 0.07 0.42 0.55 0.42 0.54 0.18 T638A 0.91 0.94 T638D 0.85 T638E 0.73 1.00 0.74 0.90 0.90 0.93 0.93 T638F 0.43 0.99 0.90 0.40 0.82 0.89 0.92 0.80 T638G 0.84 1.00 0.85 T638I 0.87 0.96 0.78 0.90 0.98 0.92 0.98 T638K 0.69 0.98 0.60 0.95 0.95 0.97 0.98 T638L 0.77 0.91 0.77 0.88 0.85 0.96 0.86 T638M 0.66 0.96 0.77 0.93 0.92 T638P 0.16 0.62 0.97 0.13 0.59 0.68 0.84 0.52 T638Q 0.71 0.74 0.93 0.95 T638R 0.89 0.79 0.99 0.93 T638S 0.87 0.98 0.94 0.98 0.95 T638T 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 T638V 0.76 0.96 0.73 0.95 0.91 T638W 0.06 0.98 0.38 T638Y 0.55 0.97 0.55 0.94 0.97 S642A 0.91 0.92 0.98 0.71 0.93 0.87 0.92 0.77 S642C 0.85 0.98 0.69 0.97 0.71 S642D 0.73 0.80 0.98 0.79 0.96 0.93 0.92 S642E 0.59 0.77 0.90 S642F 0.30 0.47 S642G 1.00 0.96 0.86 0.94 0.71 S642H 0.49 0.95 0.63 0.88 S642I 0.54 0.52 0.99 0.95 S642K 0.99 0.86 0.99 0.71 0.94 0.89 0.82 0.79 S642L 0.43 0.57 S642M 0.48 0.53 0.91 1.00 0.70 S642N 0.89 0.82 1.00 0.95 0.93 0.73 S642P 0.55 0.94 0.67 1.00 0.75 S642Q 0.74 0.99 0.86 0.91 S642R 0.97 0.99 0.85 0.98 0.98 0.75 S642S 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 S642T 0.67 0.98 0.67 0.70 S642V 0.76 0.98 0.67 1.00 0.98 0.82 S642W 0.35 0.92 0.46 S642Y 0.27 0.97 0.45 0.98 A643A 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 A643C 0.77 0.82 0.97 0.60 0.99 1.00 0.91 0.90 A643D 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 A643E 0.96 0.98 0.96 0.96 0.99 0.91 0.94 A643F 0.36 0.51 0.17 0.82 0.94 0.73 A643G 0.52 0.60 0.43 0.90 0.95 0.87 0.85 A643H 0.55 0.75 0.68 0.96 0.78 A643K 0.25 0.48 0.28 0.89 A643L 0.83 0.99 0.98 0.98 0.99 A643M 0.96 1.00 0.93 A643N 0.66 0.76 0.62 0.92 0.99 0.97 0.90 A643Q 0.48 0.58 0.44 0.92 0.99 0.80 A643R 0.64 0.77 0.91 0.62 0.93 0.93 0.96 0.77 A643S 0.87 0.88 0.88 0.92 0.99 0.97 0.87 A643T 0.92 0.96 0.95 0.93 0.90

A643V 0.89 0.92 0.87 A643W 0.43 0.54 0.44 0.88 0.96 0.89 0.89 A643Y 0.54 0.70 0.45 0.97 0.89 R645A 0.57 0.87 0.79 1.27 0.98 0.81 R645C 0.18 0.32 0.96 0.27 0.88 0.86 0.73 0.80 0.33 R645D 0.80 0.89 0.95 0.93 0.82 R645E 0.37 0.49 0.60 0.93 0.82 0.76 0.85 0.57 R645F 0.52 0.76 0.65 0.98 0.94 0.77 R645G 0.89 0.98 R645H 0.52 0.68 0.72 0.95 0.82 0.97 0.85 R645I 0.44 0.65 0.60 0.89 0.73 R645K 0.85 1.00 0.98 R645L 0.45 0.71 0.70 0.97 0.74 R645M 0.46 0.82 0.89 0.97 R645P 0.95 0.78 0.93 0.88 0.77 0.82 0.77 R645Q 0.65 0.63 0.62 0.81 0.68 0.76 0.59 R645R 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 R645S 0.69 0.73 0.89 0.91 0.98 0.86 0.91 0.80 R645T 0.56 0.79 0.71 0.98 0.90 0.79 R645V 0.42 0.63 0.53 1.00 0.93 0.71 R645W 0.30 0.66 0.56 0.95 R645Y 0.57 0.89 0.80 0.98 K649A 0.71 0.79 0.88 0.89 K649C 0.69 0.96 0.80 0.97 K649E 0.40 0.78 0.90 0.42 0.89 1.00 0.86 0.80 0.96 K649F 0.77 0.97 0.77 0.97 0.95 0.90 1.00 K649I 0.67 0.96 0.88 0.97 0.97 0.92 0.90 0.82 K649K 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 K649L 0.86 0.98 0.99 0.98 0.90 0.92 K649M 0.94 0.93 1.33 0.92 0.95 0.91 0.94 0.88 K649N 0.09 0.37 0.86 0.14 0.82 0.58 K649P 0.41 0.20 0.77 0.07 0.23 0.32 0.18 0.14 0.30 K649Q 0.82 0.93 0.91 0.91 0.83 K649S 0.89 0.98 0.85 0.97 0.96 K649T 0.48 0.97 0.98 0.73 0.99 0.99 K649W 0.94 1.00 0.95 0.84 0.92 K649Y 0.80 0.96 1.00 0.99 0.99 0.99 0.84 Q650A 0.82 0.99 0.90 0.81 0.98 0.97 0.92 1.00 Q650C 0.97 0.97 0.99 0.73 0.98 0.99 Q650D 0.94 0.68 0.95 0.93 Q650E 0.52 0.80 0.92 0.59 0.95 0.92 Q650F 0.26 0.50 0.90 0.28 0.80 0.99 0.73 Q650G 0.80 0.96 0.94 0.71 0.97 Q650H 0.61 0.91 0.91 0.77 0.94 0.93 Q650I 0.50 0.84 0.92 0.51 0.97 0.93 0.83 Q650K 0.39 0.62 0.93 0.34 0.90 0.76 Q650L 0.57 0.89 0.94 0.67 0.98 0.96 0.96 Q650M 0.50 0.79 0.91 0.55 0.97 0.91 1.00 0.97 Q650N 0.54 0.88 0.94 0.53 1.00 1.00 Q650Q 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Q650R 0.41 0.68 0.83 0.43 0.88 0.74 Q650T 0.76 0.94 0.97 0.65 0.98 0.98 Q650V 0.89 0.98 0.96 0.92 1.00 Q650W 0.26 0.53 0.82 0.26 0.86 0.90 0.79 0.96 0.75 Q650Y 0.55 0.91 0.87 0.61 K656A 0.07 0.05 0.05 0.05 0.05 0.36 0.05 0.05 0.35 K656C 0.07 0.05 0.05 0.05 0.06 0.25 0.05 0.05 0.44 K656D 0.07 0.05 0.05 0.05 0.08 0.14 0.05 0.05 0.37 K656E 0.06 0.05 0.05 0.09 0.07 0.12 0.05 0.05 0.33 K656F 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 K656G 0.05 0.05 0.05 0.05 0.05 0.35 0.05 0.05 0.28 K656I 0.08 0.05 0.05 0.05 0.08 0.31 0.05 0.05 0.37 K656K 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 K656L 0.06 0.05 0.05 0.05 0.07 0.13 0.05 0.05 0.44 K656M 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 K656P 0.07 0.05 0.05 0.05 0.05 0.16 0.05 0.05 0.43 K656Q 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 K656R 0.40 0.58 0.46 0.93 0.97 0.92 0.95 0.82 K656S 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 K656T 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 K656V 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 K656W 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 K656Y 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 T660C 0.59 0.90 T660D 0.85 0.99 0.98 1.00 0.95 T660E 0.86 0.92 0.95 0.84 0.91 0.82 0.98 T660F 0.61 0.86 0.97 0.96 0.96 0.86 T660G 0.83 0.92 0.30 1.00 0.80 0.93 0.93 0.86 T660H 0.62 0.92 0.95 0.97 0.93 0.95 0.91 0.80 T660I 0.90 0.99 0.99 0.99 0.95 0.80 T660K 0.68 0.90 0.95 0.78 0.86 0.92 0.76 T660L 0.42 0.80 0.97 0.92 0.98 0.91 0.98 0.92 0.58 T660M 0.61 0.84 0.95 0.83 0.90 0.89 0.69 T660N 0.47 0.88 0.95 0.94 0.88 T660P 0.16 0.33 0.30 0.66 0.55 0.57 0.70 0.32 T660Q 0.61 0.87 1.00 0.97 0.91 0.89 0.67 T660R 0.18 0.42 0.32 0.87 0.80 0.75 0.87 0.38 T660S 0.52 0.94 0.97 0.80 0.76 T660T 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 T660V 0.66 0.93 0.94 0.87 0.90 0.90 0.90 T660W 0.29 T660Y 0.62 0.97 0.83 P661A 0.82 0.94 0.99 0.97 1.00 P661C 0.62 P661D 0.75 0.86 P661E 0.45 0.98 1.00 0.93 0.88 0.86 P661F 0.23 0.75 P661G 0.66 0.87 0.93 0.91 0.98 0.95 0.94 P661H 0.36 0.86 0.98 0.66 0.90 P661I 0.28 0.87 P661K 0.47 0.83 0.98 0.74 P661L 0.19 0.94 0.84 P661M 0.15 0.79 0.88 0.51 P661P 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 P661Q 0.37 0.99 0.98 0.81 P661R 0.47 0.91 0.95 0.83 P661S 0.37 0.93 0.98 P661T 0.24 0.95 0.98 0.75 P661V 0.48 0.99 P661W 0.14 0.93 0.59 G662A 0.30 0.96 0.96 G662C 0.16 0.99 G662D 0.97 0.99 0.98 0.91 0.93 0.88 G662E 0.86 0.91 0.98 0.78 0.76 0.81 G662F 0.59 G662G 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 G662H 0.89 0.97 0.94 0.90 0.85 G662I 0.21 0.67 0.11 0.69 G662K 0.77 0.97 0.97 1.05 0.88 0.87 0.74 G662L 0.94 0.95 0.98 0.89 0.80 0.85 G662M 0.90 0.97 0.98 0.98 0.88 0.85 0.83 G662N 0.67 0.98 0.86 0.99 0.92 0.79 G662P 0.25 0.56 0.96 0.50 0.87 0.92 0.78 0.78 0.44 G662Q 0.95 0.91 0.92 0.87 0.84 0.80 0.63 G662R 0.76 0.91 0.99 0.92 0.88 0.84 0.71 G662S 0.84 0.90 0.95 0.74 0.74 0.80 G662T 0.99 0.95 0.80 0.92 0.89 0.80 G662V 0.54 0.87 1.00 1.00 0.95 0.81 0.94 0.87 0.67 G662W 0.99 0.97 0.93 0.92 0.75 0.88 G662Y 0.73 1.00 0.99 0.93 0.97 0.80 Q663A 0.84 0.82 0.70 0.92 0.96 0.99 Q663C 0.77 0.97 0.64 0.96 0.99 Q663D 0.96 0.78 0.98 Q663E 0.30 0.92 0.54 Q663F 0.66 0.96 0.93 0.49 0.90 0.95 Q663G 0.85 0.98 0.95 0.96 Q663H 0.50 0.94 0.98 0.61 0.90 0.99 0.92 Q663I 0.60 0.85 0.62 0.93 0.97 Q663K 0.89 1.04 0.84 0.67 0.84 0.91 0.92 0.94 0.99 Q663L 0.68 0.94 0.79 0.68 0.87 0.94 0.97 Q663M 0.70 0.97 0.77 0.71 0.88 0.97 0.96 0.99 Q663N 0.78 0.97 0.78 0.74 0.88 0.92 0.92 Q663Q 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Q663R 0.81 1.00 0.83 0.53 0.84 0.94 0.88 Q663S 0.85 0.98 0.84 0.91 0.95 0.96 Q663T 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 Q663V 0.89 0.89 0.72 0.86 0.92 0.87 Q663W 0.96 0.82 0.91 0.99 1.00 0.93 Q663Y 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 T666A 0.99 0.94 0.95 T666C 0.43 0.90 T666D 0.65 0.90 0.75 1.00 0.94 0.95 0.82 T666E 0.70 0.89 0.83 0.94 0.92 0.85 0.85 0.79 T666F 0.82 0.77 0.77 0.84 0.95 0.77 0.76 0.65 T666G 0.51 0.79 0.63 0.93 0.87 0.87 0.84 0.77 T666H 0.56 0.82 0.72 0.91 0.84 0.88 0.72 T666I 0.64 0.79 1.00 0.80 0.84 0.96 0.81 0.90 0.77 T666K 0.67 0.89 0.97 0.74 0.90 0.87 0.97 0.85 T666L 0.65 0.93 0.86 0.81 0.96 0.86 0.83 0.83 T666M 0.62 0.97 1.00 0.89 0.88 0.99 0.95 0.86 0.84 T666N 0.49 0.99 0.91 T666P 0.47 0.85 0.98 0.77 0.91 0.98 0.83 0.88 0.64 T666R 0.61 0.88 0.99 0.75 0.83 0.90 0.74 T666S 0.68 0.89 0.94 0.95 0.98 0.86 0.92 0.81 T666T 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 T666V 0.69 0.92 0.89 0.88 0.80 0.86 0.91 0.86 T666W 0.63 0.80 0.99 0.71 0.90 0.83 0.81 0.74 T666Y 0.77 0.90 0.77 0.90 0.92 0.82 0.81 0.81 R672A 0.56 0.80 0.84 0.53 0.97 0.97 0.91 0.98 0.92 R672C 0.36 0.75 0.83 0.42 R672D 0.07 0.24 0.65 0.06 0.78 R672E 0.12 0.31 0.74 0.05 0.91 0.95 0.73 R672F 0.37 0.70 0.80 0.39 0.89 0.91 0.83 R672G 0.27 0.61 0.81 0.29 0.94 R672H 0.51 0.92 0.84 0.61 0.99 0.86 0.98 R672I 0.23 0.52 0.81 0.27 0.95 0.86 R672K 0.76 0.84 0.77 0.96 R672L 0.49 0.88 0.89 0.55 0.92 0.93 R672M 0.18 0.67 0.05 0.94 0.79 R672N 0.48 0.93 0.85 0.48 0.92 0.98 R672P 0.14 0.11 0.63 0.06 0.24 0.49 0.32 0.55 0.28 R672Q 0.15 0.19 0.76 0.05 0.47 0.77 0.67 0.86 0.34 R672R 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 R672S 0.11 0.13 0.75 0.05 0.38 0.68 0.48 0.83 0.36 R672T 0.32 0.87 0.85 0.44 0.97 R672V 0.56 0.92 0.92 0.57 0.96 R672W 0.16 0.43 0.85 0.17 0.99 0.75 R672Y 0.16 0.29 0.79 0.07 0.68 0.98 0.82 0.60 R673A 0.77 0.95 0.85 0.74 0.94 0.94 R673C 0.90 0.99 0.89 0.82 0.98 0.97 R673E 0.48 0.80 0.86 0.59 0.99 0.93 0.95 R673F 0.43 0.43 0.05 0.37 0.11 0.32 0.47 0.41 R673G 0.57 0.88 0.91 0.52 R673H 0.70 0.99 0.91 0.86 0.98 0.98 R673I 0.72 0.99 0.91 0.65 1.00 R673K 0.98 0.98 0.78 0.96 R673L 0.37 0.94 0.90 0.55 R673M 0.62 0.93 0.91 0.62 0.99 1.00 R673N 0.96 0.97 0.94 0.98 R673P 0.20 0.31 0.77 0.16 0.67 0.92 0.89 0.99 0.64 R673Q 0.76 0.97 0.88 0.72 0.99 1.00 0.95 R673R 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 R673S 0.79 0.96 0.83 0.94 R673T 0.89 0.94 0.85 0.96 1.00 R673V 0.99 1.00 0.96 0.76 0.99 0.93 R673W 0.98 0.93 0.97 0.96 R674A 0.39 0.05 0.05 0.19 0.39 0.51 0.30 0.27 0.34 R674C 0.42 0.05 0.05 0.50 0.37 0.40 0.29 0.27 0.75 R674D 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 R674E 0.24 0.05 0.05 0.32 0.20 0.27 0.11 0.05 0.38 R674G 0.22 0.05 0.05 0.32 0.55 0.75 0.39 0.27 0.49 R674H 0.26 0.05 0.05 0.53 0.19 0.24 0.13 0.05 0.15 R674I 0.25 0.05 0.05 0.40 0.16 0.31 0.08 0.05 0.20 R674K 0.83 0.42 0.31 0.77 0.93 0.83 0.83 0.66 R674L 0.38 0.44 0.60 0.83 0.88 0.73 0.79 R674M 0.32 0.28 0.05 0.70 0.92 0.89 0.93 R674P 0.26 0.05 0.05 0.23 0.21 0.32 0.10 0.05 0.16 R674Q 0.28 0.43 0.94 0.71 0.82 0.62 0.63 0.38 R674R 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 R674S 0.26 0.05 0.05 0.29 0.28 0.40 0.19 0.09 0.60 R674T 0.06 0.05 0.05 0.12 2.22 R674V 0.39 0.05 0.05 0.19 0.78 0.72 0.58 0.55 R674W 0.29 0.05 0.05 0.36 0.63 0.76 0.59 0.54 0.96 R674Y 0.07 0.05 0.05 0.33 2.15 0.42 D675A 0.09 0.05 0.05 0.11 0.17 0.27 0.44 0.52 0.58 D675C 0.09 0.05 0.05 0.13 0.24 0.45 0.64 0.75 D675D 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 D675E 0.21 0.20 0.62 0.95 0.99 0.83 0.55 D675F 0.11 0.05 0.05 0.21 0.44 0.78 0.79 0.85 0.94 D675G 0.07 0.05 0.05 0.11 0.05 0.52 0.34 0.70 0.72 D675H 0.28 0.30 0.80 0.94 0.96 0.85 0.80 D675I 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 D675K 0.07 0.05 0.05 0.08 0.08 0.05 0.47 0.61 0.46 D675L 0.10 0.05 0.05 0.21 0.38 0.41 0.67 0.67 D675M 0.09 0.05 0.05 0.17 0.35 0.05 0.64 0.74 0.74 D675N 0.10 0.05 0.05 0.22 0.40 0.48 0.66 0.82 0.87 D675P 0.08 0.05 0.05 0.08 0.05 0.05 0.35 0.50 0.41 D675R 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 D675S 0.23 0.24 0.71 0.80 0.83 0.84 0.70 D675T 0.07 0.05 0.05 0.07 0.12 0.68 0.69 0.58 0.70 D675V 0.07 0.05 0.05 0.08 0.05 0.05 0.29 0.67 0.62 D675W 0.07 0.05 0.05 0.10 0.05 0.05 0.25 0.65 0.48 D675Y 0.22 0.23 0.66 0.98 0.92 0.95 0.66 D680A 0.60 0.91 0.63 0.97 D680C 0.88 0.91 0.92 D680D 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 D680E 0.77 0.98 0.75 0.99 0.99 D680F 0.30 0.90 0.88 0.44 0.97 D680G 0.61 0.92 0.93 0.63 0.93 0.98 0.99 0.96 0.87 D680H 0.41 0.95 0.91 0.51 0.99 0.90 D680I 0.28 0.95 0.87 0.37 0.85

D680K 0.84 0.97 0.86 0.75 1.00 0.98 0.92 0.84 D680L 0.25 0.71 0.92 0.27 0.88 0.95 0.78 D680M 0.50 0.86 0.61 D680N 0.43 0.90 0.91 0.46 0.95 0.99 0.99 0.94 D680P 0.09 0.15 0.05 0.05 0.25 0.91 0.31 0.16 0.33 D680Q 0.65 0.90 0.70 D680R 0.87 0.92 0.86 1.00 0.95 0.93 D680S 0.20 0.48 0.87 0.19 0.69 0.70 0.69 0.58 D680V 0.66 0.90 0.72 D680W 0.29 0.85 0.90 0.36 0.99 D680Y 0.48 0.91 0.63 T681A 0.87 0.99 0.91 0.99 T681F 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 T681G 0.48 0.70 T681H 0.49 0.89 0.93 0.56 0.93 0.91 T681K 0.52 0.99 0.92 0.61 0.99 T681L 0.67 0.93 0.77 0.95 T681M 0.53 0.97 0.61 1.00 T681N 0.59 0.93 0.99 0.65 0.94 0.97 0.95 T681P 0.44 0.92 0.95 0.56 0.96 T681Q 0.57 0.98 0.96 0.65 T681R 0.69 0.86 0.95 0.66 0.93 0.96 0.92 0.97 T681S 0.60 0.99 0.76 0.95 T681T 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 T681V 0.66 1.00 0.99 0.78 0.95 T681W 0.73 0.98 0.86 T681Y 0.61 0.80 0.96 0.57 0.73 0.93 1.15 0.96 0.90 A682A 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 A682C 0.45 0.33 0.16 0.51 0.73 0.49 0.55 0.39 A682D 0.45 0.42 0.87 0.22 0.62 0.86 0.61 0.73 0.56 A682E 0.26 0.21 0.13 0.41 0.64 0.41 0.53 0.29 A682F 0.28 0.27 0.86 0.17 0.47 0.62 0.54 0.65 0.59 A682I 0.11 0.14 0.14 0.49 0.90 0.60 0.87 0.42 A682L 0.26 0.26 0.18 0.54 0.66 0.61 0.73 0.45 A682M 0.30 0.21 0.10 0.39 0.67 0.42 0.48 A682N 0.33 0.34 0.47 0.53 0.62 0.59 0.70 0.67 A682P 0.28 0.22 0.22 0.42 0.61 0.40 0.57 0.26 A682R 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 A682S 0.29 0.24 0.12 0.45 0.61 0.45 0.54 0.51 A682T 0.33 0.30 0.94 0.16 0.48 0.65 0.47 0.57 0.28 A682V 0.34 0.41 0.80 0.22 0.64 0.93 0.60 0.72 0.52 A682W 0.37 0.28 0.15 0.49 0.61 0.45 0.57 0.35 A682Y 0.38 0.31 0.16 0.49 0.63 0.48 0.58 0.38 S683A 0.91 0.96 0.88 0.98 0.92 0.97 0.95 S683C 0.87 0.91 0.83 0.95 0.94 0.75 S683D 0.45 0.65 0.39 0.87 0.90 0.86 S683E 0.58 0.91 0.67 0.93 0.93 S683F 0.14 0.50 0.96 0.22 0.84 S683G 0.86 0.98 0.88 0.98 0.96 0.98 S683I 0.31 0.77 0.95 0.38 0.96 0.98 S683K 0.87 0.84 0.92 0.89 0.89 0.88 0.96 S683L 0.62 0.95 0.72 0.97 0.98 0.90 S683M 0.53 0.92 1.00 0.60 0.97 0.99 S683P 0.57 0.90 1.00 0.62 0.89 0.96 S683Q 0.66 0.87 0.99 0.69 0.94 0.98 0.96 0.96 S683R 0.96 0.90 0.86 0.95 0.95 0.97 0.92 S683S 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 S683V 1.00 S683W 0.51 0.88 0.82 Q684A 0.53 0.95 0.54 0.88 0.93 Q684C 0.37 0.89 0.05 Q684D 0.87 0.86 0.91 0.90 0.71 0.85 Q684E 0.86 0.94 0.75 0.88 0.99 0.92 0.70 0.90 Q684F 0.84 0.90 0.91 0.89 0.68 0.87 Q684G 0.23 0.83 0.11 Q684H 0.97 0.96 0.98 0.92 0.75 0.99 Q684I 0.66 0.98 0.61 0.85 0.85 0.89 0.71 0.83 Q684K 0.83 0.81 0.93 0.85 0.69 0.86 Q684L 0.88 0.91 0.97 0.79 0.69 0.89 Q684M 0.80 0.88 0.86 0.82 0.70 0.85 Q684N 0.69 0.92 0.12 1.00 Q684P 0.35 0.34 0.82 0.93 0.86 0.77 0.90 Q684Q 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Q684R 0.86 0.98 0.85 0.90 0.81 0.73 0.75 Q684S 0.79 1.00 0.87 0.97 0.81 0.69 0.79 Q684T 0.86 0.89 0.90 0.88 0.74 0.79 Q684W 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 K685A 0.67 0.98 0.99 K685E 0.69 0.95 0.92 0.86 0.81 K685F 0.60 0.98 0.89 0.99 0.85 0.62 K685G 0.41 0.94 0.75 0.95 0.96 K685I 0.35 0.95 0.72 K685K 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 K685L 0.65 0.98 K685M 0.53 0.99 0.97 K685N 0.37 0.94 0.76 0.93 0.95 K685P 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 K685Q 0.46 0.98 0.79 1.00 0.90 K685R 0.57 0.97 0.91 0.78 0.99 0.89 0.84 K685S 0.76 0.99 K685T 0.39 0.98 0.77 K685V 0.42 0.96 0.85 0.96 0.97 K685W 0.51 0.99 0.89 K685Y 0.53 0.98 0.81 S692C 0.77 0.94 0.95 0.90 S692D 0.82 1.00 0.93 0.71 0.96 0.84 0.97 0.94 0.94 S692E 0.77 0.98 0.77 0.94 0.92 0.88 0.96 S692F 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 S692G 0.60 0.98 0.98 0.67 0.94 0.94 0.86 0.98 0.99 S692H 0.86 S692I 0.56 0.92 0.77 1.00 S692K 0.89 1.00 0.98 0.94 0.96 0.99 S692L 0.90 0.96 S692M 0.70 0.98 0.95 S692N 0.91 0.97 0.91 0.98 0.84 0.83 0.90 S692P 0.60 0.89 0.63 0.92 0.92 0.85 0.91 S692Q 0.79 0.99 0.84 0.97 0.88 0.98 0.90 S692R 0.95 0.89 0.98 0.73 0.83 0.92 0.80 0.80 0.92 S692S 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 S692T 0.89 0.99 0.97 0.89 S692V 0.86 0.80 1.00 S692W 0.66 0.99 0.62 S692Y 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 R702C 0.56 0.90 0.52 R702D 0.24 0.79 0.20 0.89 R702F 0.28 0.80 0.16 0.96 0.55 R702G 0.46 0.57 R702H 0.38 0.94 0.94 0.47 0.95 0.81 R702I 0.57 0.91 0.61 0.95 0.94 0.75 R702K 0.99 0.90 0.83 0.94 0.99 0.98 R702L 0.51 1.00 0.69 0.99 0.96 R702M 0.53 0.86 0.59 0.91 0.97 0.87 0.73 R702N 0.53 0.96 1.00 0.49 0.94 0.94 0.77 R702P 0.05 0.05 0.05 0.05 0.05 0.80 0.05 0.05 0.28 R702Q 0.57 0.91 0.56 0.95 0.84 R702R 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 R702S 0.43 0.93 0.44 R702T 0.39 0.94 0.97 0.43 0.83 R702V 0.65 0.97 0.64 0.82 R702W 0.56 0.95 0.58 0.98 0.94 R705C 0.64 0.97 0.83 0.67 1.00 0.97 0.96 R705D 0.25 0.37 0.99 0.13 0.72 0.94 0.56 0.74 0.52 R705E 0.24 0.44 0.85 0.23 0.71 0.87 0.61 0.83 0.76 R705F 0.47 0.93 0.96 0.53 0.99 1.11 0.95 R705G 0.33 0.76 0.90 0.34 0.96 1.00 0.89 0.98 0.79 R705H 0.37 0.84 0.96 0.71 0.99 0.95 0.85 R705I 0.59 0.92 0.84 R705L 0.49 0.96 0.88 0.96 0.81 R705M 0.42 0.90 0.77 0.70 0.92 0.99 0.87 R705N 0.28 0.59 1.00 0.40 0.85 0.90 0.72 0.87 0.82 R705P 0.27 0.67 0.41 0.85 0.77 0.94 0.67 R705R 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 R705S 0.35 0.87 0.91 0.61 0.93 0.86 R705T 0.41 0.94 0.85 0.70 0.98 R705V 0.56 0.95 0.80 0.98 R705W 0.43 0.93 0.83 0.51 0.97 0.87 R705Y 0.35 0.80 0.92 0.34 0.94 0.93 0.85 0.84 0.89

[0401] Various terms, for example, Heat, CNPG, PASC, PCS, GLUC, G2 pH5, G2 pH6, G2 CC, are used to describe the mutations with respect to activity and/or stability in the table above. Up mutations have a PI of 1 or greater; neutral mutations have a PI greater than or equal to 0.5; non-deleterious mutations have a PI greater than 0.05; and deleterious mutations have a PI of 0.05. Positions at which mutations occur are classified as follows: non-fully restrictive positions have at least one neutral mutation for at least one property, while fully restrictive positions have no neutral mutations for activity and stability.

[0402] As determined during development of the present disclosure, positions 441 and 452 of H. jecorina BGL1 are fully restrictive positions. The data presented in Table 3-1 may be used to engineer any beta-glucosidase, even if the BGL to be engineered has an amino acid different from that of H. jecorina BGL1 at a particular position. For instance, the data in Table 3-1 may be used to find a substitution that will alter the desired property of a BGL by identifying the best choice substitution, including a substitution to the H. jecorina BGL1 wild type amino acid.

[0403] All BGL1 variants (3,153) were categorized as described above. Combinable mutations are mutations that can be combined to deliver proteins with appropriate performance indices for one or more desired properties. Briefly, 2,609 moderately combinable variants having a PI greater than or equal to 0.5 for at least one property were identified. In addition, 365 highly combinable variants having a PI of at least 0.5 for protein expression (HPLC) and a PI greater than 0.8 for all other properties were identified, while 213 of these highly combinable variants were found to have a PI greater than 0.8 for all properties. Non-combinable variants are those for which all PI values are <0.05. Any BGL1 variant that has one of the above substitutions relative to Hypocrea jecorina BGL1 can be improved by mutating that amino acid to one of the combinable substitutions at that position. Of the 3153 BGL1 variants listed in Table 3-1, 2,268 up variants having a PI of 1.0 or greater for at least one property were identified, while 1,836 up variants having a PI greater than 1.1 for at least one property were identified.

[0404] In some embodiments, the variants are improved variants having a PI greater than or equal to 1.0 in at least one property of interest. However, in other embodiments, the BGL1 variants of interest have a PI between 0.1 and 1.0 for expression. In particular, in instances when moderate to high levels of expression of a BGL1 variant are deleterious for protein production in a fermentator, variants with a PI between 0.1 and 1.0 for expression are desirable. Likewise, in circumstances in which an optimal ratio of enzyme concentrations is desired in the culture medium of a recombinant host cell, it may be desirable to utilize a variant having a reduced but measurable level of expression (0.1<PI<1.0).

[0405] In further embodiments, the BGL1 variants of interest have a PI between 0.1 and 1.0 for thermostability. For instance when a variant has reduced thermostability as compared to the wild type or reference BGL but improved activity under conditions of interest, then variants with a PI between 0.1 and 1.0 for thermostability are desirable. Likewise, in circumstances in which an optimal ratio of enzyme activities is desired in the culture medium of a recombinant host cell, it is desirable to utilize a variant having a reduced but appreciable level of thermostability (0.1<PI<1.0).

[0406] Likewise in some embodiments, the BGL1 variants of interest have a PI between 0.1 and 1.0 for activity. For instance when a variant has reduced activity as compared to the wild type or reference BGL but improved thermostability under conditions of interest, then variants with a PI between 0.1 and 1.0 for activity are desirable. Likewise, in circumstances in which an optimal ratio of enzyme activities is desired in the culture medium of a recombinant host cell, it is desirable to utilize a variant having a reduced but appreciable level of activity (0.1<PI<1.0).

[0407] Table 3-2 provides a summary of variants of particular interest identified from the above-described study that have a number of improved activities over wild type BGL1.

TABLE-US-00007 TABLE 3-2 G2 spiked Variant Glu Heat HPLC PASC PCS G2 pH5 G2 pH6 CNPG CC L167W wt + wt wt wt wt wt wt wt E170F + - -- + + ++ ++ wt - P176L wt -- -- wt ++ + wt + wt D177M wt -- -- + wt ++ ++ wt wt D178I wt -- -- - - - -- - -- D178K wt - -- wt wt wt - wt - D178N wt -- - - wt - -- wt - R179K wt -- -- wt ++ wt - -- -- R179S wt -- -- wt ++ wt + - -- R179V ++ -- -- wt ++ wt wt -- -- S199T wt -- -- wt ++ wt wt - - T209I wt -- -- wt +++ + + ++ wt D215S wt + -- ++ + +++ ++ +++ + Q216E wt wt wt wt wt wt wt wt wt Q216I wt -- -- + ++ ++ ++ wt wt Q216K wt - wt wt + wt wt wt wt D225Q wt wt -- wt + wt wt - - Q226W wt -- -- ++ +++ ++ +++ wt ++ Q226Y wt -- -- ++ +++ ++ +++ ++ + N238F ++ -- -- -- -- -- -- -- -- N238W ++ -- -- wt -- -- -- -- -- T242H wt ++ wt + wt ++ ++ ++ + T242S ++ ++++ +++ wt wt wt wt wt wt N263C wt wt -- + ++ +++ ++ ++ +++ N263S wt - -- ++ ++ ++ ++ ++ +++ N263T + -- -- wt + ++ + wt ++ N264D wt -- wt - - - -- - -- N264K + -- -- -- -- - -- - -- N264L + -- + -- - -- -- -- -- N264M ++ -- - - - - -- -- -- R265M ++ -- - wt wt wt - wt - R265P ++ -- -- -- - -- -- -- -- N278F wt -- -- ++ ++++ +++ ++ wt ++ T282D wt wt wt - wt wt wt wt wt T282I wt - - - - -- - - -- T282K wt wt wt - wt - wt - -- Q303E wt ++ wt wt wt wt -- wt - Q303I wt ++ - wt - wt - wt - Q303N wt +++ ++ wt - - - wt - Q303R wt ++ wt wt wt wt - wt - S312C + wt wt ++ ++ ++ ++ ++ +++ S312D wt - ++ wt wt wt -- - wt S312I wt -- wt - wt - - - -- S312K wt -- -- wt wt wt wt wt -- S312Y wt -- -- ++ ++ ++ ++ +++ ++ Q316T wt -- -- ++ +++ +++ +++ ++ ++ K320S wt +++ ++ - wt wt +++ wt -- K320Y wt +++ ++ - wt ++ wt - wt D329A wt ++ wt wt wt wt wt ++ + A338D wt +++ +++ wt ++ wt wt wt + A338I wt wt - wt ++ wt wt + wt A338K wt - - - ++ wt wt wt wt K345E wt - -- ++ ++ ++ ++ ++ ++ A347D wt + wt wt wt wt wt wt wt A347Y wt +++ + wt wt + wt ++ wt N369E wt +++ -- + - ++ wt ++ wt N369I ++ wt -- - -- - -- - -- N369T + ++ -- wt wt + wt + wt N369W wt ++ -- +++ wt ++++ ++++ ++++ wt N369Y wt ++ -- ++ wt ++ +++ +++ wt D370W ++ -- -- -- -- -- -- + -- G372A wt + -- ++ + ++ ++ ++ ++ G427C wt -- -- ++ ++ +++ +++ ++ ++ G427F wt wt -- ++ wt ++ + ++ ++ K428N - -- -- wt ++++ wt wt -- - N455D + wt -- +++ ++++ +++ ++++ ++ +++ N473S wt ++ wt wt ++ wt - wt wt S474D ++ +++ +++ wt wt wt wt -- wt S474I wt + wt wt wt wt wt wt wt S474R wt ++ ++ wt + wt wt wt wt K498A + -- - wt wt wt wt wt wt K498F wt -- -- + wt wt wt - - K498H + -- -- wt wt wt wt wt wt D521A wt wt wt wt ++ - -- wt wt D521R wt -- -- wt ++ wt -- -- - V522Y wt -- -- wt ++++ ++ wt ++ + R542N - -- - - ++ wt wt ++ +++ G547A wt +++ -- +++ ++ +++ ++++ ++ +++ G554C wt -- -- wt ++ wt + wt wt G554F wt -- -- wt ++ + + wt + K560S ++ -- -- wt wt -- -- -- wt D564T wt -- -- + wt ++ ++ + ++ D564V wt -- -- ++ + ++ +++ + - N583R wt + - wt ++ wt + wt wt V603G wt -- -- wt wt ++ ++++ wt ++++ F611A wt ++ -- ++ + +++ ++ ++ +++ F611R wt -- -- wt ++ wt ++ -- -- R645G wt ++ - wt ++ wt + + wt R645K wt wt - wt wt + + wt wt K656R + -- -- wt wt wt wt -- - P661E wt wt -- + - + wt wt - P661F wt -- -- +++ ++ +++ +++ wt ++ P661L wt - -- +++ ++ ++++ ++++ + +++ P661Q wt - -- ++ + ++ ++ wt + G662C wt ++ -- ++++ ++++ ++++ ++++ +++ ++++ G662D wt ++ ++ wt wt wt - wt wt G662F wt +++ -- ++ + ++ ++ ++ ++ G662K wt ++ -- wt wt - - wt -- G662L wt +++ wt wt wt - -- wt - G662Y wt ++ -- wt ++ wt wt wt -- T666C wt wt -- ++ ++ +++ ++ ++ ++ S683W - - -- + ++ ++ ++ ++ ++ Q684A wt -- -- - + wt wt + wt Q684C wt - -- +++ -- ++++ +++ ++++ +++ Q684D wt - wt wt wt - -- - - Q684G wt - -- ++++ -- ++++ ++++ ++++ ++++ Q684N + wt -- wt -- + wt ++ + Q684R wt wt + - - - -- - -- S692E wt -- -- wt wt wt - wt wt S692K wt wt - wt wt wt wt + wt S692L wt ++ - wt ++ wt wt ++ + ++++ PI >2 +++ 2 > PI > 1.5 ++ 1.5 > PI > 1.2 + 1.2 > PI > 1.1 wt 1.1 > PI > 0.9 - 0.9 > PI > 0.8 -- 0.8 > PI

Example 4

Expression, Activity and Performance of Additional BGL Variants

4-1. Assays

[0408] A modified HPLC assay was used to determine the protein content when studying the BGL variants in this library as compared to those described in Example 1. The specific procedure is described below. The glucose inhibition assay was also modified, thus the procedure used was described below.

HPLC Assay for Protein Content Determination

[0409] The concentration of BGL variants from pooled culture supernatants was determined by an Agilent 1200 (Agilent Technologies) HPLC equipped with a Proswift RP-2H 50.times.4.6 mm column (Dionex) calibrated at 50.degree. C. Fifty (50) .mu.L of sample was mixed with 50 .mu.L of 10% acetonitrile, and after 5 min, filtered under vacuum using a 0.22 .mu.m Millipore Multiscreen HTS 96 well filtration system. Ten (10) .mu.L of the filtered sample was loaded onto the column. Two buffers were used to construct an elution gradient having a flow rate of 1 mL/min: (1) Buffer A: 0.3% PEG1000, 0.1% TFA in deionized water; and (2) Buffer B: 64.63% acetonitrile, 35% 2-propanol, 0.3% PEG1000, 0.07% TFA in deionized water. Elution was carried out using the following program: from 0 min to 0.25 min with 5% Buffer B, followed by a gradient of 0.25 min to 1 min of from 5% Buffer B to 35% Buffer B, followed by a gradient of 1 min to 5 min of from 35% Buffer B to 55% Buffer B. A calibration curve was generated using purified wild type BGL1. Concentrations of BGL variants were determined using that standard calibration curve. To calculate performance index (PI), the concentration of a BGL variant was divided by the average concentration of wild-type BGL1 (e.g., a reference enzyme) in the same plate.

Glucose Inhibition Assay

[0410] The effect of glucose on the hydrolytic activity of beta-glucosidase was determined by conducting the CNPGase activity assay as described above in the presence of 2.5 mM glucose. The relative residual activity of the variants and the wild-type protein was determined by the ratio of the averaged specific activity in the presence of glucose and the averaged specific activity in the absence of glucose. A performance index (PI) for the BGL variants was determined by dividing the relative residual activity of a BGL variant by the relative residual activity of the wild-type BGL1 (e.g., a reference enzyme).

4-2. Generation of Additional H. jecorina BGL1 Site Evaluation Libraries

[0411] The pTTTpyrG-bgl1 plasmid containing the wild type H. jecorina BGL1 encoding sequence (SEQ ID NO: 1) was sent to BASEClear (Leiden, The Netherlands), who then generated positional libraries at each of the sites in Table 4-1 below. The sites were numbered in reference to the residue numbers of BGL1 mature protein SEQ ID NO:3.

TABLE-US-00008 TABLE 4-1 Additional Positions In The Mature BGL1 Protein Selected For The Generation Of SELs 11 38 43 60 68 110 128 146 180 181 184 201 206 217 220 221 243 245 255 258 259 260 261 266 270 283 293 300 308 377 384 392 400 406 436 442 443 444 450 457 461 463 466 468 482 485 486 491 500 507 530 532 536 550 556 565 566 567 568 575 601 602 604 605 606 607 624 630 633 639 646 655 667 671 678

[0412] For each of the 75 sites in Table 4-1, about 14 to 16 substitution variants were made. These variants were then produced as described in Example 2.

4-3 Expression, Stability, Activity and Performance of BGL1 Variants

[0413] The expression levels of the variants were measured using the HPLC assay, as described herein. PI values were calculated and listed in Table 4-2 under the column marked "HPLC." The CNPG hydrolyzing activities were also measured. Corresponding PI values were calculated and the results are listed in Table 4-2 under the column marked "CNPG." Effects of glucose on activity, or glucose inhibition, were also determined and the results are listed under the column marked "Gluc."

[0414] Thermostability measurements were listed under the column marked "Heat," specific activities in hydrolysis of PASC were listed under the column marked "PASC," and specific activities in hydrolysis of PCS were listed under the column marked "PCS." Cellobiase activities of these variants were also measured at pH 5.0, the results of which were listed under the column marked "G2 pH5." The specific activity of hydrolysis of ammonia pretreated corncob (CC) was likewise measured, and the results were listed under the column marked "CC." The PIs listed in Table 4-2 are classified based on ranges, as marked below the table in the margins. Specifically, PI is the ratio of performance of the variant to the parent or reference beta-glucosidase.

[0415] Table 4-2 lists 1501 additional substitutions tested in the second SEL library.

TABLE-US-00009 TABLE 4-2 Table 4-2 Performance of BGL1 variants Variant Gluc Heat HPLC PASC PCS G2 CNPG CC T011A wt wt - wt wt + + wt T011C wt -- - wt wt + ++ wt T011D wt - wt wt wt wt wt wt T011E wt ++ wt wt wt + + wt T011F wt -- -- -- -- -- -- ++ T011G wt wt -- wt - wt wt wt T011H wt -- -- wt wt + ++ wt T011I wt -- -- wt wt wt + wt T011K wt + - - -- - wt - T011L wt - -- wt - wt + wt T011M wt - - - - - wt wt T011N wt wt -- - -- -- - wt T011P -- -- -- -- -- -- -- -- T011Q -- -- -- -- -- -- -- -- T011R wt -- -- wt - wt wt wt T011S wt -- -- wt wt + + wt T011T -- -- -- -- -- -- -- -- T011V - wt -- wt wt wt + + T011W wt ++ wt wt - wt -- wt T011Y wt + -- wt wt + wt - N038A - - -- - wt -- - + N038C wt wt wt wt wt wt - wt N038D wt - -- wt wt + wt wt N038E + - ++ -- -- -- -- wt N038F wt - ++ wt - + ++ wt N038G -- -- -- -- -- -- -- -- N038H -- -- -- -- -- -- -- wt N038I wt -- - - -- - -- wt N038K + -- -- -- -- - - wt N038L ++ wt wt -- -- -- -- wt N038M ++ wt ++ -- -- -- -- wt N038N -- -- -- -- -- -- -- -- N038P + wt ++ -- -- -- -- wt N038Q wt wt + -- -- -- -- wt N038R wt + wt -- -- -- -- wt N038S wt -- -- - - -- wt wt N038T wt -- -- wt - wt wt wt N038V wt -- ++ wt -- wt wt wt N038W -- -- -- -- -- -- -- -- N038Y wt -- ++++ wt -- wt -- wt V043A +++ ++ ++ -- -- -- -- - V043C ++ + - -- -- -- -- - V043D wt -- + -- -- -- -- -- V043E -- -- -- -- -- -- -- -- V043F +++ -- wt -- -- -- -- -- V043G ++ ++ ++ -- -- -- -- - V043H + -- +++ -- -- -- -- -- V043I wt ++ -- -- -- -- -- - V043K -- -- -- -- -- -- -- -- V043L +++ - -- wt wt wt -- wt V043M -- -- -- -- -- -- -- -- V043N ++ +++ ++ -- -- -- -- - V043P -- -- -- -- -- -- -- wt V043Q wt ++ ++ -- - -- -- - V043R -- wt + -- -- -- -- -- V043S -- -- -- -- -- -- -- -- V043T wt -- -- - wt -- ++ - V043V -- -- -- -- -- -- -- -- V043W ++++ -- +++ -- -- -- -- -- V043Y -- -- +++ -- -- -- -- -- Q060A -- -- +++ -- -- -- + - Q060C -- ++++ -- -- -- -- -- - Q060D + ++ ++ wt wt -- -- - Q060E wt -- ++ wt wt - wt wt Q060F wt -- ++++ - -- wt wt - Q060G - -- +++ -- -- -- wt - Q060H -- wt ++++ wt -- wt wt - Q060I wt -- - -- -- -- - - Q060K -- +++ -- -- -- -- -- -- Q060L -- -- -- -- -- -- -- wt Q060M - -- ++ - - - + wt Q060N - -- +++ - - -- - wt Q060P -- -- -- -- -- -- -- -- Q060Q -- -- -- -- -- -- -- -- Q060R -- wt -- -- -- -- -- - Q060S -- -- -- -- -- -- -- -- Q060T -- -- + - -- -- wt wt Q060V + -- - -- -- -- -- wt Q060W -- -- wt -- -- -- - -- Q060Y wt -- +++ -- -- -- - wt Y068A ++ -- wt wt - wt ++ wt Y068C -- -- -- -- -- -- -- -- Y068D ++ -- -- wt - wt + wt Y068E ++ -- ++ - - -- wt wt Y068F -- -- -- -- -- -- -- -- Y068G ++ -- ++ - - - + wt Y068H ++ -- - - - -- - wt Y068I ++ -- -- - -- - - wt Y068K ++ -- -- -- -- -- wt wt Y068L ++ -- wt -- - -- ++ wt Y068M ++ -- ++ - -- - wt wt Y068N ++ -- wt wt wt wt -- wt Y068P ++ -- - -- -- -- ++ -- Y068Q -- -- -- -- -- -- -- -- Y068R ++ -- -- -- -- - - wt Y068S ++ -- wt - - wt ++ wt Y068T ++ -- wt - -- - + wt Y068V ++ -- -- wt - wt ++ + Y068W -- -- -- -- -- -- -- -- Y068Y -- -- -- -- -- -- -- -- L110A ++ -- - -- -- -- -- wt L110C ++ -- + -- - -- -- wt L110D wt -- -- -- -- -- -- wt L110E ++ -- -- -- -- -- -- wt L110F ++ -- wt -- -- -- -- wt L110G +++ -- ++++ -- -- -- -- -- L110H ++ -- -- -- -- -- -- wt L110I wt -- -- -- -- -- -- wt L110K + -- -- -- -- -- -- - L110L L110M ++ - -- wt wt - -- wt L110N wt -- -- -- -- -- -- - L110P ++ -- -- -- -- -- -- -- L110Q ++ -- ++ -- -- -- -- wt L110R + -- -- -- -- -- -- -- L110S ++ -- - -- -- -- -- - L110T L110V ++ -- -- -- -- -- -- - L110W ++ -- ++ -- -- -- -- -- L110Y +++ -- -- -- -- -- -- -- E128A wt -- -- -- -- -- - - E128C wt -- -- -- -- -- -- wt E128D wt -- - -- -- -- - - E128E E128F + wt -- -- -- -- -- -- E128G wt -- - -- -- -- - -- E128H wt -- -- -- -- -- -- -- E128I wt -- -- -- -- -- -- -- E128K - -- -- -- -- -- -- -- E128L -- -- -- -- -- -- -- -- E128M E128N wt -- + -- -- -- -- -- E128P + - -- -- -- -- -- -- E128Q ++ - -- -- -- -- -- -- E128R + +++ -- -- -- -- -- -- E128S -- -- - -- -- -- -- -- E128T E128V + -- -- -- -- -- -- -- E128W wt -- -- -- -- -- -- -- E128Y wt ++ -- -- -- -- -- -- N146A + ++ wt wt wt + - - N146C wt wt - wt wt wt wt wt N146D wt +++ -- wt + wt - wt N146E wt ++ -- wt wt ++ wt wt N146F wt -- -- wt wt ++ +++ wt N146G wt ++ -- - - wt wt wt N146H wt wt wt wt + + wt wt N146I wt -- wt wt wt wt ++ - N146K wt ++ -- - wt wt wt - N146L -- -- -- -- -- -- -- -- N146M + +++ -- - wt wt - wt N146N N146P -- -- -- -- -- -- -- - N146Q + ++ -- wt wt ++ wt wt N146R -- -- -- -- -- -- -- -- N146S wt wt wt wt + ++ ++ wt N146T wt wt -- wt wt + wt wt N146V + ++ wt - - wt - wt N146W ++ ++ -- -- -- -- -- wt N146Y wt wt ++ - + wt + - T180A wt wt -- wt wt wt wt wt T180C wt - - wt wt + ++ wt T180D wt -- -- wt wt wt - wt T180E wt -- -- wt wt wt -- ++ T180F wt -- -- wt wt wt -- ++ T180G - - -- wt wt wt -- ++ T180H wt -- -- wt wt + - ++ T180I wt - -- wt - - -- wt T180K wt -- -- wt - wt -- ++ T180L wt -- -- -- - -- -- wt T180M wt -- -- wt wt ++ - +++ T180N wt wt -- wt wt wt - + T180P wt - -- wt - - - wt T180Q wt - -- - - - -- wt T180R wt -- -- - -- -- -- + T180S wt - -- wt wt wt -- ++ T180T T180V wt wt -- wt wt wt wt wt T180W - -- -- wt wt wt -- ++ T180Y wt -- -- wt wt wt wt wt L181A wt wt -- wt wt wt wt wt L181C wt + -- wt wt wt wt wt L181D wt - -- wt wt + - - L181E wt + -- - wt wt - wt L181F + + - - wt wt - wt L181G wt wt -- - - wt wt - L181H ++ wt -- - wt wt - - L181I wt wt -- -- wt wt - - L181K - - -- - - wt - -- L181L L181M + + + wt - - - wt L181N -- -- -- -- -- -- -- -- L181P -- -- -- -- -- -- -- - L181Q wt wt wt wt wt wt wt wt L181R - - -- - -- wt -- - L181S wt + wt wt wt wt wt wt L181T + wt -- -- - - -- - L181V ++ wt -- - wt - -- wt L181W wt wt wt - - - -- - L181Y wt wt -- - - wt wt wt L184A wt - -- - wt - -- - L184C wt - wt wt wt wt wt - L184D wt wt -- wt wt wt - wt L184E - - -- - -- -- -- wt L184F wt - -- wt - wt - ++ L184G L184H -- -- -- -- -- -- -- -- L184I - -- -- wt -- wt - wt L184K L184L L184M -- wt ++ wt - wt wt wt L184N -- wt -- -- -- -- -- -- L184P -- -- -- -- -- -- -- -- L184Q - -- -- - -- - - wt L184R -- -- -- -- -- -- -- wt L184S wt -- -- wt - wt -- ++ L184T wt wt -- - - -- -- wt L184V wt - -- - -- -- -- - L184W wt -- -- -- -- -- -- +++ L184Y -- -- -- -- -- -- -- -- M201A -- -- -- -- -- -- -- -- M201C wt -- -- -- -- -- -- - M201D -- -- -- -- ++++ -- -- + M201E -- -- -- -- -- -- -- -- M201F -- -- -- -- -- -- -- + M201G -- -- wt -- -- -- -- -- M201H -- -- -- -- -- -- -- - M201I M201K -- -- -- -- -- -- -- wt M201L -- -- -- - -- wt wt + M201M M201N -- -- -- -- -- -- -- + M201P M201Q +++ -- -- -- -- -- -- - M201R -- -- -- -- -- -- -- wt M201S -- -- wt -- -- -- -- -- M201T -- -- -- -- -- -- -- -- M201V -- -- -- -- -- -- -- -- M201W -- -- -- -- -- -- -- wt M201Y -- -- -- -- -- -- -- wt K206A wt -- ++ + ++ + ++ wt K206C K206D wt -- + + wt wt wt wt K206E K206F +++ -- -- wt wt - - wt

K206G wt -- -- + ++ wt wt wt K206H wt -- -- - -- - - wt K206I K206K K206L wt wt wt wt + wt wt wt K206M wt -- - wt wt wt wt - K206N wt -- wt wt wt wt - wt K206P + -- wt - wt -- - wt K206Q - wt wt wt wt wt wt wt K206R wt wt -- wt wt wt - wt K206S + -- + wt + wt wt - K206T + -- -- wt wt wt wt wt K206V wt -- ++ wt wt wt wt wt K206W ++ -- - wt wt - - - K206Y wt -- wt wt wt -- -- wt Y217A wt wt ++ wt wt wt wt wt Y217C - -- -- wt wt wt + wt Y217D wt wt -- wt + wt + wt Y217E - -- -- wt wt wt wt wt Y217F wt -- -- wt + wt + wt Y217G wt wt +++ wt wt wt - - Y217H + wt - wt wt - - - Y217I wt - -- wt wt wt wt wt Y217K wt - wt wt wt wt wt wt Y217L wt wt -- wt wt wt wt - Y217M - - -- wt + wt wt wt Y217N - wt - wt wt - - - Y217P ++ -- -- wt wt wt wt wt Y217Q wt -- wt wt wt wt wt wt Y217R -- wt - wt wt wt wt wt Y217S -- - -- wt + wt wt wt Y217T wt - -- wt wt wt + wt Y217V - wt -- wt wt wt - wt Y217W -- -- -- -- -- -- -- -- Y217Y Q220A Q220C wt -- -- + wt + ++ wt Q220D wt wt + wt wt wt wt wt Q220E wt wt -- wt wt wt wt wt Q220F wt + -- wt wt wt - wt Q220G - - wt wt wt wt wt wt Q220H - wt ++ wt wt wt wt wt Q220I wt -- -- wt wt wt + wt Q220K -- - -- wt ++ wt + wt Q220L - wt -- wt wt wt wt wt Q220M - -- -- + + + + wt Q220N Q220P ++ wt -- wt + wt -- + Q220Q Q220R wt - -- wt + wt wt wt Q220S wt wt -- wt wt wt wt wt Q220T -- -- -- -- -- -- -- -- Q220V - - -- + wt wt + wt Q220W Q220Y wt -- -- wt + wt wt wt T221A wt -- -- ++ ++ + +++ wt T221C wt + -- wt + wt ++ wt T221D -- -- -- -- -- -- -- -- T221E wt wt ++ - wt - -- wt T221F wt -- - wt wt - wt wt T221G wt wt -- + + + ++ wt T221H wt wt wt - - -- -- -- T221I wt - -- ++ ++ + +++ wt T221K wt wt -- wt - -- - - T221L T221M wt +++ -- wt wt -- wt wt T221N wt + - - - - -- wt T221P -- -- -- -- -- -- -- - T221Q -- -- -- -- -- -- -- -- T221R -- -- -- -- -- -- -- -- T221S wt wt -- wt ++ wt ++ wt T221T T221V -- -- -- -- -- -- -- -- T221W wt -- + - - - - -- T221Y -- -- -- -- -- -- -- -- T243A wt -- +++ wt wt + wt wt T243C wt wt +++ wt wt + wt wt T243D wt -- ++ wt wt wt - wt T243E wt -- -- wt - -- -- wt T243F -- -- -- -- -- -- -- wt T243G wt - wt wt - - -- wt T243H T243I wt -- - wt wt wt wt wt T243K -- +++ -- -- -- -- -- -- T243L wt -- wt wt - - -- wt T243M - - -- wt - - wt + T243N -- - -- -- - -- -- wt T243P wt -- -- wt - -- - wt T243Q - - -- wt - - - wt T243R wt -- wt - - wt - wt T243S - -- +++ wt wt wt wt wt T243T T243V wt -- ++ wt wt wt wt + T243W -- -- -- -- -- -- -- - T243Y -- -- -- -- -- -- -- wt Q245A -- -- -- -- -- -- -- -- Q245C -- -- -- -- -- -- -- -- Q245D -- -- -- -- -- -- -- -- Q245E Q245F wt +++ - - - wt - + Q245G -- + -- - -- - wt wt Q245H - wt +++ wt wt + wt - Q245I wt wt ++ wt wt wt + - Q245K wt ++ -- -- -- -- -- wt Q245L - wt wt wt wt wt wt wt Q245M wt - ++ wt wt ++ ++ wt Q245N wt ++ ++ wt wt + wt wt Q245P + ++ -- - - -- -- wt Q245Q Q245R Q245S Q245T wt wt ++++ wt wt ++ wt - Q245V - wt ++ wt wt wt wt wt Q245W wt wt wt wt wt wt wt wt Q245Y wt wt wt wt wt wt - wt M255A wt -- wt wt -- wt + wt M255C - -- -- -- wt wt wt wt M255D -- -- -- -- -- -- -- wt M255E wt -- -- -- -- -- + wt M255F wt -- -- -- -- -- -- - M255G -- -- -- -- -- -- -- wt M255H ++ -- -- -- -- -- -- wt M255I wt -- ++++ -- -- -- -- - M255K -- -- -- -- -- -- -- - M255L wt -- -- - wt wt ++ + M255M M255N M255P wt -- -- - wt - ++ + M255Q wt -- ++++ -- - -- wt wt M255R -- -- -- -- -- -- -- - M255S -- -- -- -- -- -- -- wt M255T - -- -- - wt wt + + M255V - -- +++ - wt wt ++ wt M255W -- -- -- -- -- -- -- - M255Y -- -- wt -- -- -- -- -- T258A T258C -- -- -- + wt ++ wt wt T258D -- -- -- wt - - wt wt T258E -- -- -- wt wt ++ -- + T258F -- -- -- - -- wt - wt T258G -- -- - wt wt + ++ wt T258H -- -- -- wt -- wt -- + T258I -- -- -- wt -- +++ -- ++ T258K -- -- -- wt -- ++ -- ++ T258L -- -- -- + -- +++ -- ++ T258M -- -- -- wt -- wt -- wt T258N -- -- -- wt - wt - wt T258P -- -- -- -- -- -- -- wt T258Q -- -- -- wt - ++ -- ++ T258R -- -- -- -- -- -- -- -- T258S wt wt -- ++ ++ ++ ++ + T258T T258V -- -- -- + + +++ -- ++ T258W -- -- -- -- -- -- -- wt T258Y -- -- -- - -- -- -- + D259A -- -- -- wt - wt + wt D259C - -- -- wt -- + -- wt D259D D259E - -- +++ wt - wt wt wt D259F - -- -- - -- - wt wt D259G wt -- + - -- wt wt wt D259H - -- +++ wt -- wt wt wt D259I -- -- -- -- -- - - - D259K -- -- -- -- -- - -- wt D259L -- -- - - -- - wt wt D259M -- -- ++ -- -- -- - wt D259N wt ++ - wt - wt - wt D259P wt -- ++ - -- - wt wt D259Q -- -- -- - -- - - - D259R -- -- + -- -- -- -- wt D259S wt wt ++++ wt wt wt wt + D259T D259V D259W wt -- -- - -- wt wt wt D259Y wt -- -- - -- wt -- wt F260A -- +++ ++ wt + wt wt wt F260C -- - -- wt + wt wt wt F260D -- + - wt ++ ++ ++ + F260E -- +++ ++ + ++ ++ + wt F260F F260G -- ++ -- wt ++ ++ ++ + F260H -- wt -- wt wt wt + + F260I -- wt -- wt ++ + +++ wt F260K -- +++ -- wt wt wt -- wt F260L wt ++ ++ wt + + +++ + F260M -- -- -- -- -- -- -- wt F260N F260P -- -- -- -- -- -- -- wt F260Q -- ++ -- wt + + wt + F260R F260S -- ++ -- wt + wt wt wt F260T -- ++ -- wt + ++ +++ wt F260V -- ++ wt wt wt wt wt wt F260W ++ wt -- + wt + -- + F260Y -- ++ wt wt wt wt - wt N261A N261C wt - -- + wt ++ - wt N261D wt - wt wt wt wt + wt N261E wt ++ ++ wt wt wt wt - N261F wt + wt wt -- wt wt wt N261G wt wt -- wt wt wt - wt N261H wt wt -- wt wt wt + wt N261I wt wt - wt -- wt wt wt N261K wt ++ +++ wt - wt wt wt N261L wt + -- wt wt wt wt wt N261M - wt ++ wt - wt ++ wt N261N N261P N261Q wt - -- wt wt wt wt wt N261R -- -- -- -- -- -- -- -- N261S wt wt -- wt - wt - wt N261T wt wt wt wt - wt wt wt N261V wt wt -- wt - wt -- wt N261W wt -- -- wt -- wt -- wt N261Y wt -- -- -- -- -- -- wt L266A - -- -- wt wt + ++ + L266C - wt - wt + + ++ + L266D - -- - wt wt + ++ wt L266E - -- -- wt wt wt wt + L266F + ++ - wt wt wt wt wt L266G -- -- -- - -- - wt + L266H - wt -- wt - - - + L266I wt -- wt wt - - wt wt L266K - + - - -- - - wt L266L L266M - wt -- wt wt wt + wt L266N - -- -- + - + ++ + L266P wt -- -- - -- - - wt L266Q L266R L266S -- -- -- - -- -- -- wt L266T wt -- -- - - -- -- wt L266V -- - -- wt - - - + L266W L266Y wt wt wt wt + wt wt wt A270A A270C wt wt -- ++ ++ ++ wt + A270D wt wt ++ wt ++ wt wt wt A270E wt wt wt wt wt wt wt - A270F wt wt -- wt wt wt wt wt A270G A270H wt wt + wt - wt wt wt A270I wt wt ++ wt wt wt wt wt A270K wt wt -- + + wt wt wt A270L wt wt -- wt wt wt + wt A270M A270N wt wt -- + ++ wt wt wt A270P - -- -- wt wt wt ++ wt A270Q -- -- -- -- -- -- -- -- A270R + ++ - wt wt wt wt wt A270S wt ++ -- wt wt wt wt wt

A270T wt + - wt wt wt wt wt A270V - - -- wt wt wt ++ wt A270W wt wt ++ - - wt wt wt A270Y - -- wt wt wt wt wt wt S283A S283C -- -- -- -- -- -- -- - S283D wt wt ++ wt ++ wt - - S283E wt wt ++ - - wt wt wt S283F wt - wt wt wt + ++ + S283G wt wt ++ - wt wt - wt S283H - wt wt - - - - - S283I wt - -- - - - - - S283K wt wt -- - -- -- -- - S283L wt wt -- wt wt wt + - S283M wt wt wt - wt - wt wt S283N wt wt wt - wt - - wt S283P wt -- -- wt wt + - + S283Q wt wt -- wt - - - wt S283R wt wt -- wt - wt wt wt S283S S283T wt wt -- wt wt wt -- + S283V wt wt -- wt - - -- wt S283W S283Y - -- wt wt - wt wt wt L293A wt -- -- wt ++ wt - ++ L293C wt - -- wt wt wt -- + L293D wt ++++ -- -- -- -- -- -- L293E wt -- -- -- -- -- -- -- L293F ++ ++ -- ++++ -- ++++ +++ ++++ L293G wt -- -- -- -- -- -- +++ L293H -- -- -- -- -- -- -- -- L293I wt ++ + - wt - - wt L293K wt +++ -- -- -- -- -- +++ L293L L293M + -- -- wt + wt wt + L293N - -- -- -- -- -- -- +++ L293P -- -- -- -- -- -- -- -- L293Q - ++ -- -- -- -- -- -- L293R wt -- -- -- -- -- -- -- L293S wt -- -- -- wt -- -- ++ L293T wt wt -- wt ++ wt - wt L293V wt - -- + ++ ++ ++ wt L293W ++ -- -- -- -- -- -- -- L293Y wt - -- -- -- -- -- -- G300A wt -- - wt wt wt + wt G300C wt - -- wt ++ ++ ++ wt G300D -- -- -- wt wt wt wt wt G300E wt wt - wt - wt wt wt G300F wt -- -- wt wt ++ ++ wt G300G G300H wt -- -- wt wt wt + wt G300I wt -- -- wt wt ++ wt wt G300K wt wt -- wt wt - wt wt G300L -- -- -- -- -- -- -- -- G300M wt -- -- wt wt ++ wt + G300N wt wt -- wt wt wt wt wt G300P + -- -- -- -- -- -- -- G300Q wt wt -- wt wt wt wt wt G300R G300S + wt -- wt wt wt - - G300T wt -- -- wt wt wt wt wt G300V wt -- -- wt wt wt -- wt G300W wt wt -- wt wt ++ + wt G300Y wt - -- wt wt + wt wt S308A S308C wt - -- wt + ++ ++ wt S308D wt -- ++ wt wt wt wt wt S308E ++ + -- wt ++ wt wt ++ S308F wt wt ++ wt wt wt wt wt S308G wt - wt wt wt wt wt wt S308H - -- -- ++ +++ ++ + ++ S308I - -- -- wt wt wt wt wt S308K - - -- + wt wt ++ wt S308L wt -- -- wt wt wt + + S308M S308N wt wt -- wt wt wt wt + S308P S308Q -- -- -- -- -- -- -- -- S308R - - -- wt ++ wt wt + S308S S308T -- -- -- -- -- -- -- -- S308V wt wt +++ wt wt wt wt wt S308W wt wt + wt - wt - wt S308Y wt - -- wt wt -- -- wt A377A A377C wt -- -- wt + +++ ++ wt A377D wt -- -- wt + ++ ++ wt A377E wt -- -- wt -- wt + wt A377F wt -- -- - -- ++ ++ wt A377G wt -- -- wt -- + ++ wt A377H -- -- -- -- -- -- -- -- A377I + -- -- - -- + -- wt A377K - -- -- -- -- -- -- -- A377L wt -- -- -- -- +++ wt wt A377M A377N - -- wt -- -- -- -- -- A377P A377Q -- -- -- ++ wt +++ -- wt A377R -- -- -- -- -- -- -- -- A377S wt -- -- wt wt wt wt wt A377T wt -- -- wt wt wt ++ wt A377V wt -- -- wt -- wt wt wt A377W ++ -- -- -- -- -- -- wt A377Y wt -- -- -- -- - -- - S384A S384C + ++++ -- -- -- -- -- -- S384D + - -- -- -- -- -- - S384E ++ +++ ++++ -- -- -- -- -- S384F + -- -- -- -- -- -- -- S384G ++ -- ++ -- -- -- -- -- S384H - -- -- -- -- -- -- -- S384I - -- ++ -- -- -- -- -- S384K wt -- wt -- -- -- -- -- S384L -- -- wt -- -- -- -- -- S384M ++ -- - -- -- -- -- -- S384N S384P wt -- -- -- -- -- -- - S384Q - -- + -- -- -- -- -- S384R wt - wt -- -- -- -- -- S384S S384T - -- - -- -- -- -- -- S384V wt -- +++ -- -- -- -- -- S384W + -- ++ -- -- -- -- -- S384Y + -- -- -- -- -- -- -- F392A -- -- -- -- -- -- -- -- F392C wt -- -- + -- wt - + F392D wt -- -- - -- wt wt -- F392E wt -- -- wt -- wt wt -- F392F F392G wt - -- -- -- -- -- -- F392H wt -- -- -- -- -- -- -- F392I wt -- -- wt -- wt - -- F392K wt -- -- -- -- -- -- -- F392L wt -- -- wt - + + wt F392M wt -- -- wt wt wt wt + F392N wt -- -- wt wt wt -- ++ F392P -- -- -- -- -- -- -- -- F392Q - -- -- + -- ++ ++ wt F392R - -- -- ++ -- ++ -- +++ F392S wt -- -- -- -- - -- - F392T - -- wt -- -- - wt -- F392V F392W wt -- -- - -- - -- -- F392Y wt -- +++ wt wt + + wt N400A wt + + wt wt wt wt wt N400C wt wt -- wt wt wt wt wt N400D -- -- -- -- -- -- -- -- N400E wt wt -- wt wt wt wt wt N400F wt -- -- wt wt + wt wt N400G wt ++ -- wt wt wt -- wt N400H wt ++ wt wt - - - wt N400I -- -- -- -- -- -- -- -- N400K N400L N400M -- -- -- -- -- -- -- -- N400N N400P wt -- -- wt wt wt -- ++ N400Q wt ++ -- wt wt wt - + N400R -- -- -- -- -- -- -- -- N400S - + -- wt + + -- + N400T -- -- -- -- -- -- -- -- N400V - -- -- wt wt + - wt N400W -- -- -- -- -- -- -- -- N400Y Q406A -- -- -- -- -- -- -- -- Q406C -- -- -- -- -- -- -- -- Q406D + -- -- wt ++ ++++ wt ++ Q406E wt -- wt - -- wt ++ wt Q406F wt wt ++++ - wt wt ++ wt Q406G wt -- - -- -- -- wt wt Q406H wt wt +++ - wt ++ +++ wt Q406I Q406K -- -- -- -- -- -- -- -- Q406L -- -- -- -- -- -- -- wt Q406M - -- -- - -- +++ +++ + Q406N -- -- -- -- -- -- -- wt Q406P Q406Q Q406R - -- -- -- -- wt wt wt Q406S - -- -- - -- ++ ++ + Q406T wt -- ++++ wt wt + +++ wt Q406V -- -- -- -- -- wt -- + Q406W -- -- -- -- -- -- -- wt Q406Y -- -- -- -- -- -- -- -- T436A wt -- -- + wt + ++ wt T436C wt -- -- + -- ++ +++ wt T436D wt -- + wt wt wt + wt T436E + -- -- + -- + ++ wt T436F wt -- -- ++ -- +++ +++ -- T436G wt -- -- + - wt wt wt T436H wt -- -- wt -- + wt - T436I wt -- -- + -- ++ ++ wt T436K T436L wt -- -- - -- -- -- - T436M - -- -- + -- + +++ wt T436N wt -- -- - -- + + - T436P wt -- -- wt -- wt + - T436Q - -- -- + -- + ++ wt T436R wt -- wt wt wt wt wt wt T436S T436T T436V + -- -- wt -- wt wt wt T436W wt -- -- wt -- +++ wt -- T436Y wt -- -- ++ -- ++++ +++ -- G442A -- -- -- -- -- -- -- -- G442C - -- -- - - - wt wt G442D - -- - wt wt wt ++ - G442E -- -- wt wt wt wt + wt G442F -- -- -- - - - wt - G442G G442H -- -- -- -- -- -- -- wt G442I -- -- -- -- -- -- ++ - G442K wt -- -- -- -- -- -- wt G442L -- -- -- -- -- - - -- G442M -- -- -- -- -- -- -- -- G442N -- -- -- -- -- -- -- -- G442P - -- - -- -- -- -- -- G442Q -- -- -- - -- wt ++ - G442R -- -- -- -- - -- wt - G442S -- -- -- -- - -- wt - G442T -- -- -- -- -- -- wt -- G442V -- -- ++ -- -- -- wt - G442W -- -- -- -- -- -- wt - G442Y -- -- -- -- - -- wt wt Y443A -- -- -- -- -- -- -- -- Y443C - -- wt -- -- -- - -- Y443D - -- ++++ -- -- -- -- -- Y443E - -- -- -- -- -- ++ -- Y443F - -- -- - -- wt +++ wt Y443G -- -- -- -- -- -- -- + Y443H wt -- -- -- -- -- + - Y443I - -- ++ -- -- -- + -- Y443K -- -- -- -- -- -- -- -- Y443L -- -- wt -- -- -- - -- Y443M -- -- -- -- -- -- +++ wt Y443N -- -- -- -- -- -- ++ -- Y443P -- -- + -- -- -- wt -- Y443Q wt -- -- -- -- -- ++ -- Y443R -- -- wt -- -- -- -- -- Y443S -- -- ++ -- -- -- -- -- Y443T wt -- ++++ -- -- -- wt -- Y443V -- -- -- -- -- -- ++ -- Y443W -- -- -- -- -- -- -- -- Y443Y I444A -- -- -- -- - -- -- wt I444C -- -- -- - ++ wt -- ++ I444D -- -- -- -- -- -- - wt I444E -- -- -- wt wt +++ -- +++ I444F -- -- -- wt -- +++ - ++ I444G -- -- -- -- -- - -- ++ I444H -- -- -- -- -- -- -- wt

I444I I444K -- -- -- - -- wt -- ++ I444L - -- -- wt wt + + wt I444M wt -- -- wt wt + wt wt I444N -- -- -- - -- + -- ++ I444P -- -- -- -- -- -- -- + I444Q wt -- -- - -- - wt wt I444R -- -- -- -- -- - -- ++ I444S -- -- -- -- -- - -- ++ I444T -- -- -- wt -- wt -- ++ I444V -- -- -- wt + ++ -- ++ I444W -- -- -- wt -- +++ -- +++ I444Y - -- -- wt wt +++ -- +++ A450A A450C + - -- -- -- -- -- wt A450D -- -- -- -- -- -- -- -- A450E wt - -- + + ++ ++ ++ A450F wt -- -- ++ wt ++ +++ + A450G wt -- -- wt wt - -- + A450H wt ++ - - -- -- -- wt A450I wt -- -- wt - wt - ++ A450K -- ++ -- - - - - wt A450L wt -- -- + wt wt + wt A450M - wt wt wt wt + ++ + A450N A450P -- + -- + wt + ++ + A450Q wt ++ -- wt - wt + + A450R wt ++ - wt -- wt wt wt A450S - -- -- wt - - - wt A450T wt -- -- + wt + ++ + A450V -- -- -- + wt + ++ + A450W wt -- -- + wt + ++ + A450Y + -- -- wt - - -- wt D457A wt + ++ wt wt wt + wt D457C wt -- -- ++ ++ ++ +++ wt D457D D457E wt - wt wt wt wt + wt D457F wt -- -- wt wt wt + wt D457G - -- ++ wt wt wt ++ wt D457H wt -- -- - - -- wt - D457I wt -- - - -- -- wt wt D457K wt -- -- - - - - -- D457L wt -- wt - - - wt wt D457M - -- wt - -- -- wt wt D457N wt -- -- - -- -- -- wt D457P -- -- -- -- -- -- -- wt D457Q wt -- -- wt - wt wt wt D457R -- -- -- -- -- -- -- + D457S wt -- -- wt wt wt + wt D457T wt -- -- wt + wt ++ wt D457V - -- + wt - wt + wt D457W -- -- -- -- -- -- -- - D457Y wt -- - - - -- -- wt N461A wt -- -- + - ++ ++ wt N461C wt - -- ++ ++ +++ +++ wt N461D wt wt - + + wt wt wt N461E N461F wt wt -- + wt + wt wt N461G wt wt ++ wt wt + + wt N461H - - wt wt - wt wt wt N461I wt -- -- wt wt wt wt wt N461K -- -- -- -- -- -- -- -- N461L wt wt - wt wt wt wt wt N461M N461N N461P wt -- -- + -- ++ wt wt N461Q N461R wt - -- wt wt wt wt wt N461S wt ++ ++ wt wt wt wt - N461T wt -- + wt wt wt + wt N461V wt -- + + + + ++ wt N461W wt -- -- wt wt + + wt N461Y + wt wt wt wt + wt wt N463A -- -- -- -- -- -- -- -- N463C -- -- -- -- ++++ -- -- wt N463D - -- wt wt - - wt - N463E wt -- -- + ++ wt + wt N463F -- -- -- -- -- -- -- -- N463G wt -- -- + wt wt + wt N463H -- -- -- -- -- -- -- -- N463I wt -- -- wt wt wt + wt N463K wt -- -- +++ +++ ++++ +++ ++ N463L N463M - -- - wt wt wt ++ wt N463N N463P - -- -- wt ++ wt ++ wt N463Q N463R wt -- -- ++ +++ +++ + ++ N463S wt -- -- + wt + +++ + N463T - -- -- + + + ++ wt N463V wt -- - + wt wt ++ wt N463W N463Y - -- -- wt wt - wt wt V466A -- -- -- -- -- -- -- -- V466C wt wt wt wt wt wt - wt V466D -- -- -- -- -- -- -- wt V466E -- -- -- -- -- -- -- wt V466F wt -- -- wt - - wt wt V466G wt -- -- wt wt wt - wt V466H -- -- -- -- -- -- -- wt V466I wt -- ++ wt wt wt + wt V466K -- -- -- -- -- -- -- -- V466L wt -- wt + wt wt ++ wt V466M -- -- -- -- -- -- -- wt V466N -- -- -- -- -- -- -- wt V466P wt -- -- wt wt wt + wt V466Q wt wt -- - -- -- -- wt V466R -- -- -- -- -- -- -- wt V466S wt -- -- +++ ++ ++++ ++++ ++ V466T + + -- wt wt wt wt + V466V V466W -- -- -- -- -- -- -- wt V466Y -- -- -- -- -- -- -- wt A468A A468C wt wt -- ++ ++ +++ +++ + A468D wt - -- wt + wt wt - A468E wt -- wt wt wt wt + wt A468F wt - -- ++ +++ ++ ++ wt A468G wt ++ -- + wt wt wt wt A468H wt wt -- wt wt wt wt wt A468I wt -- -- + - ++ ++ wt A468K wt -- -- wt - wt wt wt A468L A468M -- ++ -- -- -- -- -- -- A468N - -- -- wt -- wt wt wt A468P wt -- -- -- -- -- -- -- A468Q wt -- -- + ++ wt + wt A468R wt -- wt wt -- wt wt wt A468S wt -- -- + ++++ + ++ wt A468T + - -- + ++++ wt wt wt A468V wt -- -- wt -- wt wt wt A468W wt wt -- wt ++ wt wt wt A468Y wt -- -- + +++ wt wt wt S482A wt -- -- ++ + +++ wt ++ S482C wt -- wt wt wt + + - S482D - -- -- -- -- -- -- ++ S482E +++ -- -- -- -- -- -- -- S482F -- -- -- -- -- -- -- -- S482G -- -- -- -- -- -- -- -- S482H wt -- -- -- -- -- -- -- S482I - - -- ++ - ++++ -- ++++ S482K -- -- -- -- -- -- -- -- S482L -- -- -- -- -- -- -- -- S482M -- -- -- -- -- -- -- -- S482N -- -- -- -- -- -- -- -- S482P -- -- -- wt - +++ -- ++++ S482Q -- -- -- -- -- -- -- -- S482R -- -- -- -- -- -- -- -- S482S S482T S482V wt -- -- wt -- - -- + S482W -- -- -- -- -- -- -- -- S482Y -- -- -- -- -- -- -- -- A485A A485C A485D wt -- -- wt wt wt wt wt A485E - -- -- wt - wt wt wt A485F wt -- -- - -- -- -- ++ A485G -- -- -- -- -- -- -- +++ A485H -- -- -- -- -- -- -- -- A485I wt -- -- wt wt wt - + A485K -- -- -- - -- - -- ++ A485L wt -- -- wt wt ++ -- ++ A485M wt -- -- wt wt wt -- + A485N -- -- -- -- -- -- -- +++ A485P wt - ++ wt - wt + wt A485Q wt wt -- -- -- -- -- wt A485R A485S -- -- -- -- -- -- -- +++ A485T - -- -- ++ + ++ - ++ A485V -- -- -- -- -- -- -- -- A485W -- -- -- wt -- ++ -- +++ A485Y wt -- -- -- -- -- -- -- I486A I486C wt + - wt wt wt wt + I486D -- -- -- -- -- -- -- +++ I486E -- -- -- -- -- -- -- wt I486F -- wt -- ++ wt wt ++ ++ I486G -- -- -- -- -- -- -- + I486H -- -- -- -- -- -- -- ++ I486I I486K -- -- -- -- -- -- -- +++ I486L - -- -- - -- -- -- wt I486M - -- -- wt - - + wt I486N -- -- -- -- -- -- -- ++ I486P I486Q -- -- -- -- -- -- -- ++ I486R -- -- -- -- -- -- -- +++ I486S -- -- -- -- -- -- -- ++ I486T I486V wt wt -- + wt + +++ ++ I486W - -- -- ++ -- wt - +++ I486Y -- ++ -- -- -- -- ++ ++ I491A wt -- -- -- -- -- -- -- I491C wt -- -- wt wt + + wt I491D wt wt -- -- -- -- -- -- I491E wt - -- -- -- -- -- -- I491F - -- -- wt wt + ++ wt I491G wt -- -- -- -- -- -- -- I491H wt -- -- wt - wt wt - I491I I491K -- -- -- -- -- -- -- -- I491L wt - -- wt wt wt wt + I491M - wt ++ wt wt wt wt wt I491N wt -- -- -- -- -- -- -- I491P wt -- -- -- -- -- -- -- I491Q wt wt -- -- -- -- -- -- I491R wt -- -- -- -- -- -- -- I491S wt -- -- -- -- -- -- -- I491T -- -- -- -- -- -- -- -- I491V wt -- -- wt wt + wt wt I491W I491Y + -- -- wt - wt - wt V500A V500C wt -- wt - wt - - - V500D -- -- -- -- -- -- -- wt V500E -- -- -- -- -- -- -- - V500F wt -- -- - - - -- + V500G -- -- -- -- -- -- -- -- V500H V500I wt - ++ wt wt wt wt - V500K -- -- -- -- -- -- -- wt V500L wt wt wt wt - - - + V500M wt -- - - - - - wt V500N -- -- -- -- -- -- -- ++ V500P -- -- -- -- -- -- -- + V500Q -- -- -- wt -- ++++ -- ++ V500R -- -- -- -- -- -- -- wt V500S wt -- -- - -- -- -- ++ V500T - -- -- - -- - -- + V500V V500W -- -- -- -- -- -- -- - V500Y -- -- -- -- -- -- -- wt S507A S507C wt -- -- -- -- -- -- wt S507D -- -- -- -- -- -- -- - S507E -- -- -- -- -- -- -- wt S507F + ++ -- -- -- -- -- wt S507G + -- ++ + wt + - wt S507H -- -- -- -- -- -- -- wt S507I -- -- -- -- -- -- -- - S507K -- -- -- -- -- -- -- wt S507L -- -- -- -- -- -- -- - S507M -- -- -- -- -- -- -- -- S507N -- -- -- - -- wt -- wt S507P S507Q ++ -- -- -- -- wt wt - S507R -- -- -- -- -- -- -- - S507S S507T - -- -- wt wt wt wt wt S507V -- -- -- -- -- -- -- wt

S507W -- -- -- -- -- -- -- - S507Y wt - ++++ wt wt wt ++ - Y530A wt - - wt wt + wt wt Y530C -- -- ++++ wt wt wt + wt Y530D -- -- -- -- -- -- -- - Y530E - -- +++ wt wt wt wt wt Y530F wt wt ++ + wt ++ ++ + Y530G - -- -- wt wt ++ wt wt Y530H wt -- ++ wt wt wt wt - Y530I -- -- wt wt wt + ++ wt Y530K ++ - -- -- -- -- -- - Y530L wt -- -- wt wt wt - + Y530M -- -- +++ wt wt wt + wt Y530N - -- -- wt wt wt -- wt Y530P Y530Q Y530R wt -- -- - wt wt -- wt Y530S + wt -- wt wt + - + Y530T wt -- -- wt wt + wt + Y530V wt -- -- wt wt + wt wt Y530W - -- ++ - - -- -- - Y530Y I532A I532C wt -- -- -- wt -- -- ++ I532D -- -- -- -- -- -- -- + I532E -- -- -- -- -- -- -- ++++ I532F wt wt -- wt wt wt -- ++ I532G -- -- -- -- -- -- -- +++ I532H -- -- -- -- -- -- -- + I532I I532K -- -- -- -- -- -- -- + I532L - -- -- wt wt wt ++ wt I532M - -- -- wt wt wt wt wt I532N I532P -- -- -- -- -- -- -- +++ I532Q -- -- -- -- -- -- -- ++ I532R -- -- -- -- -- -- -- +++ I532S -- -- -- -- -- -- -- +++ I532T -- -- -- - -- -- -- +++ I532V wt - - wt wt wt wt + I532W wt -- -- -- -- -- -- +++ I532Y wt -- -- wt -- ++ -- +++ P536A P536C wt + -- +++ ++ +++ ++++ + P536D wt wt -- + + + wt ++ P536E wt - -- ++ + ++ wt + P536F -- -- -- + - wt -- ++ P536G wt + - wt wt + wt + P536H - -- -- wt -- - -- + P536I - -- -- + wt ++ ++ + P536K -- -- -- -- -- -- -- -- P536L - -- -- -- -- -- -- -- P536M -- -- -- -- -- -- -- -- P536N -- -- -- -- -- -- -- -- P536P P536Q - + -- + wt + ++ wt P536R -- -- -- -- -- -- -- -- P536S wt -- -- wt - -- -- ++ P536T -- wt -- wt wt + -- ++ P536V wt wt wt + wt ++ ++ + P536W wt -- -- wt wt + wt + P536Y - wt -- wt wt - wt wt S550A - wt - wt wt - wt wt S550C wt wt - wt wt + + wt S550D wt wt -- wt wt wt wt + S550E wt ++ -- wt wt wt wt wt S550F wt + -- wt wt wt + wt S550G wt wt -- wt wt wt wt wt S550H wt wt -- wt wt wt wt + S550I - wt -- + wt + ++ ++ S550K wt wt -- wt wt wt - + S550L S550M wt wt -- wt - wt + wt S550N wt + -- wt wt wt wt wt S550P wt wt wt - - - wt wt S550Q wt + -- wt - wt - ++ S550R - wt -- wt -- - wt + S550S S550T wt wt -- + wt + ++ + S550V wt wt -- ++++ wt ++++ ++++ +++ S550W - -- -- wt - - wt wt S550Y F556A F556C wt - -- + -- wt wt wt F556D wt wt -- wt wt wt wt - F556E wt wt -- wt ++ + wt wt F556F F556G wt ++ -- wt ++ wt wt wt F556H wt -- -- + +++ wt wt wt F556I wt -- -- ++ -- wt wt wt F556K wt -- -- + ++ wt wt - F556L wt - -- ++ - + wt + F556M -- -- -- wt ++ wt ++ wt F556N wt - -- wt wt wt wt wt F556P -- -- -- -- -- -- -- -- F556Q -- -- -- -- -- -- -- -- F556R wt wt -- + wt wt wt wt F556S wt - -- wt ++ wt - wt F556T F556V wt wt -- + ++ + - wt F556W wt - - wt - - - - F556Y wt wt - wt wt wt wt wt A565A A565C -- -- + wt + ++ ++ - A565D wt wt +++ wt wt wt - wt A565E wt - +++ wt wt + + wt A565F wt - -- wt + ++ ++ wt A565G -- -- -- + ++ ++ ++ + A565H -- - -- wt wt wt wt wt A565I -- -- wt wt wt + ++ wt A565K - wt -- wt ++ +++ + wt A565L -- -- ++ wt wt wt wt wt A565M wt wt -- -- - - -- wt A565N - -- ++ wt wt wt wt wt A565P -- -- -- -- -- -- -- wt A565Q - wt wt wt + ++ + wt A565R -- -- -- -- -- -- -- wt A565S wt wt +++ wt wt + + wt A565T - - +++ wt wt wt + wt A565V - - - wt + + + wt A565W - wt ++ wt wt wt wt wt A565Y wt -- ++ wt wt + ++ wt N566A wt - -- wt + wt + wt N566C N566D wt + -- wt wt wt wt - N566E wt wt -- wt - wt + wt N566F - -- -- + ++ wt + + N566G wt ++ ++ wt - + wt wt N566H - -- -- + ++++ + ++ - N566I wt -- -- wt +++ wt + wt N566K wt -- -- wt wt wt ++ - N566L - - -- + +++ wt wt wt N566M -- -- -- -- -- -- -- -- N566N N566P wt wt -- + +++ wt -- wt N566Q - -- -- wt wt wt ++ wt N566R wt - wt wt wt wt wt - N566S wt - wt wt - wt wt wt N566T N566V N566W wt wt -- + ++++ wt wt wt N566Y -- -- -- -- -- -- -- -- I567A -- -- -- -- -- -- -- -- I567C wt wt - wt wt ++ wt - I567D wt wt -- wt wt wt -- wt I567E wt wt -- wt wt + wt + I567F wt wt - wt + ++ wt + I567G -- -- -- -- -- -- -- - I567H -- -- -- -- -- -- -- -- I567I I567K wt - +++ wt wt ++ + wt I567L -- -- -- -- -- -- -- wt I567M -- - wt wt wt ++ ++ wt I567N -- -- -- wt - wt - wt I567P -- -- -- -- -- -- -- wt I567Q -- - -- wt + ++ ++ wt I567R - - ++ wt wt + wt - I567S wt -- -- wt + wt wt ++ I567T wt wt wt wt wt + wt wt I567V wt + ++++ wt wt + wt wt I567W -- -- -- -- -- -- -- -- I567Y + ++ -- wt wt wt -- wt T568A wt -- ++ wt wt + wt wt T568C wt -- -- - -- -- -- wt T568D -- -- -- -- -- -- -- -- T568E wt ++ +++ wt + + wt wt T568F -- -- -- -- -- -- -- - T568G - - wt wt wt wt wt wt T568H - wt wt wt wt wt wt wt T568I T568K -- ++ + + ++ + ++ wt T568L wt wt + wt wt wt - wt T568M wt + wt wt wt wt wt wt T568N T568P wt - -- - wt -- -- wt T568Q - - wt wt wt - wt wt T568R wt wt ++ wt wt wt wt - T568S wt wt ++ - - - wt wt T568T T568V -- -- -- -- -- -- -- -- T568W wt wt -- - wt - -- - T568Y -- -- -- -- -- -- -- wt Y575A wt ++ -- + - -- - ++ Y575C wt +++ -- - -- -- -- ++ Y575D -- -- -- -- -- -- -- ++ Y575E -- -- -- -- -- -- -- -- Y575F Y575G Y575H -- -- -- -- -- -- -- wt Y575I Y575K -- + -- ++ -- - + +++ Y575L wt wt -- - - -- -- ++ Y575M -- -- -- -- -- -- -- -- Y575N -- -- -- -- -- -- -- +++ Y575P -- -- -- -- -- -- -- +++ Y575Q -- -- -- -- -- -- -- ++ Y575R -- + -- wt -- -- -- ++ Y575S -- -- -- -- -- -- -- ++ Y575T -- -- -- -- -- -- -- ++ Y575V -- -- -- - -- -- -- ++ Y575W wt - -- wt - wt ++ + Y575Y A601A A601C wt wt -- ++ + ++ ++ + A601D wt ++ ++ + wt wt wt wt A601E wt wt - wt - wt wt wt A601F A601G wt ++ -- wt wt wt -- ++ A601H wt + -- wt wt wt - wt A601I wt wt wt - - - - wt A601K wt wt wt wt wt - wt wt A601L wt + -- wt wt wt wt + A601M wt wt -- + wt + wt ++ A601N wt - -- wt wt wt + wt A601P wt wt -- wt wt wt wt + A601Q -- -- -- -- -- -- -- ++ A601R -- -- -- -- -- -- -- wt A601S A601T -- -- -- -- -- -- -- ++ A601V -- -- -- -- -- -- -- ++ A601W wt wt -- wt wt wt + + A601Y wt wt -- + + + + ++ V602A wt - wt wt wt wt - wt V602C wt wt ++ wt wt wt - - V602D wt wt -- - - -- -- - V602E wt wt -- - - wt -- ++ V602F wt + -- wt wt wt wt + V602G wt -- -- wt wt + + wt V602H wt - wt - wt - -- - V602I - -- wt wt wt - - - V602K wt + ++ - - - -- -- V602L - -- -- wt wt + ++ wt V602M - -- wt - wt - wt wt V602N - -- -- wt wt wt ++ + V602P wt - -- wt - - - wt V602Q wt wt -- - - - -- - V602R wt -- wt - wt -- - - V602S wt wt -- wt wt wt wt wt V602T - - -- wt wt + + + V602V V602W wt wt -- wt - - -- wt V602Y wt - -- wt - - -- - P604A P604C wt wt ++ wt wt ++ ++ - P604D P604E wt ++ -- wt ++++ wt - wt P604F wt ++ -- -- -- -- -- -- P604G wt wt -- ++ wt wt wt wt P604H wt - -- wt + wt - - P604I wt wt -- wt wt wt wt - P604K - -- -- ++ - wt wt -

P604L wt wt -- wt wt wt wt wt P604M wt wt -- + wt + + wt P604N wt - -- + ++++ wt wt wt P604P P604Q - - -- wt wt wt + - P604R P604S wt - -- wt + wt wt wt P604T wt - ++ wt -- wt + wt P604V wt ++ -- wt ++ wt wt wt P604W wt -- -- wt -- wt -- wt P604Y wt wt -- + ++++ + wt - G605A - -- -- -- -- -- -- wt G605C + - -- wt +++ ++++ - + G605D -- -- -- -- -- -- -- -- G605E -- -- -- -- + +++ -- + G605F -- -- -- -- ++ -- -- wt G605G G605H ++ -- -- -- -- -- -- - G605I -- -- -- -- -- -- -- - G605K wt -- -- -- -- -- -- wt G605L - -- -- -- wt - -- + G605M -- - -- -- - wt -- wt G605N -- -- -- -- -- -- -- - G605P G605Q - -- -- -- -- -- -- wt G605R wt - -- wt ++ ++++ -- wt G605S wt -- -- -- wt wt + + G605T wt wt -- -- + -- -- wt G605V -- -- -- -- -- -- -- wt G605W -- -- -- -- -- -- -- wt G605Y wt -- -- -- -- -- -- wt G606A wt -- -- -- - -- -- ++ G606C wt -- -- - wt -- ++ + G606D ++ -- -- -- - -- + + G606E wt -- -- -- + - ++ + G606F -- -- -- -- -- -- -- wt G606G G606H wt - -- -- + - wt ++ G606I wt -- -- - ++ ++ + ++ G606K wt -- -- - ++ +++ wt + G606L wt - -- - + + ++ ++ G606M wt -- -- - ++ + ++ ++ G606N wt wt -- -- + wt - + G606P + -- -- -- -- -- -- wt G606Q wt -- -- - ++ ++++ - ++ G606R -- -- -- -- -- -- -- + G606S wt - -- -- + wt wt + G606T -- -- -- -- -- -- -- -- G606V - -- -- -- ++ ++ ++ ++ G606W wt -- -- -- wt wt -- + G606Y wt - -- -- wt wt -- ++ P607A -- -- -- -- -- -- -- -- P607C wt wt -- + wt ++ ++ wt P607D wt wt -- wt wt ++ + ++ P607E wt - -- wt ++ ++ + wt P607F + +++ -- wt ++ ++ wt wt P607G wt ++ -- wt + ++ + ++ P607H wt + -- wt wt + wt wt P607I - + -- ++ wt +++ ++ ++ P607K ++ ++ -- wt wt + wt wt P607L wt wt -- wt + + wt + P607M wt - -- wt wt wt wt wt P607N wt wt wt wt wt + wt wt P607P P607Q wt ++ -- wt wt ++ + + P607R - + -- wt wt wt wt ++ P607S wt + -- wt + + wt wt P607T wt -- - wt wt wt wt wt P607V P607W wt wt -- -- -- -- -- wt P607Y wt - -- wt wt wt wt + S624A - -- ++ wt wt - wt wt S624C - -- -- wt wt wt + wt S624D wt -- - wt + wt + wt S624E - -- -- + wt + ++ wt S624F wt -- -- ++ + ++ ++ + S624G -- -- -- + wt + ++ + S624H - - -- wt wt wt wt + S624I -- -- -- ++ ++ ++ +++ + S624K - wt - wt wt wt + wt S624L - -- -- + wt wt wt + S624M S624N -- -- -- + wt + wt + S624P wt -- -- wt + ++ -- + S624Q wt wt -- + - + - + S624R wt wt -- + wt wt wt + S624S S624T - - -- + wt ++ wt + S624V -- -- -- ++ + +++ wt + S624W -- -- -- + wt wt wt + S624Y - -- - wt wt - wt wt A630A A630C - wt -- + + +++ ++ + A630D wt -- -- ++ + ++ ++ + A630E A630F -- -- -- -- -- -- -- -- A630G wt -- -- + + ++ + wt A630H + ++ -- wt wt wt wt + A630I -- -- -- -- -- -- -- -- A630K - + ++ wt wt + wt - A630L -- -- -- -- -- -- -- -- A630M - - -- wt wt ++ ++ wt A630N wt wt -- wt wt + wt wt A630P A630Q wt + -- + ++ +++ ++ + A630R wt ++ - wt wt wt wt wt A630S wt wt -- wt wt ++ + + A630T wt wt -- wt wt ++ ++ + A630V -- -- -- ++ -- ++++ -- + A630W ++ + -- -- -- -- -- - A630Y + ++ -- wt wt ++ + ++ A633A A633C + wt -- ++ ++ ++ + + A633D -- -- -- -- -- -- -- wt A633E -- -- -- -- -- -- -- wt A633F -- -- -- -- -- -- -- -- A633G -- -- -- -- -- -- -- -- A633H -- -- -- -- -- -- -- wt A633I wt - -- wt wt + wt + A633K -- -- -- -- -- -- -- wt A633L wt - - wt wt ++ ++ wt A633M -- -- -- -- -- -- -- wt A633N -- -- -- -- -- -- ++ wt A633P wt wt -- - wt -- wt + A633Q -- -- -- -- -- -- -- wt A633R -- -- -- -- -- -- -- wt A633S wt -- -- - wt - ++ wt A633T wt - -- wt wt + + wt A633V wt wt wt wt + ++ ++ + A633W A633Y -- -- -- -- -- -- -- wt Y639A -- -- -- -- -- -- -- -- Y639C -- -- -- -- -- -- -- -- Y639D Y639E Y639F + wt - wt wt wt - wt Y639G + - ++ wt wt + wt wt Y639H Y639I wt wt -- wt wt wt wt - Y639K wt ++ ++ wt wt + wt wt Y639L wt -- + wt wt + + wt Y639M wt - +++ wt wt + wt - Y639N wt - -- wt - wt - wt Y639P + ++ -- wt wt wt - - Y639Q wt wt - wt wt wt wt - Y639R wt + -- wt wt wt - wt Y639S wt -- wt wt wt wt + wt Y639T wt ++ -- wt + wt - wt Y639V + wt - wt + + wt wt Y639W wt wt wt wt wt wt - - Y639Y T646A wt wt ++ wt wt + wt wt T646C wt wt ++ wt wt + wt wt T646D -- -- -- -- -- -- -- -- T646E wt wt -- wt - wt + wt T646F wt wt -- wt wt wt wt wt T646G wt ++ -- wt wt wt wt wt T646H - -- -- ++ -- ++++ -- wt T646I T646K wt -- -- -- -- -- -- -- T646L - -- -- wt wt wt ++ wt T646M -- -- -- -- -- -- -- -- T646N wt - - wt wt wt + wt T646P wt wt -- wt wt wt -- - T646Q wt wt -- wt wt + - wt T646R wt wt -- wt wt + wt - T646S wt + -- wt wt wt wt wt T646T T646V wt wt -- wt wt wt + wt T646W T646Y wt wt -- wt wt wt + wt A655A A655C wt wt -- wt + + wt wt A655D + + ++ wt wt + - wt A655E wt wt wt wt wt + wt wt A655F A655G wt wt -- wt ++ + + wt A655H + wt ++ wt wt wt - wt A655I A655K wt - - wt wt wt wt wt A655L wt wt -- + wt ++ + wt A655M wt wt wt wt wt + wt wt A655N wt +++ -- wt + + + wt A655P -- -- -- -- -- -- -- wt A655Q wt ++ -- wt wt ++ ++ + A655R wt wt - + + + + wt A655S wt ++ - wt wt wt wt + A655T wt -- -- wt wt wt wt + A655V wt wt wt wt wt wt wt wt A655W wt -- -- wt + + + + A655Y + + ++ wt wt wt - wt A667A A667C A667D -- -- -- -- -- -- -- -- A667E -- -- -- wt - wt -- + A667F wt -- -- ++ + ++ wt + A667G wt -- -- + wt wt + + A667H - wt - wt - - - wt A667I - wt wt wt - - - wt A667K -- -- -- wt wt wt - ++ A667L wt - -- ++ + ++ - ++ A667M - wt -- wt - - - wt A667N -- -- -- wt - -- -- wt A667P -- -- -- wt wt wt -- ++ A667Q A667R - wt -- + + ++ -- ++ A667S -- -- -- + wt wt wt + A667T - - -- wt wt wt wt wt A667V - wt -- wt wt + -- + A667W -- -- -- - - -- - + A667Y - -- -- ++ ++ ++ -- ++ I671A wt + -- wt wt wt wt wt I671C wt wt +++ + + ++ wt wt I671D -- -- -- -- -- -- -- - I671E -- -- -- -- -- -- -- wt I671F wt - +++ wt wt ++ wt wt I671G -- -- -- -- -- -- -- wt I671H wt wt - wt wt wt wt wt I671I I671K wt + - wt + + - wt I671L wt wt ++++ wt wt + + wt I671M -- -- -- -- -- -- -- - I671N -- -- -- -- -- -- -- wt I671P -- -- -- -- -- -- -- wt I671Q -- -- -- -- -- -- -- wt I671R -- -- -- -- -- -- -- -- I671S -- -- -- -- -- -- -- wt I671T + wt -- -- -- -- -- wt I671V -- -- -- -- -- -- -- wt I671W -- -- -- -- -- -- -- - I671Y -- -- -- -- -- -- -- wt Y678A - -- -- ++ + ++ + + Y678C - wt -- ++ + ++ ++ + Y678D -- -- -- wt - - -- ++ Y678E -- - -- wt - - -- wt Y678F wt wt -- ++ wt ++ ++ wt Y678G - wt -- wt wt wt -- wt Y678H - -- -- wt + ++ -- ++ Y678I - wt -- + wt ++ ++ + Y678K -- - -- wt wt - -- ++ Y678L -- -- wt wt wt - wt wt Y678M - -- - wt - - wt wt Y678N - -- - wt - - wt wt Y678P -- -- -- -- -- -- -- -- Y678Q - wt -- ++ + ++ wt ++ Y678R - - -- wt wt +++ -- ++ Y678S - wt -- wt wt - -- wt Y678T - - -- wt - wt wt wt Y678V wt wt + wt wt wt - wt Y678W - wt -- wt wt -- -- + Y678Y

++++ PI >2 +++ 2 > PI > 1.5 ++ 1.5 > PI > 1.2 + 1.2 > PI > 1.1 wt 1.1 > PI > 0.9 - 0.9 > PI > 0.8 -- 0.8 > PI

[0416] Some variants of interest were manually selected. Table 4-3 includes 35 additional such variants from the second SEL library.

TABLE-US-00010 TABLE 4-3 Variant Inh Heat HPLC PASC PCS G2 CNPG CC L266Y wt wt -- wt + wt wt wt I567S wt -- -- wt + wt wt ++ A270D wt wt -- wt ++ wt wt wt S550D wt wt -- wt wt wt wt + T258S wt wt -- ++ ++ ++ ++ + P536D wt wt -- + + + wt ++ P536V wt wt -- + wt ++ ++ + F260D -- + -- wt ++ ++ ++ + F260G -- ++ -- wt ++ ++ ++ + Y530F wt wt -- + wt ++ ++ + S624N -- -- -- + wt + wt + P607Q wt ++ -- wt wt ++ + + G606M wt -- -- - ++ + ++ ++ Q406H wt wt -- - wt ++ +++ wt N400Q wt ++ -- wt wt wt - + G300M wt -- -- wt wt ++ wt + N038L ++ wt -- -- -- -- -- wt N038M ++ wt -- -- -- -- -- wt A601Y wt wt -- + + + + ++ L293V wt - -- + ++ ++ ++ wt T568K -- ++ -- + ++ + ++ wt S308E ++ + -- wt ++ wt wt ++ A630Y + ++ -- wt wt ++ + ++ N461D wt wt -- + + wt wt wt N146D wt +++ -- wt + wt - wt A450E wt - -- + + ++ ++ ++ V043L +++ - -- wt wt wt -- wt Q220A wt -- -- -- -- -- -- -- A655Q wt ++ -- wt wt ++ ++ + S482A wt -- -- ++ + +++ wt ++ A667L wt - -- ++ + ++ - ++ A485T - -- -- ++ + ++ - ++ K206A wt -- -- + ++ + ++ wt Y678Q - wt -- ++ + ++ wt ++ ++++ PI > 2 +++ 2 > PI > 1.5 ++ 1.5 > PI > 1.2 + 1.2 > PI > 1.1 wt 1.1 > PI > 0.9 - 0.9 > PI > 0.8 -- 0.8 > PI

[0417] The results of the substitutions from the first (Example 3) and second (Example 4) SEL screens were analyzed for various activities as described above and grouped accordingly to those variants that had two, three, four, five, or six (or more) activities. Variants possessing these multiple activities are shown below in Table 4-4 to Table 4-7:

TABLE-US-00011 TABLE 4-4 Variants with Two Improved Activities PCS + HPLC + PCS + Gluc + G2 + Heat + PASC + PASC + Heat + Heat + PASC + Gluc + Gluc + Gluc + G2 G2 CC Heat CC HPLC G2 PCS PCS CC CC HPLC CC PSC I567Q I567K I567S L266F L266A N261E N261C N566L F556G S550Q P536F S384G G606D R179V A565F I567R G606E I567Y I567E N261K T258C N566P F260S P607R F392C S384W Y068V A565K A565E G606H A270R S283F N400A F392Q N566W P604E N400Q S624L N038E A565Q A565S G606N S384C S283P V602K S624E A270K P604V V602F S624R N038M Gluc + G2 A565V A565Y G606S A630W T258E L293I P607C A270N N146D A601G S624W N038P A377I F556E F392Y L293A E128R T258I N461S P604M F556H Y639T A601L I486F V043H N461Y F260I Q406H S308R N146M T258K D457A A377Q F556K T221C L293K I486W V043W P607E Q406T I444C N146V T258Q V043Q N461A P604N N473S Y575C A667G Y068E HPLC + PASC G605R P604C M201D N146W P536T Q303N N461F N461D N583R Y575R A667S Y068G K206D G300C N038F R542N L181F P536W K320S N461P N463E R645G A450Q Y068M A377C T568A V043C I532Y G662D T436A K206G G662Y I486C L110C Heat + PASC A377D N461G Y639P Y530T T436C A468Q I486Y L110G A468G S308C Y639L S507F P607D Heat + T436F A468Y A655S L110Q G2 N146H Y639M Q245P Q406M P607H T436I Q245F L110W N146S T243A Q406S T011E T436M D329A A655H A655C T243C HPLC + CC V602T T011Y T436Q N264L A655G Q245H D259S G300M N146E T436Y P176L Q245M T243V A630S Q220C T209I Q245T A630T A655L T646A T180H T646H HPLC + T646C T180M Y678F PCS S283D I671F A450M A468I A270D I671L I444E D177M N146Y I444F P661E I444N I444W I444Y V500Q A633I S482P A667V A485L A485W Y678R V603G

TABLE-US-00012 TABLE 4-5 Variants with Three Improved Activities Heat + HPLC + PCS Heat + HPLC + G2 PASC + PCS + G2 Heat + PASC + CC Gluc + Heat + HPLC Heat + PCS + G2 F260A I567V N566H Y575A S384E F260T S474R N566G F556V Y575K L181M P607S D564T A630K P604Y Gluc + Heat + CC V043A A655N PASC + PCS + CC Y639K L293V A630H V043G I671K N566F Q245N A630G V466T V043N Gluc + PASC + PCS HPLC + PCS + G2 K320Y N461C Gluc + Heat + G2 Q060D A468T A565C A347Y N463T P607K A655Y Heat + PCS + CC Heat + G2 + CC Heat + PASC + G2 D457C N146A T242S S692L P536G P536Q Q220M N146Q S474D Gluc + PCS + G2 P607Q N369E T221A N369T Gluc + HPLC + PCS Y639V A655Q N369W T221G Gluc + PASC + G2 K206S Heat + HPLC + PASC N369Y T221I T436E Gluc + G2 + CC A601D Gluc + PCS + CC A655R Gluc + HPLC + G2 Y530S L293M A468F Y639G Q684N Q220P A468S Q216I D564V

TABLE-US-00013 TABLE 4-6 Variants with Four Improved Activities Heat + PASC + G2 + CC Gluc + PASC + PCS + G2 P607I E170F A450P Heat + HPLC + PCS + CC T242H A338D Heat + HPLC + PCS + G2 Gluc + Heat + PCS + CC T568E S308E Gluc + Heat + G2 + CC Gluc + HPLC + PASC + G2 A630Y S507G Gluc + Heat + HPLC + G2 A655D

TABLE-US-00014 TABLE 4-7 Variants with Five Improved Activities Heat + HPLC + PCS + PASC + G2 Heat + PASC + PCS + G2 + CC F260E P536C T568K A630Q Heat + HPLC + PCS + G2 + CC D215S F260L G372A Gluc + PASC + PCS + G2 + CC G547A A633C F611A S312C G662C N455D G662F Gluc + Heat + PASC + G2 + CC L293F

[0418] In summary, Table 4-8 lists all variants having two or more improved activities selected from (1) Heat (thermostability), (2) HPLC (protein expression), (3) PCS, (4) (PASC), (5) G2 (cellobiohydrolase activity), (6) beta-glucosidase activity measured by G2+CC or CC hydrolysis.

TABLE-US-00015 TABLE 4-8 I567Q I567K I567S L266A N261C N566L S550Q S384G F260T A565F I567R G606E I567E T258C N566P P607R S384W P607S A565K A565E G606H S283F F392Q N566W N400Q N038E A655N A565Q A565S G606N S283P S624E A270K V602F N038M I671K A565V A565Y G606S T258E P607C A270N A601G N038P P607I F556E F392Y L293A T258I P604M F556H A601L V043H A450P F260I Q406H S308R T258K A377Q F556K L293K V043W T242H P607E Q406T I444C T258Q N461A P604N Y575C Y068E T568E G605R P604C M201D P536T N461F N461D Y575R Y068G A630Y G300C N038F R542N P536W N461P N463E A450Q Y068M A655D A377C T568A D259S I532Y T436A K206G I486C L110C E170F A377D N461G T243V Y530T T436C A468Q I486Y L110G A338D S308C Y639L L266F P607D T436F A468Y A655S L110Q S308E N146H Y639M I567Y Q406M T436I F556G Q245F L110W S507G N146S T243A A270R Q406S T436M F260S D329A A655H F260E A655C T243C S384C V602T T436Q P604E P536F N264L T568K A655G Q245H A630W G300M T436Y P604V F392C N566H F260L P176L Q245M E128R A630S Q220C N146D S624L F556V A633C T209I Q245T N146M A630T A655L Y639T S624R P604Y S312C S283D T646A N146V T180H T646H T221C S624W L293V N455D A270D T646C N146W T180M Y678F N473S I486F A630G P536C N146Y I671F L181F A450M A468I N583R I486W N461C A630Q N261E I671L V043C I444E D177M R645G A667G N463T D215S N261K P607H Y639P I444F P661E G662Y A667S D457C G372A N400A T011E S507F I444N P536G P536Q S384E Q220M G547A V602K T011Y Q245P I444W P607Q N369E L181M T221A F611A L293I N146E R179V I444Y A655Q N369W V043A T221G G662C N461S G606D F260A V500Q I567V N369Y V043G T221I G662F D457A Y068V S474R A633I N566G P607K V043N A655R L293F V043Q A377I D564T S482P A630K N146A Q060D A468F Q303N N461Y N566F A667V Y639K N146Q A655Y A468S K320S K206D A565C A485L Q245N N369T T242S Q216I G662D A468G A601D A485W K320Y Y639G S474D D564V L293M Y575A A630H Y678R A347Y K206S Y530S S692L Q220P Y575K V466T V603G T436E A468T Q684N Y639V

Example 5

BGL1 Combinatorial Library Variants and Activities Thereof

5.1 Assays:

HPLC Assay for Protein Content Determination

[0419] The concentration of each BGL polypeptide (wild type or variant) in pooled culture supernatants was determined using an Agilent 1200 (Agilent Technologies) HPLC equipped with a Shodex HIC PH-814 PHM gel 75.times.8 mm column (Phenomenex) equilibrated at 35.degree. C. Forty five (45) .mu.L of a supernatant was incubated with 15 .mu.L of 80 ppm recombinantly expressed S. plicatus glycosidase EndoH (e.g. NEB P0702L) in 200 mM of sodium acetate buffer, at pH 5.0, and incubated at 37.degree. C. overnight with shaking at 900 rpm. Sixty (60) .mu.L 1.6 M (NH.sub.4).sub.2SO.sub.4 was added to the supernatant, and after 5 min, the mixture was filtered under vacuum using a 0.22 .mu.m Millipore Multiscreen HTS 96 well filtration system. Forty (40) .mu.L of the filtered sample was loaded onto the column. Two elution buffers were employed to build an elution gradient: (1) Buffer A: 16 mM NaH.sub.2PO.sub.4, pH 6.75, 800 mM (NH.sub.4).sub.2SO.sub.4; and (2) Buffer B: 16 mM NaH.sub.2PO.sub.4, pH 6.75. Elution was carried out at a flow rate of 1.8 mL/min, using the following program: 0% buffer B from 0 min to 0.5 min, followed by a gradient of 0% buffer B to 50% from 0.5 min to 1 min, followed by 50% buffer B to 100% from 1 min to 6 min, followed by 100% buffer B from 6 to 8 min. Protein concentrations of BGL variants were determined using a calibration curve generated with purified wild-type BGL1. To calculate performance index (PI), the concentration of a BGL variant was divided by the average concentration of wild-type BGL1 (e.g., a reference enzyme) in the same plate.

Using CNPGase Activity Assay to Determine Required Sample Dilution for Assays

[0420] The activity of the BGL variants towards chloro-nitrophenol-.beta.-D-glucoside (CNPG) was measured to determine the BGL1 production levels. Five (5) .mu.L of supernatant were added to 95 .mu.L of 1 mM CNPG in a 50 mM sodium acetate buffer, pH5, and OD.sub.405 readings were recorded in a microplate reader for 3 min. Based on the CNPG activities, and relative to the activity of a wild type BGL1 control, the supernatants were diluted to a level of between 25 and 300 ppm BGL1.

CNPGase Activity Assay

[0421] The activity of the BGL variants towards chloro-nitrophenol-.beta.-D-glucoside (CNPG) was determined. Culture supernatants expressing BGL variants were diluted 5, 6.67, 10 and 20-fold in a 50 mM sodium acetate buffer, pH 5.0, containing 0.1 mg/mL bovine serum albumin (BSA). Fifty (50) .mu.L aliquots of diluted supernatants were added to 50 .mu.L of 2 mM CNPG in a 50 mM sodium acetate buffer, pH 5.0, achieving a final concentration of 1 mM CNPG. Kinetics of CNP release was determined by monitoring 013405, which was recorded in a microtiter plate reader (Spectramax, Molecular Devices) for 3 min. Average specific activities for the wild-type BGL1 and BGL variants were calculated by dividing the averaged CNPG hydrolyzing activity by the BGL polypeptide concentration. A performance index (PI) was calculated by dividing the specific activity of a BGL variant by the average specific activity of wild-type BGL1 (e.g., a reference enzyme) on the same plate.

Thermostability Assay

[0422] Residual activity of BGL1 variants after heat incubation was determined using the CNPG assay. Culture supernatants expressing BGL variants were diluted 5, 6.67, 10 and 20-fold in a 50 mM sodium acetate buffer, pH 5.0, containing 0.1 mg/mL BSA. Eighty (80) .mu.L aliquots were incubated in quadruplicate in a skirted 96-well PCR plate in a thermocycler at 66.degree. C. for 1 hr. After 5 min of cooling on ice, the residual specific activity of each of the wild type and BGL1 variants was determined as described above. The residual activity of the variants and the wild type BGL1 was determined by the ratio of the averaged specific activity after incubation and the averaged specific activity before incubation. A performance index (PI) for the BGL variants was determined by dividing the residual activity of a BGL1 variant by the relative residual activity of the wild-type BGL1 (e.g., a reference enzyme).

Glucose Inhibition Assay

[0423] The effect of glucose on the hydrolytic activity of beta-glucosidase was determined by repeating the CNPGase activity assay as described above in the presence of 18.75 mM glucose. The relative residual activity of the variants and the wild-type protein was determined by the ratio of the averaged specific activity in the presence of glucose and the averaged specific activity in the absence of glucose. A performance index (PI) for the BGL variants was determined by dividing the relative residual activity of a BGL variant by the relative residual activity of the wild-type BGL1 (e.g., a reference enzyme).

Specific Activity in a Phosphoric Acid Swollen Cellulose (PASC) Hydrolysis Assay

[0424] Phosphoric acid swollen cellulose (PASC) was prepared from Avicel according to published methods (see, e.g., Walseth, Tappi, 35:228, 1971; and Wood, Biochem. J., 121:353-362, 1971). This material was diluted with a sodium acetate buffer and water to achieve a 1% w/v mixture, wherein the final concentration of sodium acetate was 50 mM, and pH was 5.0. One hundred and fifty (150) .mu.L of a 1% suspension of PASC in a 50 mM sodium acetate buffer, pH 5.0, was dispensed into a 96-well microtiterplate (Costar Flat Bottom PS). Ten (10) .mu.L of a culture supernatant from a bgl1-deleted strain containing 0.75 mg/mL protein was added to the PASC. Then 5, 10, 20, or 40 .mu.L of 8-fold diluted (in 50 mM sodium acetate buffer pH 5.0) pooled culture supernatants from H. jecorina cells expressing either wild-type BGL1 or a BGL variant were added to the PASC/deletion mutant supernatant mixture. Compensating volumes of sodium acetate buffer were added to make up for differences in total volume. The microtiter plate was sealed and incubated in a thermostatted incubator at 50.degree. C. with continuous shaking at 900 rpm. After two hours, the hydrolysis reaction was stopped by the addition of 100 .mu.L of a 100 mM glycine buffer, pH 10, to each well. The plates were sealed and centrifuged at 3000 rpm at room temperature for 5 min. The hydrolysis reaction products in the supernatant were analyzed by the ABTS assay. A dose response curve was generated for wild-type BGL1 protein. To calculate performance index (PI), the (average) total sugar produced by a variant BGL was divided by the (average) total sugar produced by the wild-type BGL1 (e.g., a reference enzyme) at the same dose.

Specific Activity in a Dilute Acid Pretreated Corn Stover (PCS) Hydrolysis Assay

[0425] Corn stover was pretreated with 2% w/w H.sub.2SO.sub.4 (see, Schell et al., J Appl. Biochem. Biotechnol., 105:69-86, 2003), followed by multiple washes with deinonized water to obtain a paste of pH 4.5. A sodium acetate buffer (pH 5.0) was then added (to a final concentration of 50 mM sodium acetate) and, if necessary, this mixture was further titrated to pH 5.0 using 1 N NaOH. The cellulose concentration in the reaction mixture was approximately 7%. Sixty five (65) .mu.L of this cellulose suspension was added per well into a 96-well microtiter plate (Nunc Flat Bottom PS). Ten (10) .mu.L of a culture supernatant from a bgl1-deleted strain containing 10 mg/mL protein was added to the PCS. Then 5, 10, 20, or 40 .mu.L of 2-fold diluted (in a 50 mM sodium acetate buffer, pH 5.0) pooled culture supernatants from H. jecorina cells expressing either wild-type BGL1 or a BGL variant were added to the PCS/deletion mutant supernatant mixture. Compensating volumes of sodium acetate buffer were added to make up for the differences in total volume. After sealing, the plates were placed in a thermostatted incubator at 50.degree. C. with continuous shaking at 900 rpm. After 16 hours the plates were placed on ice for 5 min and the hydrolysis reaction was stopped by the addition of 100 .mu.L of a 100 mM glycine buffer, pH 10, to each well. The plates were sealed and centrifuged at 3,000 rpm for 5 min at room temperature. The hydrolysis reaction products that were present in the supernatants were analyzed by the ABTS assay (above). A dose response curve was generated from a purified wild-type BGL1. To calculate performance index (PI), the (average) total sugar produced by a variant BGL was divided by the (average) total sugar produced by the wild-type BGL1 (e.g., a reference enzyme) at the same dose.

Cellobiase Activity Assay

[0426] The cellobiose hydrolyzing capability of wild-type BGL1 and the BGL variants at pH 5.0 was tested. Varying amounts (5, 10, 15, or 20 .mu.L) of 4-fold diluted (in a 50 mM sodium acetate buffer, pH 5.0) pooled culture supernatants from H. jecorina cells expressing either wild-type BGL1 or BGL variants were added to 80 .mu.L of a 16.4 mM (5.63 mg/mL) cellobiose solution in a 50 mM sodium acetate buffer, pH 5.0. Compensating volumes of sodium acetate buffer were added to make up for the differences in the total volume. The microtiter plate was sealed and incubated in a thermostatted incubator at 50.degree. C. with continuous shaking at 900 rpm. After 30 min, the plates were cooled on ice and 100 .mu.L of a 100 mM glycine buffer, pH 10, was added to each well. The hydrolysis reaction products were analyzed by the ABTS assay (above). A dose response curve was generated using purified wild-type BGL1. To calculate performance index (PI), the (average) total sugar produced by a variant BGL was divided by the (average) total sugar produced by the wild-type BGL1 (e.g., a reference enzyme) at the same dose.

Specific Beta-Glucosidase Activity in an Ammonia Pretreated Corncob Hydrolysis Assay

[0427] Corn cob was ground to pass a 0.9 mm screen and pretreated as described in PCT Patent Application Publication WO 2006110901. Pretreated corn cob was used as a 7% cellulose suspension in a 50 mM sodium acetate buffer, pH 5.0. Sixty five (65) .mu.L of the suspension was added per well into a 96-well microtiter plate (Nunc Flat Bottom PS). Ten (10) .mu.L of a T. reesei strain overexpressing T. reesei endoxylanase gene xyn3, Fusarium verticillioides .beta.-xylosidase gene Fv3A, F. verticillioides .beta.-xylosidase gene Fv43D, and F. verticillioides .alpha.-arabinofuranosidase gene Fv51A containing 0.76 mg/mL protein in 50 mM sodium acetate buffer, pH 5.0, was added to the pretreated corn cob. Varying amounts (5, 10, 20, or 40 .mu.L) of pooled culture supernatants from H. jecorina cells expressing the wild-type BGL1 or BGL variants were added. Compensating volumes of sodium acetate buffer were added to make up for the differences in total volume. The microtiter plate was sealed and incubated in a thermostatted incubator at 50.degree. C. with continuous shaking at 900 rpm. After 24 hrs, the hydrolysis reaction was stopped by the addition of 100 .mu.L of a 100 mM glycine buffer, pH 10, to each well. After mixing, the plate was centrifuged for 5 min at 3,000 rpm. The hydrolysis reaction products were analyzed by the ABTS assay (above). A dose response curve was generated using purified wild-type BGL1. To calculate performance index (PI), the (average) total sugar produced by a variant BGL was divided by the (average) total sugar produced by the wild-type BGL1 (e.g., a reference enzyme) at the same dose.

5-2. Generation of Hypocrea jecorina BGL Combinatorial Variants

[0428] Combinatorial BGL variants were constructed or purchased from commercial vendors, e.g., Sloning Biotechnology GmbH (Puchheim, Germany), BaseClear (Leiden, The Netherlands). Table 5-1 lists substitutions that were selected for inclusion in BGL combinatorial libraries. The amino acid residue numbers were assigned in reference to the reference wild type BGL1 mature amino acid sequence, SEQ ID NO:3.

TABLE-US-00016 TABLE 5-1 BGL1 substitutions selected for construction of combinatorial variants. K51A Q226Y Q303R N369Y S550N G662F E92V N238F Q303N N369W G554C G662K L167W N238W S312D D370W G554F G662L E170F T242H S312I G372A K560S G662Y P176L T242S S312K G427C D564T T666C D177M N263C S312Y G427F D564V S683W D178I N263T S312C K428N N583R Q684A D178K N263S Q316T N455D V603G Q684C D178N N264D K320S N473S F611A Q684D R179K N264K K320Y S474D F611R Q684G R179S N264L D329A S474I R636E Q684N R179V N264M A338D S474R R645G Q684R S199T R265M A338I K498F R645K S692E T209I R265P A338K K498H K656R S692K D215S N278F K345E K498A P661E S692L Q216E T282D A347D D521A P661F Q216I T282I A347Y D521R P661L Q216K T282K N369E V522Y P661Q D225Q Q303E N369I R542N G662C Q226W Q303I N369T G547A G662D

[0429] Combinatorial variants derived from pTTTpyrG-bgl1 were generated in E. coli and plated onto 2.times.TY agar plates (16 g/L Bacto Tryptone (Difco, USA), 10 g/L Bacto Yeast Extract (Difco, USA), 5 g/L NaCl, 16 g/L Bacto Agar (Difco, USA)) with 100 .mu.g/mL ampicillin. After overnight incubation at 37.degree. C., E. coli colonies harboring the bgl1 variants were picked from the 2.times.TYagar plates containing 100 .mu.g/mL ampicillin and grown for 24 hr at 37.degree. C. in a microtiterplate containing 1 mL of a 2.times.TYmedium with 100 .mu.g/mL ampicillin and 50 .mu.g/mL kanamycin. Bacterial cultures were used for purification of plasmid DNA.

[0430] Purified pTTTpyrG-bgl1 derived plasmids encoding bgl1 combinatorial variants were used in H. jecorina transformations at concentrations of 150-300 ng/.mu.L. These replicative plasmids expressing bgl1 variants under the cbh1 promoter conferred transformed H. jecorina cells for growth on acetamide. Five (5) .mu.L of plasmids was used for fungal transformation as described in, for example, U.S. Patent Application Publication US2006/0094080 A1. Protoplasts of H. jecorina strain (.DELTA.eg1, .DELTA.eg2, .DELTA.cbh1, .DELTA.cbh2, .DELTA.bgl1) were transformed with individual pTTTpyrG-bgl1 constructs (i.e., including a single BGL1 variant per transformation) and grown in 24-well microtiter plates on selective medium containing acetamide at 28.degree. C. for 7 d.

[0431] Spores from the initial population of H. jecorina transformants of individual variants were harvested and reselected on acetamide agar plates. Spores were harvested using saline physiological solution, re-arrayed in 96 microtiter plates, and used for inoculation of a number of production media to generate BGL variant samples. For this purpose, 96-well filter plates (Corning, Art. No. 3505) containing in each well 250 .mu.L of a glycine production medium, containing 4.7 g/L (NH.sub.4).sub.2SO.sub.4; 33 g/L 1,4-piperazinebis(propanesulfonic acid) pH 5.5; 6.0 g/L glycine; 5.0 g/L KH.sub.2PO.sub.4; 1.0 g/L CaCl.sub.2.times.2H.sub.2O; 1.0 g/L MgSO.sub.4.times.7H.sub.2O; 2.5 mL/L of 400.times. T. reesei trace elements, containing 5 g/L FeSO.sub.4.times.7H.sub.2O, 1.4 g/L ZnSO.sub.4.times.7H.sub.2O, 1.6 g/L MnSO.sub.4.times.H.sub.2O, 3.7 g/L CoCl.sub.2.times.6H.sub.2O; 20 g/L Glucose; and 6.5 g/L Sophorose, were inoculated in quadruplicate with spore suspensions of H. jecorina transformants. Plates were incubated at 28.degree. C. and 80% humidity for 6 to 8 d. Culture supernatants were harvested by vacuum filtration. Residual glucose in these supernatants filtrates were measured using the hexokinase assay as described in Example 1A.

[0432] Combinations of substitutions were tested for the various activities as described above. Results of this testing is shown below in Table 5-2.

TABLE-US-00017 TABLE 5-2 Performance of Combinatorial BGL variants Variant Gluc Heat HPLC PASC PCS G2 CNPG CC L167W | D225Q wt - -- wt ++ wt + wt D177M | D225Q | D564T | Q626F | -- -- -- Q684A D177M | D225Q | Q684R -- -- -- wt - wt + wt L167W | D225Q | Q626F | Q684R -- -- -- ++ wt + ++ + L167W | D177M | D225Q | Q626F | wt -- -- + wt + ++ -- Q684G L167W | D177M | Q626F wt -- -- ++ ++ wt wt ++ D177M | D564T | Q684C -- -- -- - -- wt ++ wt L167W | D225Q | Q626F | Q684D ++ ++ -- wt wt wt wt wt L167W | D225Q | D564V | Q684N wt -- -- + -- - wt wt L167W | D177M | D225Q | D564T | -- -- -- Q684A L167W | D177M | D564V | Q684R - -- -- +++ -- wt + ++ L167W | D177M | D225Q | D564V | wt -- -- ++ + wt wt + Q684G L167W | D225Q | D564V -- -- -- +++ wt wt wt ++ D177M | D225Q | D564T | Q626F | -- -- -- ++ -- wt ++ ++ Q684N D177M | D225Q | D564T | Q684N wt -- -- ++ +++ wt ++ + L167W | D225Q | Q684N ++++ +++ -- wt wt wt -- wt L167W | D177M | Q626F | Q684N ++ -- -- wt -- - -- wt Q684D - -- -- L167W | D177M | Q626F | Q684G + -- -- +++ -- + wt ++ L167W | D225Q | D564T | Q626F | - -- -- ++ -- wt + wt Q684A D177M | Q626F | Q684R - -- -- ++ ++++ wt wt ++ D225Q | Q626F | Q684R wt - -- wt wt wt + wt L167W | Q626F wt -- -- + - - ++ + D177M | D225Q | D564T | Q626F | -- -- -- Q684R L167W | D177M | D564T | Q626F | ++ -- -- ++ -- -- -- + Q684N D225Q | D564V | Q684A +++ wt -- D225Q | D564T | Q626F ++++ wt -- Q684R wt wt -- wt -- - - wt Q684A -- ++++ -- L167W | Q626F | Q684D + -- -- + -- wt - + D564T wt ++ -- wt ++ wt - wt D225Q | D564V | Q626F | Q684R - wt -- +++ -- wt wt ++ L167W | D177M | D225Q | D564T | -- -- -- Q626F | Q684A D225Q | D564T | Q684A wt - -- wt - - wt + L167W | D225Q | D564T | Q626F | ++++ ++++ -- Q684C L167W | D177M | D564T | Q684R ++ -- -- ++ -- wt - ++ D177M | D225Q | D564V | Q684R wt -- -- +++ -- wt - ++ L167W | D564T | Q626F wt wt -- ++ wt ++ ++ ++ L167W | D177M | D225Q | D564V | - -- -- wt - - wt + Q626F | Q684N L167W | D177M | D225Q | D564T | ++ -- -- wt wt wt - wt Q684D L167W | D225Q | D564V | Q626F | ++++ ++++ -- wt + wt - wt Q684N L167W | D177M | D564T wt ++ -- L167W | D177M | D564V | Q626F | +++ -- -- ++ -- + wt ++ Q684A L167W | D177M | D225Q | D564T | -- ++ -- Q626F | Q684G L167W | D177M | D564T | Q684N ++++ ++++ -- +++ -- ++ wt ++ L167W | D225Q | D564T | Q626F | - -- -- Q684D D177M | D564V | Q684D + -- -- D177M | D225Q | D564V | Q684G wt -- -- wt ++++ - wt + L167W | D177M | D225Q | Q626F | -- -- -- Q684C D177M | D564T | Q626F | Q684A + -- -- wt ++ - + wt D177M | D564T | Q626F | Q684R -- -- -- L167W | D177M | D225Q | Q684D ++ -- -- ++ wt wt -- ++ L167W | D177M | D564V | Q626F | -- -- -- Q684N D177M | D225Q | D564T | Q626F | -- -- -- Q684D Q626F | Q684D ++++ ++++ -- L167W | D177M | D564T | Q626F | + -- -- ++ + ++ -- - Q684G L167W | D177M | D564V | Q684G wt -- -- + wt ++ wt - D177M | D225Q | D564T | Q684A -- -- -- + + ++ -- -- L167W | D177M | D225Q | D564V + -- -- + wt ++ wt - Q626F | Q684N ++ wt +++ wt wt wt ++ -- L167W | D177M | D225Q | D564V | ++ -- -- ++ ++ ++ -- + Q626F | Q684R D177M | D225Q | D564V | Q626F | wt -- -- ++ + ++ wt wt Q684N N369I | D370W -- -- -- N264M | R265P | N369I | D370W +++ ++++ -- R179V | N238F | D370W ++++ ++++ -- R179V | R265P | N369I | K656R -- ++++ -- R179V | N238F | K656R ++ ++++ -- R179V | R265P wt ++++ -- R179V | N238W | N264M | R265P | ++++ wt -- N369I | D370W R179V | N238W | R265P ++++ -- -- N264M + -- -- R179V | N264M | D370W ++ ++++ -- N238F | N264M | R265M | N369I ++++ +++ -- wt - - -- + R179V | R265M | K656R -- ++++ -- R179V | N238F | R265M ++ ++++ -- R179V | N238W | N264M | N369I | -- -- -- D370W | K656R R179V | R265P | D370W | K656R ++++ ++++ -- R179V | N369I | D370W + wt -- R179V | N238W | N264M | R265M | ++++ ++++ -- N369I R179V | N369I | D370W | K656R ++ ++++ -- R179V | N238F | R265P -- +++ -- R179V | N264M | R265M (+P229S) -- ++++ -- R179V | N238W | N264M | D370W -- ++++ -- N238F | R265M | D370W | K656R -- +++ -- R179V | N264M | R265P | K656R ++++ ++++ -- R179V | N238W | R265M + wt -- R179V | R265M | N369I | K656R wt ++++ -- R179V | R265M | N369I ++ ++++ -- R179V | N238F -- wt -- R179V | N264M | R265M | D370W | + ++++ -- K656R R179V | N238W | N264M | R265M | -- ++ -- K656R R179V | N238F | R265P | D370W | -- ++++ -- K656R R179V | N264M | R265M | N369I + ++++ -- R179V | N238W | N264M ++++ ++++ -- R179V | N238F | N264M | R265P | - ++ -- N369I N238W | N264M | R265M | D370W ++++ +++ -- R179V | N238W | N264M | N369I -- ++++ -- N264M | R265P | N369I -- +++ -- R179V | N238F | N264M | R265P | -- ++++ -- N369I | D370W N238W | R265P | D370W | K656R wt ++++ -- R179V | N238W | R265P | D370W ++ +++ -- R179V | N238W | N264M | D370W | ++++ ++++ -- K656R R179V | N238F | N264M | R265M | -- -- -- N369I | D370W | K656R R179V | N264M | R265M | K656R -- ++++ -- (+A157T) R179V | N264M | R265M | N369I | -- + -- D370W R179V | N238F | N264M | R265P | - -- -- K656R N264M | R265P ++++ ++++ -- N264M | N369I | D370W ++++ -- -- R265P | D370W (+G662F) +++ ++ -- N238F | R265M | N369I | D370W - ++++ -- R179V | N264M | R265P | N369I | ++++ ++++ -- D370W R265M | N369I +++ ++++ -- R179V | R265M | D370W +++ ++++ -- N238W | N264M | R265P ++++ ++++ -- R179V | N264M | N369I | D370W | -- +++ -- K656R R179V | N238W | N264M | R265P ++++ +++ -- N264M | N369I ++++ ++ -- wt - - -- wt R265M | K560S ++++ -- -- ++ ++ - -- wt N238W | R265P | K656R ++++ - -- - ++ -- -- ++ N264M | R265P (+G662F) ++ -- -- wt + wt -- ++ N238F | R265M | N369I ++++ +++ -- -- -- -- -- wt R179V | R265P | N369I ++ - -- ++ wt -- -- ++ K345E | N369T | P661E - + wt wt wt wt wt wt N263C | K345E | N369E | P661L | - + -- S683W K345E | N369E | P661E | S683W wt +++ -- + ++ ++ ++ ++ K345E | P661E | S683W - +++ -- + + ++ ++ +++ K345E | N369E | G372A | S683W ++ +++ -- ++ ++++ +++ +++ wt N263C | N369T ++ ++ -- ++ +++ +++ +++ ++ K428N | S683W -- -- -- wt ++++ +++ +++ wt K345E | K428N | S683W - -- -- wt ++++ ++ +++ wt K345E | N369T | G372A | P661E | -- wt -- + +++ +++ ++ ++++ S683W N263C | N369E | P661E - +++ -- N263C | K345E | N369E - ++ -- ++ +++ ++++ +++ +++ N263C | N369T | P661E wt ++ -- ++ ++++ ++++ +++ ++++ N369T | K428N | P661L | S683W -- +++ -- N263C | K345E | K428N | S683W -- +++ -- N263C | K345E | N369E | G372A | + + -- K428N | P661E | S683W N263C | N369T | G372A | P661E | + wt -- S683W K345E | N369T | P661E | S683W -- wt -- K345E | P661L -- ++++ -- N263C | K345E | N369T | G372A | ++ +++ -- K428N | P661E | S683W N369E | S683W + ++++ -- ++ ++ +++ +++ wt N369T | G372A | P661E -- ++++ -- N263C | K345E | K428N | P661E -- + -- N263C | K345E | N369E | G372A ++ ++ -- G372A | P661E | S683W ++ ++ -- wt + +++ +++ ++ N263C | P661L | S683W +++ ++++ -- K345E | N369E | S683W wt +++ -- ++ ++ ++++ +++ + N369T | G372A | P661L | S683W wt +++ -- wt ++++ +++ wt -- N263C | K345E | N369T | K428N wt +++ -- ++ ++ ++++ +++ ++ N263C | K345E | N369T | P661L wt wt -- N263C | N369T | G372A | K428N | wt ++ -- P661L | S683W K345E | N369E | G372A | P661E wt wt -- wt ++ ++ ++ +++ K428N | P661L | S683W -- -- -- K345E | N369E | P661L - + -- wt ++ ++ ++ +++ K345E | K428N | P661L | S683W -- +++ -- K345E | N369T | G372A | K428N | - ++ -- - ++++ wt - -- P661L | S683W N369T | G372A | K428N | S683W ++ +++ -- + + +++ ++ ++ N263C | K345E | N369T | G372A | ++ ++++ -- K428N N369T | P661L | S683W wt wt -- wt ++++ +++ +++ ++++ N263C | G372A | K428N -- +++ -- N263C | K428N | P661L | S683W - wt -- N263C | N369E | K428N | P661E wt +++ -- +++ ++++ ++++ ++++ ++++ N263C | N369E | G372A | K428N | wt +++ -- P661L | S683W N263C | K345E | N369T | G372A | P661E K345E | N369E | K428N | P661L - +++ -- wt -- ++ - wt N263C | K345E | N369E | K428N | -- -- -- S683W K345E | G372A | K428N | P661E + + -- N263C | K345E | N369E | P661L wt ++ -- wt - wt - + K345E | P661L | S683W -- wt -- N263C | N369T | S683W wt ++ -- ++ ++++ ++++ ++++ ++ N263C | G372A ++ + -- + ++ ++ +++ + N263C | K345E | N369E | G372A | ++ +++ -- +++ +++ ++++ ++++ ++ P661E K320S | R363E wt wt -- +++ +++ ++++ +++ ++ E170F | S312Y | N369Y | G372A | wt V603G Q226Y | G372A | V603G | F611A -- E170F | Q226Y | S312Y | G372A | -- P661F T242S | S312Y | N369Y | G372A | -- P661F Q226Y | T242S | S312Y | N369Y | wt V603G | F611A E170F | Q226Y | G372A | T666C + E170F | Q226Y | S312Y | N369Y | -- V603G | F611A | P661F | T666C Q226Y | T242S | S312Y | G372A | wt F611A S312Y | G372A | V603G -- E170F | T242S | S312Y | G372A | V603G E170F | Q226Y | N369Y | F611A | -- T666C E170F | Q226Y | T242S | S312Y | ++++ F611A | P661F Q226Y | S312Y | G372A | V603G | --

P661F | T666C E170F | Q226Y | T242S | N369Y | -- V603G Q226Y | T242S | N369Y | G372A -- E170F | Q226Y | S312Y | V603G | -- F611A | T666C E170F | Q226Y | T242S | N369Y | -- G372A | F611A E170F | S312Y | F611A | P661F | -- T666C Q226Y | T242S | S312Y | N369Y | -- V603G | F611A | T666C E170F | Q226Y | S312Y | N369Y | -- -- F611A | P661F | T666C T242S | N369Y | V603G - -- wt - wt Q226Y | N369Y | V603G | F611A | -- T666C T242S | V603G | F611A | T666C -- E170F | T242S | S312Y | T666C -- Q226Y | S312Y | V603G | F611A | -- P661F | T666C E170F | T242S | V603G -- E170F | T242S | S312Y | F611A | -- P661F | T666C N369Y | G372A -- S312Y | G372A | V603G | F611A | -- T666C Q226Y | T242S | S312Y | N369Y | -- T666C Q226Y | T242S | N369Y | P661F -- Q226Y | T242S | V603G -- E170F | S312Y | G372A | V603G | wt T666C E170F | T242S | N369Y | G372A | -- V603G | F611A E170F | Q226Y | T242S | S312Y | -- V603G | F611A E170F | Q226Y | N369Y | F611A -- E170F | Q226Y | T242S | F611A | -- P661F E170F | Q226Y | T242S | G372A | + V603G T242S | N369Y | G372A | T666C -- E170F | Q226Y | T242S | S312Y | -- V603G | F611A | P661F E170F | Q226Y | N369Y | V603G | -- F611A E170F | S312Y | V603G | F611A | -- T666C E170F | Q226Y | S312Y | G372A | T666C E170F | S312Y | G372A | V603G | wt P661F | T666C Q226Y | N369Y | G372A | V603G | -- P661F T242S | S312Y | N369Y | V603G | -- F611A | P661F | T666C S312Y | N369Y | V603G | T666C -- E170F | T242S | N369Y | T666C wt Q226Y | F611A | T666C -- T242S | S312Y | N369Y | G372A | -- -- V603G | F611A Q226Y | T242S | N369Y | G372A | -- F611A | T666C T242S | S312Y | N369Y | G372A | V603G | P661F E170F | Q226Y | F611A | T666C -- S312Y | P661F -- E170F | T242S | V603G | F611A -- T242S | S312Y | N369Y | F611A -- E170F | Q226Y | T242S | S312Y | wt G372A | V603G | F611A | P661F | T666C E170F | V603G + ++ -- wt ++ wt wt + Q226Y | S312Y | V603G | F611A | -- -- P661F E170F | T242S | N369Y | G372A | ++++ -- -- + - V603G | T666C E170F | T242S | F611A +++ -- -- - -- E170F | Q226Y | N369Y | V603G | +++ -- - ++ -- T666C E170F | Q226Y | T242S | S312Y | -- -- V603G | F611A | T666C E170F | G372A | F611A | P661F -- -- E170F | T242S | S312Y | N369Y | ++++ -- G372A E170F | Q226Y | T242S | N369Y | - -- -- + -- T666C Q226Y | T242S | G372A | F611A -- -- Q226Y | T242S | N369Y | G372A | wt -- V603G | P661F | T666C Q226Y | G372A | V603G | T666C -- -- +++ + wt E170F | Q226Y | S312Y | G372A | -- -- V603G | F611A | T666C E170F | Q226Y | T242S | S312Y | +++ -- N369Y | G372A | V603G Q226Y | T242S | S312Y | N369Y | -- -- -- +++ -- V603G E170F | Q226Y | T242S | V603G | -- -- F611A | T666C E170F | Q226Y | S312Y | N369Y | -- -- G372A | V603G | T666C E170F | Q226Y | T242S | V603G | ++ -- F611A E170F | T242S | S312Y | G372A | -- -- F611A | P661F | T666C E170F | S312Y | V603G | F611A | -- -- P661F N369Y | V603G | P661F -- -- E170F | Q226Y | T242S | S312Y | ++ -- N369Y | G372A | F611A | T666C E170F | Q226Y | V603G | P661F -- -- T242S | S312Y | N369Y | G372A | -- -- F611A | P661F S312Y | F611A | P661F -- -- E170F | Q226Y | S312Y | G372A | -- -- F611A | T666C Q226Y | S312Y | G372A | F611A - -- E170F | Q226Y | P661F +++ -- E170F | V603G | P661F | T666C ++ -- Q226Y | S312Y | N369Y | G372A | -- -- V603G | F611A | T666C E170F | Q226Y | G372A | F611A | P661F E170F | T242S | S312Y | V603G | -- -- P661F | T666C E170F | Q226Y | T242S | S312Y | -- -- N369Y | G372A E170F | Q226Y | T242S | S312Y | -- -- N369Y | F611A | P661F E170F | G372A | V603G | F611A | + -- P661F | T666C E170F | Q226Y | S312Y | G372A | -- -- V603G | F611A | P661F Q226Y | S312Y | G372A | V603G | -- -- F611A | T666C E170F | T242S | N369Y | V603G | -- -- F611A Q226Y | T242S | S312Y | V603G | + -- F611A | P661F | T666C T242S | S312Y | N369Y | G372A | -- -- -- +++ -- F611A | T666C Q226Y | G372A | F611A | P661F | -- -- T666C E170F | Q226Y | S312Y ++ -- -- ++++ -- T242S | S312Y wt -- +++ wt -- E170F | Q226Y | T242S | N369Y | -- -- V603G | F611A | P661F | T666C Q226Y | T242S | S312Y | N369Y | -- -- G372A | F611A E170F | S312Y | G372A | F611A | -- -- P661F E170F | Q226Y | T242S | S312Y | -- -- G372A | F611A | T666C E170F | Q226Y | T242S | G372A | ++ V603G | P661F | T666C E170F | Q226Y | T242S | V603G | wt T666C Q226Y | T242S | V603G | P661F | -- T666C E170F | T242S | S312Y | G372A | ++ T666C E170F | Q226Y | T242S | V603G | + P661F | T666C Q226Y | T242S | G372A | V603G | -- F611A S312Y | N369Y | G372A | V603G | -- P661F E170F | T242S | V603G | T666C wt E170F | Q226Y | T242S | S312Y | -- G372A | P661F E170F | S312Y | G372A | V603G | wt F611A | P661F | T666C E170F | T242S | N369Y | G372A | wt F611A | T666C Q226Y | S312Y | G372A | F611P | -- P661F | T666C E170F | Q226Y | T242S | S312Y | -- V603G | T666C E170F | Q226Y | T242S wt Q226Y | S312Y | N369Y | G372A | -- T666C Q226Y | T242S | V603G | F611A | -- T666C S312Y | G372A | P661F -- V603G | P661F | T666C -- E170F | S312Y | N369Y | G372A | + V603G | P661F E170F | Q226Y | S312Y | G372A | ++++ V603G | P661F | T666C Q226Y | S312Y | G372A -- T242S | S312Y | V603G | F611A -- E170F | Q226Y | S312Y | N369Y | + G372A | F611A Q226Y -- Q226Y | N369Y | V603G | P661F | -- T666C E170F | G372A + S312Y | N369Y | G372A | V603G wt T242S | S312Y | G372A -- T242S | N369Y | G372A | F611A | -- T666C E170F | S312Y | N369Y | T666C - E170F | F611P - Q226Y | T242S | S312Y | G372A | -- V603G Q226Y | T242S | N369Y | G372A | - V603G | F611A | P661F E170F | Q226Y | T242S | S312Y | -- G372A | V603G Q226Y | G372A | F611A | P661F -- T242S | S312Y | G372A | V603G | -- F611A | P661F | T666C Q226Y | V603G | T666C -- T242S | S312Y | F611A -- E170F | Q226Y | T242S | N369Y | + G372A | P661F Q226Y | T242S | S312Y | P661F -- E170F | T242S | N369Y | F611A | -- P661F Q226Y | T242S | N369Y | G372A | -- V603G E170F | T242S | G372A | P661F wt E170F | S312Y | V603G | P661F wt E170F | T242S | S312Y | V603G -- E170F | T242S | N369Y | V603G | wt T666C T242S -- E170F | T242S | S312Y | G372A | -- F611P | P661F | T666C T242S | P661F -- E170F | T242S | S312Y wt E170F | N369Y | G372A | T666C wt Q226Y | S312Y | V603G | F611A ++++ Q226Y | T242S | S312Y | G372A | wt V603G | F611A | T666C N369Y | V603G | F611A | P661F + S312Y | T666C -- E170F | T242S | N369Y | G372A | wt T666C Q226Y | T242S | N369Y | G372A | -- V603G | F611A | T666C S312Y | P661F | T666C + E170F | Q226Y | T242S | S312Y | -- F611A | P661F | T666C F611A | T666C -- E170F | V603G | F611A | T666C -- N369Y | G372A | V603G | T666C -- E170F | S312Y | N369Y | G372A | -- F611A | P661F T242S | N369Y | F611A | P661F --

Q226Y | S312Y | G372A | P661F -- Q226Y | T242S | S312Y | F611A | +++ P661F | T666C E170F | T242S | G372A | F611A | -- P661F | T666C E170F | T242S | G372A -- Q226Y | G372A | P661F | T666C -- E170F | T242S | S312Y | G372A | -- V603G | F611A | P661F | T666C E170F | T242S | S312Y | N369Y | -- G372A | V603G | F611A | T666C Q226Y | T242S | V603G | T666C -- G372A | T666C E170F | Q226Y | T242S | S312Y | wt N369Y | G372A | V603G | F611A | P661F | T666C E170F | Q226Y | T242S | N369Y | wt G372A | V603G | F611A | P661F Q226Y | T242S | S312Y | N369Y | -- G372A | P661F | T666C E170F | Q226Y | S312Y | N369Y | +++ G372A T242S | S312Y | N369Y | G372A | -- F611A E170F | T242S | G372A | P661F | +++ T666C E170F | N369Y | G372A | V603G | ++ F611A | P661F | T666C E170F | Q226Y | T242S | S312Y | -- N369Y | G372A | T666C Q226Y | T242S | T666C -- E170F | Q226Y | G372A | V603G | -- P661F | T666C Q226Y | T242S | S312Y | V603G -- E170F -- E170F | T242S | S312Y | F611A -- E170F | Q226Y | T242S | S312Y | - N369Y | V603G | F611A | P661F N369Y | G372A | F611A | T666C wt Q226Y | T242S | S312Y | N369Y | ++ V603G | T666C E170F | Q226Y | S312Y | V603G -- Q226Y | T242S | S312Y | N369Y | + G372A | V603G | F611A E170F | S312Y | P661F -- N369Y | G372A | V603G | F611A | ++ T666C Q226Y | S312Y | N369Y | G372A | -- F611A S312Y | N369Y | G372A | V603G | -- T666C E170F | Q226Y | S312Y | N369Y | -- G372A | P661F | T666C E170F | S312Y | N369Y | V603G | -- F611A | T666C E170F | Q226Y | N369Y | P661F | -- T666C S312Y | N369Y | V603G | F611A | -- P661F | T666C T666C -- Q226Y | F611A ++++ Q226Y | T242S | S312Y | N369Y | ++ G372A | F611A | P661F | T666C Q226Y | N369Y | V603G | F611A | -- P661F N369Y -- Q226Y | S312Y | V603G | P661F | -- T666C N369Y | F611A | P661F -- Q226Y | S312Y | N369Y | G372A | - P661F | T666C E170F | T242S | T666C -- Q226Y | N369Y | G372A | T666C wt E170F | Q226Y | T242S | S312Y | ++ G372A | F611A | P661F | T666C E170F | G372A | P661F | T666C wt Q226Y | T242S | N369Y | G372A | -- F611A | P661F E170F | S312Y | N369Y wt Q226Y | T242S | G372A | V603G | ++ T666C E170F | T242S | G372A | V603G | ++++ F611A | P661F | T666C Q226Y | T242S | N369Y -- T242S | S312Y | G372A | F611A | -- T666C G372A | F611A | T666C E170F | T242S | S312Y | G372A | -- F611A | P661F E170F | Q226Y | T242S | P661F - S312Y | N369Y | F611A -- E170F | Q226Y | T242S | N369Y | wt G372A | V603G | P661F E170F | T242S | N369Y | G372A | -- F611A | P661F | T666C Q226Y | S312Y | G372A | F611A | T666C Q226Y | T242S | G372A | T666C -- S312Y | G372A | T666C wt E170F | Q226Y | T242S | S312Y | -- V603G E170F | T242S | G372A | T666C -- E170F | Q226Y | G372A | F611A wt Q226Y | T242S | S312Y | V603G | -- F611A | T666C E170F | Q226Y | S312Y | N369Y -- T242S | S312Y | G372A | V603G -- E170F | Q226Y | V603G | T666C -- E170F | S312Y | V603G | T666C -- E170F | Q226Y | T242S | N369Y | -- F611A E170F | Q226Y | N369Y | G372A | -- V603G | F611A E170F | Q226Y | T242S | G372A | -- V603G | F611A S312Y | N369Y | V603G -- E170F | G372A | V603G | T666C wt E170F | Q226Y | T242S | F611A | -- T666C E170F | Q226Y | S312Y | N369Y | -- F611A E170F | G372A | T666C + N369Y | V603G -- G372A | V603G | F611A | P661F T242S | N369Y | T666C -- E170F | T242S | N369Y | G372A | wt V603G | F611A | T666C S312Y | G372A -- E170F | T242S | S312Y | N369Y | ++++ F611A | P661F E170F | Q226Y | T242S | S312Y | wt - -- ++ + +++ +++ + G372A | V603G | P661F | T666C E170F | Q226Y | N369Y | G372A +++ +++ ++ wt -- wt - wt Q226Y | T242S | G372A | P661F +++ ++ wt wt wt wt wt wt T242S | T666C wt wt -- ++ ++ +++ +++ ++ E170F | Q226Y | N369Y | G372A | ++ wt wt ++ ++ ++ +++ wt P661F T242S | N369Y | P661F - +++ ++ wt - wt wt wt Q226Y | T666C - wt -- ++ +++ +++ +++ + Q216E | T2821 | S312D | S692K ++ ++ ++ wt wt wt wt wt Q216K | T282K | S312D | A622K | wt wt + wt wt wt wt + S692L Q2161 | T282K | S312K | A622K ++++ +++ -- wt wt wt wt wt Q216E | T282K | S692L wt wt - wt wt wt wt wt Q216E | S312K | S692K wt wt wt ++ ++ +++ +++ + D178K | A338K | S474D | G662L - wt -- wt ++ wt wt wt N264L | A3381 | S474R | G662D ++ wt -- wt ++ wt - + D178N | N264K | A338D | S474R | wt wt -- wt ++ wt - + G662K D1781 | N264D | Q3031 | A338K | wt + -- wt wt wt ++ wt G662L D1781 | Q303E | A338I + wt -- wt wt wt wt + P176L | Q226W | K320S | G662F -- -- -- P176L | Q226W | K320S | V522Y | -- -- -- G662F P176L | Q226W | K320Y | R363E -- ++++ -- wt + + ++ wt P176L | G662F -- -- -- + ++ ++ ++ ++ P176L | Q316T | K320Y | V522Y -- wt -- Q226W | R363E | V522Y wt -- -- -- -- -- -- wt Q226W | Q316T | R363E -- -- -- - wt wt + wt P176L | Q226W | Q316T | K320Y | -- -- -- ++ + ++ ++ ++ R363E P176L | Q226W | Q316T | K320S | -- -- -- ++ wt ++ ++ ++ V522Y | G662C Q316T | K320Y | V522Y wt + +++ wt wt + wt wt Q316T | K320S | G662F wt -- -- wt wt wt wt wt R363E | V522Y | G662F - -- -- ++ wt ++ + ++ Q226W | K320S | V522Y | G662F wt -- -- wt - wt wt wt Q316T | K320Y | R363E ++ -- -- P176L | Q316T | G662C ++++ ++++ -- Q316T | K320Y | R363E | V522Y | ++ -- -- + wt wt -- ++ G662F Q226W | K320Y | G662C -- ++++ -- P176L | Q226W | K320S | G662C -- -- -- K320Y | G662C - -- -- Q316T | K320S | V522Y | G662F -- -- -- + wt + - ++ P176L | Q226W | K320S | R363E | -- -- -- +++ ++ +++ + +++ G662F Q316T | K320Y | G662F + -- -- -- -- -- -- ++ Q226W | K320S | R363E wt -- wt wt wt wt wt wt P176L | Q226W | K320Y | R363E | wt -- -- V522Y | G662C P176L | Q226W | Q316T | K320Y | - -- -- +++ ++ +++ ++ ++ V522Y Q226W | K320Y wt - -- + - wt -- + P176L | V522Y -- -- -- + -- wt -- + Q226W | K320Y | V522Y -- -- -- +++ wt +++ -- +++ P176L | Q316T | K320S | R363E | ++++ ++++ -- wt ++ +++ -- + G662F Q226W | Q316T | K320S | G662C wt -- -- P176L | Q226W | K320Y | R363E | -- -- -- ++ + ++ ++ ++ V522Y Q226W | K320Y | R363E -- -- -- +++ ++ +++ ++ +++ Q226W | Q316T | V522Y | G662F +++ + -- Q316T | K320Y | R363E | G662F wt -- -- +++ + +++ - ++ P176L | Q226W | Q316T | K320S | -- -- -- R363E | G662F P176L | Q226W | Q316T | R363E | wt -- -- G662C Q226W | Q316T | K320Y | R363E | wt -- -- G662F Q316T | K320S | V522Y - -- -- + wt + wt + P176L | Q226W | G547A | G662C +++ + -- ++ + ++ + ++ Q316T | K320S | R363E | G662F wt -- -- wt wt wt wt wt R363E | G662C wt -- -- + wt wt wt ++ P176L | Q226W | R363E | V522Y -- -- -- wt - wt -- ++ Q226W | Q316T | R363E | V522Y | -- -- -- +++ ++ ++++ ++ +++ G662F P176L | G662C +++ -- -- P176L | K320S | V522Y | G662C - + -- ++ wt ++ wt ++ Q226W | K320S | R363E | V522Y | - -- -- ++ wt ++ -- +++ G662F P176L | K320S | R363E | G662C - -- -- ++ ++ +++ +++ + R363E | G547A | G662C wt -- -- ++ ++ ++ +++ + Q316T | V522Y | G662F wt -- -- wt wt wt wt + G662C ++++ ++++ -- Q226W | G662C -- -- -- wt - wt -- + Q226W | K320Y | R363E | G662F P176L | Q226W | R363E - -- -- -- -- -- -- ++ Q226W | K320S | G662C wt -- -- ++ + ++ ++ + P176L | Q316T | K320Y | V522Y | - -- wt wt wt + + wt G547A | G662F P176L | Q226W | Q316T | K320Y | -- -- -- + ++ ++ + ++ R363E | G662F P176L | Q226W | K320S | V522Y wt -- -- wt wt wt wt + P176L | Q226W | Q316T | K320S | -- -- -- + ++ ++ -- ++ G662F Q226W | K320S | R363E | G662C -- +++ -- P176L | Q316T ++++ ++++ -- P176L | Q316T | K320S | R363E | -- -- -- ++ +++ ++++ -- ++ V522Y | G662C Q226W | Q316T | R363E | G662F - -- -- wt wt wt -- ++ K320Y | R363E | G662C wt -- -- ++ +++ ++++ ++++ ++ K51A | T242H | D329A wt + + wt wt wt wt wt D329A | A347Y | R542N wt + + wt wt wt wt wt A347Y | R542N +++ ++ wt wt wt wt wt wt A347Y | R542K - + wt wt wt wt wt wt K51A | A347Y | R542N | R645K wt wt ++ wt wt wt wt wt K51A | T242H | D329A | R542N wt wt -- wt - wt wt wt K51A | T242H | D329A | A347Y | wt -- -- wt wt wt ++ + R542N | R645G D329A | A347Y wt wt -- wt wt wt + wt E170F | G372A +++ -- -- -- T242S | N369L wt wt wt + D215S | S312Y ++ + wt + N263T | G372A wt wt wt + N263T | E170F wt wt wt - D215S | S548W + wt wt wt N263T | E170F | G372A ++++ -- -- -- N369T | G372A - wt wt wt Q226Y | V603G | F611A ++++ -- -- --

E170F | S312Y | N369Y -- ++ wt +++ D215S | 263S | S312Y | K498F | ++++ -- -- -- R586V ++++ PI > 2 +++ 2 > PI > 1.5 ++ 1.5 > PI > 1.2 + 1.2 > PI > 1.1 wt 1.1 > P I> 0.9 - 0.9 > PI > 0.8 -- 0.8 > PI blank Not tested

[0433] The results of combinatorial substitutions were further analyzed to determine those variants that had at least two, three, four, five, or six (or more) improved activities over wild type BGL1. Variants possessing these multiple improved activities are shown below in Table 5-3 to Table 5-6.

TABLE-US-00018 TABLE 5-3 Variants Comprising Combination of Substitutions with At Least Two Improved Activities HPLC + PCS Inh + Heat L167W | D225Q L167W | D225Q | Q626F | Q684D T242S | S312Y L167W | D225Q | Q684N D178K | A338K | S474D | G662L L167W | D225Q | D564T | Q626F | Q684C Heat + G2 Q626F | Q684D K345E | N369E | K428N | P661L N264M | R265P | N369I | D370W Q316T | K320Y | V522Y R179V | N238F | D370W Inh + G2 R179V | N238F | K656R E170F | T242S | N369Y | G372A | V603G | T666C R179V | N264M | D370W E170F | Q226Y | N369Y | V603G | T666C R179V | N238F | R265M E170F | Q226Y | S312Y R179V | R265P | D370W | K656R PASC + CC R179V | N238W | N264M | R265M | N369I L167W | D177M | D564V | Q684R R179V | N369I | D370W | K656R L167W | D225Q | D564V R179V | N264M | R265P | K656R D177M | D225Q | D564T | Q626F | Q684N R179V | R265M | N369I L167W | Q626F R179V | N264M | R265M | D370W | K656R D225Q | D564V | Q626F | Q684R R179V | N264M | R265M | N369I D177M | D225Q | D564V | Q684R R179V | N238W | N264M Q226W | K320Y N238W | N264M | R265M | D370W P176L | V522Y R179V | N238W | R265P | D370W R363E | G662C R179V | N238W | N264M | D370W | K656R PASC + G2 N264M | R265P L167W | D177M | D225Q | Q626F | Q684G R265P | D370W (+G662F) L167W | D177M | D564V | Q684G R179V | N264M | R265P | N369I | D370W D215S | S312Y R265M | N369I E170F | S312Y | N369Y R179V | R265M | D370W Heat + CC N238W | N264M | R265P N263C | K345E | N369E | P661L R179V | N238W | N264M | R265P Inh + CC N264M | N369I D178I | Q303E | A338I N238F | R265M | N369I Q316T | K320Y | G662F N263C | K345E | N369E | G372A | K428N | P661E | S683W N263C | K345E | N369T | G372A | K428N | P661E | S683W N263C | K345E | N369E | G372A N263C | P661L | S683W N263C | K345E | N369T | G372A | K428N K345E | G372A | K428N | P661E E170F | Q226Y | N369Y | G372A Q226Y | T242S | G372A | P661F Q216E | T282I | S312D | S692K Q216I | T282K | S312K | A622K P176L | Q316T | G662C Q226W | Q316T | V522Y | G662F P176L | Q316T A347Y | R542N

TABLE-US-00019 TABLE 5-4 Variants Comprising Combination of Substitutions with At Least Three Improved Activities PASC + G2 + CC Inh + PASC + CC L167W | D225Q | Q626F | Q684R L167W | D177M | D564T | Q626F | Q684N L167W | D564T | Q626F L167W | Q626F | Q684D P176L | Q226W | Q316T | K320S | V522Y | G662C L167W | D177M | D564T | Q684R R363E | V522Y | G662F L167W | D177M | D225Q | Q684D Q316T | K320S | V522Y | G662F R179V | R265P | N369I Q226W | K320Y | V522Y Q316T | K320Y | R363E | V522Y | G662F Q316T | K320S | V522Y Inh + HPLC + PCS Q226W | K320S | R363E | V522Y | G662F D177M | D564T | Q626F | Q684A HPLC + PCS + CC Heat + HPLC + PCS D177M | D225Q | D564V | Q684G K345E | N369T | G372A | K428N | P661L | S683W D178N | N264K | A338D | S474R | G662K Inh + PASC + G2 HPLC + PCS + G2 L167W | D177M | D225Q | D564V K428N | S683W K345E | K428N | S683W Q226Y | G372A | V603G | T666C Inh + Heat + CC N238F | N264M | R265M | N369I

TABLE-US-00020 TABLE 5-5 Variants Comprising Combination of Substitutions with At Least Four Improved Activities HPLC + PASC + PCS + CC Inh + HPLC + PSC + CC L167W | D177M | Q626F N238W | R265P | K656R L167W | D177M | D225Q | D564V N264M | R265P (+G662F) | Q684G N264L | A338I | S474R | G662D D177M | D225Q | D564T | Q684N HPLC + PASC + PCS + G2 D177M | Q626F | Q684R D177M | D225Q | D564T | Q684A Inh + Heat + HPLC + PCS D177M | D225Q | D564V | Q626F L167W | D225Q | D564V | Q626F | Q684N | Q684N Inh + HPLC + PASC + PCS Inh + PASC + G2 + CC R265M | K560S L167W | D177M | Q626F | Q684G HPLC + PCS + G2 + CC L167W | D177M | D564V | Q626F K345E | N369E | G372A | P661E | Q684A N369T | P661L | S683W Heat + PASC + G2 + CC P176L | K320S | V522Y | G662C Heat + HPLC + PCS + G2 N369T | G372A | P661L | S683W P176L | Q226W | K320Y | R363E

TABLE-US-00021 TABLE 5-6 Variants wi with Combination of Substitutions with At Least Five, Six, or Seven Improved Activities HPLC + PASC + PCS + G2 + CC Heat + HPLC + PCS + G2 + CC K345E | N369T | G372A | P661E | S683W K345E | N369E | P661L K320S | R363E Inh + HPLC + PASC + PCS + G2 E170F | Q226Y | T242S | S312Y | G372A | L167W | D177M | D564T | Q626F | Q684G V603G | P661F | T666C E170F | Q226Y | N369Y | G372A | P661F T242S | T666C Inh + Heat + PASC + G2 + CC Q226Y | T666C L167W | D177M | D564T | Q684N Q216E | S312K | S692K Inh + Heat + HPLC + PCS + CC P176L | G662F E170F | V603G P176L | Q226W | Q316T | K320Y | R363E Inh + Heat + HPLC + PASC + PCS + G2 + CC P176L | Q226W | K320S | R363E | G662F N263C | N369T P176L | Q226W | Q316T | K320Y | V522Y N369T | G372A | K428N | S683W P176L | Q226W | K320Y | R363E | V522Y N263C | G372A Q226W | K320Y | R363E N263C | K345E | N369E | G372A | P661E Q316T | K320Y | R363E | G662F P176L | Q226W | G547A | G662C Q226W | Q316T | R363E | V522Y | G662F Inh + Heat + HPLC + PASC + PCS + G2 P176L | K320S | R363E | G662C K345E | N369E | G372A | S683W R363E | G547A | G662C N369E | S683W Q226W | K320S | G662C Inh + Heat + HPLC + PCS + G2 + CC P176L | Q226W | Q316T | K320Y | R363E | G372A | P661E | S683W G662F P176L | Q316T | K320S | R363E | G662F P176L | Q226W | Q316T | K320S | G662F Inh + HPLC + PASC + PCS + G2 + CC P176L | Q316T | K320S | R363E | V522Y | L167W | D177M | D225Q | D564V | Q626F G662C | Q684R K320Y | R363E | G662C Heat + HPLC + PASC + PCS + G2 + CC K345E | N369E | P661E | S683W K345E | P661E | S683W N263C | K345E | N369E N263C | N369T | P661E K345E | N369E | S683W N263C | K345E | N369T | K428N N263C | N369E | K428N | P661E N263C | N369T | S683W

Example 6

BGL1 Combinatorial Variants Exhibiting Reduced Glucose Inhibition

[0434] A number of BGL variants were selected and tested for their capacity to hydrolyze CNPG in the presence of glucose at a range of concentrations. A culture supernatant of a H. jecorina strain producing wild type BGL1 or a BGL variant was diluted to a minimal CNPG activity of 20 mOD/min. The wild type BGL1 or the BGL variant supernatant was then mixed with various amounts of glucose, to a final glucose concentration of between 0 and 25 mM.

[0435] The assay was initiated by the addition of 1 mM CNPG in a 50 mM sodium acetate buffer, pH 5.0. Kinetic measurements were made by recording OD405 nm in a SpectraMax plate reader (Molecular devices) for 3 min.

[0436] IC50 values were measured using the formula y=a/(1+x/b), wherein y represents the specific CNPG activity (in mOD/min), x represents the inhibitory substrate concentration (in mM glucose), a represents the maximum reaction rate (CNPG activity, in mOD/min), and b represents the inhibitor concentration at which the enzyme activity was reduced by half. To calculate reduction in inhibition, the IC50 value obtained for a given BGL variant was divided by the IC50 value obtained for the wild type BGL1.

TABLE-US-00022 TABLE 6-1 Performance Index of BGL variants In a Glucose Inhibition Activity Assay. Reduction in BGL Variant Inhibition* G372A + N263T + E170F | G372A + E170F + N264M | R265P | G662F + N264M | N369I + N263T | G372A + R265M | K560S + N264M | N369I | D370W ++ R179V | R265P | N369I ++ E170F | S312Y | N369Y ++ E170F | N263T +++ R265P | D370W | G662F +++ N238W | R265P | K656R +++ N238F | N264M | R265M +++ | N369I N238F ++++ E170F | N263T | G372A ++++ N238F | R265M | N369I ++++ *`+`, `++`, `+++`, and `++++` indicate a 1.2- to 2-fold, 2- to 3-fold, 3- to 6-fold, 6- to 10-fold reduction in glucose inhibition, respectively, as compared to the glucose inhibition observed with the wild type BGL1.

[0437] Various modifications and variations of the present disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific preferred embodiments, it should be understood that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure which are understood by those skilled in the art are intended to be within the scope of the claims.

Sequence CWU 1

1

4512238DNAHypocrea jecorina 1atgcgctacc gcaccgctgc cgctttagcc ttagccaccg gccccttcgc cagagccgat 60agccacagca cctccggcgc tagtgctgaa gctgttgtcc ctcctgctgg caccccttgg 120ggcaccgcct acgacaaggc caaggccgcc ctcgccaagc tcaacctcca ggacaaggtc 180ggcatcgtca gcggcgtcgg ctggaacggc ggtccctgcg tcggcaacac cagccccgcc 240agcaagatca gctaccccag cctctgcctc caggacggcc ccctcggcgt ccgctacagc 300accggcagca ccgccttcac ccctggcgtc caggccgcca gcacctggga cgtcaacctc 360atccgcgagc gcggccagtt catcggcgaa gaggtcaagg ccagcggcat ccacgtcatc 420ctcggtcccg ttgctggtcc cttaggcaag accccccagg gcggtcgcaa ctgggagggc 480ttcggcgtcg acccctacct caccggcatt gccatgggcc agaccatcaa cggcatccag 540agcgtcggcg tccaggccac cgccaagcac tacatcctca acgagcaaga gttaaaccgc 600gagactatca gcagcaaccc cgacgaccgc accctccacg agttatacac ctggcccttc 660gccgacgccg tccaggccaa cgtcgccagc gtcatgtgca gctacaacaa ggtcaacacc 720acctgggcct gcgaggacca gtacaccctc cagaccgtcc tcaaggacca gctcggcttc 780cccggctacg tcatgaccga ctggaacgcc cagcacacca ccgtccagag cgccaacagc 840ggcctcgaca tgagcatgcc cggcaccgac ttcaacggca acaaccgcct ctggggccct 900gccctcacca acgccgtcaa cagcaaccag gtccccacct cccgcgtcga cgacatggtc 960acccgcatcc tcgccgcctg gtacttaacc ggccaagacc aggctggcta tcccagcttc 1020aacatcagcc gcaacgtcca gggcaaccac aagaccaacg tccgcgccat tgcccgcgac 1080ggcatcgtcc tcctcaagaa cgacgccaac atcctccccc tcaagaagcc cgcctctatc 1140gccgtcgtcg gcagcgccgc catcatcggc aaccacgccc gcaacagccc cagctgcaac 1200gacaagggct gcgatgacgg tgccctcggc atgggctggg gctctggcgc cgtcaactac 1260ccctacttcg tcgcccccta cgacgccatc aacacccgcg ccagcagcca gggcacccag 1320gtcaccctca gcaacaccga caatacttct tctggcgctt ctgctgctag aggcaaggac 1380gtcgccatcg tttttatcac tgccgattct ggcgaaggct acatcaccgt cgagggcaac 1440gccggcgacc gcaacaacct cgacccctgg cacaacggca atgccctcgt ccaggccgtt 1500gctggtgcta acagcaacgt catcgtcgtc gtccacagcg tcggcgccat catcctcgag 1560cagatcctcg ccctccccca ggtcaaggcc gtcgtctggg ccggcttacc cagccaggaa 1620agcggcaacg ccttagtcga cgtcctctgg ggtgacgttt ccccctctgg caagctcgtc 1680tacaccattg ccaagagccc caacgactac aacacccgca ttgtcagcgg cggcagcgac 1740agcttcagcg agggcctctt catcgactac aagcacttcg acgacgccaa cattaccccc 1800cgctacgagt tcggctacgg cctcagctac accaagttca actacagccg cctcagcgtc 1860ctcagcaccg ccaagagcgg ccctgccact ggtgctgtcg tccctggtgg cccttctgac 1920ctcttccaga acgtcgccac ggtcaccgtc gacattgcca actccggcca ggtcactggc 1980gccgaggtcg cccagctcta catcacctac cccagcagcg cccctcgcac tcctcccaag 2040cagctcagag gcttcgctaa gttaaactta acccctggcc agagcggcac cgccaccttt 2100aacatccgca gacgcgacct cagctactgg gacaccgcca gccagaagtg ggtcgtcccc 2160agcggcagct tcggcatctc cgtcggcgcc agctcccgcg acatccgcct caccagcacc 2220ctcagcgtcg cctgatga 22382744PRTTrichoderma reesei 2Met Arg Tyr Arg Thr Ala Ala Ala Leu Ala Leu Ala Thr Gly Pro Phe 1 5 10 15 Ala Arg Ala Asp Ser His Ser Thr Ser Gly Ala Ser Ala Glu Ala Val 20 25 30 Val Pro Pro Ala Gly Thr Pro Trp Gly Thr Ala Tyr Asp Lys Ala Lys 35 40 45 Ala Ala Leu Ala Lys Leu Asn Leu Gln Asp Lys Val Gly Ile Val Ser 50 55 60 Gly Val Gly Trp Asn Gly Gly Pro Cys Val Gly Asn Thr Ser Pro Ala 65 70 75 80 Ser Lys Ile Ser Tyr Pro Ser Leu Cys Leu Gln Asp Gly Pro Leu Gly 85 90 95 Val Arg Tyr Ser Thr Gly Ser Thr Ala Phe Thr Pro Gly Val Gln Ala 100 105 110 Ala Ser Thr Trp Asp Val Asn Leu Ile Arg Glu Arg Gly Gln Phe Ile 115 120 125 Gly Glu Glu Val Lys Ala Ser Gly Ile His Val Ile Leu Gly Pro Val 130 135 140 Ala Gly Pro Leu Gly Lys Thr Pro Gln Gly Gly Arg Asn Trp Glu Gly 145 150 155 160 Phe Gly Val Asp Pro Tyr Leu Thr Gly Ile Ala Met Gly Gln Thr Ile 165 170 175 Asn Gly Ile Gln Ser Val Gly Val Gln Ala Thr Ala Lys His Tyr Ile 180 185 190 Leu Asn Glu Gln Glu Leu Asn Arg Glu Thr Ile Ser Ser Asn Pro Asp 195 200 205 Asp Arg Thr Leu His Glu Leu Tyr Thr Trp Pro Phe Ala Asp Ala Val 210 215 220 Gln Ala Asn Val Ala Ser Val Met Cys Ser Tyr Asn Lys Val Asn Thr 225 230 235 240 Thr Trp Ala Cys Glu Asp Gln Tyr Thr Leu Gln Thr Val Leu Lys Asp 245 250 255 Gln Leu Gly Phe Pro Gly Tyr Val Met Thr Asp Trp Asn Ala Gln His 260 265 270 Thr Thr Val Gln Ser Ala Asn Ser Gly Leu Asp Met Ser Met Pro Gly 275 280 285 Thr Asp Phe Asn Gly Asn Asn Arg Leu Trp Gly Pro Ala Leu Thr Asn 290 295 300 Ala Val Asn Ser Asn Gln Val Pro Thr Ser Arg Val Asp Asp Met Val 305 310 315 320 Thr Arg Ile Leu Ala Ala Trp Tyr Leu Thr Gly Gln Asp Gln Ala Gly 325 330 335 Tyr Pro Ser Phe Asn Ile Ser Arg Asn Val Gln Gly Asn His Lys Thr 340 345 350 Asn Val Arg Ala Ile Ala Arg Asp Gly Ile Val Leu Leu Lys Asn Asp 355 360 365 Ala Asn Ile Leu Pro Leu Lys Lys Pro Ala Ser Ile Ala Val Val Gly 370 375 380 Ser Ala Ala Ile Ile Gly Asn His Ala Arg Asn Ser Pro Ser Cys Asn 385 390 395 400 Asp Lys Gly Cys Asp Asp Gly Ala Leu Gly Met Gly Trp Gly Ser Gly 405 410 415 Ala Val Asn Tyr Pro Tyr Phe Val Ala Pro Tyr Asp Ala Ile Asn Thr 420 425 430 Arg Ala Ser Ser Gln Gly Thr Gln Val Thr Leu Ser Asn Thr Asp Asn 435 440 445 Thr Ser Ser Gly Ala Ser Ala Ala Arg Gly Lys Asp Val Ala Ile Val 450 455 460 Phe Ile Thr Ala Asp Ser Gly Glu Gly Tyr Ile Thr Val Glu Gly Asn 465 470 475 480 Ala Gly Asp Arg Asn Asn Leu Asp Pro Trp His Asn Gly Asn Ala Leu 485 490 495 Val Gln Ala Val Ala Gly Ala Asn Ser Asn Val Ile Val Val Val His 500 505 510 Ser Val Gly Ala Ile Ile Leu Glu Gln Ile Leu Ala Leu Pro Gln Val 515 520 525 Lys Ala Val Val Trp Ala Gly Leu Pro Ser Gln Glu Ser Gly Asn Ala 530 535 540 Leu Val Asp Val Leu Trp Gly Asp Val Ser Pro Ser Gly Lys Leu Val 545 550 555 560 Tyr Thr Ile Ala Lys Ser Pro Asn Asp Tyr Asn Thr Arg Ile Val Ser 565 570 575 Gly Gly Ser Asp Ser Phe Ser Glu Gly Leu Phe Ile Asp Tyr Lys His 580 585 590 Phe Asp Asp Ala Asn Ile Thr Pro Arg Tyr Glu Phe Gly Tyr Gly Leu 595 600 605 Ser Tyr Thr Lys Phe Asn Tyr Ser Arg Leu Ser Val Leu Ser Thr Ala 610 615 620 Lys Ser Gly Pro Ala Thr Gly Ala Val Val Pro Gly Gly Pro Ser Asp 625 630 635 640 Leu Phe Gln Asn Val Ala Thr Val Thr Val Asp Ile Ala Asn Ser Gly 645 650 655 Gln Val Thr Gly Ala Glu Val Ala Gln Leu Tyr Ile Thr Tyr Pro Ser 660 665 670 Ser Ala Pro Arg Thr Pro Pro Lys Gln Leu Arg Gly Phe Ala Lys Leu 675 680 685 Asn Leu Thr Pro Gly Gln Ser Gly Thr Ala Thr Phe Asn Ile Arg Arg 690 695 700 Arg Asp Leu Ser Tyr Trp Asp Thr Ala Ser Gln Lys Trp Val Val Pro 705 710 715 720 Ser Gly Ser Phe Gly Ile Ser Val Gly Ala Ser Ser Arg Asp Ile Arg 725 730 735 Leu Thr Ser Thr Leu Ser Val Ala 740 3713PRTTrichoderma reesei 3Val Val Pro Pro Ala Gly Thr Pro Trp Gly Thr Ala Tyr Asp Lys Ala 1 5 10 15 Lys Ala Ala Leu Ala Lys Leu Asn Leu Gln Asp Lys Val Gly Ile Val 20 25 30 Ser Gly Val Gly Trp Asn Gly Gly Pro Cys Val Gly Asn Thr Ser Pro 35 40 45 Ala Ser Lys Ile Ser Tyr Pro Ser Leu Cys Leu Gln Asp Gly Pro Leu 50 55 60 Gly Val Arg Tyr Ser Thr Gly Ser Thr Ala Phe Thr Pro Gly Val Gln 65 70 75 80 Ala Ala Ser Thr Trp Asp Val Asn Leu Ile Arg Glu Arg Gly Gln Phe 85 90 95 Ile Gly Glu Glu Val Lys Ala Ser Gly Ile His Val Ile Leu Gly Pro 100 105 110 Val Ala Gly Pro Leu Gly Lys Thr Pro Gln Gly Gly Arg Asn Trp Glu 115 120 125 Gly Phe Gly Val Asp Pro Tyr Leu Thr Gly Ile Ala Met Gly Gln Thr 130 135 140 Ile Asn Gly Ile Gln Ser Val Gly Val Gln Ala Thr Ala Lys His Tyr 145 150 155 160 Ile Leu Asn Glu Gln Glu Leu Asn Arg Glu Thr Ile Ser Ser Asn Pro 165 170 175 Asp Asp Arg Thr Leu His Glu Leu Tyr Thr Trp Pro Phe Ala Asp Ala 180 185 190 Val Gln Ala Asn Val Ala Ser Val Met Cys Ser Tyr Asn Lys Val Asn 195 200 205 Thr Thr Trp Ala Cys Glu Asp Gln Tyr Thr Leu Gln Thr Val Leu Lys 210 215 220 Asp Gln Leu Gly Phe Pro Gly Tyr Val Met Thr Asp Trp Asn Ala Gln 225 230 235 240 His Thr Thr Val Gln Ser Ala Asn Ser Gly Leu Asp Met Ser Met Pro 245 250 255 Gly Thr Asp Phe Asn Gly Asn Asn Arg Leu Trp Gly Pro Ala Leu Thr 260 265 270 Asn Ala Val Asn Ser Asn Gln Val Pro Thr Ser Arg Val Asp Asp Met 275 280 285 Val Thr Arg Ile Leu Ala Ala Trp Tyr Leu Thr Gly Gln Asp Gln Ala 290 295 300 Gly Tyr Pro Ser Phe Asn Ile Ser Arg Asn Val Gln Gly Asn His Lys 305 310 315 320 Thr Asn Val Arg Ala Ile Ala Arg Asp Gly Ile Val Leu Leu Lys Asn 325 330 335 Asp Ala Asn Ile Leu Pro Leu Lys Lys Pro Ala Ser Ile Ala Val Val 340 345 350 Gly Ser Ala Ala Ile Ile Gly Asn His Ala Arg Asn Ser Pro Ser Cys 355 360 365 Asn Asp Lys Gly Cys Asp Asp Gly Ala Leu Gly Met Gly Trp Gly Ser 370 375 380 Gly Ala Val Asn Tyr Pro Tyr Phe Val Ala Pro Tyr Asp Ala Ile Asn 385 390 395 400 Thr Arg Ala Ser Ser Gln Gly Thr Gln Val Thr Leu Ser Asn Thr Asp 405 410 415 Asn Thr Ser Ser Gly Ala Ser Ala Ala Arg Gly Lys Asp Val Ala Ile 420 425 430 Val Phe Ile Thr Ala Asp Ser Gly Glu Gly Tyr Ile Thr Val Glu Gly 435 440 445 Asn Ala Gly Asp Arg Asn Asn Leu Asp Pro Trp His Asn Gly Asn Ala 450 455 460 Leu Val Gln Ala Val Ala Gly Ala Asn Ser Asn Val Ile Val Val Val 465 470 475 480 His Ser Val Gly Ala Ile Ile Leu Glu Gln Ile Leu Ala Leu Pro Gln 485 490 495 Val Lys Ala Val Val Trp Ala Gly Leu Pro Ser Gln Glu Ser Gly Asn 500 505 510 Ala Leu Val Asp Val Leu Trp Gly Asp Val Ser Pro Ser Gly Lys Leu 515 520 525 Val Tyr Thr Ile Ala Lys Ser Pro Asn Asp Tyr Asn Thr Arg Ile Val 530 535 540 Ser Gly Gly Ser Asp Ser Phe Ser Glu Gly Leu Phe Ile Asp Tyr Lys 545 550 555 560 His Phe Asp Asp Ala Asn Ile Thr Pro Arg Tyr Glu Phe Gly Tyr Gly 565 570 575 Leu Ser Tyr Thr Lys Phe Asn Tyr Ser Arg Leu Ser Val Leu Ser Thr 580 585 590 Ala Lys Ser Gly Pro Ala Thr Gly Ala Val Val Pro Gly Gly Pro Ser 595 600 605 Asp Leu Phe Gln Asn Val Ala Thr Val Thr Val Asp Ile Ala Asn Ser 610 615 620 Gly Gln Val Thr Gly Ala Glu Val Ala Gln Leu Tyr Ile Thr Tyr Pro 625 630 635 640 Ser Ser Ala Pro Arg Thr Pro Pro Lys Gln Leu Arg Gly Phe Ala Lys 645 650 655 Leu Asn Leu Thr Pro Gly Gln Ser Gly Thr Ala Thr Phe Asn Ile Arg 660 665 670 Arg Arg Asp Leu Ser Tyr Trp Asp Thr Ala Ser Gln Lys Trp Val Val 675 680 685 Pro Ser Gly Ser Phe Gly Ile Ser Val Gly Ala Ser Ser Arg Asp Ile 690 695 700 Arg Leu Thr Ser Thr Leu Ser Val Ala 705 710 4805PRTHansenula anomala 4Asn Thr Ser Ala Pro Gln Ala Ser Asn Asp Asp Pro Phe Asn His Ser 1 5 10 15 Pro Ser Phe Tyr Pro Thr Pro Gln Gly Gly Arg Ile Asn Asp Gly Lys 20 25 30 Trp Gln Ala Ala Phe Tyr Arg Ala Arg Glu Leu Val Asp Gln Met Ser 35 40 45 Ile Ala Glu Lys Val Asn Leu Thr Thr Gly Val Gly Ser Ala Ser Gly 50 55 60 Pro Cys Ser Gly Asn Thr Gly Ser Val Pro Arg Leu Asn Ile Ser Ser 65 70 75 80 Ile Cys Val Gln Asp Gly Pro Leu Ser Val Arg Ala Ala Asp Leu Thr 85 90 95 Asp Val Phe Pro Cys Gly Met Ala Ala Ser Ser Ser Phe Asn Lys Gln 100 105 110 Leu Ile Tyr Asp Arg Ala Val Ala Ile Gly Ser Glu Phe Lys Gly Lys 115 120 125 Gly Ala Asp Ala Ile Leu Gly Pro Val Tyr Gly Pro Met Gly Val Lys 130 135 140 Ala Ala Gly Gly Arg Gly Trp Glu Gly His Gly Pro Asp Pro Tyr Leu 145 150 155 160 Glu Gly Val Ile Ala Tyr Leu Gln Thr Ile Gly Ile Gln Ser Gln Gly 165 170 175 Val Val Ser Thr Ala Lys His Leu Ile Gly Asn Glu Gln Glu His Phe 180 185 190 Arg Phe Ala Lys Lys Asp Lys His Ala Gly Lys Ile Asp Pro Gly Met 195 200 205 Phe Asn Thr Ser Ser Ser Leu Ser Ser Glu Ile Asp Asp Arg Ala Met 210 215 220 His Glu Ile Tyr Leu Trp Pro Phe Ala Glu Ala Val Arg Gly Gly Val 225 230 235 240 Ser Ser Ile Met Cys Ser Tyr Asn Lys Leu Asn Gly Ser His Ala Cys 245 250 255 Gln Asn Ser Tyr Leu Leu Asn Tyr Leu Leu Lys Glu Glu Leu Gly Phe 260 265 270 Gln Gly Phe Val Met Thr Asp Trp Gly Ala Leu Tyr Ser Gly Ile Asp 275 280 285 Ala Ala Asn Ala Gly Leu Asp Met Asp Met Pro Cys Glu Ala Gln Tyr 290 295 300 Phe Gly Gly Asn Leu Thr Thr Ala Val Leu Asn Gly Thr Leu Pro Gln 305 310 315 320 Asp Arg Leu Asp Asp Met Ala Thr Arg Ile Leu Ser Ala Leu Ile Tyr 325 330 335 Ser Gly Val His Asn Pro Asp Gly Pro Asn Tyr Asn Ala Gln Thr Phe 340 345 350 Leu Thr Glu Gly His Glu Tyr Phe Lys Gln Gln Glu Gly Asp Ile Val 355 360 365 Val Leu Asn Lys His Val Asp Val Arg Ser Asp Ile Asn Arg Ala Val 370 375 380 Ala Leu Arg Ser Ala Val Glu Gly Val Val Leu Leu Lys Asn Glu His 385 390 395 400 Glu Thr Leu Pro Leu Gly Arg Glu Lys Val Lys Arg Ile Ser Ile Leu 405 410 415 Gly Gln Ala Ala Gly Asp Asp Ser Lys Gly Thr Ser Cys Ser Leu Arg 420 425 430 Gly Cys Gly Ser Gly Ala Ile Gly Thr Gly Tyr Gly Ser Gly Ala Gly 435 440 445 Thr Phe Ser Tyr Phe Val Thr Pro Ala Asp Gly Ile Gly Ala Arg Ala 450 455 460 Gln Gln Glu Lys Ile Ser Tyr Glu Phe Ile Gly Asp Ser Trp Asn Gln 465 470 475 480 Ala Ala Ala Met Asp Ser Ala Leu Tyr Ala Asp Ala Ala Ile Glu Val

485 490 495 Ala Asn Ser Val Ala Gly Glu Glu Ile Gly Asp Val Asp Gly Asn Tyr 500 505 510 Gly Asp Leu Asn Asn Leu Thr Leu Trp His Asn Ala Val Pro Leu Ile 515 520 525 Lys Asn Ile Ser Ser Ile Asn Asn Asn Thr Ile Val Ile Val Thr Ser 530 535 540 Gly Gln Gln Ile Asp Leu Glu Pro Phe Ile Asp Asn Glu Asn Val Thr 545 550 555 560 Ala Val Ile Tyr Ser Ser Tyr Leu Gly Gln Asp Phe Gly Thr Val Leu 565 570 575 Ala Lys Val Leu Phe Gly Asp Glu Asn Pro Ser Gly Lys Leu Pro Phe 580 585 590 Thr Ile Ala Lys Asp Val Asn Asp Tyr Ile Pro Val Ile Glu Lys Val 595 600 605 Asp Val Pro Asp Pro Val Asp Lys Phe Thr Glu Ser Ile Tyr Val Asp 610 615 620 Tyr Arg Tyr Phe Asp Lys Tyr Asn Lys Pro Val Arg Tyr Glu Phe Gly 625 630 635 640 Tyr Gly Leu Ser Tyr Ser Asn Phe Ser Leu Ser Asp Ile Glu Ile Gln 645 650 655 Thr Leu Gln Pro Phe Ser Glu Asn Ala Glu Pro Ala Ala Asn Tyr Ser 660 665 670 Glu Thr Tyr Gln Tyr Lys Gln Ser Asn Met Asp Pro Ser Glu Tyr Thr 675 680 685 Val Pro Glu Gly Phe Lys Glu Leu Ala Asn Tyr Thr Tyr Pro Tyr Ile 690 695 700 His Asp Ala Ser Ser Ile Lys Ala Asn Ser Ser Tyr Asp Tyr Pro Glu 705 710 715 720 Gly Tyr Ser Thr Glu Gln Leu Asp Gly Pro Lys Ser Leu Ala Ala Gly 725 730 735 Gly Leu Gly Gly Asn His Thr Cys Gly Met Leu Val Thr Leu Ser Leu 740 745 750 Leu Lys Ser Gln Ile Lys Val Leu Met Leu Val Gly Leu His Leu Asn 755 760 765 Cys Met Leu Asp Ile Gln Ile Met Met Asn Ser Gln His Leu Gln Cys 770 775 780 Asn Tyr Val Asp Leu Lys Arg Cys Phe Trp Ile Lys Ile Ile Leu Lys 785 790 795 800 Leu Phe Leu Leu Asn 805 5847PRTPiromyces sp. 5Thr Ser Trp Ser Glu Ala Asp Glu Lys Ala Lys Ser Phe Met Ser Asp 1 5 10 15 Leu Ser Glu Ser Glu Lys Ile Asp Ile Val Thr Gly Tyr Met Asn Met 20 25 30 Gln Gly Thr Cys Val Gly Asn Ile Lys Pro Leu Asp Arg Lys Asn Phe 35 40 45 Lys Gly Leu Cys Leu Gln Asp Gly Pro Ala Gly Val Arg Phe Asn Gly 50 55 60 Gly Thr Ser Thr Thr Trp Gln Ala Gly Ile Asn Asn Ala Ala Thr Phe 65 70 75 80 Asn Lys Asp Leu Leu Tyr Lys Ile Gly Lys Asp Gln Gly Ala Glu Phe 85 90 95 Tyr Ala Lys Gly Ile Asn Ile Ala Leu Ala Pro Ser Met Asn Ile Leu 100 105 110 Arg Ala Pro Ala Ser Gly Arg Val Trp Glu Asn Phe Gly Glu Asp Pro 115 120 125 Tyr Leu Ser Gly Val Cys Gly Ala Gln Ile Thr Lys Gly Tyr Gln Asp 130 135 140 Ser Gly Val Ile Val Ala Ala Lys His Tyr Val Ala Asn Asp Ile Glu 145 150 155 160 His Asn Arg Glu Ala Ser Ser Ser Asn Met Asp Asp Gln Thr Leu Met 165 170 175 Glu Ile His Val Glu Pro Phe Tyr Arg Thr Ile Lys Asp Gly Asp Ala 180 185 190 Gly Ser Val Met Ala Ser Tyr Asn Ala Val Asn Asn Ile Tyr Val Val 195 200 205 Gln Asn Lys Lys Val Leu Thr Glu Ile Leu Lys Glu Gly Ile Gly Phe 210 215 220 Gln Gly Phe Val Met Ser Asp Trp Trp Ala Ile His Asp Leu Glu Gly 225 230 235 240 Ser Phe Asn Ala Gly Met Asp Met Asn Met Pro Gly Gly Lys Ala Trp 245 250 255 Gly Pro Asp Tyr Val Asn Asn Ser Phe Trp Gly Ser Asn Ile Ser Asn 260 265 270 Ala Ile Arg Ser Gly Gln Val Ser Ser Ser Arg Leu Asp Asp Ala Val 275 280 285 Arg Arg Ile Ile Arg Thr Leu Tyr Arg Phe Asp Gln Met Ser Gly Tyr 290 295 300 Pro Asn Val Asn Leu Lys Ala Pro Ser Met His Ala Asp Thr Asn Arg 305 310 315 320 Gln Ala Ala Ile Glu Ser Ser Val Leu Leu Lys Asn Ala Asp Asp Ile 325 330 335 Leu Pro Leu Thr Lys Lys Tyr Arg Lys Ile Ala Ile Ile Gly Lys Asp 340 345 350 Ala Asp Lys Ala Gln Ser Cys Thr Asp Thr Ala Cys Ser Gly Gly Asn 355 360 365 Ile Ile Gln Gly Trp Gly Ser Gly Thr Thr Asp Phe Thr Gly Ile Ser 370 375 380 Asp Pro Ile Thr Ala Ile Lys Asn Arg Ala Ser Lys Glu Gly Ile Ser 385 390 395 400 Ile Val Ser Ser Ile Ser Asp Ser Ala Asn Glu Gly Ala Asn Val Ala 405 410 415 Lys Asp Ala Asp Val Ala Val Val Phe Val Arg Ala Thr Ser Gly Glu 420 425 430 Glu Tyr Ile Val Val Asp Asn Asn Lys Gly Asp Arg Asn Asn Leu Asp 435 440 445 Leu Trp His Gly Gly Asn Asp Leu Val Lys Ser Val Ala Ala Val Asn 450 455 460 Lys Asn Thr Val Val Val Ile His Ala Pro Ala Thr Val Asn Leu Pro 465 470 475 480 Phe Leu Asn Asn Val Lys Ala Ile Ile His Ala Gly Met Pro Gly Ala 485 490 495 Glu Ser Gly Asn Ala Ile Ala Ser Ile Leu Phe Gly Asp Ser Asn Pro 500 505 510 Ser Gly His Leu Pro Phe Thr Trp Ala Ala Arg Glu Asp Tyr Cys Cys 515 520 525 Asp Val Ser Tyr Pro Ala Glu Leu Pro His Gly Gly Asn Ser Lys Thr 530 535 540 Ala Tyr Asp Tyr Lys Glu Gly Leu Phe Val Gly Tyr Arg Trp Phe Asp 545 550 555 560 Lys Lys Asn Lys Thr Pro Ile Phe Pro Phe Gly His Gly Leu Ser Tyr 565 570 575 Thr Thr Phe Asp Tyr Ser Asn Leu Ser Val Ser Leu Lys Lys Ser Gly 580 585 590 Thr Gln Val Thr Gly Leu Glu Ala Thr Val Thr Val Ala Asn Thr Gly 595 600 605 Ser Tyr Glu Gly Ala Thr Val Pro Met Leu Phe Leu Gly Phe Pro Ala 610 615 620 Val Ser Glu Leu Gly Asp Tyr Pro Val Arg Asn Leu Lys Ala Phe Glu 625 630 635 640 Lys Val Asn Leu Lys Ala Gly Glu Lys Lys Thr Val Thr Leu Thr Val 645 650 655 Asp Gln His Gly Leu Ser Tyr Tyr Asn Thr Ser Lys Lys Ser Phe Val 660 665 670 Val Pro Thr Gly Gly Glu Phe Thr Val Tyr Val Gly Lys Ser Ala Gly 675 680 685 Asp Leu Pro Leu Lys Lys Ala Ile Lys Asn Thr Gln Gly Thr Asn Glu 690 695 700 Ser Ser Ser Ser Val Gly Asp Glu Asn Asn Asn Asn Pro Asn Asn Asn 705 710 715 720 Ala Asp Cys Ser Val Asn Gly Tyr Lys Cys Cys Ser Asn Ser Asn Ala 725 730 735 Glu Val Val Tyr Thr Asp Gly Asp Gly Asn Trp Gly Val Glu Asn Gly 740 745 750 Gln Trp Cys Ile Ile Lys Glu Gln Gln Gln Gln Gln Thr Cys Phe Ser 755 760 765 Ile Lys Leu Gly Tyr Pro Cys Cys Lys Gly Asn Glu Val Ala Tyr Thr 770 775 780 Asp Asn Asp Gly Gln Trp Gly Phe Glu Asn Gly Gln Trp Cys Gly Ile 785 790 795 800 Ala Thr Ala Thr Ser Gly Ala Gly Gly Cys Pro Tyr Thr Ser Lys Asn 805 810 815 Gly Tyr Pro Val Cys Gln Thr Thr Thr Lys Val Glu Tyr Val Asp Ser 820 825 830 Asp Lys Trp Gly Val Glu Asn Gly Asn Trp Cys Ile Met Cys Asn 835 840 845 6 847PRTCoccidioides immitis 6Ala Pro Pro Gly Val Gly Ala Leu Asp Asp Arg Ala Glu Leu Pro Asp 1 5 10 15 Gly Phe His Ser Pro Gln Tyr Tyr Pro Ala Pro Arg Gly Leu Gly Ala 20 25 30 Gly Met Glu Glu Ala Tyr Ser Lys Ala His Thr Val Val Ser Lys Met 35 40 45 Thr Leu Ala Gly Lys Val Asn Leu Thr Thr Gly Thr Gly Phe Leu Met 50 55 60 Ala Leu Val Gly Gln Thr Gly Ser Ala Leu Arg Phe Gly Ile Pro Arg 65 70 75 80 Leu Cys Leu Gln Asp Gly Pro Leu Gly Leu Arg Asn Thr Asp His Asn 85 90 95 Thr Ala Phe Pro Ala Gly Ile Ser Val Gly Ala Thr Phe Asp Lys Lys 100 105 110 Leu Met Tyr Glu Arg Gly Cys Ala Met Gly Glu Glu Phe Arg Gly Lys 115 120 125 Gly Ala Asn Val His Leu Gly Pro Ser Val Gly Pro Leu Gly Arg Lys 130 135 140 Pro Arg Gly Gly Arg Asn Trp Glu Gly Phe Gly Ser Asp Pro Ser Leu 145 150 155 160 Gln Ala Ile Ala Ala Val Glu Thr Ile Lys Gly Val Gln Ser Lys Gly 165 170 175 Val Ile Ala Thr Ile Lys His Leu Val Gly Asn Glu Gln Glu Met Tyr 180 185 190 Arg Met Thr Asn Ile Val Gln Arg Ala Tyr Ser Ala Asn Ile Asp Asp 195 200 205 Arg Thr Met His Glu Leu Tyr Leu Trp Pro Phe Ala Glu Ser Val Arg 210 215 220 Ala Gly Val Gly Ala Val Met Met Ala Tyr Asn Asp Val Asn Gly Ser 225 230 235 240 Ala Ser Cys Gln Asn Ser Lys Leu Ile Asn Gly Ile Leu Lys Asp Glu 245 250 255 Leu Gly Phe Gln Gly Phe Val Met Thr Asp Trp Tyr Ala Gln Ile Gly 260 265 270 Gly Val Ser Ser Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp 275 280 285 Gly Ser Val Pro Leu Ser Gly Thr Ser Phe Trp Ala Ser Glu Leu Ser 290 295 300 Arg Ser Ile Leu Asn Gly Thr Val Ala Leu Asp Arg Leu Asn Asp Met 305 310 315 320 Val Thr Arg Ile Val Ala Thr Trp Phe Lys Phe Gly Gln Asp Lys Asp 325 330 335 Phe Pro Leu Pro Asn Phe Ser Ser Tyr Thr Gln Asn Ala Lys Gly Leu 340 345 350 Leu Tyr Pro Gly Ala Leu Phe Ser Pro Leu Gly Val Val Asn Gln Phe 355 360 365 Val Asn Val Gln Ala Asp His His Lys Leu Ala Arg Val Ile Ala Arg 370 375 380 Glu Ser Ile Thr Leu Leu Lys Asn Glu Asp Asn Leu Leu Pro Leu Asp 385 390 395 400 Pro Asn Arg Ala Ile Lys Tyr Ser Glu Gln Met Pro Gly Thr Asn Pro 405 410 415 Arg Gly Ile Asn Ala Cys Pro Asp Lys Gly Cys Asn Lys Gly Val Leu 420 425 430 Thr Met Gly Trp Gly Ser Gly Thr Ser Asn Leu Pro Tyr Leu Val Thr 435 440 445 Pro Glu Asp Ala Ile Arg Asn Ile Ser Lys Asn Thr Glu Phe His Ile 450 455 460 Thr Asp Lys Phe Pro Asn Asn Val Gln Pro Gly Pro Asp Asp Val Ala 465 470 475 480 Ile Val Phe Val Asn Ala Asp Ser Gly Glu Asn Tyr Ile Ile Val Glu 485 490 495 Ser Asn Pro Gly Asp Arg Thr Val Ala Gln Met Lys Leu Trp His Asn 500 505 510 Gly Asp Glu Leu Ile Glu Ser Ala Ala Lys Lys Phe Ser Asn Val Val 515 520 525 Val Val Val Val His Thr Val Gly Pro Ile Ile Met Glu Lys Trp Ile 530 535 540 Asp Leu Leu Arg Ser Arg Val Ser Cys Leu Pro Asp Phe Gln Asp Lys 545 550 555 560 Lys Leu Glu Ile Leu Leu Leu Ile Ser Cys Ser Glu Thr Ser Val Arg 565 570 575 Val Ala Ala Ser Ile Tyr Asp Thr Glu Ser Arg Ile Gly Leu Ser Asp 580 585 590 Ser Val Ser Leu Ile Asn Gln Arg Phe Gly Gln Ile Gln Asp Thr Phe 595 600 605 Thr Glu Gly Leu Phe Ile Asp Tyr Arg His Phe Gln Lys Glu Asn Ile 610 615 620 Thr Pro Arg Tyr His Phe Gly Tyr Gly Leu Ser Tyr Thr Thr Phe Asn 625 630 635 640 Phe Thr Glu Pro Arg Leu Glu Ser Val Thr Thr Leu Ser Glu Tyr Pro 645 650 655 Pro Ala Arg Lys Pro Lys Ala Gly Asp Arg His Thr Pro Thr Ile Ser 660 665 670 His Leu Leu Gln Lys Trp Pro Gly Pro Lys Thr Leu Thr Gly Ser Gly 675 680 685 Ala Tyr Leu Tyr Pro Tyr Leu Asp Asn Pro Ser Ala Ile Lys Pro Lys 690 695 700 Pro Gly Tyr Pro Tyr Pro Glu Ala Ile Gln Pro Asn Leu Asn Leu Asn 705 710 715 720 Pro Arg Ala Gly Gly Ser Glu Ala Val Thr Arg Arg Tyr Gly Met Leu 725 730 735 Arg Ser Arg Phe Pro Leu Lys Leu Leu Ile Leu Glu Arg Asn Pro Val 740 745 750 Arg Ala Val Ala Gln Leu Tyr Val Glu Leu Pro Thr Asp Asp Glu His 755 760 765 Pro Thr Pro Lys Leu Gln Leu Arg Gln Phe Glu Lys Thr Ala Thr Leu 770 775 780 Glu Pro Gly Gln Ser Glu Val Leu Lys Met Glu Ile Thr Arg Lys Asp 785 790 795 800 Val Ser Ile Trp Asp Thr Met Val Gln Asp Trp Lys Val Pro Ala Thr 805 810 815 Gly Lys Gly Ile Lys Leu Trp Ile Gly Ala Ser Val Gly Asp Leu Lys 820 825 830 Ala Val Cys Glu Thr Gly Lys Gly Lys Ser Cys His Val Leu Asn 835 840 845 7 863PRTSaccharomycopsis fibuligera 7Leu Pro Val Gln Thr His Asn Leu Thr Asp Asn Gln Gly Phe Asp Glu 1 5 10 15 Glu Ser Ser Gln Trp Ile Ser Pro His Tyr Tyr Pro Thr Pro Gln Gly 20 25 30 Gly Arg Leu Gln Gly Val Trp Gln Asp Ala Tyr Thr Lys Ala Lys Ala 35 40 45 Leu Val Ser Gln Met Thr Ile Val Glu Lys Val Asn Leu Thr Thr Gly 50 55 60 Thr Gly Trp Gln Leu Gly Pro Cys Val Gly Asn Thr Gly Ser Val Pro 65 70 75 80 Arg Phe Gly Ile Pro Asn Leu Cys Leu Gln Asp Gly Pro Leu Gly Val 85 90 95 Arg Leu Thr Asp Phe Ser Thr Gly Tyr Pro Ser Gly Met Ala Thr Gly 100 105 110 Ala Thr Phe Asn Lys Asp Leu Phe Leu Gln Arg Gly Gln Ala Leu Gly 115 120 125 His Glu Phe Asn Ser Lys Gly Val His Ile Ala Leu Gly Pro Ala Val 130 135 140 Gly Pro Leu Gly Val Lys Ala Arg Gly Gly Arg Asn Phe Glu Ala Phe 145 150 155 160 Gly Ser Asp Pro Tyr Leu Gln Gly Ile Ala Ala Ala Ala Thr Ile Lys 165 170 175 Gly Leu Gln Glu Asn Asn Val Met Ala Cys Val Lys His Phe Ile Gly 180 185 190 Asn Glu Gln Asp Ile Tyr Arg Gln Pro Ser Asn Ser Lys Val Asp Pro 195 200 205 Glu Tyr Asp Pro Ala Thr Lys Glu Ser Ile Ser Ala Asn Ile Pro Asp 210 215 220 Arg Ala Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ser Ile Arg 225 230 235 240 Ala Gly Val Gly Ser Val Met Cys Ser Tyr Asn Arg Val Asn Asn Thr 245 250 255 Tyr Ser Cys Glu Asn Ser Tyr Met Ile Asn His Leu Leu Lys Glu Glu 260 265 270 Leu Gly Phe Gln Gly Phe Val Val Ser Asp Trp Ala Ala Gln Met

Ser 275 280 285 Gly Ala Tyr Ser Ala Ile Ser Gly Leu Asp Met Ser Met Pro Gly Glu 290 295 300 Leu Leu Gly Gly Trp Asn Thr Gly Lys Ser Tyr Trp Gly Gln Asn Leu 305 310 315 320 Thr Lys Ala Val Tyr Asn Glu Thr Val Pro Ile Glu Arg Leu Asp Asp 325 330 335 Met Ala Thr Arg Ile Leu Ala Ala Leu Tyr Ala Thr Asn Ser Phe Pro 340 345 350 Thr Lys Asp Arg Leu Pro Asn Phe Ser Ser Phe Thr Thr Lys Glu Tyr 355 360 365 Gly Asn Glu Phe Phe Val Asp Lys Thr Ser Pro Val Val Lys Val Asn 370 375 380 His Phe Val Asp Pro Ser Asn Asp Phe Thr Glu Asp Thr Ala Leu Lys 385 390 395 400 Val Ala Glu Glu Ser Ile Val Leu Leu Lys Asn Glu Lys Asn Thr Leu 405 410 415 Pro Ile Ser Pro Asn Lys Val Arg Lys Leu Leu Leu Ser Gly Ile Ala 420 425 430 Ala Gly Pro Asp Pro Lys Gly Tyr Glu Cys Ser Asp Gln Ser Cys Val 435 440 445 Asp Gly Ala Leu Phe Glu Gly Trp Gly Ser Gly Ser Val Gly Tyr Pro 450 455 460 Lys Tyr Gln Val Thr Pro Phe Glu Glu Ile Ser Ala Asn Ala Arg Lys 465 470 475 480 Asn Lys Met Gln Phe Asp Tyr Ile Arg Glu Ser Phe Asp Leu Thr Gln 485 490 495 Val Ser Thr Val Ala Ser Asp Ala His Met Ser Ile Val Val Val Ser 500 505 510 Ala Val Ser Gly Glu Gly Tyr Leu Ile Ile Asp Gly Asn Arg Gly Asp 515 520 525 Lys Asn Asn Val Thr Leu Trp His Asn Ser Asp Asn Leu Ile Lys Ala 530 535 540 Val Ala Glu Asn Cys Ala Asn Thr Val Val Val Ile Thr Ser Thr Gly 545 550 555 560 Gln Val Asp Val Glu Ser Phe Ala Asp His Pro Asn Val Thr Ala Ile 565 570 575 Val Trp Ala Gly Pro Leu Gly Asp Arg Ser Gly Thr Ala Ile Ala Asn 580 585 590 Ile Leu Phe Gly Asn Ala Asn Pro Ser Gly His Leu Pro Phe Thr Val 595 600 605 Ala Lys Ser Asn Asp Asp Tyr Ile Pro Ile Val Thr Tyr Asn Pro Pro 610 615 620 Asn Gly Glu Pro Glu Asp Asn Thr Leu Ala Glu His Asp Leu Leu Val 625 630 635 640 Asp Tyr Arg Tyr Phe Glu Glu Lys Asn Ile Glu Pro Arg Tyr Ala Phe 645 650 655 Gly Tyr Gly Leu Ser Tyr Asn Glu Tyr Lys Val Ser Asn Ala Lys Val 660 665 670 Ser Ala Ala Lys Lys Val Asp Glu Glu Leu Pro Gln Pro Lys Leu Tyr 675 680 685 Leu Ala Glu Tyr Ser Tyr Asn Lys Thr Glu Glu Ile Asn Asn Pro Glu 690 695 700 Asp Ala Phe Phe Pro Ser Asn Ala Arg Arg Ile Gln Glu Phe Leu Tyr 705 710 715 720 Pro Tyr Leu Asp Ser Asn Val Thr Leu Lys Asp Gly Asn Tyr Glu Tyr 725 730 735 Pro Asp Gly Tyr Ser Thr Glu Gln Arg Thr Thr Pro Ile Gln Pro Gly 740 745 750 Gly Gly Leu Gly Gly Asn Asp Ala Leu Trp Glu Val Ala Tyr Lys Val 755 760 765 Glu Val Asp Val Gln Asn Leu Gly Asn Ser Thr Asp Lys Phe Val Pro 770 775 780 Gln Leu Tyr Leu Lys His Pro Glu Asp Gly Lys Phe Glu Thr Pro Val 785 790 795 800 Gln Leu Arg Gly Phe Glu Lys Val Glu Leu Ser Pro Gly Glu Lys Lys 805 810 815 Thr Val Glu Phe Glu Leu Leu Arg Arg Asp Leu Ser Val Trp Asp Thr 820 825 830 Thr Arg Gln Ser Trp Ile Val Glu Ser Gly Thr Tyr Glu Ala Leu Ile 835 840 845 Gly Val Ala Val Asn Asp Ile Lys Thr Ser Val Leu Phe Thr Ile 850 855 860 8859PRTSaccharomycopsis fibuligera 8Val Pro Ile Gln Asn Tyr Thr Gln Ser Pro Ser Gln Arg Asp Glu Ser 1 5 10 15 Ser Gln Trp Val Ser Pro His Tyr Tyr Pro Thr Pro Gln Gly Gly Arg 20 25 30 Leu Gln Asp Val Trp Gln Glu Ala Tyr Ala Arg Ala Lys Ala Ile Val 35 40 45 Gly Gln Met Thr Ile Val Glu Lys Val Asn Leu Thr Thr Gly Thr Gly 50 55 60 Trp Gln Leu Asp Pro Cys Val Gly Asn Thr Gly Ser Val Pro Arg Phe 65 70 75 80 Gly Ile Pro Asn Leu Cys Leu Gln Asp Gly Pro Leu Gly Val Arg Phe 85 90 95 Ala Asp Phe Val Thr Gly Tyr Pro Ser Gly Leu Ala Thr Gly Ala Thr 100 105 110 Phe Asn Lys Asp Leu Phe Leu Gln Arg Gly Gln Ala Leu Gly His Glu 115 120 125 Phe Asn Ser Lys Gly Val His Ile Ala Leu Gly Pro Ala Val Gly Pro 130 135 140 Leu Gly Val Lys Ala Arg Gly Gly Arg Asn Phe Glu Ala Phe Gly Ser 145 150 155 160 Asp Pro Tyr Leu Gln Gly Thr Ala Ala Ala Ala Thr Ile Lys Gly Leu 165 170 175 Gln Glu Asn Asn Val Met Ala Cys Val Lys His Phe Ile Gly Asn Glu 180 185 190 Gln Glu Lys Tyr Arg Gln Pro Asp Asp Ile Asn Pro Ala Thr Asn Gln 195 200 205 Thr Thr Lys Glu Ala Ile Ser Ala Asn Ile Pro Asp Arg Ala Met His 210 215 220 Ala Leu Tyr Leu Trp Pro Phe Ala Asp Ser Val Arg Ala Gly Val Gly 225 230 235 240 Ser Val Met Cys Ser Tyr Asn Arg Val Asn Asn Thr Tyr Ala Cys Glu 245 250 255 Asn Ser Tyr Met Met Asn His Leu Leu Lys Glu Glu Leu Gly Phe Gln 260 265 270 Gly Phe Val Val Ser Asp Trp Gly Ala Gln Leu Ser Gly Val Tyr Ser 275 280 285 Ala Ile Ser Gly Leu Asp Met Ser Met Pro Gly Glu Val Tyr Gly Gly 290 295 300 Trp Asn Thr Gly Thr Ser Phe Trp Gly Gln Asn Leu Thr Lys Ala Ile 305 310 315 320 Tyr Asn Glu Thr Val Pro Ile Glu Arg Leu Asp Asp Met Ala Thr Arg 325 330 335 Ile Leu Ala Ala Leu Tyr Ala Thr Asn Ser Phe Pro Thr Glu Asp His 340 345 350 Leu Pro Asn Phe Ser Ser Trp Thr Thr Lys Glu Tyr Gly Asn Lys Tyr 355 360 365 Tyr Ala Asp Asn Thr Thr Glu Ile Val Lys Val Asn Tyr Asn Val Asp 370 375 380 Pro Ser Asn Asp Phe Thr Glu Asp Thr Ala Leu Lys Val Ala Glu Glu 385 390 395 400 Ser Ile Val Leu Leu Lys Asn Glu Asn Asn Thr Leu Pro Ile Ser Pro 405 410 415 Glu Lys Ala Lys Arg Leu Leu Leu Ser Gly Ile Ala Ala Gly Pro Asp 420 425 430 Pro Ile Gly Tyr Gln Cys Glu Asp Gln Ser Cys Thr Asn Gly Ala Leu 435 440 445 Phe Gln Gly Trp Gly Ser Gly Ser Val Gly Ser Pro Lys Tyr Gln Val 450 455 460 Thr Pro Phe Glu Glu Ile Ser Tyr Leu Ala Arg Lys Asn Lys Met Gln 465 470 475 480 Phe Asp Tyr Ile Arg Glu Ser Tyr Asp Leu Ala Gln Val Thr Lys Val 485 490 495 Ala Ser Asp Ala His Leu Ser Ile Val Val Val Ser Ala Ala Ser Gly 500 505 510 Glu Gly Tyr Ile Thr Val Asp Gly Asn Gln Gly Asp Arg Lys Asn Leu 515 520 525 Thr Leu Trp Asn Asn Gly Asp Lys Leu Ile Glu Thr Val Ala Glu Asn 530 535 540 Cys Ala Asn Thr Val Val Val Val Thr Ser Thr Gly Gln Ile Asn Phe 545 550 555 560 Glu Gly Phe Ala Asp His Pro Asn Val Thr Ala Ile Val Trp Ala Gly 565 570 575 Pro Leu Gly Asp Arg Ser Gly Thr Ala Ile Ala Asn Ile Leu Phe Gly 580 585 590 Lys Ala Asn Pro Ser Gly His Leu Pro Phe Thr Ile Ala Lys Thr Asp 595 600 605 Asp Asp Tyr Ile Pro Ile Glu Thr Tyr Ser Pro Ser Ser Gly Glu Pro 610 615 620 Glu Asp Asn His Leu Val Glu Asn Asp Leu Leu Val Asp Tyr Arg Tyr 625 630 635 640 Phe Glu Glu Lys Asn Ile Glu Pro Arg Tyr Ala Phe Gly Tyr Gly Leu 645 650 655 Ser Tyr Asn Glu Tyr Glu Val Ser Asn Ala Lys Val Ser Ala Ala Lys 660 665 670 Lys Val Asp Glu Glu Leu Pro Glu Pro Ala Thr Tyr Leu Ser Glu Phe 675 680 685 Ser Tyr Gln Asn Ala Lys Asp Ser Lys Asn Pro Ser Asp Ala Phe Ala 690 695 700 Pro Ala Asp Leu Asn Arg Val Asn Glu Tyr Leu Tyr Pro Tyr Leu Asp 705 710 715 720 Ser Asn Val Thr Leu Lys Asp Gly Asn Tyr Glu Tyr Pro Asp Gly Tyr 725 730 735 Ser Thr Glu Gln Arg Thr Thr Pro Asn Gln Pro Gly Gly Gly Leu Gly 740 745 750 Gly Asn Asp Ala Leu Trp Glu Val Ala Tyr Asn Ser Thr Asp Lys Phe 755 760 765 Val Pro Gln Gly Asn Ser Thr Asp Lys Phe Val Pro Gln Leu Tyr Leu 770 775 780 Lys His Pro Glu Asp Gly Lys Phe Glu Thr Pro Ile Gln Leu Arg Gly 785 790 795 800 Phe Glu Lys Val Glu Leu Ser Pro Gly Glu Lys Lys Thr Val Asp Leu 805 810 815 Arg Leu Leu Arg Arg Asp Leu Ser Val Trp Asp Thr Thr Arg Gln Ser 820 825 830 Trp Ile Val Glu Ser Gly Thr Tyr Glu Ala Leu Ile Gly Val Ala Val 835 840 845 Asn Asp Ile Lys Thr Ser Val Leu Phe Thr Ile 850 855 9786PRTSeptoria lycopersici 9Leu Ser His Glu Asp Gln Ser Lys His Phe Thr Thr Ile Pro Thr Phe 1 5 10 15 Pro Thr Pro Asp Ser Thr Gly Glu Gly Trp Lys Ala Ala Phe Glu Lys 20 25 30 Ala Ala Asp Ala Val Ser Arg Leu Asn Leu Thr Gln Lys Val Ala Leu 35 40 45 Thr Thr Gly Thr Thr Ala Gly Leu Ser Cys Asn Gly Asn Ile Ala Pro 50 55 60 Ile Pro Glu Ile Asn Phe Ser Gly Leu Cys Leu Ala Asp Gly Pro Val 65 70 75 80 Ser Val Arg Ile Ala Asp Leu Ala Thr Val Phe Pro Ala Gly Leu Thr 85 90 95 Ala Ala Ala Thr Trp Asp Arg Gln Leu Ile Tyr Glu Arg Ala Arg Ala 100 105 110 Leu Gly Ser Glu Phe Arg Gly Lys Gly Ser Gln Val His Leu Gly Pro 115 120 125 Ala Ser Gly Ala Leu Gly Arg His Pro Leu Gly Gly Arg Asn Trp Glu 130 135 140 Ser Phe Ser Pro Asp Pro Tyr Leu Ser Gly Val Ala Met Asp Phe Ser 145 150 155 160 Ile Arg Gly Ile Gln Glu Met Gly Val Gln Ala Asn Arg Lys His Phe 165 170 175 Ile Gly Asn Glu Gln Glu Thr Gln Arg Ser Asn Thr Phe Thr Asp Asp 180 185 190 Gly Thr Glu Ile Gln Ala Ile Ser Ser Asn Ile Asp Asp Arg Thr Met 195 200 205 His Glu Leu Tyr Leu Trp Pro Phe Ala Asn Ala Val Arg Ser Gly Val 210 215 220 Ala Ser Val Met Cys Ser Tyr Asn Arg Leu Asn Gln Thr Tyr Ala Cys 225 230 235 240 Glu Asn Ser Lys Leu Met Asn Gly Ile Leu Lys Gly Glu Leu Gly Phe 245 250 255 Gln Gly Tyr Val Val Ser Asp Trp Tyr Ala Thr His Ser Gly Val Glu 260 265 270 Ser Val Asn Ala Gly Leu Asp Met Thr Met Pro Gly Pro Leu Asp Ser 275 280 285 Pro Ser Thr Ala Leu Arg Pro Pro Pro Ser Tyr Leu Gly Gly Asn Leu 290 295 300 Thr Glu Ala Val Leu Asn Gly Thr Ile Pro Glu Ala Arg Val Asp Asp 305 310 315 320 Met Ala Arg Arg Ile Leu Met Pro Tyr Phe Phe Leu Gly Gln Asp Thr 325 330 335 Asp Phe Pro Thr Val Asp Pro Ser Thr Gly Phe Val Phe Ala Arg Thr 340 345 350 Tyr Asn Tyr Pro Asp Glu Tyr Leu Thr Leu Gly Gly Leu Asp Pro Tyr 355 360 365 Asn Pro Pro Pro Ala Arg Asp Val Arg Gly Asn His Ser Asp Ile Val 370 375 380 Arg Lys Val Ala Ala Ala Gly Thr Val Leu Leu Lys Asn Val Asn Asn 385 390 395 400 Val Leu Pro Leu Lys Glu Pro Lys Ser Val Gly Ile Phe Gly Asn Gly 405 410 415 Ala Ala Asp Val Thr Glu Gly Leu Thr Phe Thr Gly Asp Asp Ser Gly 420 425 430 Pro Trp Gly Ala Asp Ile Gly Ala Leu Ser Val Gly Gly Gly Ser Gly 435 440 445 Ala Gly Arg His Thr His Leu Val Ser Pro Leu Ala Ala Ile Arg Lys 450 455 460 Arg Thr Glu Ser Val Gly Gly Arg Val Gln Tyr Leu Leu Ser Asn Ser 465 470 475 480 Arg Ile Val Asn Asp Asp Phe Thr Ser Ile Tyr Pro Thr Pro Glu Val 485 490 495 Cys Leu Val Phe Leu Lys Thr Trp Ala Arg Glu Gly Thr Asp Arg Leu 500 505 510 Ser Tyr Glu Asn Asp Trp Asn Ser Thr Ala Val Val Asn Asn Val Ala 515 520 525 Arg Arg Cys Pro Asn Thr Ile Val Val Thr His Ser Gly Gly Ile Asn 530 535 540 Thr Met Pro Trp Ala Asp Asn Ala Asn Val Thr Ala Ile Leu Ala Ala 545 550 555 560 His Tyr Pro Gly Gln Glu Asn Gly Asn Ser Ile Met Asp Ile Leu Tyr 565 570 575 Gly Asp Val Asn Pro Ser Gly Arg Leu Pro Tyr Thr Ile Pro Lys Leu 580 585 590 Ala Thr Asp Tyr Asp Phe Pro Val Val Asn Ile Thr Asn Glu Ala Gln 595 600 605 Asp Pro Tyr Val Trp Gln Ala Asp Phe Thr Glu Gly Leu Leu Ile Asp 610 615 620 Tyr Arg His Phe Asp Ala Arg Asn Ile Thr Pro Leu Tyr Glu Phe Gly 625 630 635 640 Tyr Gly Leu Ser Tyr Thr Thr Phe Glu Ile Glu Gly Val Ala Asn Leu 645 650 655 Val Ala Lys Ser Ala Lys Leu Ser Ala Phe Pro Ala Ser Thr Asp Ile 660 665 670 Ser His Pro Gly Gly Asn Pro Asp Leu Trp Glu Glu Val Val Ser Val 675 680 685 Thr Ala Ala Val Lys Asn Thr Gly Ser Val Ser Gly Ser Gln Val Val 690 695 700 Gln Leu Tyr Ile Ser Leu Pro Ala Asp Gly Ile Pro Glu Asn Ser Pro 705 710 715 720 Met Gln Val Leu Arg Gly Phe Glu Lys Val Asp Leu Gln Pro Gly Gln 725 730 735 Ser Lys Ser Val Glu Phe Ser Ile Met Arg Arg Asp Leu Ser Phe Trp 740 745 750 Asn Thr Thr Ala Gln Asp Trp Glu Ile Pro Asn Gly Gln Ile Glu Phe 755 760 765 Arg Val Gly Phe Ser Ser Arg Asp Ile Lys Ser Ile Val Ser Arg Ser 770 775 780 Phe Leu 785 10747PRTKuraishia capsulata 10Lys Asn Ile Ser Lys Ala Glu Met Glu Asn Leu Glu His Trp Trp Ser 1 5 10 15 Tyr Gly Arg Ser Asp Pro Val Tyr Pro Ser Pro Glu Ile Ser Gly Leu 20 25 30 Gly Asp Trp Gln Phe Ala Tyr Gln Arg Ala Arg Glu Ile Val Ala Leu 35 40 45 Met Thr Asn Glu Glu Lys Thr Asn Leu Thr Phe Gly Ser Ser Gly Asp 50 55 60 Thr Gly Cys Ser Gly Met

Ile Ser Asp Val Pro Asp Val Asp Phe Pro 65 70 75 80 Gly Leu Cys Leu Gln Asp Ala Gly Asn Gly Val Arg Gly Thr Asp Met 85 90 95 Val Asn Ala Tyr Ala Ser Gly Leu His Val Gly Ala Ser Trp Asn Arg 100 105 110 Gln Leu Ala Tyr Asp Arg Ala Val Tyr Met Gly Ala Glu Phe Arg His 115 120 125 Lys Gly Val Asn Val Leu Leu Gly Pro Val Val Gly Pro Ile Gly Arg 130 135 140 Val Ala Thr Gly Gly Arg Asn Trp Glu Gly Phe Thr Asn Asp Pro Tyr 145 150 155 160 Leu Ala Gly Ala Leu Val Tyr Glu Thr Thr Lys Gly Ile Gln Glu Asn 165 170 175 Val Ile Ala Cys Thr Lys His Phe Ile Gly Asn Glu Gln Glu Thr Asn 180 185 190 Arg Asn Pro Ser Gly Thr Tyr Asn Gln Ser Val Ser Ala Asn Ile Asp 195 200 205 Asp Lys Thr Met His Glu Leu Tyr Leu Trp Pro Phe Gln Asp Ser Val 210 215 220 Arg Ala Gly Leu Gly Ser Ile Met Gly Ser Tyr Asn Arg Val Asn Asn 225 230 235 240 Ser Tyr Ala Cys Lys Asn Ser Lys Val Leu Asn Gly Leu Leu Lys Ser 245 250 255 Glu Leu Gly Phe Gln Gly Phe Val Val Ser Asp Trp Gly Gly Gln His 260 265 270 Thr Gly Ile Ala Ser Ala Asn Ala Gly Leu Asp Met Ala Met Pro Ser 275 280 285 Ser Thr Tyr Trp Glu Glu Gly Leu Ile Glu Ala Val Lys Asn Gly Thr 290 295 300 Val Asp Gln Ser Arg Leu Asp Asp Met Ala Thr Arg Ile Ile Ala Ala 305 310 315 320 Trp Tyr Lys Tyr Ala Arg Leu Asp Asp Pro Gly Phe Gly Met Pro Val 325 330 335 Ser Leu Ala Glu Asp His Glu Leu Val Asp Ala Arg Asp Pro Ala Ala 340 345 350 Ala Ser Thr Ile Phe Gln Gly Ala Val Glu Gly His Val Leu Val Lys 355 360 365 Asn Glu Asn Ala Leu Pro Leu Lys Lys Pro Lys Tyr Ile Ser Leu Phe 370 375 380 Gly Tyr Asp Gly Val Ser Thr Asp Val Asn Thr Val Gly Gly Gly Phe 385 390 395 400 Ser Phe Phe Ser Phe Asp Val Lys Ala Ile Glu Asn Lys Thr Leu Ile 405 410 415 Ser Gly Gly Gly Ser Gly Thr Asn Thr Pro Ser Tyr Val Asp Ala Pro 420 425 430 Phe Asn Ala Phe Val Ala Lys Ala Arg Glu Asp Asn Thr Phe Leu Ser 435 440 445 Trp Asp Phe Thr Ser Ala Glu Pro Val Ala Asn Pro Ala Ser Asp Ala 450 455 460 Cys Ile Asp Phe Ile Asn Ala Ala Ala Ser Glu Gly Tyr Asp Arg Pro 465 470 475 480 Asn Leu Ala Asp Lys Tyr Ser Asp Lys Leu Val Glu Ala Val Ala Ser 485 490 495 Gln Cys Ser Asn Thr Ile Val Val Ile His Asn Ala Gly Ile Arg Leu 500 505 510 Val Asp Asn Trp Ile Glu His Glu Asn Val Thr Gly Val Ile Leu Ala 515 520 525 His Leu Pro Gly Gln Asp Thr Gly Thr Ser Leu Ile Glu Val Leu Tyr 530 535 540 Gly Asn Gln Ser Pro Ser Gly Arg Leu Pro Tyr Thr Val Ala Lys Lys 545 550 555 560 Ala Ser Asp Tyr Gly Gly Leu Leu Trp Pro Thr Glu Pro Glu Gly Asp 565 570 575 Leu Asp Leu Tyr Phe Pro Gln Ser Asn Phe Thr Glu Gly Val Tyr Ile 580 585 590 Asp Tyr Lys Tyr Phe Ile Gln Lys Asn Ile Thr Pro Arg Tyr Glu Phe 595 600 605 Gly Tyr Gly Leu Thr Tyr Thr Thr Phe Asp Tyr Ser Glu Leu Glu Val 610 615 620 Asp Ala Ile Thr Asn Gln Ser Tyr Leu Pro Pro Asp Cys Thr Ile Glu 625 630 635 640 Glu Gly Gly Ala Lys Ser Leu Trp Asp Ile Val Ala Thr Val Lys Phe 645 650 655 Thr Val Thr Asn Thr Gly Asp Val Ala Ala Ala Glu Val Pro Gln Leu 660 665 670 Tyr Val Gly Ile Pro Asn Gly Pro Pro Lys Val Leu Arg Gly Phe Asp 675 680 685 Lys Lys Leu Ile His Pro Gly Gln Ser Glu Glu Phe Val Phe Glu Leu 690 695 700 Thr Arg Arg Asp Leu Ser Thr Trp Asp Val Val Ala Gln Asn Trp Gly 705 710 715 720 Leu Gln Ala Gly Thr Tyr Gln Phe Tyr Val Gly Arg Ser Val Phe Asp 725 730 735 Val Pro Leu Thr Ser Ala Leu Val Phe Thr Asn 740 745 11740PRTTrichoderma reesei 11Ala Lys Gly Val Ser Gln Ile Pro Ser Thr His Ser Ser Gln Ser Lys 1 5 10 15 Gly Asn Gly Pro Trp Ala His Ala Tyr Arg Arg Ala Glu Lys Leu Val 20 25 30 Arg Gln Met Thr Leu Glu Glu Lys Ala Asn Ile Thr Arg Gly Phe Thr 35 40 45 Gly Asp Asn Val Cys Ala Gly Asn Thr Gly Ser Val Pro Arg Leu Gly 50 55 60 Trp Pro Gly Met Cys Val His Asp Ala Gly Asn Gly Val Arg Ala Thr 65 70 75 80 Asp Leu Val Asn Ser Tyr Pro Ser Gly Ile His Val Gly Ala Ser Trp 85 90 95 Asp Arg Asn Leu Thr Tyr Glu Arg Gly Leu His Met Gly Gly Glu Phe 100 105 110 Lys Ala Lys Gly Val Asn Val Pro Leu Gly Pro Asn Ala Gly Pro Leu 115 120 125 Gly Arg Thr Pro Leu Gly Gly Arg Asn Trp Glu Gly Phe Ser Ile Asp 130 135 140 Pro Tyr Leu Ser Gly Gln Leu Asn Ala Glu Thr Ile Thr Gly Met Gln 145 150 155 160 Asp Ala Gly Val Ile Ala Asn Ile Lys His Phe Ile Ala Asn Glu Gln 165 170 175 Glu Thr Leu Arg Arg Pro Tyr Phe Gly Val Glu Ala Val Ser Ala Asn 180 185 190 Ile Asp Asp Arg Thr Leu His Glu Tyr Tyr Leu Trp Pro Phe Met Asp 195 200 205 Ser Val His Ala Gly Val Gly Ser Val Met Cys Ser Tyr Asn Arg Ile 210 215 220 Asn Asn Thr Tyr Gly Cys Met Asn Asp Lys Leu Met Asn Gly Ile Leu 225 230 235 240 Lys Ala Glu Leu Gly Phe Gln Gly Phe Val Met Leu Asp Trp Asn Ala 245 250 255 Gln His Asp Leu Gln Ser Ala Asn Ala Gly Leu Asp Met Val Met Pro 260 265 270 Leu Gly Gly Ser Trp Gly Lys Asn Leu Thr Asp Ala Val Ala Asn Gly 275 280 285 Thr Val Ser Glu Ser Arg Ile Thr Asp Met Ala Thr Arg Ile Ile Ala 290 295 300 Ala Trp Tyr Leu Val Gly Gln Asp Gly Asn Asn Phe Pro Val Pro Gly 305 310 315 320 Ile Gly Leu Lys Gln Leu Thr Lys Pro His Glu Gln Val Asp Ala Arg 325 330 335 Asp Pro Ala Ser Lys Pro Val Leu Leu Glu Gly Ala Ile Ala Gly His 340 345 350 Val Leu Val Lys Asn Glu Asn Asn Ala Leu Pro Phe Asn Lys Lys Leu 355 360 365 Thr Met Ile Ser Val Phe Gly Tyr Asp Ala Thr Ile Pro Arg Thr Lys 370 375 380 Asn Thr Asp Ile Leu Phe Gln Leu Gly Tyr Thr Ser Ser Pro Glu Met 385 390 395 400 Ala Gln Ala Val Leu Gly Asn Glu Ala His Phe Asp Gln Ala Ala Lys 405 410 415 Gly Gly Thr Ile Met Thr Gly Gly Arg Ala Gly Ala Asn Ala Pro Ser 420 425 430 Tyr Ile Asp Asp Pro Leu Ala Ala Ile Gln Arg Arg Ala Arg Lys Asp 435 440 445 Asp Thr Trp Val Asn Trp Asp Leu Asp Ser Phe Asn Pro Glu Val Asn 450 455 460 Ala Ala Ser Asp Ala Cys Leu Val Phe Ile Asn Ala Ile Ala Thr Glu 465 470 475 480 Gly Trp Asp Arg Asp Gly Leu His Asp Asp Phe Ser Asp Gly Leu Val 485 490 495 Leu Asn Val Ala Ala Asn Cys Ser Asn Thr Ile Val Val Val His Ala 500 505 510 Ala Gly Thr Arg Leu Val Asp Gln Trp Ile Glu His Pro Asn Val Thr 515 520 525 Ala Ala Val Ile Ala His Leu Pro Gly Gln Asp Ser Gly Arg Ala Leu 530 535 540 Val Lys Leu Leu Tyr Gly Glu Ala Asn Phe Ser Gly Lys Leu Pro Tyr 545 550 555 560 Thr Ile Ala Lys Asn Glu Ser Asp Tyr Ser Val Tyr Thr Pro Cys Gln 565 570 575 Arg Arg Ser Pro Glu Asp Thr Asp Pro Gln Cys Asp Phe Thr Glu Gly 580 585 590 Val Tyr Leu Asp Tyr Arg Ala Phe Asp Ala Asn Asn Met Thr Pro Arg 595 600 605 Phe Glu Phe Gly Tyr Gly Leu Ser Tyr Thr Ser Phe Asn Tyr Ser Ala 610 615 620 Leu Ser Ile Lys Lys Ala Lys Gly Leu Arg Gln Ser Arg Cys Thr Asp 625 630 635 640 Asp Leu Trp Gln Ala Ala Ala Gln Val Thr Ala Ser Ile Thr Asn Ser 645 650 655 Gly Gly Met Ser Gly Ser Glu Val Ala Gln Leu Tyr Leu Ala Ile Pro 660 665 670 Asn Ser Pro Pro Lys Gln Leu Arg Gly Phe Asn Lys Leu Leu Leu Arg 675 680 685 Pro His Glu Ser Gly Thr Val His Phe Gly Leu Thr Lys Arg Asp Leu 690 695 700 Ser Val Trp Asp Val Val Ser Gln Ser Trp Val Ile Gln Glu Gly Glu 705 710 715 720 Tyr Lys Val Phe Val Gly Ala Ser Ser Arg Asp Ile Arg Leu Ser Gly 725 730 735 Lys Leu His Ile 740 12818PRTUromyces fabae 12Ala Thr Thr Ser Pro Ser Glu Asn Gln Asn Gln Ser Tyr Asn Pro Gln 1 5 10 15 Ile Glu Gly Leu Thr Val Gln Pro Ser Thr Val Ala Asn Gly Leu Arg 20 25 30 Ile Asn Ser Asn Ser Leu Ile Ser Asn Phe Asp Phe Glu Ile Ile Gln 35 40 45 Pro Pro Pro Gly Tyr Glu Glu Trp Thr Ser Pro Val Val Leu Pro Ala 50 55 60 Pro Val Gln Ser Gly Leu Ser Pro Trp Ser Glu Ser Ile Val Arg Ala 65 70 75 80 Arg Ala Phe Val Ala Gln Leu Thr Ile Glu Glu Lys Val Asn Leu Thr 85 90 95 Thr Gly Ala Gly Thr Gln Gly Arg Cys Val Gly Glu Thr Gly Thr Val 100 105 110 Pro Arg Leu Gly Phe Asn Gln Pro Ile Cys Leu Gln Asp Gly Pro Val 115 120 125 Gly Ile Arg Tyr Thr Asp Phe Asn Ser Val Phe Pro Ala Ala Ile Asn 130 135 140 Val Ala Ala Thr Phe Asp Lys Gln Leu Met Phe Lys Arg Ala Gln Ala 145 150 155 160 Met Ala Glu Glu Phe Arg Gly Lys Gly Ala Asn Val Val Leu Ala Pro 165 170 175 Met Thr Asn Leu Met Arg Thr Pro Gln Ala Gly Arg Ala Trp Glu Gly 180 185 190 Tyr Gly Ser Asp Pro Tyr Leu Ser Gly Val Ala Thr Val Gln Ser Val 195 200 205 Leu Gly Ile Gln Ser Thr Arg Ala Ser Ala Cys Val Lys His Tyr Ile 210 215 220 Gly Asn Glu Gln Glu His Tyr Arg Gly Gly Ser Gly Ala Thr Ala Ser 225 230 235 240 Ser Ser Asn Ile Asp Asp Arg Thr Leu Arg Glu Leu Tyr Glu Trp Pro 245 250 255 Phe Ala Glu Ala Ile His Ala Gly Val Asp Tyr Ile Met Cys Ser Tyr 260 265 270 Asn Arg Val Asn Gln Thr Tyr Ala Cys Glu Asn Ser Lys Leu Ile Asn 275 280 285 Gly Ile Ala Lys Gly Glu His Lys Phe Gln Gly Val Met Val Thr Asp 290 295 300 Trp Ala Ala Ala Glu Ser Gly Val Arg Thr Ala Leu Ala Gly Thr Asp 305 310 315 320 Met Asn Met Pro Gly Phe Met Ala Tyr Gly Gln Pro Ser Glu Pro Asn 325 330 335 Pro Ser Thr Ala Asn Gly Ser Tyr Trp Gly Leu Arg Met Ile Glu Ala 340 345 350 Val Lys Asn Gly Thr Val Pro Met Glu Arg Leu Asp Asp Met Val Thr 355 360 365 Arg Val Ile Ser Thr Tyr Tyr Lys Gln Gly Gln Asp Lys Ser Asp Tyr 370 375 380 Pro Lys Leu Asn Phe Met Ser Met Gly Gln Gly Thr Pro Ala Glu Gln 385 390 395 400 Ala Val Ser Asn His His Val Asn Val Gln Lys Asp His Tyr Leu Ile 405 410 415 Ile Arg Gln Ile Ala Thr Ala Ser Thr Ile Leu Leu Lys Asn Val Asn 420 425 430 His Thr Leu Pro Leu Lys Ser Pro Asp Lys Met Arg Ser Val Val Val 435 440 445 Val Gly Ser Asp Ala Gly Asp Asn Pro Gln Gly Pro Asn Ser Cys Val 450 455 460 Asp Arg Gly Cys Asn Arg Gly Ile Leu Ala Ile Gly Trp Gly Ser Gly 465 470 475 480 Thr Ala Asn Phe Ala His Leu Thr Ala Pro Ala Thr Ser Ile Gln Asn 485 490 495 Tyr Leu Leu Gln Ser Asn Pro Thr Ile Thr Tyr Arg Ser Ile Phe Asp 500 505 510 Asp Tyr Ala Tyr Asp Glu Ile Ala Lys Ala Ala Ser Thr Ala Asp Val 515 520 525 Ser Ile Val His Val Ser Ser Asp Ser Gly Glu Gly Tyr Leu Thr Val 530 535 540 Glu Gly Asn Gln Gly Asp Arg Ser Asn Thr Ser Leu Trp Asn Lys Gly 545 550 555 560 Asp Glu Leu Ile Leu Lys Ala Ala Glu Ala Cys Asn Asn Val Val Val 565 570 575 Val Ile His Ser Val Gly Pro Val Asp Met Glu Ala Trp Ile Asn His 580 585 590 Pro Asn Val Thr Ala Val Leu Leu Ala Gly Leu Pro Gly Gln Glu Ala 595 600 605 Gly Ser Ala Glu Val Asp Val Leu Trp Gly Ser Thr Asn Pro Ser Gly 610 615 620 Arg Leu Pro Tyr Thr Ile Ala Lys Lys Pro Ser Asp Tyr Pro Ala Glu 625 630 635 640 Leu Leu Tyr Glu Ser Asn Met Thr Val Pro Gln Ile Asn Tyr Ser Glu 645 650 655 Arg Leu Asn Ile Asp Tyr Arg His Phe Asp Thr Tyr Asn Ile Glu Pro 660 665 670 Arg Phe Glu Phe Gly Phe Gly Leu Ser Tyr Thr Thr Phe Ala Trp Asn 675 680 685 Ser Leu Lys Phe Ser Ser Ser Phe Gln Leu Gln Lys Thr Ser Pro Val 690 695 700 Ile Val Pro Pro Asn Leu Asp Leu Tyr Gln Asp Val Ile Glu Phe Glu 705 710 715 720 Phe Gln Val Thr Asn Ser Gly Pro Phe Asp Gly Ser Glu Val Ala Gln 725 730 735 Leu Tyr Val Asp Phe Pro Asn Gln Val Asn Glu Pro Pro Lys Val Leu 740 745 750 Arg Gly Phe Glu Arg Ala Tyr Ile Pro Ser Lys Gln Ser Lys Thr Ile 755 760 765 Glu Ile Lys Leu Arg Val Lys Asp Leu Ser Phe Trp Asp Val Ile Thr 770 775 780 Gln Ser Trp Gln Ile Pro Asp Gly Lys Phe Asn Phe Met Ile Gly Ser 785 790 795 800 Ser Ser Arg Lys Ile Ile Phe Thr Gln Glu Ile Ser Leu Gln His Ser 805 810 815 His Met 13715PRTAspergillus terreus 13Leu Thr Thr Trp Asp Ala Ala Tyr Glu Lys Ala Leu Ala Asp Leu Ala 1 5 10 15 Ser Leu Thr Gln Ser Glu Lys Val Gly Val Val Ser Gly Ile Thr Trp 20 25 30 Glu Gly Gly Pro Cys Val Gly Asn Thr Tyr Ala Pro Glu Ser Ile Ala 35 40 45 Tyr Pro Ser Leu Cys Leu Gln Asp Gly Pro Leu Gly Ile Arg Phe Ala 50

55 60 Asn Pro Val Thr Ala Phe Pro Ala Gly Ile Asn Ala Gly Ala Thr Trp 65 70 75 80 Asp Arg Glu Leu Leu Arg Ala Arg Gly Ala Ala Met Gly Glu Glu Ala 85 90 95 Lys Gly Leu Gly Val His Val Gln Leu Ala Pro Val Ala Gly Ala Leu 100 105 110 Gly Lys Ile Pro Ser Ala Gly Arg Asn Trp Glu Gly Phe Thr Ser Asp 115 120 125 Pro Tyr Leu Ser Gly Ile Ala Met Ala Glu Thr Ile His Gly Met Gln 130 135 140 Gly Ser Gly Val Gln Ala Cys Ala Lys His Tyr Ile Leu Asn Glu Gln 145 150 155 160 Glu His Ser Arg Glu Thr Ile Ser Ser Asn Val Asp Asp Arg Thr Met 165 170 175 His Glu Val Tyr Leu Trp Pro Phe Tyr Asp Ala Val Lys Ala Asn Val 180 185 190 Ala Ser Val Met Cys Ser Tyr Asn Lys Ile Asn Gly Thr Trp Ala Cys 195 200 205 Glu Asn Glu Gly Ile Leu Asp Thr Leu Leu Lys Gln Glu Leu Gly Phe 210 215 220 Arg Gly Tyr Val Met Ser Asp Trp Asn Ala Gln His Ser Thr Val Ala 225 230 235 240 Ser Ala Asn Thr Gly Leu Asp Met Thr Met Pro Gly Ser Asp Phe Ser 245 250 255 Gln Pro Pro Gly Ser Ile Tyr Trp Asn Glu Asn Leu Ala Glu Ala Val 260 265 270 Ala Asn Gly Ser Val Pro Gln Ala Arg Val Asp Asp Met Val Thr Arg 275 280 285 Ile Leu Ala Ala Trp Tyr Leu Leu Glu Gln Asp Gln Gly Tyr Pro Ala 290 295 300 Val Ala Phe Asp Ser Arg Asn Gly Gly Lys Ala Ser Val Asp Val Thr 305 310 315 320 Ala Asp His Ala Asp Ile Ala Arg Thr Val Ala Arg Asp Ser Ile Val 325 330 335 Leu Leu Lys Asn Ser Asn Asn Thr Leu Pro Leu Arg Asn Pro Ser Ser 340 345 350 Ile Ala Val Val Gly Ser Asp Ala Ile Val Asn Pro Asp Gly Pro Asn 355 360 365 Ala Cys Thr Asp Arg Gly Cys Asn Val Gly Thr Leu Ala Gln Gly Trp 370 375 380 Gly Ser Gly Thr Ala Glu Phe Pro Tyr Leu Val Ala Pro Leu Asp Ala 385 390 395 400 Ile Gln Glu Arg Ser Ser Gly Asn Gly Thr Lys Val Val Thr Ser Thr 405 410 415 Thr Asp Asp Ala Thr Ala Gly Ala Asp Ala Ala Ala Ser Ala Asp Ile 420 425 430 Ala Ile Val Phe Ile Ser Ser Asp Ser Gly Glu Gly Tyr Ile Thr Val 435 440 445 Glu Gly His Gln Gly Asp Arg Asn Asn Leu Asp Pro Trp His Gly Gly 450 455 460 Asn Asp Leu Val Lys Ala Val Ala Ala Val Asn Lys Lys Thr Ile Val 465 470 475 480 Val Val His Ser Thr Gly Pro Val Val Leu Glu Thr Ile Leu Ala Gln 485 490 495 Pro Asn Val Val Ala Val Val Trp Ala Gly Ile Pro Gly Gln Glu Ser 500 505 510 Gly Asn Ala Leu Ala Asp Val Leu Tyr Gly Asp Val Ser Pro Ser Gly 515 520 525 Lys Leu Pro Tyr Thr Ile Gly Lys Ser Glu Ala Asp Tyr Gly Thr Thr 530 535 540 Trp Val Ala Asn Gly Ala Asp Asp Asp Phe Pro Glu Gly Leu Phe Ile 545 550 555 560 Asp Tyr Arg His Phe Asp Lys Asn Glu Ile Glu Pro Arg Tyr Glu Phe 565 570 575 Gly Phe Gly Leu Ser Tyr Thr Arg Phe Asn Phe Ser Asn Leu Ala Ile 580 585 590 Asn Ile Asp Ala Thr Ser Gly Pro Thr Ser Gly Ala Val Asp Val Gly 595 600 605 Gly Ala Ala Asp Leu Tyr Asp Ser Val Gly Thr Ile Ser Ala Thr Val 610 615 620 Thr Asn Val Gly Gly Val Ser Gly Ala Glu Val Ala Gln Leu Tyr Ile 625 630 635 640 Gly Phe Pro Ser Ser Ala Pro Glu Thr Pro Pro Lys Gln Leu Arg Gly 645 650 655 Phe Gln Lys Leu Pro Leu Ala Gly Gly Ala Asp Gly Val Ala Glu Phe 660 665 670 Glu Leu Thr Arg Arg Asp Ile Ser Tyr Trp Asp Val Gly Gln Gln Lys 675 680 685 Trp Val Val Pro Glu Gly Ser Phe Gln Val Tyr Val Gly Ala Ser Ser 690 695 700 Arg Asp Ile Arg Leu Asp Gly Ser Phe Thr Val 705 710 715 14706PRTChaetomium globosum 14Leu Glu Ala Ala Asp Trp Ala Ala Ala Glu Ala Ser Ala Lys Thr Ala 1 5 10 15 Leu Ala Lys Met Ser Gln Gln Asp Lys Ile Ser Ile Val Thr Gly Ile 20 25 30 Gly Trp Asp Lys Gly Pro Cys Val Gly Asn Thr Ala Ala Ile Asn Ser 35 40 45 Ile Asn Tyr Pro Gln Leu Cys Leu Gln Asp Gly Pro Leu Gly Ile Arg 50 55 60 Phe Gly Thr Gly Ser Thr Ala Phe Thr Pro Gly Val Gln Ala Ala Ser 65 70 75 80 Thr Trp Asp Thr Glu Leu Met Arg Gln Arg Gly Glu Tyr Leu Gly Ala 85 90 95 Glu Ala Lys Gly Cys Gly Ile His Val Leu Leu Gly Pro Val Ala Gly 100 105 110 Ala Leu Gly Lys Ile Pro His Gly Gly Arg Asn Trp Glu Gly Phe Gly 115 120 125 Thr Asp Pro Tyr Leu Ala Gly Ile Ala Met Ala Glu Thr Ile Glu Gly 130 135 140 Leu Gln Ser Ala Gly Val Gln Ala Cys Ala Lys His Tyr Ile Val Asn 145 150 155 160 Glu Gln Glu Leu Asn Arg Glu Thr Ile Ser Ser Asp Val Asp Asp Arg 165 170 175 Thr Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val His Ala 180 185 190 Asn Val Ala Ser Val Met Cys Ser Tyr Asn Lys Ile Asn Gly Ser Trp 195 200 205 Gly Cys Glu Asn Asp His Ala Gln Asn Gly Leu Leu Lys Lys Glu Leu 210 215 220 Gly Phe Lys Gly Tyr Val Val Ser Asp Trp Asn Ala Gln His Thr Thr 225 230 235 240 Asp Gly Ala Ala Asn Asn Gly Met Asp Met Thr Met Pro Gly Ser Asp 245 250 255 Tyr Asn Gly Asn Asn Val Leu Trp Gly Pro Gln Leu Ser Asn Ala Val 260 265 270 Asn Ser Asn Arg Val Ser Arg Asp Arg Leu Asp Asp Met Ala Lys Arg 275 280 285 Ile Leu Thr Ser Trp Tyr Leu Leu Gly Gln Asn Ser Gly Tyr Pro Asn 290 295 300 Ile Asn Ile Asn Ala Asn Val Gln Gly Asn His Lys Glu Asn Val Arg 305 310 315 320 Ala Val Ala Arg Asp Gly Ile Val Leu Leu Lys Asn Asp Glu Gly Val 325 330 335 Leu Pro Leu Lys Lys Pro Gly Lys Val Ala Leu Val Gly Ser Ala Ala 340 345 350 Ser Val Asn Ser Ala Gly Pro Asn Ala Cys Val Asp Lys Gly Cys Asn 355 360 365 Thr Gly Ala Leu Gly Met Gly Trp Gly Ser Gly Ser Val Asn Tyr Pro 370 375 380 Tyr Phe Val Ala Pro Tyr Asp Ala Leu Lys Thr Arg Ala Gln Ala Asp 385 390 395 400 Gly Thr Thr Leu Ser Leu His Asn Ser Asp Ser Thr Asn Gly Val Ser 405 410 415 Gly Val Val Ser Gly Ala Asp Val Ala Ile Val Val Ile Thr Ala Asp 420 425 430 Ser Gly Glu Gly Tyr Ile Thr Val Glu Gly His Ala Gly Asp Arg Asn 435 440 445 His Leu Asp Pro Trp His Asp Gly Asn Ala Leu Val Lys Ala Val Ala 450 455 460 Ala Ala Asn Lys Asn Thr Ile Val Val Val His Ser Thr Gly Pro Ile 465 470 475 480 Ile Leu Glu Thr Ile Leu Ala Thr Glu Gly Val Lys Ala Val Val Trp 485 490 495 Ala Gly Leu Pro Ser Gln Glu Asn Gly Asn Ala Leu Val Asp Val Leu 500 505 510 Tyr Gly Leu Thr Ser Pro Ser Gly Lys Leu Val Tyr Ser Ile Ala Lys 515 520 525 Arg Pro Glu Asp Tyr Gly Thr Ala Pro Ser Lys Gly Ser Asn Asp Lys 530 535 540 Phe Thr Glu Gly Leu Phe Val Asp Tyr Arg His Phe Asp Asn Ala Lys 545 550 555 560 Ile Glu Pro Arg Tyr Glu Phe Gly Phe Gly Leu Ser Tyr Thr Glu Phe 565 570 575 Thr Tyr Ala Asp Leu Ser Val Thr Ser Thr Val Thr Ala Gly Pro Ala 580 585 590 Ser Gly Glu Thr Ile Pro Gly Gly Ala Ala Asp Leu Trp Glu Thr Val 595 600 605 Ala Thr Val Thr Ala Ser Ile Thr Asn Ser Gly Glu Val Glu Gly Ala 610 615 620 Glu Val Ala Gln Leu Tyr Ile Thr Leu Pro Ser Ala Ala Pro Ser Thr 625 630 635 640 Pro Pro Lys Gln Leu Arg Gly Phe Ala Lys Leu Lys Leu Glu Pro Gly 645 650 655 Ala Ser Gly Val Ala Thr Phe Asn Leu Arg Arg Arg Asp Leu Ser Tyr 660 665 670 Trp Asp Ala Gly Arg Gly Gln Trp Val Val Pro Ala Gly Glu Phe Thr 675 680 685 Val Ser Val Gly Ala Ser Ser Arg Asp Val Arg Leu Thr Gly Ser Leu 690 695 700 Thr Ala 705 15856PRTTrichoderma reesei 15Asn Pro Tyr Pro Pro Pro His Ser Asn Gln Ala Tyr Ser Pro Pro Phe 1 5 10 15 Tyr Pro Ser Pro Trp Met Asp Pro Ser Ala Pro Gly Trp Glu Gln Ala 20 25 30 Tyr Ala Gln Ala Lys Glu Phe Val Ser Gly Leu Thr Leu Leu Glu Lys 35 40 45 Val Asn Leu Thr Thr Gly Val Gly Trp Met Gly Glu Lys Cys Val Gly 50 55 60 Asn Val Gly Thr Val Pro Arg Leu Gly Met Arg Ser Leu Cys Met Gln 65 70 75 80 Asp Gly Pro Leu Gly Leu Arg Phe Asn Thr Tyr Asn Ser Ala Phe Ser 85 90 95 Val Gly Leu Thr Ala Ala Ala Ser Trp Ser Arg His Leu Trp Val Asp 100 105 110 Arg Gly Thr Ala Leu Gly Ser Glu Ala Lys Gly Lys Gly Val Asp Val 115 120 125 Leu Leu Gly Pro Val Ala Gly Pro Leu Gly Arg Asn Pro Asn Gly Gly 130 135 140 Arg Asn Val Glu Gly Phe Gly Ser Asp Pro Tyr Leu Ala Gly Leu Ala 145 150 155 160 Leu Ala Asp Thr Val Thr Gly Ile Gln Asn Ala Gly Thr Ile Ala Cys 165 170 175 Ala Lys His Phe Leu Leu Asn Glu Gln Glu His Phe Arg Gln Val Gly 180 185 190 Glu Ala Asn Gly Tyr Gly Tyr Pro Ile Thr Glu Ala Leu Ser Ser Asn 195 200 205 Val Asp Asp Lys Thr Ile His Glu Val Tyr Gly Trp Pro Phe Gln Asp 210 215 220 Ala Val Lys Ala Gly Val Gly Ser Phe Met Cys Ser Tyr Asn Gln Val 225 230 235 240 Asn Asn Ser Tyr Ala Cys Gln Asn Ser Lys Leu Ile Asn Gly Leu Leu 245 250 255 Lys Glu Glu Tyr Gly Phe Gln Gly Phe Val Met Ser Asp Trp Gln Ala 260 265 270 Gln His Thr Gly Val Ala Ser Ala Val Ala Gly Leu Asp Met Thr Met 275 280 285 Pro Gly Asp Thr Ala Phe Asn Thr Gly Ala Ser Tyr Phe Gly Ser Asn 290 295 300 Leu Thr Leu Ala Val Leu Asn Gly Thr Val Pro Glu Trp Arg Ile Asp 305 310 315 320 Asp Met Val Met Arg Ile Met Ala Pro Phe Phe Lys Val Gly Lys Thr 325 330 335 Val Asp Ser Leu Ile Asp Thr Asn Phe Asp Ser Trp Thr Asn Gly Glu 340 345 350 Tyr Gly Tyr Val Gln Ala Ala Val Asn Glu Asn Trp Glu Lys Val Asn 355 360 365 Tyr Gly Val Asp Val Arg Ala Asn His Ala Asn His Ile Arg Glu Val 370 375 380 Gly Ala Lys Gly Thr Val Ile Phe Lys Asn Asn Gly Ile Leu Pro Leu 385 390 395 400 Lys Lys Pro Lys Phe Leu Thr Val Ile Gly Glu Asp Ala Gly Gly Asn 405 410 415 Pro Ala Gly Pro Asn Gly Cys Gly Asp Arg Gly Cys Asp Asp Gly Thr 420 425 430 Leu Ala Met Glu Trp Gly Ser Gly Thr Thr Asn Phe Pro Tyr Leu Val 435 440 445 Thr Pro Asp Ala Ala Leu Gln Ser Gln Ala Leu Gln Asp Gly Thr Arg 450 455 460 Tyr Glu Ser Ile Leu Ser Asn Tyr Ala Ile Ser Gln Thr Gln Ala Leu 465 470 475 480 Val Ser Gln Pro Asp Ala Ile Ala Ile Val Phe Ala Asn Ser Asp Ser 485 490 495 Gly Glu Gly Tyr Ile Asn Val Asp Gly Asn Glu Gly Asp Arg Lys Asn 500 505 510 Leu Thr Leu Trp Lys Asn Gly Asp Asp Leu Ile Lys Thr Val Ala Ala 515 520 525 Val Asn Pro Lys Thr Ile Val Val Ile His Ser Thr Gly Pro Val Ile 530 535 540 Leu Lys Asp Tyr Ala Asn His Pro Asn Ile Ser Ala Ile Leu Trp Ala 545 550 555 560 Gly Ala Pro Gly Gln Glu Ser Gly Asn Ser Leu Val Asp Ile Leu Tyr 565 570 575 Gly Lys Gln Ser Pro Gly Arg Thr Pro Phe Thr Trp Gly Pro Ser Leu 580 585 590 Glu Ser Tyr Gly Val Ser Val Met Thr Thr Pro Asn Asn Gly Asn Gly 595 600 605 Ala Pro Gln Asp Asn Phe Asn Glu Gly Ala Phe Ile Asp Tyr Arg Tyr 610 615 620 Phe Asp Lys Val Ala Pro Gly Lys Pro Arg Ser Ser Asp Lys Ala Pro 625 630 635 640 Thr Tyr Glu Phe Gly Phe Gly Leu Ser Trp Ser Thr Phe Lys Phe Ser 645 650 655 Asn Leu His Ile Gln Lys Asn Asn Val Gly Pro Met Ser Pro Pro Asn 660 665 670 Gly Lys Thr Ile Ala Ala Pro Ser Leu Gly Ser Phe Ser Lys Asn Leu 675 680 685 Lys Asp Tyr Gly Phe Pro Lys Asn Val Arg Arg Ile Lys Glu Phe Ile 690 695 700 Tyr Pro Tyr Leu Ser Thr Thr Thr Ser Gly Lys Glu Ala Ser Gly Asp 705 710 715 720 Ala His Tyr Gly Gln Thr Ala Lys Glu Phe Leu Pro Ala Gly Ala Leu 725 730 735 Asp Gly Ser Pro Gln Pro Arg Ser Ala Ala Ser Gly Glu Pro Gly Gly 740 745 750 Asn Arg Gln Leu Tyr Asp Ile Leu Tyr Thr Val Thr Ala Thr Ile Thr 755 760 765 Asn Thr Gly Ser Val Met Asp Asp Ala Val Pro Gln Leu Tyr Leu Ser 770 775 780 His Gly Gly Pro Asn Glu Pro Pro Lys Val Leu Arg Gly Phe Asp Arg 785 790 795 800 Ile Glu Arg Ile Ala Pro Gly Gln Ser Val Thr Phe Lys Ala Asp Leu 805 810 815 Thr Arg Arg Asp Leu Ser Asn Trp Asp Thr Lys Lys Gln Gln Trp Val 820 825 830 Ile Thr Asp Tyr Pro Lys Thr Val Tyr Val Gly Ser Ser Ser Arg Asp 835 840 845 Leu Pro Leu Ser Ala Arg Leu Pro 850 855 16859PRTPenicillium brasilianum 16Ile Ala Gln Pro Ile Gln Lys His Glu Pro Gly Phe Leu His Gly Pro 1 5 10 15 Gln Ala Ile Glu Ser Phe Ser Glu Pro Phe Tyr Pro Ser Pro Trp Met 20 25 30 Asn Pro His Ala Glu Gly Trp Glu Ala Ala Tyr Gln Lys Ala Gln Asp 35 40 45 Phe Val Ser Gln Leu Thr Ile Leu Glu Lys Ile Asn Leu Thr Thr Gly 50 55 60 Val Gly Trp Glu Asn Gly Pro Cys Val Gly Asn Thr Gly Ser Ile Pro 65

70 75 80 Arg Leu Gly Phe Lys Gly Phe Cys Thr Gln Asp Ser Pro Gln Gly Val 85 90 95 Arg Phe Ala Asp Tyr Ser Ser Ala Phe Thr Ser Ser Gln Met Ala Ala 100 105 110 Ala Thr Phe Asp Arg Ser Ile Leu Tyr Gln Arg Gly Gln Ala Met Ala 115 120 125 Gln Glu His Lys Ala Lys Gly Ile Thr Ile Gln Leu Gly Pro Val Ala 130 135 140 Gly Pro Leu Gly Arg Ile Pro Glu Gly Gly Arg Asn Trp Glu Gly Phe 145 150 155 160 Ser Pro Asp Pro Val Leu Thr Gly Ile Ala Met Ala Glu Thr Ile Lys 165 170 175 Gly Met Gln Asp Thr Gly Val Ile Ala Cys Ala Lys His Tyr Ile Gly 180 185 190 Asn Glu Gln Glu His Phe Arg Gln Val Gly Glu Ala Ala Gly His Gly 195 200 205 Tyr Thr Ile Ser Asp Thr Ile Ser Ser Asn Ile Asp Asp Arg Ala Met 210 215 220 His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala Gly Val 225 230 235 240 Gly Ser Phe Met Cys Ser Tyr Ser Gln Ile Asn Asn Ser Tyr Gly Cys 245 250 255 Gln Asn Ser Gln Thr Leu Asn Lys Leu Leu Lys Ser Glu Leu Gly Phe 260 265 270 Gln Gly Phe Val Met Ser Asp Trp Gly Ala His His Ser Gly Val Ser 275 280 285 Ser Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Thr Glu Phe 290 295 300 Asp Ser Gly Leu Ser Phe Trp Gly Ser Asn Leu Thr Ile Ala Ile Leu 305 310 315 320 Asn Gly Thr Val Pro Glu Trp Arg Leu Asp Asp Met Ala Met Arg Ile 325 330 335 Met Ala Ala Tyr Phe Lys Val Gly Leu Thr Ile Glu Asp Gln Pro Asp 340 345 350 Val Asn Phe Asn Ala Trp Thr His Asp Thr Tyr Gly Tyr Lys Tyr Ala 355 360 365 Tyr Ser Lys Glu Asp Tyr Glu Gln Val Asn Trp His Val Asp Val Arg 370 375 380 Ser Asp His Asn Lys Leu Ile Arg Glu Thr Ala Ala Lys Gly Thr Val 385 390 395 400 Leu Leu Lys Asn Asn Phe His Ala Leu Pro Leu Lys Gln Pro Arg Phe 405 410 415 Val Ala Val Val Gly Gln Asp Ala Gly Pro Asn Pro Lys Gly Pro Asn 420 425 430 Gly Cys Ala Asp Arg Gly Cys Asp Gln Gly Thr Leu Ala Met Gly Trp 435 440 445 Gly Ser Gly Ser Thr Glu Phe Pro Tyr Leu Val Thr Pro Asp Thr Ala 450 455 460 Ile Gln Ser Lys Val Leu Glu Tyr Gly Gly Arg Tyr Glu Ser Ile Phe 465 470 475 480 Asp Asn Tyr Asp Asp Asn Ala Ile Leu Ser Leu Val Ser Gln Pro Asp 485 490 495 Ala Thr Cys Ile Val Phe Ala Asn Ala Asp Ser Gly Glu Gly Tyr Ile 500 505 510 Thr Val Asp Asn Asn Trp Gly Asp Arg Asn Asn Leu Thr Leu Trp Gln 515 520 525 Asn Ala Asp Gln Val Ile Ser Thr Val Ser Ser Arg Cys Asn Asn Thr 530 535 540 Ile Val Val Leu His Ser Val Gly Pro Val Leu Leu Asn Gly Ile Tyr 545 550 555 560 Glu His Pro Asn Ile Thr Ala Ile Val Trp Ala Gly Met Pro Gly Glu 565 570 575 Glu Ser Gly Asn Ala Leu Val Asp Ile Leu Trp Gly Asn Val Asn Pro 580 585 590 Ala Gly Arg Thr Pro Phe Thr Trp Ala Lys Ser Arg Glu Asp Tyr Gly 595 600 605 Thr Asp Ile Met Tyr Glu Pro Asn Asn Gly Gln Arg Ala Pro Gln Gln 610 615 620 Asp Phe Thr Glu Ser Ile Tyr Leu Asp Tyr Arg His Phe Asp Lys Ala 625 630 635 640 Gly Ile Glu Pro Ile Tyr Glu Phe Gly Phe Gly Leu Ser Tyr Thr Thr 645 650 655 Phe Glu Tyr Ser Asp Leu Arg Val Val Lys Lys Tyr Val Gln Pro Tyr 660 665 670 Ser Pro Thr Thr Gly Thr Gly Ala Gln Ala Pro Ser Ile Gly Gln Pro 675 680 685 Pro Ser Gln Asn Leu Asp Thr Tyr Lys Phe Pro Ala Thr Tyr Lys Tyr 690 695 700 Ile Lys Thr Phe Ile Tyr Pro Tyr Leu Asn Ser Thr Val Ser Leu Arg 705 710 715 720 Ala Ala Ser Lys Asp Pro Glu Tyr Gly Arg Thr Asp Phe Ile Pro Pro 725 730 735 His Ala Arg Asp Gly Ser Pro Gln Pro Leu Asn Pro Ala Gly Asp Pro 740 745 750 Val Ala Ser Gly Gly Asn Asn Met Leu Tyr Asp Glu Leu Tyr Glu Val 755 760 765 Thr Ala Gln Ile Lys Asn Thr Gly Asp Val Ala Gly Asp Glu Val Val 770 775 780 Gln Leu Tyr Val Asp Leu Gly Gly Asp Asn Pro Pro Arg Gln Leu Arg 785 790 795 800 Asn Phe Asp Arg Phe Tyr Leu Leu Pro Gly Gln Ser Ser Thr Phe Arg 805 810 815 Ala Thr Leu Thr Arg Arg Asp Leu Ser Asn Trp Asp Ile Glu Ala Gln 820 825 830 Asn Trp Arg Val Thr Glu Ser Pro Lys Arg Val Tyr Val Gly Arg Ser 835 840 845 Ser Arg Asp Leu Pro Leu Ser Ser Gln Leu Glu 850 855 17849PRTPericonia sp. 17Gln Ala Pro Phe Pro Asn Gly Ser Ser Pro Leu Asn Asp Ile Thr Ser 1 5 10 15 Pro Pro Phe Tyr Pro Ser Pro Trp Met Asp Pro Ser Ala Ala Gly Trp 20 25 30 Ala Glu Ala Tyr Thr Lys Ala Gln Ala Phe Val Arg Gln Leu Thr Leu 35 40 45 Leu Glu Lys Val Asn Leu Thr Thr Gly Val Gly Trp Glu Gly Glu Ala 50 55 60 Cys Val Gly Asn Thr Gly Ser Ile Pro Arg Leu Gly Phe Pro Gly Phe 65 70 75 80 Cys Thr Gln Asp Ser Pro Leu Gly Val Arg Phe Ala Asp Tyr Val Ser 85 90 95 Ala Phe Thr Ala Gly Gly Thr Ile Ala Ala Ser Trp Asp Arg Ser Glu 100 105 110 Phe Tyr Arg Arg Gly Tyr Gln Met Gly Val Glu His Arg Gly Lys Gly 115 120 125 Val Asp Val Gln Leu Gly Pro Val Val Gly Pro Ile Gly Arg His Pro 130 135 140 Lys Gly Gly Arg Asn Trp Glu Gly Phe Ser Pro Asp Pro Val Leu Ser 145 150 155 160 Gly Ile Ala Val Ala Glu Thr Val Lys Gly Ile Gln Asp Ala Gly Val 165 170 175 Ile Ala Cys Thr Lys His Phe Ile Leu Asn Glu Gln Glu His Phe Arg 180 185 190 Gln Pro Gly Asn Val Gly Asp Phe Gly Phe Val Asp Ala Val Ser Ala 195 200 205 Asn Leu Ala Asp Lys Thr Leu His Glu Leu Tyr Leu Trp Pro Phe Ala 210 215 220 Asp Ala Val Arg Ala Gly Thr Gly Ser Ile Met Cys Ser Tyr Asn Lys 225 230 235 240 Ala Asn Asn Ser Gln Val Cys Gln Asn Ser Tyr Leu Gln Asn Tyr Ile 245 250 255 Leu Lys Gly Glu Leu Gly Phe Gln Gly Phe Thr Met Ser Asp Trp Asp 260 265 270 Ala Gln His Ser Gly Val Ala Ser Thr Leu Ala Gly Leu Asp Met Asn 275 280 285 Met Pro Gly Asp Thr Asp Phe Asp Ser Gly Phe Ser Phe Trp Gly Pro 290 295 300 Asn Met Thr Leu Ser Ile Ile Asn Gly Thr Val Pro Glu Trp Arg Leu 305 310 315 320 Asp Asp Ala Ala Thr Arg Ile Met Ala Ala Tyr Tyr Leu Val Gly Arg 325 330 335 Asp Arg His Ala Val Pro Val Asn Phe Asn Ser Trp Ser Lys Asp Thr 340 345 350 Tyr Gly Tyr Gln His Ala Tyr Ala Lys Val Gly Tyr Gly Leu Ile Asn 355 360 365 Gln His Val Asp Val Arg Ala Asp His Phe Lys Ser Ile Arg Thr Ala 370 375 380 Ala Ala Lys Ser Thr Val Leu Leu Lys Asn Asn Gly Val Leu Pro Leu 385 390 395 400 Lys Gly Thr Glu Lys Tyr Thr Ala Val Phe Gly Asn Asp Ala Gly Glu 405 410 415 Ala Gln Tyr Gly Pro Asn Gly Cys Ala Asp His Gly Cys Asp Asn Gly 420 425 430 Thr Leu Ala Met Gly Trp Gly Ser Gly Thr Ala Asp Tyr Pro Tyr Leu 435 440 445 Val Thr Pro Leu Glu Ala Ile Lys Arg Thr Val Gly Asp His Gly Gly 450 455 460 Val Ile Ala Ser Val Thr Asp Asn Tyr Ala Phe Ser Gln Ile Met Ala 465 470 475 480 Leu Ala Lys Gln Ala Thr His Ala Ile Val Phe Val Asn Ala Asp Ser 485 490 495 Gly Glu Gly Tyr Ile Thr Val Asp Gly Asn Glu Gly Asp Arg Asn Asn 500 505 510 Leu Thr Leu Trp Gln Asn Gly Glu Glu Leu Val Arg Asn Val Ser Gly 515 520 525 Tyr Cys Asn Asn Thr Ile Val Val Ile His Ser Val Gly Pro Val Leu 530 535 540 Val Asp Ser Phe Asn Asn Ser Pro Asn Val Ser Ala Ile Leu Trp Ala 545 550 555 560 Gly Leu Pro Gly Gln Glu Ser Gly Asn Ala Ile Thr Asp Val Leu Tyr 565 570 575 Gly Arg Val Asn Pro Gly Gly Lys Leu Pro Phe Thr Ile Gly Lys Ser 580 585 590 Ala Glu Glu Tyr Gly Pro Asp Ile Ile Tyr Glu Pro Thr Ala Gly His 595 600 605 Gly Ser Pro Gln Ala Asn Phe Glu Glu Gly Val Phe Ile Asp Tyr Arg 610 615 620 Ser Phe Asp Lys Lys Asn Ile Thr Pro Val Tyr Glu Phe Gly Phe Gly 625 630 635 640 Leu Ser Tyr Thr Asn Phe Ser Tyr Ser Asn Leu Val Val Thr Arg Val 645 650 655 Asn Ala Pro Ala Tyr Val Pro Thr Thr Gly Asn Thr Thr Ala Ala Pro 660 665 670 Thr Leu Gly Asn Ser Ser Lys Asp Ala Ser Asp Tyr Gln Trp Pro Ala 675 680 685 Asn Leu Thr Tyr Val Asn Lys Tyr Ile Tyr Pro Tyr Leu Asn Ser Thr 690 695 700 Asp Leu Lys Glu Ala Ser Asn Asp Pro Glu Tyr Gly Ile Glu His Glu 705 710 715 720 Tyr Pro Glu Gly Ala Thr Asp Gly Ser Pro Gln Pro Arg Ile Ala Ala 725 730 735 Gly Gly Gly Pro Gly Gly Asn Pro Gln Leu Trp Asp Val Leu Tyr Lys 740 745 750 Val Thr Ala Thr Val Thr Asn Asn Gly Ala Val Ala Gly Asp Glu Val 755 760 765 Ala Gln Leu Tyr Val Ser Leu Gly Gly Pro Glu Asp Pro Pro Val Val 770 775 780 Leu Arg Asn Phe Asp Arg Leu Thr Ile Ala Pro Gly Gln Ser Val Glu 785 790 795 800 Phe Thr Ala Asp Ile Thr Arg Arg Asp Val Ser Asn Trp Asp Thr Val 805 810 815 Ser Gln Asn Trp Val Ile Ser Asn Ser Thr Lys Thr Val Tyr Val Gly 820 825 830 Ala Ser Ser Arg Lys Leu Pro Leu Lys Ala Thr Leu Pro Ser Ser Ser 835 840 845 Tyr 18857PRTPhaeosphaeria avenaria 18Gln Gln Tyr Pro Thr Ser Asn Thr Ser Ser Pro Ala Ala Asn Ser Ser 1 5 10 15 Ser Pro Leu Asp Asn Ala Val Ser Pro Pro Phe Tyr Pro Ser Pro Trp 20 25 30 Ile Glu Gly Leu Gly Asp Trp Glu Ala Ala Tyr Gln Lys Ala Gln Ala 35 40 45 Phe Val Ser Gln Leu Thr Leu Leu Glu Lys Val Asn Leu Thr Thr Gly 50 55 60 Thr Gly Trp Gln Ser Asp His Cys Val Gly Asn Thr Gly Gly Val Pro 65 70 75 80 Arg Leu Asn Phe Thr Gly Ile Cys Asn Gln Asp Ala Pro Leu Gly Val 85 90 95 Arg Phe Ala Asp Tyr Val Ser Ala Phe Pro Ser Gly Gly Thr Ile Ala 100 105 110 Ala Ala Trp Asp Arg Gly Glu Trp Tyr Leu Arg Gly Tyr Gln Met Gly 115 120 125 Ser Glu His Arg Ser Lys Gly Val Asp Val Gln Leu Gly Pro Val Val 130 135 140 Gly Pro Leu Gly Arg Asn Pro Lys Gly Gly Arg Asn Trp Glu Gly Phe 145 150 155 160 Ser Pro Asp Pro Tyr Leu Ser Gly Ile Ala Ser Ala Glu Ser Val Arg 165 170 175 Gly Ile Gln Asp Ala Gly Val Ile Ala Cys Thr Lys His Tyr Ile Met 180 185 190 Asn Glu Gln Glu His Phe Arg Gln Pro Gly Asn Phe Glu Asp Gln Gly 195 200 205 Phe Val Asp Ala Leu Ser Ser Asn Leu Asp Asp Lys Thr Leu His Glu 210 215 220 Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala Gly Thr Gly Ser 225 230 235 240 Ile Met Cys Ser Tyr Asn Lys Val Asn Asn Ser Gln Ala Cys Gln Asn 245 250 255 Ser Tyr Leu Gln Asn Tyr Ile Leu Lys Gly Glu Leu Gly Phe Gln Gly 260 265 270 Phe Ile Met Ser Asp Trp Asp Ala Gln His Ser Gly Val Ala Ser Thr 275 280 285 Phe Ala Gly Leu Asp Met Thr Met Pro Gly Asp Thr Asp Phe Asn Ser 290 295 300 Gly Lys Thr Phe Trp Gly Thr Asn Phe Thr Thr Ser Ile Leu Asn Gly 305 310 315 320 Thr Val Pro Gln Trp Arg Leu Asp Asp Ala Val Thr Arg Ile Met Ala 325 330 335 Ala Phe Tyr Tyr Val Gly Arg Asp Lys Ala Arg Ile Pro Val Asn Phe 340 345 350 Asp Ser Trp Ser Arg Asp Thr Tyr Gly Phe Asp His Tyr Tyr Gly Lys 355 360 365 Ala Gly Tyr Ser Gln Ile Asn Ser His Val Asp Val Arg Ala Asp His 370 375 380 Phe Arg Ser Ile Arg Arg Thr Ala Ala Met Ser Thr Val Leu Leu Lys 385 390 395 400 Asn Glu Gly Ala Leu Pro Leu Thr Gly Ser Glu Lys Trp Thr Ala Val 405 410 415 Phe Gly Asp Asp Ala Gly Glu Gly Gln Leu Gly Pro Asn Gly Phe Pro 420 425 430 Asp His Gly Gly Asn Asn Gly Thr Leu Ala Met Gly Trp Gly Ser Gly 435 440 445 Thr Ser Asp Tyr Pro Tyr Leu Val Thr Pro Leu Glu Ser Ile Lys Ala 450 455 460 Thr Val Ala Gln Asn Gly Gly Ile Val Thr Ser Val Thr Asp Asn Trp 465 470 475 480 Ala Tyr Thr Gln Ile Gln Thr Leu Ala Lys Gln Ala Ser Val Ala Ile 485 490 495 Val Phe Val Asn Ala Asp Ser Gly Glu Gly Tyr Ile Thr Val Asp Gly 500 505 510 Asn Ala Gly Asp Arg Asn Asn Leu Thr Leu Trp Gln Asp Gly Asp Thr 515 520 525 Leu Ile Lys Asn Val Ser Ser Leu Cys Asn Asn Thr Ile Val Val Ile 530 535 540 His Ser Val Gly Pro Val Leu Val Asn Ser Phe Tyr Asp Ser Glu Asn 545 550 555 560 Val Thr Ala Ile Leu Trp Ala Gly Leu Pro Gly Gln Glu Ser Gly Asn 565 570 575 Ala Ile Ala Asp Ile Leu Tyr Gly Arg His Asn Pro Gly Gly Lys Leu 580 585 590 Pro Phe Thr Ile Gly Ser Asp Ala Ala Glu Tyr Gly Pro Asp Leu Ile 595 600 605 Tyr Glu Pro Thr Asn Asn Ser Ser Ser Pro Gln Asp Asn Phe Glu Glu 610 615 620 Gly Val Phe Ile Asp Tyr Arg Ala Phe Asp Lys Gln Asn Val Thr Pro 625 630 635 640 Ile Tyr Glu Phe Gly Phe Gly Leu Ser Tyr Thr Lys Phe Ser Tyr Ser 645 650 655 Asn Leu Thr Val Lys Lys Ala Asn Ala Gly Ala Tyr Thr Pro Ala Thr 660

665 670 Gly Gln Ser Lys Ala Ala Pro Thr Leu Gly Asn Phe Ser Thr Asp Ala 675 680 685 Ser Gln Tyr Gln Trp Pro Ser Asp Phe Thr Tyr Ile Asp Thr Phe Ile 690 695 700 Tyr Pro Tyr Leu Asn Ser Thr Asp Leu Lys Thr Ala Ser Gln Asp Pro 705 710 715 720 Glu Tyr Gly Leu Asn Tyr Thr Trp Pro Ala Gly Ala Thr Asp Gly Thr 725 730 735 Pro Gln Ala Arg Ile Pro Ala Gly Gly Ala Pro Gly Gly Asn Pro Gln 740 745 750 Leu Trp Asp Val Leu Phe Ser Val Glu Ala Thr Ile Thr Asn Asn Gly 755 760 765 Thr Val Pro Gly Asp Glu Val Val Gln Leu Tyr Val Ser Leu Gly Asn 770 775 780 Pro Asp Asp Pro Lys Ile Val Leu Arg Gly Phe Asp Arg Leu Ser Ile 785 790 795 800 Gln Pro Gly Lys Thr Ala Thr Phe His Ala Asp Ile Thr Arg Arg Asp 805 810 815 Val Ser Asn Trp Asp Val Ala Ser Gln Asn Trp Val Ile Thr Ser Ala 820 825 830 Pro Lys Thr Val Tyr Val Gly Ala Ser Ser Arg Lys Leu Pro Leu Thr 835 840 845 Ala Thr Leu Asp Thr Ser Asp Phe Gln 850 855 19854PRTAspergillus fumigatus 19Gln Val Phe Asp Asn Ser His Gly Asn Asn Gln Glu Leu Ala Phe Ser 1 5 10 15 Pro Pro Phe Tyr Pro Ser Pro Trp Ala Asp Gly Gln Gly Glu Trp Ala 20 25 30 Asp Ala His Arg Arg Ala Val Glu Ile Val Ser Gln Met Thr Leu Ala 35 40 45 Glu Lys Val Asn Leu Thr Thr Gly Thr Gly Trp Glu Met Asp Arg Cys 50 55 60 Val Gly Gln Thr Gly Ser Val Pro Arg Leu Gly Ile Asn Trp Gly Leu 65 70 75 80 Cys Gly Gln Asp Ser Pro Leu Gly Ile Arg Phe Ser Asp Leu Asn Ser 85 90 95 Ala Phe Pro Ala Gly Thr Asn Val Ala Ala Thr Trp Asp Lys Thr Leu 100 105 110 Ala Tyr Leu Arg Gly Lys Ala Met Gly Glu Glu Phe Asn Asp Lys Gly 115 120 125 Val Asp Ile Leu Leu Gly Pro Ala Ala Gly Pro Leu Gly Lys Tyr Pro 130 135 140 Asp Gly Gly Arg Ile Trp Glu Gly Phe Ser Pro Asp Pro Ala Leu Thr 145 150 155 160 Gly Val Leu Phe Ala Glu Thr Ile Lys Gly Ile Gln Asp Ala Gly Val 165 170 175 Ile Ala Thr Ala Lys His Tyr Ile Leu Asn Glu Gln Glu His Phe Arg 180 185 190 Gln Val Gly Glu Ala Gln Gly Tyr Gly Tyr Asn Ile Thr Glu Thr Ile 195 200 205 Ser Ser Asn Val Asp Asp Lys Thr Met His Glu Leu Tyr Leu Trp Pro 210 215 220 Phe Ala Asp Ala Val Arg Ala Gly Val Gly Ala Val Met Cys Ser Tyr 225 230 235 240 Asn Gln Ile Asn Asn Ser Tyr Gly Cys Gln Asn Ser Gln Thr Leu Asn 245 250 255 Lys Leu Leu Lys Ala Glu Leu Gly Phe Gln Gly Phe Val Met Ser Asp 260 265 270 Trp Ser Ala His His Ser Gly Val Gly Ala Ala Leu Ala Gly Leu Asp 275 280 285 Met Ser Met Pro Gly Asp Ile Ser Phe Asp Asp Gly Leu Ser Phe Trp 290 295 300 Gly Thr Asn Leu Thr Val Ser Val Leu Asn Gly Thr Val Pro Ala Trp 305 310 315 320 Arg Val Asp Asp Met Ala Val Arg Ile Met Thr Ala Tyr Tyr Lys Val 325 330 335 Gly Arg Asp Arg Leu Arg Ile Pro Pro Asn Phe Ser Ser Trp Thr Arg 340 345 350 Asp Glu Tyr Gly Trp Glu His Ser Ala Val Ser Glu Gly Ala Trp Thr 355 360 365 Lys Val Asn Asp Phe Val Asn Val Gln Arg Ser His Ser Gln Ile Ile 370 375 380 Arg Glu Ile Gly Ala Ala Ser Thr Val Leu Leu Lys Asn Thr Gly Ala 385 390 395 400 Leu Pro Leu Thr Gly Lys Glu Val Lys Val Gly Val Leu Gly Glu Asp 405 410 415 Ala Gly Ser Asn Pro Trp Gly Ala Asn Gly Cys Pro Asp Arg Gly Cys 420 425 430 Asp Asn Gly Thr Leu Ala Met Ala Trp Gly Ser Gly Thr Ala Asn Phe 435 440 445 Pro Tyr Leu Val Thr Pro Glu Gln Ala Ile Gln Arg Glu Val Ile Ser 450 455 460 Asn Gly Gly Asn Val Phe Ala Val Thr Asp Asn Gly Ala Leu Ser Gln 465 470 475 480 Met Ala Asp Val Ala Ser Gln Ser Ser Val Ser Leu Val Phe Val Asn 485 490 495 Ala Asp Ser Gly Glu Gly Phe Ile Ser Val Asp Gly Asn Glu Gly Asp 500 505 510 Arg Lys Asn Leu Thr Leu Trp Lys Asn Gly Glu Ala Val Ile Asp Thr 515 520 525 Val Val Ser His Cys Asn Asn Thr Ile Val Val Ile His Ser Val Gly 530 535 540 Pro Val Leu Ile Asp Arg Trp Tyr Asp Asn Pro Asn Val Thr Ala Ile 545 550 555 560 Ile Trp Ala Gly Leu Pro Gly Gln Glu Ser Gly Asn Ser Leu Val Asp 565 570 575 Val Leu Tyr Gly Arg Val Asn Pro Ser Ala Lys Thr Pro Phe Thr Trp 580 585 590 Gly Lys Thr Arg Glu Ser Tyr Gly Ala Pro Leu Leu Thr Glu Pro Asn 595 600 605 Asn Gly Asn Gly Ala Pro Gln Asp Asp Phe Asn Glu Gly Val Phe Ile 610 615 620 Asp Tyr Arg His Phe Asp Lys Arg Asn Glu Thr Pro Ile Tyr Glu Phe 625 630 635 640 Gly His Gly Leu Ser Tyr Thr Thr Phe Gly Tyr Ser His Leu Arg Val 645 650 655 Gln Ala Leu Asn Ser Ser Ser Ser Ala Tyr Val Pro Thr Ser Gly Glu 660 665 670 Thr Lys Pro Ala Pro Thr Tyr Gly Glu Ile Gly Ser Ala Ala Asp Tyr 675 680 685 Leu Tyr Pro Glu Gly Leu Lys Arg Ile Thr Lys Phe Ile Tyr Pro Trp 690 695 700 Leu Asn Ser Thr Asp Leu Glu Asp Ser Ser Asp Asp Pro Asn Tyr Gly 705 710 715 720 Trp Glu Asp Ser Glu Tyr Ile Pro Glu Gly Ala Arg Asp Gly Ser Pro 725 730 735 Gln Pro Leu Leu Lys Ala Gly Gly Ala Pro Gly Gly Asn Pro Thr Leu 740 745 750 Tyr Gln Asp Leu Val Arg Val Ser Ala Thr Ile Thr Asn Thr Gly Asn 755 760 765 Val Ala Gly Tyr Glu Val Pro Gln Leu Tyr Val Ser Leu Gly Gly Pro 770 775 780 Asn Glu Pro Arg Val Val Leu Arg Lys Phe Asp Arg Ile Phe Leu Ala 785 790 795 800 Pro Gly Glu Gln Lys Val Trp Thr Thr Thr Leu Asn Arg Arg Asp Leu 805 810 815 Ala Asn Trp Asp Val Glu Ala Gln Asp Trp Val Ile Thr Lys Tyr Pro 820 825 830 Lys Lys Val His Val Gly Ser Ser Ser Arg Lys Leu Pro Leu Arg Ala 835 840 845 Pro Leu Pro Arg Val Tyr 850 20842PRTAspergillus oryzae 20Lys Asp Asp Leu Ala Tyr Ser Pro Pro Phe Tyr Pro Ser Pro Trp Ala 1 5 10 15 Asp Gly Gln Gly Glu Trp Ala Glu Val Tyr Lys Arg Ala Val Asp Ile 20 25 30 Val Ser Gln Met Thr Leu Thr Glu Lys Val Asn Leu Thr Thr Gly Thr 35 40 45 Gly Trp Gln Leu Glu Arg Cys Val Gly Gln Thr Gly Ser Val Pro Arg 50 55 60 Leu Asn Ile Pro Ser Leu Cys Leu Gln Asp Ser Pro Leu Gly Ile Arg 65 70 75 80 Phe Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn Val Ala Ala 85 90 95 Thr Trp Asp Lys Thr Leu Ala Tyr Leu Arg Gly Gln Ala Met Gly Glu 100 105 110 Glu Phe Ser Asp Lys Gly Ile Asp Val Gln Leu Gly Pro Ala Ala Gly 115 120 125 Pro Leu Gly Ala His Pro Asp Gly Gly Arg Asn Trp Glu Gly Phe Ser 130 135 140 Pro Asp Pro Ala Leu Thr Gly Val Leu Phe Ala Glu Thr Ile Lys Gly 145 150 155 160 Ile Gln Asp Ala Gly Val Ile Ala Thr Ala Lys His Tyr Ile Met Asn 165 170 175 Glu Gln Glu His Phe Arg Gln Gln Pro Glu Ala Ala Gly Tyr Gly Phe 180 185 190 Asn Val Ser Asp Ser Leu Ser Ser Asn Val Asp Asp Lys Thr Met His 195 200 205 Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala Gly Val Gly 210 215 220 Ala Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr Gly Cys Glu 225 230 235 240 Asn Ser Glu Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu Gly Phe Gln 245 250 255 Gly Phe Val Met Ser Asp Trp Thr Ala His His Ser Gly Val Gly Ala 260 265 270 Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Val Thr Phe Asp 275 280 285 Ser Gly Thr Ser Phe Trp Gly Ala Asn Leu Thr Val Gly Val Leu Asn 290 295 300 Gly Thr Ile Pro Gln Trp Arg Val Asp Asp Met Ala Val Arg Ile Met 305 310 315 320 Ala Ala Tyr Tyr Lys Val Gly Arg Asp Thr Lys Tyr Thr Pro Pro Asn 325 330 335 Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly Phe Ala His Asn His Val 340 345 350 Ser Glu Gly Ala Tyr Glu Arg Val Asn Glu Phe Val Asp Val Gln Arg 355 360 365 Asp His Ala Asp Leu Ile Arg Arg Ile Gly Ala Gln Ser Thr Val Leu 370 375 380 Leu Lys Asn Lys Gly Ala Leu Pro Leu Ser Arg Lys Glu Lys Leu Val 385 390 395 400 Ala Leu Leu Gly Glu Asp Ala Gly Ser Asn Ser Trp Gly Ala Asn Gly 405 410 415 Cys Asp Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met Ala Trp Gly 420 425 430 Ser Gly Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu Gln Ala Ile 435 440 445 Gln Asn Glu Val Leu Gln Gly Arg Gly Asn Val Phe Ala Val Thr Asp 450 455 460 Ser Trp Ala Leu Asp Lys Ile Ala Ala Ala Ala Arg Gln Ala Ser Val 465 470 475 480 Ser Leu Val Phe Val Asn Ser Asp Ser Gly Glu Gly Tyr Leu Ser Val 485 490 495 Asp Gly Asn Glu Gly Asp Arg Asn Asn Ile Thr Leu Trp Lys Asn Gly 500 505 510 Asp Asn Val Val Lys Thr Ala Ala Asn Asn Cys Asn Asn Thr Val Val 515 520 525 Ile Ile His Ser Val Gly Pro Val Leu Ile Asp Glu Trp Tyr Asp His 530 535 540 Pro Asn Val Thr Gly Ile Leu Trp Ala Gly Leu Pro Gly Gln Glu Ser 545 550 555 560 Gly Asn Ser Ile Ala Asp Val Leu Tyr Gly Arg Val Asn Pro Gly Ala 565 570 575 Lys Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ser Tyr Gly Ser Pro 580 585 590 Leu Val Lys Asp Ala Asn Asn Gly Asn Gly Ala Pro Gln Ser Asp Phe 595 600 605 Thr Gln Gly Val Phe Ile Asp Tyr Arg His Phe Asp Lys Phe Asn Glu 610 615 620 Thr Pro Ile Tyr Glu Phe Gly Tyr Gly Leu Ser Tyr Thr Thr Phe Glu 625 630 635 640 Leu Ser Asp Leu His Val Gln Pro Leu Asn Ala Ser Arg Tyr Thr Pro 645 650 655 Thr Ser Gly Met Thr Glu Ala Ala Lys Asn Phe Gly Glu Ile Gly Asp 660 665 670 Ala Ser Glu Tyr Val Tyr Pro Glu Gly Leu Glu Arg Ile His Glu Phe 675 680 685 Ile Tyr Pro Trp Ile Asn Ser Thr Asp Leu Lys Ala Ser Ser Asp Asp 690 695 700 Ser Asn Tyr Gly Trp Glu Asp Ser Lys Tyr Ile Pro Glu Gly Ala Thr 705 710 715 720 Asp Gly Ser Ala Gln Pro Arg Leu Pro Ala Ser Gly Gly Ala Gly Gly 725 730 735 Asn Pro Gly Leu Tyr Glu Asp Leu Phe Arg Val Ser Val Lys Val Lys 740 745 750 Asn Thr Gly Asn Val Ala Gly Asp Glu Val Pro Gln Leu Tyr Val Ser 755 760 765 Leu Gly Gly Pro Asn Glu Pro Lys Val Val Leu Arg Lys Phe Glu Arg 770 775 780 Ile His Leu Ala Pro Ser Gln Glu Ala Val Trp Thr Thr Thr Leu Thr 785 790 795 800 Arg Arg Asp Leu Ala Asn Trp Asp Val Ser Ala Gln Asp Trp Thr Val 805 810 815 Thr Pro Tyr Pro Lys Thr Ile Tyr Val Gly Asn Ser Ser Arg Lys Leu 820 825 830 Pro Leu Gln Ala Ser Leu Pro Lys Ala Gln 835 840 21841PRTAspergillus aculeatus 21Asp Glu Leu Ala Phe Ser Pro Pro Phe Tyr Pro Ser Pro Trp Ala Asn 1 5 10 15 Gly Gln Gly Glu Trp Ala Glu Ala Tyr Gln Arg Ala Val Ala Ile Val 20 25 30 Ser Gln Met Thr Leu Asp Glu Lys Val Asn Leu Thr Thr Gly Thr Gly 35 40 45 Trp Glu Leu Glu Lys Cys Val Gly Gln Thr Gly Gly Val Pro Arg Leu 50 55 60 Asn Ile Gly Gly Met Cys Leu Gln Asp Ser Pro Leu Gly Ile Arg Asp 65 70 75 80 Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn Val Ala Ala Thr 85 90 95 Trp Asp Lys Asn Leu Ala Tyr Leu Arg Gly Gln Ala Met Gly Gln Glu 100 105 110 Phe Ser Asp Lys Gly Ile Asp Val Gln Leu Gly Pro Ala Ala Gly Pro 115 120 125 Leu Gly Arg Ser Pro Asp Gly Gly Arg Asn Trp Glu Gly Phe Ser Pro 130 135 140 Asp Pro Ala Leu Thr Gly Val Leu Phe Ala Glu Thr Ile Lys Gly Ile 145 150 155 160 Gln Asp Ala Gly Val Val Ala Thr Ala Lys His Tyr Ile Leu Asn Glu 165 170 175 Gln Glu His Phe Arg Gln Val Ala Glu Ala Ala Gly Tyr Gly Phe Asn 180 185 190 Ile Ser Asp Thr Ile Ser Ser Asn Val Asp Asp Lys Thr Ile His Glu 195 200 205 Met Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala Gly Val Gly Ala 210 215 220 Ile Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr Gly Cys Gln Asn 225 230 235 240 Ser Tyr Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu Gly Phe Gln Gly 245 250 255 Phe Val Met Ser Asp Trp Gly Ala His His Ser Gly Val Gly Ser Ala 260 265 270 Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Ile Thr Phe Asp Ser 275 280 285 Ala Thr Ser Phe Trp Gly Thr Asn Leu Thr Ile Ala Val Leu Asn Gly 290 295 300 Thr Val Pro Gln Trp Arg Val Asp Asp Met Ala Val Arg Ile Met Ala 305 310 315 320 Ala Tyr Tyr Lys Val Gly Arg Asp Arg Leu Tyr Gln Pro Pro Asn Phe 325 330 335 Ser Ser Trp Thr Arg Asp Glu Tyr Gly Phe Lys Tyr Phe Tyr Pro Gln 340 345 350 Glu Gly Pro Tyr Glu Lys Val Asn His Phe Val Asn Val Gln Arg Asn 355 360 365 His Ser Glu Val Ile Arg Lys Leu Gly Ala Asp Ser Thr Val Leu Leu 370 375 380 Lys Asn Asn Asn Ala Leu Pro Leu Thr Gly Lys Glu Arg Lys Val Ala 385 390 395 400 Ile Leu Gly Glu Asp Ala Gly Ser Asn Ser Tyr Gly Ala Asn Gly Cys

405 410 415 Ser Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met Ala Trp Gly Ser 420 425 430 Gly Thr Ala Glu Phe Pro Tyr Leu Val Thr Pro Glu Gln Ala Ile Gln 435 440 445 Ala Glu Val Leu Lys His Lys Gly Ser Val Tyr Ala Ile Thr Asp Asn 450 455 460 Trp Ala Leu Ser Gln Val Glu Thr Leu Ala Lys Gln Ala Ser Val Ser 465 470 475 480 Leu Val Phe Val Asn Ser Asp Ala Gly Glu Gly Tyr Ile Ser Val Asp 485 490 495 Gly Asn Glu Gly Asp Arg Asn Asn Leu Thr Leu Trp Lys Asn Gly Asp 500 505 510 Asn Leu Ile Lys Ala Ala Ala Asn Asn Cys Asn Asn Thr Ile Val Val 515 520 525 Ile His Ser Val Gly Pro Val Leu Val Asp Glu Trp Tyr Asp His Pro 530 535 540 Asn Val Thr Ala Ile Leu Trp Ala Gly Leu Pro Gly Gln Glu Ser Gly 545 550 555 560 Asn Ser Leu Ala Asp Val Leu Tyr Gly Arg Val Asn Pro Gly Ala Lys 565 570 575 Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ala Tyr Gly Asp Tyr Leu 580 585 590 Val Arg Glu Leu Asn Asn Gly Asn Gly Ala Pro Gln Asp Asp Phe Ser 595 600 605 Glu Gly Val Phe Ile Asp Tyr Arg Gly Phe Asp Lys Arg Asn Glu Thr 610 615 620 Pro Ile Tyr Glu Phe Gly His Gly Leu Ser Tyr Thr Thr Phe Asn Tyr 625 630 635 640 Ser Gly Leu His Ile Gln Val Leu Asn Ala Ser Ser Asn Ala Gln Val 645 650 655 Ala Thr Glu Thr Gly Ala Ala Pro Thr Phe Gly Gln Val Gly Asn Ala 660 665 670 Ser Asp Tyr Val Tyr Pro Glu Gly Leu Thr Arg Ile Ser Lys Phe Ile 675 680 685 Tyr Pro Trp Leu Asn Ser Thr Asp Leu Lys Ala Ser Ser Gly Asp Pro 690 695 700 Tyr Tyr Gly Val Asp Thr Ala Glu His Val Pro Glu Gly Ala Thr Asp 705 710 715 720 Gly Ser Pro Gln Pro Val Leu Pro Ala Gly Gly Gly Ser Gly Gly Asn 725 730 735 Pro Arg Leu Tyr Asp Glu Leu Ile Arg Val Ser Val Thr Val Lys Asn 740 745 750 Thr Gly Arg Val Ala Gly Asp Ala Val Pro Gln Leu Tyr Val Ser Leu 755 760 765 Gly Gly Pro Asn Glu Pro Lys Val Val Leu Arg Lys Phe Asp Arg Leu 770 775 780 Thr Leu Lys Pro Ser Glu Glu Thr Val Trp Thr Thr Thr Leu Thr Arg 785 790 795 800 Arg Asp Leu Ser Asn Trp Asp Val Ala Ala Gln Asp Trp Val Ile Thr 805 810 815 Ser Tyr Pro Lys Lys Val His Val Gly Ser Ser Ser Arg Gln Leu Pro 820 825 830 Leu His Ala Ala Leu Pro Lys Val Gln 835 840 22841PRTAspergillus niger 22Asp Glu Leu Ala Tyr Ser Pro Pro Tyr Tyr Pro Ser Pro Trp Ala Asn 1 5 10 15 Gly Gln Gly Asp Trp Ala Gln Ala Tyr Gln Arg Ala Val Asp Ile Val 20 25 30 Ser Gln Met Thr Leu Asp Glu Lys Val Asn Leu Thr Thr Gly Thr Gly 35 40 45 Trp Glu Leu Glu Leu Cys Val Gly Gln Thr Gly Gly Val Pro Arg Leu 50 55 60 Gly Val Pro Gly Met Cys Leu Gln Asp Ser Pro Leu Gly Val Arg Asp 65 70 75 80 Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Met Asn Val Ala Ala Thr 85 90 95 Trp Asp Lys Asn Leu Ala Tyr Leu Arg Gly Lys Ala Met Gly Gln Glu 100 105 110 Phe Ser Asp Lys Gly Ala Asp Ile Gln Leu Gly Pro Ala Ala Gly Pro 115 120 125 Leu Gly Arg Ser Pro Asp Gly Gly Arg Asn Trp Glu Gly Phe Ser Pro 130 135 140 Asp Pro Ala Leu Ser Gly Val Leu Phe Ala Glu Thr Ile Lys Gly Ile 145 150 155 160 Gln Asp Ala Gly Val Val Ala Thr Ala Lys His Tyr Ile Ala Tyr Glu 165 170 175 Gln Glu His Phe Arg Gln Ala Pro Glu Ala Gln Gly Phe Gly Phe Asn 180 185 190 Ile Ser Glu Ser Gly Ser Ala Asn Leu Asp Asp Lys Thr Met His Glu 195 200 205 Leu Tyr Leu Trp Pro Phe Ala Asp Ala Ile Arg Ala Gly Ala Gly Ala 210 215 220 Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr Gly Cys Gln Asn 225 230 235 240 Ser Tyr Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu Gly Phe Gln Gly 245 250 255 Phe Val Met Ser Asp Trp Ala Ala His His Ala Gly Val Ser Gly Ala 260 265 270 Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Val Asp Tyr Asp Ser 275 280 285 Gly Thr Ser Tyr Trp Gly Thr Asn Leu Thr Ile Ser Val Leu Asn Gly 290 295 300 Thr Val Pro Gln Trp Arg Val Asp Asp Met Ala Val Arg Ile Met Ala 305 310 315 320 Ala Tyr Tyr Lys Val Gly Arg Asp Arg Leu Trp Thr Pro Pro Asn Phe 325 330 335 Ser Ser Trp Thr Arg Asp Glu Tyr Gly Tyr Lys Tyr Tyr Tyr Val Ser 340 345 350 Glu Gly Pro Tyr Glu Lys Val Asn Gln Tyr Val Asn Val Gln Arg Asn 355 360 365 His Ser Glu Leu Ile Arg Arg Ile Gly Ala Asp Ser Thr Val Leu Leu 370 375 380 Lys Asn Asp Gly Ala Leu Pro Leu Thr Gly Lys Glu Arg Leu Val Ala 385 390 395 400 Leu Ile Gly Glu Asp Ala Gly Ser Asn Pro Tyr Gly Ala Asn Gly Cys 405 410 415 Ser Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met Gly Trp Gly Ser 420 425 430 Gly Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu Gln Ala Ile Ser 435 440 445 Asn Glu Val Leu Lys His Lys Asn Gly Val Phe Thr Ala Thr Asp Asn 450 455 460 Trp Ala Ile Asp Gln Ile Glu Ala Leu Ala Lys Thr Ala Ser Val Ser 465 470 475 480 Leu Val Phe Val Asn Ala Asp Ser Gly Glu Gly Tyr Ile Asn Val Asp 485 490 495 Gly Asn Leu Gly Asp Arg Arg Asn Leu Thr Leu Trp Arg Asn Gly Asp 500 505 510 Asn Val Ile Lys Ala Ala Ala Ser Asn Cys Asn Asn Thr Ile Val Val 515 520 525 Ile His Ser Val Gly Pro Val Leu Val Asn Glu Trp Tyr Asp Asn Pro 530 535 540 Asn Val Thr Ala Ile Leu Trp Gly Gly Leu Pro Gly Gln Glu Ser Gly 545 550 555 560 Asn Ser Leu Ala Asp Val Leu Tyr Gly Arg Val Asn Pro Gly Ala Lys 565 570 575 Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ala Tyr Gln Asp Tyr Leu 580 585 590 Val Thr Glu Pro Asn Asn Gly Asn Gly Ala Pro Gln Glu Asp Phe Val 595 600 605 Glu Gly Val Phe Ile Asp Tyr Arg Gly Phe Asp Lys Arg Asn Glu Thr 610 615 620 Pro Ile Tyr Glu Phe Gly Tyr Gly Leu Ser Tyr Thr Thr Phe Asn Tyr 625 630 635 640 Ser Asn Leu Glu Val Gln Val Leu Ser Ala Pro Ala Tyr Glu Pro Ala 645 650 655 Ser Gly Glu Thr Glu Ala Ala Pro Thr Phe Gly Glu Val Gly Asn Ala 660 665 670 Ser Asp Tyr Leu Tyr Pro Ser Gly Leu Gln Arg Ile Thr Lys Phe Ile 675 680 685 Tyr Pro Trp Leu Asn Gly Thr Asp Leu Glu Ala Ser Ser Gly Asp Ala 690 695 700 Ser Tyr Gly Gln Asp Ser Ser Asp Tyr Leu Pro Glu Gly Ala Thr Asp 705 710 715 720 Gly Ser Ala Gln Pro Ile Leu Pro Ala Gly Gly Gly Pro Gly Gly Asn 725 730 735 Pro Arg Leu Tyr Asp Glu Leu Ile Arg Val Ser Val Thr Ile Lys Asn 740 745 750 Thr Gly Lys Val Ala Gly Asp Glu Val Pro Gln Leu Tyr Val Ser Leu 755 760 765 Gly Gly Pro Asn Glu Pro Lys Ile Val Leu Arg Gln Phe Glu Arg Ile 770 775 780 Thr Leu Gln Pro Ser Glu Glu Thr Lys Trp Ser Thr Thr Leu Thr Arg 785 790 795 800 Arg Asp Leu Ala Asn Trp Asn Val Glu Lys Gln Asp Trp Glu Ile Thr 805 810 815 Ser Tyr Pro Lys Met Val Phe Val Gly Ser Ser Ser Arg Lys Leu Pro 820 825 830 Leu Arg Ala Ser Leu Pro Thr Val His 835 840 23838PRTTalaromyces emersonii 23Glu Asn Leu Ala Tyr Ser Pro Pro Phe Tyr Pro Ser Pro Trp Ala Asn 1 5 10 15 Gly Gln Gly Asp Trp Ala Glu Ala Tyr Gln Lys Ala Val Gln Phe Val 20 25 30 Ser Gln Leu Thr Leu Ala Glu Lys Val Asn Leu Thr Thr Gly Thr Gly 35 40 45 Trp Glu Gln Asp Arg Cys Val Gly Gln Val Gly Ser Ile Pro Arg Leu 50 55 60 Gly Phe Pro Gly Leu Cys Met Gln Asp Ser Pro Leu Gly Val Arg Asp 65 70 75 80 Thr Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn Val Ala Ala Thr 85 90 95 Trp Asp Arg Asn Leu Ala Tyr Arg Arg Gly Val Ala Met Gly Glu Glu 100 105 110 His Arg Gly Lys Gly Val Asp Val Gln Leu Gly Pro Val Ala Gly Pro 115 120 125 Leu Gly Arg Ser Pro Asp Ala Gly Arg Asn Trp Glu Gly Phe Ala Pro 130 135 140 Asp Pro Val Leu Thr Gly Asn Met Met Ala Ser Thr Ile Gln Gly Ile 145 150 155 160 Gln Asp Ala Gly Val Ile Ala Cys Ala Lys His Phe Ile Leu Tyr Glu 165 170 175 Gln Glu His Phe Arg Gln Gly Ala Gln Asp Gly Tyr Asp Ile Ser Asp 180 185 190 Ser Ile Ser Ala Asn Ala Asp Asp Lys Thr Met His Glu Leu Tyr Leu 195 200 205 Trp Pro Phe Ala Asp Ala Val Arg Ala Gly Val Gly Ser Val Met Cys 210 215 220 Ser Tyr Asn Gln Val Asn Asn Ser Tyr Ala Cys Ser Asn Ser Tyr Thr 225 230 235 240 Met Asn Lys Leu Leu Lys Ser Glu Leu Gly Phe Gln Gly Phe Val Met 245 250 255 Thr Asp Trp Gly Gly His His Ser Gly Val Gly Ser Ala Leu Ala Gly 260 265 270 Leu Asp Met Ser Met Pro Gly Asp Ile Ala Phe Asp Ser Gly Thr Ser 275 280 285 Phe Trp Gly Thr Asn Leu Thr Val Ala Val Leu Asn Gly Ser Ile Pro 290 295 300 Glu Trp Arg Val Asp Asp Met Ala Val Arg Ile Met Ser Ala Tyr Tyr 305 310 315 320 Lys Val Gly Arg Asp Arg Tyr Ser Val Pro Ile Asn Phe Asp Ser Trp 325 330 335 Thr Leu Asp Thr Tyr Gly Pro Glu His Tyr Ala Val Gly Gln Gly Gln 340 345 350 Thr Lys Ile Asn Glu His Val Asp Val Arg Gly Asn His Ala Glu Ile 355 360 365 Ile His Glu Ile Gly Ala Ala Ser Ala Val Leu Leu Lys Asn Lys Gly 370 375 380 Gly Leu Pro Leu Thr Gly Thr Glu Arg Phe Val Gly Val Phe Gly Lys 385 390 395 400 Asp Ala Gly Ser Asn Pro Trp Gly Val Asn Gly Cys Ser Asp Arg Gly 405 410 415 Cys Asp Asn Gly Thr Leu Ala Met Gly Trp Gly Ser Gly Thr Ala Asn 420 425 430 Phe Pro Tyr Leu Val Thr Pro Glu Gln Ala Ile Gln Arg Glu Val Leu 435 440 445 Ser Arg Asn Gly Thr Phe Thr Gly Ile Thr Asp Asn Gly Ala Leu Ala 450 455 460 Glu Met Ala Ala Ala Ala Ser Gln Ala Asp Thr Cys Leu Val Phe Ala 465 470 475 480 Asn Ala Asp Ser Gly Glu Gly Tyr Ile Thr Val Asp Gly Asn Glu Gly 485 490 495 Asp Arg Lys Asn Leu Thr Leu Trp Gln Gly Ala Asp Gln Val Ile His 500 505 510 Asn Val Ser Ala Asn Cys Asn Asn Thr Val Val Val Leu His Thr Val 515 520 525 Gly Pro Val Leu Ile Asp Asp Trp Tyr Asp His Pro Asn Val Thr Ala 530 535 540 Ile Leu Trp Ala Gly Leu Pro Gly Gln Glu Ser Gly Asn Ser Leu Val 545 550 555 560 Asp Val Leu Tyr Gly Arg Val Asn Pro Gly Lys Thr Pro Phe Thr Trp 565 570 575 Gly Arg Ala Arg Asp Asp Tyr Gly Ala Pro Leu Ile Val Lys Pro Asn 580 585 590 Asn Gly Lys Gly Ala Pro Gln Gln Asp Phe Thr Glu Gly Ile Phe Ile 595 600 605 Asp Tyr Arg Arg Phe Asp Lys Tyr Asn Ile Thr Pro Ile Tyr Glu Phe 610 615 620 Gly Phe Gly Leu Ser Tyr Thr Thr Phe Glu Phe Ser Gln Leu Asn Val 625 630 635 640 Gln Pro Ile Asn Ala Pro Pro Tyr Thr Pro Ala Ser Gly Phe Thr Lys 645 650 655 Ala Ala Gln Ser Phe Gly Gln Pro Ser Asn Ala Ser Asp Asn Leu Tyr 660 665 670 Pro Ser Asp Ile Glu Arg Val Pro Leu Tyr Ile Tyr Pro Trp Leu Asn 675 680 685 Ser Thr Asp Leu Lys Ala Ser Ala Asn Asp Pro Asp Tyr Gly Leu Pro 690 695 700 Thr Glu Lys Tyr Val Pro Pro Asn Ala Thr Asn Gly Asp Pro Gln Pro 705 710 715 720 Ile Asp Pro Ala Gly Gly Ala Pro Gly Gly Asn Pro Ser Leu Tyr Glu 725 730 735 Pro Val Ala Arg Val Thr Thr Ile Ile Thr Asn Thr Gly Lys Val Thr 740 745 750 Gly Asp Glu Val Pro Gln Leu Tyr Val Ser Leu Gly Gly Pro Asp Asp 755 760 765 Ala Pro Lys Val Leu Arg Gly Phe Asp Arg Ile Thr Leu Ala Pro Gly 770 775 780 Gln Gln Tyr Leu Trp Thr Thr Thr Leu Thr Arg Arg Asp Ile Ser Asn 785 790 795 800 Trp Asp Pro Val Thr Gln Asn Trp Val Val Thr Asn Tyr Thr Lys Thr 805 810 815 Ile Tyr Val Gly Asn Ser Ser Arg Asn Leu Pro Leu Gln Ala Pro Leu 820 825 830 Lys Pro Tyr Pro Gly Ile 835 24824PRTThermoascus aurentiacus 24Lys Asp Asp Leu Ala Tyr Ser Pro Pro Phe Tyr Pro Ser Pro Trp Met 1 5 10 15 Asn Gly Asn Gly Glu Trp Ala Glu Ala Tyr Arg Arg Ala Val Asp Phe 20 25 30 Val Ser Gln Leu Thr Leu Ala Glu Lys Val Asn Leu Thr Thr Gly Val 35 40 45 Gly Trp Met Gln Glu Lys Cys Val Gly Glu Thr Gly Ser Ile Pro Arg 50 55 60 Leu Gly Phe Arg Gly Leu Cys Leu Gln Asp Ser Pro Leu Gly Val Arg 65 70 75 80 Phe Ala Asp Tyr Val Ser Ala Phe Pro Ala Gly Val Asn Val Ala Ala 85 90 95 Thr Trp Asp Lys Asn Leu Ala Tyr Leu Arg Gly Lys Ala Met Gly Glu 100 105 110 Glu His Arg Gly Lys Gly Val Asp Val Gln Leu Gly Pro Val Ala Gly 115 120 125 Pro Leu Gly Arg His Pro Asp Gly Gly Arg Asn Trp Glu Gly Phe Ser 130 135 140 Pro Asp Pro Val Leu Thr Gly Val Leu Met Ala Glu Thr Ile Lys Gly 145 150 155 160 Ile Gln Asp Ala Gly Val Ile Ala Cys Ala Lys His Phe Ile Gly Asn 165 170 175 Glu Met Glu His Phe Arg Gln Ala Gly Glu

Ala Val Gly Tyr Gly Phe 180 185 190 Asp Ile Thr Glu Ser Val Ser Ser Asn Ile Asp Asp Lys Thr Leu His 195 200 205 Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala Gly Val Gly 210 215 220 Ser Phe Met Cys Ser Tyr Asn Gln Val Asn Asn Ser Tyr Ser Cys Ser 225 230 235 240 Asn Ser Tyr Leu Leu Asn Lys Leu Leu Lys Ser Glu Leu Asp Phe Gln 245 250 255 Gly Phe Val Met Ser Asp Trp Gly Ala His His Ser Gly Val Gly Ala 260 265 270 Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Thr Ala Phe Gly 275 280 285 Thr Gly Lys Ser Phe Trp Gly Thr Asn Leu Thr Ile Ala Val Leu Asn 290 295 300 Gly Thr Val Pro Glu Trp Arg Val Asp Asp Met Ala Val Arg Ile Met 305 310 315 320 Ala Ala Phe Tyr Lys Val Gly Arg Asp Arg Tyr Gln Val Pro Val Asn 325 330 335 Phe Asp Ser Trp Thr Lys Asp Glu Tyr Gly Tyr Glu His Ala Leu Val 340 345 350 Gly Gln Asn Tyr Val Lys Val Asn Asp Lys Val Asp Val Arg Ala Asp 355 360 365 His Ala Asp Ile Ile Arg Gln Ile Gly Ser Ala Ser Val Val Leu Leu 370 375 380 Lys Asn Asp Gly Gly Leu Pro Leu Thr Gly Tyr Glu Lys Phe Thr Gly 385 390 395 400 Val Phe Gly Glu Asp Ala Gly Ser Asn Arg Trp Gly Ala Asp Gly Cys 405 410 415 Ser Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met Gly Trp Gly Ser 420 425 430 Gly Thr Ala Asp Phe Pro Tyr Leu Val Thr Pro Glu Gln Ala Ile Gln 435 440 445 Asn Glu Ile Leu Ser Lys Gly Lys Gly Leu Asp Ser Val Ser Ile Val 450 455 460 Phe Val Asn Ala Asp Ser Gly Glu Gly Tyr Ile Asn Val Asp Gly Asn 465 470 475 480 Glu Gly Asp Arg Lys Asn Leu Thr Leu Trp Lys Gly Gly Glu Glu Val 485 490 495 Ile Lys Thr Val Ala Ala Asn Cys Asn Asn Thr Ile Val Val Met His 500 505 510 Thr Val Gly Pro Val Leu Ile Asp Glu Trp Tyr Asp Asn Pro Asn Val 515 520 525 Thr Ala Ile Val Trp Ala Gly Leu Pro Gly Gln Glu Ser Gly Asn Ser 530 535 540 Leu Val Asp Val Leu Tyr Gly Arg Val Ser Pro Gly Gly Lys Thr Pro 545 550 555 560 Phe Thr Trp Gly Lys Thr Arg Glu Ser Tyr Gly Ala Pro Leu Leu Thr 565 570 575 Lys Pro Asn Asn Gly Lys Gly Ala Pro Gln Asp Asp Phe Thr Glu Gly 580 585 590 Val Phe Ile Asp Tyr Arg Arg Phe Asp Lys Tyr Asn Glu Thr Pro Ile 595 600 605 Tyr Glu Phe Gly Phe Gly Leu Ser Tyr Thr Thr Phe Glu Tyr Ser Asn 610 615 620 Ile Tyr Val Gln Pro Leu Asn Ala Arg Pro Tyr Thr Pro Ala Ser Gly 625 630 635 640 Ser Thr Lys Ala Ala Pro Thr Phe Gly Asn Ile Ser Thr Asp Tyr Ala 645 650 655 Asp Tyr Leu Tyr Pro Glu Asp Ile His Lys Val Pro Leu Tyr Ile Tyr 660 665 670 Pro Trp Leu Asn Thr Thr Asp Pro Glu Glu Val Leu Arg Arg Ser Arg 675 680 685 Leu Thr Glu Met Lys Ala Glu Asp Tyr Ile Pro Ser Gly Ala Thr Asp 690 695 700 Gly Ser Pro Gln Pro Ile Leu Pro Ala Gly Gly Ala Pro Gly Gly Asn 705 710 715 720 Pro Gly Leu Tyr Asp Glu Met Tyr Arg Val Ser Ala Ile Ile Thr Asn 725 730 735 Thr Gly Asn Val Val Gly Asp Glu Val Pro Gln Leu Tyr Val Ser Leu 740 745 750 Gly Gly Pro Asp Asp Pro Lys Val Val Leu Arg Asn Phe Asp Arg Ile 755 760 765 Thr Leu His Pro Gly Gln Gln Thr Met Trp Thr Thr Thr Leu Thr Arg 770 775 780 Arg Asp Ile Ser Asn Trp Asp Pro Ala Ser Gln Asn Trp Val Val Thr 785 790 795 800 Lys Tyr Pro Lys Thr Val Tyr Ile Gly Ser Ser Ser Arg Lys Leu His 805 810 815 Leu Gln Ala Pro Leu Pro Pro Tyr 820 25825PRTHansenula anomala 25Met Leu Leu Pro Leu Tyr Gly Leu Ala Ser Phe Leu Val Leu Ser Gln 1 5 10 15 Ala Ala Leu Val Asn Thr Ser Ala Pro Gln Ala Ser Asn Asp Asp Pro 20 25 30 Phe Asn His Ser Pro Ser Phe Tyr Pro Thr Pro Gln Gly Gly Arg Ile 35 40 45 Asn Asp Gly Lys Trp Gln Ala Ala Phe Tyr Arg Ala Arg Glu Leu Val 50 55 60 Asp Gln Met Ser Ile Ala Glu Lys Val Asn Leu Thr Thr Gly Val Gly 65 70 75 80 Ser Ala Ser Gly Pro Cys Ser Gly Asn Thr Gly Ser Val Pro Arg Leu 85 90 95 Asn Ile Ser Ser Ile Cys Val Gln Asp Gly Pro Leu Ser Val Arg Ala 100 105 110 Ala Asp Leu Thr Asp Val Phe Pro Cys Gly Met Ala Ala Ser Ser Ser 115 120 125 Phe Asn Lys Gln Leu Ile Tyr Asp Arg Ala Val Ala Ile Gly Ser Glu 130 135 140 Phe Lys Gly Lys Gly Ala Asp Ala Ile Leu Gly Pro Val Tyr Gly Pro 145 150 155 160 Met Gly Val Lys Ala Ala Gly Gly Arg Gly Trp Glu Gly His Gly Pro 165 170 175 Asp Pro Tyr Leu Glu Gly Val Ile Ala Tyr Leu Gln Thr Ile Gly Ile 180 185 190 Gln Ser Gln Gly Val Val Ser Thr Ala Lys His Leu Ile Gly Asn Glu 195 200 205 Gln Glu His Phe Arg Phe Ala Lys Lys Asp Lys His Ala Gly Lys Ile 210 215 220 Asp Pro Gly Met Phe Asn Thr Ser Ser Ser Leu Ser Ser Glu Ile Asp 225 230 235 240 Asp Arg Ala Met His Glu Ile Tyr Leu Trp Pro Phe Ala Glu Ala Val 245 250 255 Arg Gly Gly Val Ser Ser Ile Met Cys Ser Tyr Asn Lys Leu Asn Gly 260 265 270 Ser His Ala Cys Gln Asn Ser Tyr Leu Leu Asn Tyr Leu Leu Lys Glu 275 280 285 Glu Leu Gly Phe Gln Gly Phe Val Met Thr Asp Trp Gly Ala Leu Tyr 290 295 300 Ser Gly Ile Asp Ala Ala Asn Ala Gly Leu Asp Met Asp Met Pro Cys 305 310 315 320 Glu Ala Gln Tyr Phe Gly Gly Asn Leu Thr Thr Ala Val Leu Asn Gly 325 330 335 Thr Leu Pro Gln Asp Arg Leu Asp Asp Met Ala Thr Arg Ile Leu Ser 340 345 350 Ala Leu Ile Tyr Ser Gly Val His Asn Pro Asp Gly Pro Asn Tyr Asn 355 360 365 Ala Gln Thr Phe Leu Thr Glu Gly His Glu Tyr Phe Lys Gln Gln Glu 370 375 380 Gly Asp Ile Val Val Leu Asn Lys His Val Asp Val Arg Ser Asp Ile 385 390 395 400 Asn Arg Ala Val Ala Leu Arg Ser Ala Val Glu Gly Val Val Leu Leu 405 410 415 Lys Asn Glu His Glu Thr Leu Pro Leu Gly Arg Glu Lys Val Lys Arg 420 425 430 Ile Ser Ile Leu Gly Gln Ala Ala Gly Asp Asp Ser Lys Gly Thr Ser 435 440 445 Cys Ser Leu Arg Gly Cys Gly Ser Gly Ala Ile Gly Thr Gly Tyr Gly 450 455 460 Ser Gly Ala Gly Thr Phe Ser Tyr Phe Val Thr Pro Ala Asp Gly Ile 465 470 475 480 Gly Ala Arg Ala Gln Gln Glu Lys Ile Ser Tyr Glu Phe Ile Gly Asp 485 490 495 Ser Trp Asn Gln Ala Ala Ala Met Asp Ser Ala Leu Tyr Ala Asp Ala 500 505 510 Ala Ile Glu Val Ala Asn Ser Val Ala Gly Glu Glu Ile Gly Asp Val 515 520 525 Asp Gly Asn Tyr Gly Asp Leu Asn Asn Leu Thr Leu Trp His Asn Ala 530 535 540 Val Pro Leu Ile Lys Asn Ile Ser Ser Ile Asn Asn Asn Thr Ile Val 545 550 555 560 Ile Val Thr Ser Gly Gln Gln Ile Asp Leu Glu Pro Phe Ile Asp Asn 565 570 575 Glu Asn Val Thr Ala Val Ile Tyr Ser Ser Tyr Leu Gly Gln Asp Phe 580 585 590 Gly Thr Val Leu Ala Lys Val Leu Phe Gly Asp Glu Asn Pro Ser Gly 595 600 605 Lys Leu Pro Phe Thr Ile Ala Lys Asp Val Asn Asp Tyr Ile Pro Val 610 615 620 Ile Glu Lys Val Asp Val Pro Asp Pro Val Asp Lys Phe Thr Glu Ser 625 630 635 640 Ile Tyr Val Asp Tyr Arg Tyr Phe Asp Lys Tyr Asn Lys Pro Val Arg 645 650 655 Tyr Glu Phe Gly Tyr Gly Leu Ser Tyr Ser Asn Phe Ser Leu Ser Asp 660 665 670 Ile Glu Ile Gln Thr Leu Gln Pro Phe Ser Glu Asn Ala Glu Pro Ala 675 680 685 Ala Asn Tyr Ser Glu Thr Tyr Gln Tyr Lys Gln Ser Asn Met Asp Pro 690 695 700 Ser Glu Tyr Thr Val Pro Glu Gly Phe Lys Glu Leu Ala Asn Tyr Thr 705 710 715 720 Tyr Pro Tyr Ile His Asp Ala Ser Ser Ile Lys Ala Asn Ser Ser Tyr 725 730 735 Asp Tyr Pro Glu Gly Tyr Ser Thr Glu Gln Leu Asp Gly Pro Lys Ser 740 745 750 Leu Ala Ala Gly Gly Leu Gly Gly Asn His Thr Cys Gly Met Leu Val 755 760 765 Thr Leu Ser Leu Leu Lys Ser Gln Ile Lys Val Leu Met Leu Val Gly 770 775 780 Leu His Leu Asn Cys Met Leu Asp Ile Gln Ile Met Met Asn Ser Gln 785 790 795 800 His Leu Gln Cys Asn Tyr Val Asp Leu Lys Arg Cys Phe Trp Ile Lys 805 810 815 Ile Ile Leu Lys Leu Phe Leu Leu Asn 820 825 26867PRTPiromyces sp. 26Met Lys Ile Gln Asn Ile Leu Val Ala Leu Thr Cys Gly Leu Val Ser 1 5 10 15 Gln Val Phe Ala Thr Ser Trp Ser Glu Ala Asp Glu Lys Ala Lys Ser 20 25 30 Phe Met Ser Asp Leu Ser Glu Ser Glu Lys Ile Asp Ile Val Thr Gly 35 40 45 Tyr Met Asn Met Gln Gly Thr Cys Val Gly Asn Ile Lys Pro Leu Asp 50 55 60 Arg Lys Asn Phe Lys Gly Leu Cys Leu Gln Asp Gly Pro Ala Gly Val 65 70 75 80 Arg Phe Asn Gly Gly Thr Ser Thr Thr Trp Gln Ala Gly Ile Asn Asn 85 90 95 Ala Ala Thr Phe Asn Lys Asp Leu Leu Tyr Lys Ile Gly Lys Asp Gln 100 105 110 Gly Ala Glu Phe Tyr Ala Lys Gly Ile Asn Ile Ala Leu Ala Pro Ser 115 120 125 Met Asn Ile Leu Arg Ala Pro Ala Ser Gly Arg Val Trp Glu Asn Phe 130 135 140 Gly Glu Asp Pro Tyr Leu Ser Gly Val Cys Gly Ala Gln Ile Thr Lys 145 150 155 160 Gly Tyr Gln Asp Ser Gly Val Ile Val Ala Ala Lys His Tyr Val Ala 165 170 175 Asn Asp Ile Glu His Asn Arg Glu Ala Ser Ser Ser Asn Met Asp Asp 180 185 190 Gln Thr Leu Met Glu Ile His Val Glu Pro Phe Tyr Arg Thr Ile Lys 195 200 205 Asp Gly Asp Ala Gly Ser Val Met Ala Ser Tyr Asn Ala Val Asn Asn 210 215 220 Ile Tyr Val Val Gln Asn Lys Lys Val Leu Thr Glu Ile Leu Lys Glu 225 230 235 240 Gly Ile Gly Phe Gln Gly Phe Val Met Ser Asp Trp Trp Ala Ile His 245 250 255 Asp Leu Glu Gly Ser Phe Asn Ala Gly Met Asp Met Asn Met Pro Gly 260 265 270 Gly Lys Ala Trp Gly Pro Asp Tyr Val Asn Asn Ser Phe Trp Gly Ser 275 280 285 Asn Ile Ser Asn Ala Ile Arg Ser Gly Gln Val Ser Ser Ser Arg Leu 290 295 300 Asp Asp Ala Val Arg Arg Ile Ile Arg Thr Leu Tyr Arg Phe Asp Gln 305 310 315 320 Met Ser Gly Tyr Pro Asn Val Asn Leu Lys Ala Pro Ser Met His Ala 325 330 335 Asp Thr Asn Arg Gln Ala Ala Ile Glu Ser Ser Val Leu Leu Lys Asn 340 345 350 Ala Asp Asp Ile Leu Pro Leu Thr Lys Lys Tyr Arg Lys Ile Ala Ile 355 360 365 Ile Gly Lys Asp Ala Asp Lys Ala Gln Ser Cys Thr Asp Thr Ala Cys 370 375 380 Ser Gly Gly Asn Ile Ile Gln Gly Trp Gly Ser Gly Thr Thr Asp Phe 385 390 395 400 Thr Gly Ile Ser Asp Pro Ile Thr Ala Ile Lys Asn Arg Ala Ser Lys 405 410 415 Glu Gly Ile Ser Ile Val Ser Ser Ile Ser Asp Ser Ala Asn Glu Gly 420 425 430 Ala Asn Val Ala Lys Asp Ala Asp Val Ala Val Val Phe Val Arg Ala 435 440 445 Thr Ser Gly Glu Glu Tyr Ile Val Val Asp Asn Asn Lys Gly Asp Arg 450 455 460 Asn Asn Leu Asp Leu Trp His Gly Gly Asn Asp Leu Val Lys Ser Val 465 470 475 480 Ala Ala Val Asn Lys Asn Thr Val Val Val Ile His Ala Pro Ala Thr 485 490 495 Val Asn Leu Pro Phe Leu Asn Asn Val Lys Ala Ile Ile His Ala Gly 500 505 510 Met Pro Gly Ala Glu Ser Gly Asn Ala Ile Ala Ser Ile Leu Phe Gly 515 520 525 Asp Ser Asn Pro Ser Gly His Leu Pro Phe Thr Trp Ala Ala Arg Glu 530 535 540 Asp Tyr Cys Cys Asp Val Ser Tyr Pro Ala Glu Leu Pro His Gly Gly 545 550 555 560 Asn Ser Lys Thr Ala Tyr Asp Tyr Lys Glu Gly Leu Phe Val Gly Tyr 565 570 575 Arg Trp Phe Asp Lys Lys Asn Lys Thr Pro Ile Phe Pro Phe Gly His 580 585 590 Gly Leu Ser Tyr Thr Thr Phe Asp Tyr Ser Asn Leu Ser Val Ser Leu 595 600 605 Lys Lys Ser Gly Thr Gln Val Thr Gly Leu Glu Ala Thr Val Thr Val 610 615 620 Ala Asn Thr Gly Ser Tyr Glu Gly Ala Thr Val Pro Met Leu Phe Leu 625 630 635 640 Gly Phe Pro Ala Val Ser Glu Leu Gly Asp Tyr Pro Val Arg Asn Leu 645 650 655 Lys Ala Phe Glu Lys Val Asn Leu Lys Ala Gly Glu Lys Lys Thr Val 660 665 670 Thr Leu Thr Val Asp Gln His Gly Leu Ser Tyr Tyr Asn Thr Ser Lys 675 680 685 Lys Ser Phe Val Val Pro Thr Gly Gly Glu Phe Thr Val Tyr Val Gly 690 695 700 Lys Ser Ala Gly Asp Leu Pro Leu Lys Lys Ala Ile Lys Asn Thr Gln 705 710 715 720 Gly Thr Asn Glu Ser Ser Ser Ser Val Gly Asp Glu Asn Asn Asn Asn 725 730 735 Pro Asn Asn Asn Ala Asp Cys Ser Val Asn Gly Tyr Lys Cys Cys Ser 740 745 750 Asn Ser Asn Ala Glu Val Val Tyr Thr Asp Gly Asp Gly Asn Trp Gly 755 760 765 Val Glu Asn Gly Gln Trp Cys Ile Ile Lys Glu Gln Gln Gln Gln Gln 770 775 780 Thr Cys Phe Ser Ile Lys Leu Gly Tyr Pro Cys Cys Lys Gly Asn Glu 785 790 795 800 Val Ala Tyr Thr Asp Asn Asp Gly Gln Trp Gly Phe Glu Asn Gly Gln 805 810 815 Trp Cys Gly Ile Ala Thr Ala Thr Ser Gly Ala Gly Gly Cys Pro Tyr 820 825 830 Thr

Ser Lys Asn Gly Tyr Pro Val Cys Gln Thr Thr Thr Lys Val Glu 835 840 845 Tyr Val Asp Ser Asp Lys Trp Gly Val Glu Asn Gly Asn Trp Cys Ile 850 855 860 Met Cys Asn 865 27870PRTCoccidioides immitis 27Met Ser Pro Thr Ile Trp Ile Ala Thr Leu Leu Tyr Trp Phe Ala Phe 1 5 10 15 Gln Ala Arg Lys Ser Val Ala Ala Pro Pro Gly Val Gly Ala Leu Asp 20 25 30 Asp Arg Ala Glu Leu Pro Asp Gly Phe His Ser Pro Gln Tyr Tyr Pro 35 40 45 Ala Pro Arg Gly Leu Gly Ala Gly Met Glu Glu Ala Tyr Ser Lys Ala 50 55 60 His Thr Val Val Ser Lys Met Thr Leu Ala Gly Lys Val Asn Leu Thr 65 70 75 80 Thr Gly Thr Gly Phe Leu Met Ala Leu Val Gly Gln Thr Gly Ser Ala 85 90 95 Leu Arg Phe Gly Ile Pro Arg Leu Cys Leu Gln Asp Gly Pro Leu Gly 100 105 110 Leu Arg Asn Thr Asp His Asn Thr Ala Phe Pro Ala Gly Ile Ser Val 115 120 125 Gly Ala Thr Phe Asp Lys Lys Leu Met Tyr Glu Arg Gly Cys Ala Met 130 135 140 Gly Glu Glu Phe Arg Gly Lys Gly Ala Asn Val His Leu Gly Pro Ser 145 150 155 160 Val Gly Pro Leu Gly Arg Lys Pro Arg Gly Gly Arg Asn Trp Glu Gly 165 170 175 Phe Gly Ser Asp Pro Ser Leu Gln Ala Ile Ala Ala Val Glu Thr Ile 180 185 190 Lys Gly Val Gln Ser Lys Gly Val Ile Ala Thr Ile Lys His Leu Val 195 200 205 Gly Asn Glu Gln Glu Met Tyr Arg Met Thr Asn Ile Val Gln Arg Ala 210 215 220 Tyr Ser Ala Asn Ile Asp Asp Arg Thr Met His Glu Leu Tyr Leu Trp 225 230 235 240 Pro Phe Ala Glu Ser Val Arg Ala Gly Val Gly Ala Val Met Met Ala 245 250 255 Tyr Asn Asp Val Asn Gly Ser Ala Ser Cys Gln Asn Ser Lys Leu Ile 260 265 270 Asn Gly Ile Leu Lys Asp Glu Leu Gly Phe Gln Gly Phe Val Met Thr 275 280 285 Asp Trp Tyr Ala Gln Ile Gly Gly Val Ser Ser Ala Leu Ala Gly Leu 290 295 300 Asp Met Ser Met Pro Gly Asp Gly Ser Val Pro Leu Ser Gly Thr Ser 305 310 315 320 Phe Trp Ala Ser Glu Leu Ser Arg Ser Ile Leu Asn Gly Thr Val Ala 325 330 335 Leu Asp Arg Leu Asn Asp Met Val Thr Arg Ile Val Ala Thr Trp Phe 340 345 350 Lys Phe Gly Gln Asp Lys Asp Phe Pro Leu Pro Asn Phe Ser Ser Tyr 355 360 365 Thr Gln Asn Ala Lys Gly Leu Leu Tyr Pro Gly Ala Leu Phe Ser Pro 370 375 380 Leu Gly Val Val Asn Gln Phe Val Asn Val Gln Ala Asp His His Lys 385 390 395 400 Leu Ala Arg Val Ile Ala Arg Glu Ser Ile Thr Leu Leu Lys Asn Glu 405 410 415 Asp Asn Leu Leu Pro Leu Asp Pro Asn Arg Ala Ile Lys Tyr Ser Glu 420 425 430 Gln Met Pro Gly Thr Asn Pro Arg Gly Ile Asn Ala Cys Pro Asp Lys 435 440 445 Gly Cys Asn Lys Gly Val Leu Thr Met Gly Trp Gly Ser Gly Thr Ser 450 455 460 Asn Leu Pro Tyr Leu Val Thr Pro Glu Asp Ala Ile Arg Asn Ile Ser 465 470 475 480 Lys Asn Thr Glu Phe His Ile Thr Asp Lys Phe Pro Asn Asn Val Gln 485 490 495 Pro Gly Pro Asp Asp Val Ala Ile Val Phe Val Asn Ala Asp Ser Gly 500 505 510 Glu Asn Tyr Ile Ile Val Glu Ser Asn Pro Gly Asp Arg Thr Val Ala 515 520 525 Gln Met Lys Leu Trp His Asn Gly Asp Glu Leu Ile Glu Ser Ala Ala 530 535 540 Lys Lys Phe Ser Asn Val Val Val Val Val Val His Thr Val Gly Pro 545 550 555 560 Ile Ile Met Glu Lys Trp Ile Asp Leu Leu Arg Ser Arg Val Ser Cys 565 570 575 Leu Pro Asp Phe Gln Asp Lys Lys Leu Glu Ile Leu Leu Leu Ile Ser 580 585 590 Cys Ser Glu Thr Ser Val Arg Val Ala Ala Ser Ile Tyr Asp Thr Glu 595 600 605 Ser Arg Ile Gly Leu Ser Asp Ser Val Ser Leu Ile Asn Gln Arg Phe 610 615 620 Gly Gln Ile Gln Asp Thr Phe Thr Glu Gly Leu Phe Ile Asp Tyr Arg 625 630 635 640 His Phe Gln Lys Glu Asn Ile Thr Pro Arg Tyr His Phe Gly Tyr Gly 645 650 655 Leu Ser Tyr Thr Thr Phe Asn Phe Thr Glu Pro Arg Leu Glu Ser Val 660 665 670 Thr Thr Leu Ser Glu Tyr Pro Pro Ala Arg Lys Pro Lys Ala Gly Asp 675 680 685 Arg His Thr Pro Thr Ile Ser His Leu Leu Gln Lys Trp Pro Gly Pro 690 695 700 Lys Thr Leu Thr Gly Ser Gly Ala Tyr Leu Tyr Pro Tyr Leu Asp Asn 705 710 715 720 Pro Ser Ala Ile Lys Pro Lys Pro Gly Tyr Pro Tyr Pro Glu Ala Ile 725 730 735 Gln Pro Asn Leu Asn Leu Asn Pro Arg Ala Gly Gly Ser Glu Ala Val 740 745 750 Thr Arg Arg Tyr Gly Met Leu Arg Ser Arg Phe Pro Leu Lys Leu Leu 755 760 765 Ile Leu Glu Arg Asn Pro Val Arg Ala Val Ala Gln Leu Tyr Val Glu 770 775 780 Leu Pro Thr Asp Asp Glu His Pro Thr Pro Lys Leu Gln Leu Arg Gln 785 790 795 800 Phe Glu Lys Thr Ala Thr Leu Glu Pro Gly Gln Ser Glu Val Leu Lys 805 810 815 Met Glu Ile Thr Arg Lys Asp Val Ser Ile Trp Asp Thr Met Val Gln 820 825 830 Asp Trp Lys Val Pro Ala Thr Gly Lys Gly Ile Lys Leu Trp Ile Gly 835 840 845 Ala Ser Val Gly Asp Leu Lys Ala Val Cys Glu Thr Gly Lys Gly Lys 850 855 860 Ser Cys His Val Leu Asn 865 870 28880PRTSaccharomycopsis fibuligera 28Met Leu Leu Ile Leu Glu Leu Leu Val Leu Ile Ile Gly Leu Gly Val 1 5 10 15 Ala Leu Pro Val Gln Thr His Asn Leu Thr Asp Asn Gln Gly Phe Asp 20 25 30 Glu Glu Ser Ser Gln Trp Ile Ser Pro His Tyr Tyr Pro Thr Pro Gln 35 40 45 Gly Gly Arg Leu Gln Gly Val Trp Gln Asp Ala Tyr Thr Lys Ala Lys 50 55 60 Ala Leu Val Ser Gln Met Thr Ile Val Glu Lys Val Asn Leu Thr Thr 65 70 75 80 Gly Thr Gly Trp Gln Leu Gly Pro Cys Val Gly Asn Thr Gly Ser Val 85 90 95 Pro Arg Phe Gly Ile Pro Asn Leu Cys Leu Gln Asp Gly Pro Leu Gly 100 105 110 Val Arg Leu Thr Asp Phe Ser Thr Gly Tyr Pro Ser Gly Met Ala Thr 115 120 125 Gly Ala Thr Phe Asn Lys Asp Leu Phe Leu Gln Arg Gly Gln Ala Leu 130 135 140 Gly His Glu Phe Asn Ser Lys Gly Val His Ile Ala Leu Gly Pro Ala 145 150 155 160 Val Gly Pro Leu Gly Val Lys Ala Arg Gly Gly Arg Asn Phe Glu Ala 165 170 175 Phe Gly Ser Asp Pro Tyr Leu Gln Gly Ile Ala Ala Ala Ala Thr Ile 180 185 190 Lys Gly Leu Gln Glu Asn Asn Val Met Ala Cys Val Lys His Phe Ile 195 200 205 Gly Asn Glu Gln Asp Ile Tyr Arg Gln Pro Ser Asn Ser Lys Val Asp 210 215 220 Pro Glu Tyr Asp Pro Ala Thr Lys Glu Ser Ile Ser Ala Asn Ile Pro 225 230 235 240 Asp Arg Ala Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ser Ile 245 250 255 Arg Ala Gly Val Gly Ser Val Met Cys Ser Tyr Asn Arg Val Asn Asn 260 265 270 Thr Tyr Ser Cys Glu Asn Ser Tyr Met Ile Asn His Leu Leu Lys Glu 275 280 285 Glu Leu Gly Phe Gln Gly Phe Val Val Ser Asp Trp Ala Ala Gln Met 290 295 300 Ser Gly Ala Tyr Ser Ala Ile Ser Gly Leu Asp Met Ser Met Pro Gly 305 310 315 320 Glu Leu Leu Gly Gly Trp Asn Thr Gly Lys Ser Tyr Trp Gly Gln Asn 325 330 335 Leu Thr Lys Ala Val Tyr Asn Glu Thr Val Pro Ile Glu Arg Leu Asp 340 345 350 Asp Met Ala Thr Arg Ile Leu Ala Ala Leu Tyr Ala Thr Asn Ser Phe 355 360 365 Pro Thr Lys Asp Arg Leu Pro Asn Phe Ser Ser Phe Thr Thr Lys Glu 370 375 380 Tyr Gly Asn Glu Phe Phe Val Asp Lys Thr Ser Pro Val Val Lys Val 385 390 395 400 Asn His Phe Val Asp Pro Ser Asn Asp Phe Thr Glu Asp Thr Ala Leu 405 410 415 Lys Val Ala Glu Glu Ser Ile Val Leu Leu Lys Asn Glu Lys Asn Thr 420 425 430 Leu Pro Ile Ser Pro Asn Lys Val Arg Lys Leu Leu Leu Ser Gly Ile 435 440 445 Ala Ala Gly Pro Asp Pro Lys Gly Tyr Glu Cys Ser Asp Gln Ser Cys 450 455 460 Val Asp Gly Ala Leu Phe Glu Gly Trp Gly Ser Gly Ser Val Gly Tyr 465 470 475 480 Pro Lys Tyr Gln Val Thr Pro Phe Glu Glu Ile Ser Ala Asn Ala Arg 485 490 495 Lys Asn Lys Met Gln Phe Asp Tyr Ile Arg Glu Ser Phe Asp Leu Thr 500 505 510 Gln Val Ser Thr Val Ala Ser Asp Ala His Met Ser Ile Val Val Val 515 520 525 Ser Ala Val Ser Gly Glu Gly Tyr Leu Ile Ile Asp Gly Asn Arg Gly 530 535 540 Asp Lys Asn Asn Val Thr Leu Trp His Asn Ser Asp Asn Leu Ile Lys 545 550 555 560 Ala Val Ala Glu Asn Cys Ala Asn Thr Val Val Val Ile Thr Ser Thr 565 570 575 Gly Gln Val Asp Val Glu Ser Phe Ala Asp His Pro Asn Val Thr Ala 580 585 590 Ile Val Trp Ala Gly Pro Leu Gly Asp Arg Ser Gly Thr Ala Ile Ala 595 600 605 Asn Ile Leu Phe Gly Asn Ala Asn Pro Ser Gly His Leu Pro Phe Thr 610 615 620 Val Ala Lys Ser Asn Asp Asp Tyr Ile Pro Ile Val Thr Tyr Asn Pro 625 630 635 640 Pro Asn Gly Glu Pro Glu Asp Asn Thr Leu Ala Glu His Asp Leu Leu 645 650 655 Val Asp Tyr Arg Tyr Phe Glu Glu Lys Asn Ile Glu Pro Arg Tyr Ala 660 665 670 Phe Gly Tyr Gly Leu Ser Tyr Asn Glu Tyr Lys Val Ser Asn Ala Lys 675 680 685 Val Ser Ala Ala Lys Lys Val Asp Glu Glu Leu Pro Gln Pro Lys Leu 690 695 700 Tyr Leu Ala Glu Tyr Ser Tyr Asn Lys Thr Glu Glu Ile Asn Asn Pro 705 710 715 720 Glu Asp Ala Phe Phe Pro Ser Asn Ala Arg Arg Ile Gln Glu Phe Leu 725 730 735 Tyr Pro Tyr Leu Asp Ser Asn Val Thr Leu Lys Asp Gly Asn Tyr Glu 740 745 750 Tyr Pro Asp Gly Tyr Ser Thr Glu Gln Arg Thr Thr Pro Ile Gln Pro 755 760 765 Gly Gly Gly Leu Gly Gly Asn Asp Ala Leu Trp Glu Val Ala Tyr Lys 770 775 780 Val Glu Val Asp Val Gln Asn Leu Gly Asn Ser Thr Asp Lys Phe Val 785 790 795 800 Pro Gln Leu Tyr Leu Lys His Pro Glu Asp Gly Lys Phe Glu Thr Pro 805 810 815 Val Gln Leu Arg Gly Phe Glu Lys Val Glu Leu Ser Pro Gly Glu Lys 820 825 830 Lys Thr Val Glu Phe Glu Leu Leu Arg Arg Asp Leu Ser Val Trp Asp 835 840 845 Thr Thr Arg Gln Ser Trp Ile Val Glu Ser Gly Thr Tyr Glu Ala Leu 850 855 860 Ile Gly Val Ala Val Asn Asp Ile Lys Thr Ser Val Leu Phe Thr Ile 865 870 875 880 29876PRTSaccharomycopsis fibuligera 29Met Leu Met Ile Val Gln Leu Leu Val Phe Ala Leu Gly Leu Ala Val 1 5 10 15 Ala Val Pro Ile Gln Asn Tyr Thr Gln Ser Pro Ser Gln Arg Asp Glu 20 25 30 Ser Ser Gln Trp Val Ser Pro His Tyr Tyr Pro Thr Pro Gln Gly Gly 35 40 45 Arg Leu Gln Asp Val Trp Gln Glu Ala Tyr Ala Arg Ala Lys Ala Ile 50 55 60 Val Gly Gln Met Thr Ile Val Glu Lys Val Asn Leu Thr Thr Gly Thr 65 70 75 80 Gly Trp Gln Leu Asp Pro Cys Val Gly Asn Thr Gly Ser Val Pro Arg 85 90 95 Phe Gly Ile Pro Asn Leu Cys Leu Gln Asp Gly Pro Leu Gly Val Arg 100 105 110 Phe Ala Asp Phe Val Thr Gly Tyr Pro Ser Gly Leu Ala Thr Gly Ala 115 120 125 Thr Phe Asn Lys Asp Leu Phe Leu Gln Arg Gly Gln Ala Leu Gly His 130 135 140 Glu Phe Asn Ser Lys Gly Val His Ile Ala Leu Gly Pro Ala Val Gly 145 150 155 160 Pro Leu Gly Val Lys Ala Arg Gly Gly Arg Asn Phe Glu Ala Phe Gly 165 170 175 Ser Asp Pro Tyr Leu Gln Gly Thr Ala Ala Ala Ala Thr Ile Lys Gly 180 185 190 Leu Gln Glu Asn Asn Val Met Ala Cys Val Lys His Phe Ile Gly Asn 195 200 205 Glu Gln Glu Lys Tyr Arg Gln Pro Asp Asp Ile Asn Pro Ala Thr Asn 210 215 220 Gln Thr Thr Lys Glu Ala Ile Ser Ala Asn Ile Pro Asp Arg Ala Met 225 230 235 240 His Ala Leu Tyr Leu Trp Pro Phe Ala Asp Ser Val Arg Ala Gly Val 245 250 255 Gly Ser Val Met Cys Ser Tyr Asn Arg Val Asn Asn Thr Tyr Ala Cys 260 265 270 Glu Asn Ser Tyr Met Met Asn His Leu Leu Lys Glu Glu Leu Gly Phe 275 280 285 Gln Gly Phe Val Val Ser Asp Trp Gly Ala Gln Leu Ser Gly Val Tyr 290 295 300 Ser Ala Ile Ser Gly Leu Asp Met Ser Met Pro Gly Glu Val Tyr Gly 305 310 315 320 Gly Trp Asn Thr Gly Thr Ser Phe Trp Gly Gln Asn Leu Thr Lys Ala 325 330 335 Ile Tyr Asn Glu Thr Val Pro Ile Glu Arg Leu Asp Asp Met Ala Thr 340 345 350 Arg Ile Leu Ala Ala Leu Tyr Ala Thr Asn Ser Phe Pro Thr Glu Asp 355 360 365 His Leu Pro Asn Phe Ser Ser Trp Thr Thr Lys Glu Tyr Gly Asn Lys 370 375 380 Tyr Tyr Ala Asp Asn Thr Thr Glu Ile Val Lys Val Asn Tyr Asn Val 385 390 395 400 Asp Pro Ser Asn Asp Phe Thr Glu Asp Thr Ala Leu Lys Val Ala Glu 405 410 415 Glu Ser Ile Val Leu Leu Lys Asn Glu Asn Asn Thr Leu Pro Ile Ser 420 425 430 Pro Glu Lys Ala Lys Arg Leu Leu Leu Ser Gly Ile Ala Ala Gly Pro 435 440 445 Asp Pro Ile Gly Tyr Gln Cys Glu Asp Gln Ser Cys Thr Asn Gly Ala 450 455 460 Leu Phe Gln Gly Trp Gly Ser Gly Ser Val Gly Ser Pro Lys Tyr Gln 465 470 475 480 Val Thr Pro Phe Glu Glu Ile Ser Tyr Leu Ala Arg Lys Asn Lys Met 485 490 495 Gln Phe Asp Tyr Ile Arg Glu Ser Tyr Asp Leu Ala Gln Val Thr Lys 500

505 510 Val Ala Ser Asp Ala His Leu Ser Ile Val Val Val Ser Ala Ala Ser 515 520 525 Gly Glu Gly Tyr Ile Thr Val Asp Gly Asn Gln Gly Asp Arg Lys Asn 530 535 540 Leu Thr Leu Trp Asn Asn Gly Asp Lys Leu Ile Glu Thr Val Ala Glu 545 550 555 560 Asn Cys Ala Asn Thr Val Val Val Val Thr Ser Thr Gly Gln Ile Asn 565 570 575 Phe Glu Gly Phe Ala Asp His Pro Asn Val Thr Ala Ile Val Trp Ala 580 585 590 Gly Pro Leu Gly Asp Arg Ser Gly Thr Ala Ile Ala Asn Ile Leu Phe 595 600 605 Gly Lys Ala Asn Pro Ser Gly His Leu Pro Phe Thr Ile Ala Lys Thr 610 615 620 Asp Asp Asp Tyr Ile Pro Ile Glu Thr Tyr Ser Pro Ser Ser Gly Glu 625 630 635 640 Pro Glu Asp Asn His Leu Val Glu Asn Asp Leu Leu Val Asp Tyr Arg 645 650 655 Tyr Phe Glu Glu Lys Asn Ile Glu Pro Arg Tyr Ala Phe Gly Tyr Gly 660 665 670 Leu Ser Tyr Asn Glu Tyr Glu Val Ser Asn Ala Lys Val Ser Ala Ala 675 680 685 Lys Lys Val Asp Glu Glu Leu Pro Glu Pro Ala Thr Tyr Leu Ser Glu 690 695 700 Phe Ser Tyr Gln Asn Ala Lys Asp Ser Lys Asn Pro Ser Asp Ala Phe 705 710 715 720 Ala Pro Ala Asp Leu Asn Arg Val Asn Glu Tyr Leu Tyr Pro Tyr Leu 725 730 735 Asp Ser Asn Val Thr Leu Lys Asp Gly Asn Tyr Glu Tyr Pro Asp Gly 740 745 750 Tyr Ser Thr Glu Gln Arg Thr Thr Pro Asn Gln Pro Gly Gly Gly Leu 755 760 765 Gly Gly Asn Asp Ala Leu Trp Glu Val Ala Tyr Asn Ser Thr Asp Lys 770 775 780 Phe Val Pro Gln Gly Asn Ser Thr Asp Lys Phe Val Pro Gln Leu Tyr 785 790 795 800 Leu Lys His Pro Glu Asp Gly Lys Phe Glu Thr Pro Ile Gln Leu Arg 805 810 815 Gly Phe Glu Lys Val Glu Leu Ser Pro Gly Glu Lys Lys Thr Val Asp 820 825 830 Leu Arg Leu Leu Arg Arg Asp Leu Ser Val Trp Asp Thr Thr Arg Gln 835 840 845 Ser Trp Ile Val Glu Ser Gly Thr Tyr Glu Ala Leu Ile Gly Val Ala 850 855 860 Val Asn Asp Ile Lys Thr Ser Val Leu Phe Thr Ile 865 870 875 30803PRTSeptoria lycopersici 30Met Val Ser Ser Leu Phe Asn Ile Ala Ala Leu Ala Gly Ala Val Ile 1 5 10 15 Ala Leu Ser His Glu Asp Gln Ser Lys His Phe Thr Thr Ile Pro Thr 20 25 30 Phe Pro Thr Pro Asp Ser Thr Gly Glu Gly Trp Lys Ala Ala Phe Glu 35 40 45 Lys Ala Ala Asp Ala Val Ser Arg Leu Asn Leu Thr Gln Lys Val Ala 50 55 60 Leu Thr Thr Gly Thr Thr Ala Gly Leu Ser Cys Asn Gly Asn Ile Ala 65 70 75 80 Pro Ile Pro Glu Ile Asn Phe Ser Gly Leu Cys Leu Ala Asp Gly Pro 85 90 95 Val Ser Val Arg Ile Ala Asp Leu Ala Thr Val Phe Pro Ala Gly Leu 100 105 110 Thr Ala Ala Ala Thr Trp Asp Arg Gln Leu Ile Tyr Glu Arg Ala Arg 115 120 125 Ala Leu Gly Ser Glu Phe Arg Gly Lys Gly Ser Gln Val His Leu Gly 130 135 140 Pro Ala Ser Gly Ala Leu Gly Arg His Pro Leu Gly Gly Arg Asn Trp 145 150 155 160 Glu Ser Phe Ser Pro Asp Pro Tyr Leu Ser Gly Val Ala Met Asp Phe 165 170 175 Ser Ile Arg Gly Ile Gln Glu Met Gly Val Gln Ala Asn Arg Lys His 180 185 190 Phe Ile Gly Asn Glu Gln Glu Thr Gln Arg Ser Asn Thr Phe Thr Asp 195 200 205 Asp Gly Thr Glu Ile Gln Ala Ile Ser Ser Asn Ile Asp Asp Arg Thr 210 215 220 Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asn Ala Val Arg Ser Gly 225 230 235 240 Val Ala Ser Val Met Cys Ser Tyr Asn Arg Leu Asn Gln Thr Tyr Ala 245 250 255 Cys Glu Asn Ser Lys Leu Met Asn Gly Ile Leu Lys Gly Glu Leu Gly 260 265 270 Phe Gln Gly Tyr Val Val Ser Asp Trp Tyr Ala Thr His Ser Gly Val 275 280 285 Glu Ser Val Asn Ala Gly Leu Asp Met Thr Met Pro Gly Pro Leu Asp 290 295 300 Ser Pro Ser Thr Ala Leu Arg Pro Pro Pro Ser Tyr Leu Gly Gly Asn 305 310 315 320 Leu Thr Glu Ala Val Leu Asn Gly Thr Ile Pro Glu Ala Arg Val Asp 325 330 335 Asp Met Ala Arg Arg Ile Leu Met Pro Tyr Phe Phe Leu Gly Gln Asp 340 345 350 Thr Asp Phe Pro Thr Val Asp Pro Ser Thr Gly Phe Val Phe Ala Arg 355 360 365 Thr Tyr Asn Tyr Pro Asp Glu Tyr Leu Thr Leu Gly Gly Leu Asp Pro 370 375 380 Tyr Asn Pro Pro Pro Ala Arg Asp Val Arg Gly Asn His Ser Asp Ile 385 390 395 400 Val Arg Lys Val Ala Ala Ala Gly Thr Val Leu Leu Lys Asn Val Asn 405 410 415 Asn Val Leu Pro Leu Lys Glu Pro Lys Ser Val Gly Ile Phe Gly Asn 420 425 430 Gly Ala Ala Asp Val Thr Glu Gly Leu Thr Phe Thr Gly Asp Asp Ser 435 440 445 Gly Pro Trp Gly Ala Asp Ile Gly Ala Leu Ser Val Gly Gly Gly Ser 450 455 460 Gly Ala Gly Arg His Thr His Leu Val Ser Pro Leu Ala Ala Ile Arg 465 470 475 480 Lys Arg Thr Glu Ser Val Gly Gly Arg Val Gln Tyr Leu Leu Ser Asn 485 490 495 Ser Arg Ile Val Asn Asp Asp Phe Thr Ser Ile Tyr Pro Thr Pro Glu 500 505 510 Val Cys Leu Val Phe Leu Lys Thr Trp Ala Arg Glu Gly Thr Asp Arg 515 520 525 Leu Ser Tyr Glu Asn Asp Trp Asn Ser Thr Ala Val Val Asn Asn Val 530 535 540 Ala Arg Arg Cys Pro Asn Thr Ile Val Val Thr His Ser Gly Gly Ile 545 550 555 560 Asn Thr Met Pro Trp Ala Asp Asn Ala Asn Val Thr Ala Ile Leu Ala 565 570 575 Ala His Tyr Pro Gly Gln Glu Asn Gly Asn Ser Ile Met Asp Ile Leu 580 585 590 Tyr Gly Asp Val Asn Pro Ser Gly Arg Leu Pro Tyr Thr Ile Pro Lys 595 600 605 Leu Ala Thr Asp Tyr Asp Phe Pro Val Val Asn Ile Thr Asn Glu Ala 610 615 620 Gln Asp Pro Tyr Val Trp Gln Ala Asp Phe Thr Glu Gly Leu Leu Ile 625 630 635 640 Asp Tyr Arg His Phe Asp Ala Arg Asn Ile Thr Pro Leu Tyr Glu Phe 645 650 655 Gly Tyr Gly Leu Ser Tyr Thr Thr Phe Glu Ile Glu Gly Val Ala Asn 660 665 670 Leu Val Ala Lys Ser Ala Lys Leu Ser Ala Phe Pro Ala Ser Thr Asp 675 680 685 Ile Ser His Pro Gly Gly Asn Pro Asp Leu Trp Glu Glu Val Val Ser 690 695 700 Val Thr Ala Ala Val Lys Asn Thr Gly Ser Val Ser Gly Ser Gln Val 705 710 715 720 Val Gln Leu Tyr Ile Ser Leu Pro Ala Asp Gly Ile Pro Glu Asn Ser 725 730 735 Pro Met Gln Val Leu Arg Gly Phe Glu Lys Val Asp Leu Gln Pro Gly 740 745 750 Gln Ser Lys Ser Val Glu Phe Ser Ile Met Arg Arg Asp Leu Ser Phe 755 760 765 Trp Asn Thr Thr Ala Gln Asp Trp Glu Ile Pro Asn Gly Gln Ile Glu 770 775 780 Phe Arg Val Gly Phe Ser Ser Arg Asp Ile Lys Ser Ile Val Ser Arg 785 790 795 800 Ser Phe Leu 31763PRTKuraishia capsulata 31Met Lys Ser Thr Ile Ile Ile Leu Ser Val Leu Ala Ala Ala Thr Ala 1 5 10 15 Lys Asn Ile Ser Lys Ala Glu Met Glu Asn Leu Glu His Trp Trp Ser 20 25 30 Tyr Gly Arg Ser Asp Pro Val Tyr Pro Ser Pro Glu Ile Ser Gly Leu 35 40 45 Gly Asp Trp Gln Phe Ala Tyr Gln Arg Ala Arg Glu Ile Val Ala Leu 50 55 60 Met Thr Asn Glu Glu Lys Thr Asn Leu Thr Phe Gly Ser Ser Gly Asp 65 70 75 80 Thr Gly Cys Ser Gly Met Ile Ser Asp Val Pro Asp Val Asp Phe Pro 85 90 95 Gly Leu Cys Leu Gln Asp Ala Gly Asn Gly Val Arg Gly Thr Asp Met 100 105 110 Val Asn Ala Tyr Ala Ser Gly Leu His Val Gly Ala Ser Trp Asn Arg 115 120 125 Gln Leu Ala Tyr Asp Arg Ala Val Tyr Met Gly Ala Glu Phe Arg His 130 135 140 Lys Gly Val Asn Val Leu Leu Gly Pro Val Val Gly Pro Ile Gly Arg 145 150 155 160 Val Ala Thr Gly Gly Arg Asn Trp Glu Gly Phe Thr Asn Asp Pro Tyr 165 170 175 Leu Ala Gly Ala Leu Val Tyr Glu Thr Thr Lys Gly Ile Gln Glu Asn 180 185 190 Val Ile Ala Cys Thr Lys His Phe Ile Gly Asn Glu Gln Glu Thr Asn 195 200 205 Arg Asn Pro Ser Gly Thr Tyr Asn Gln Ser Val Ser Ala Asn Ile Asp 210 215 220 Asp Lys Thr Met His Glu Leu Tyr Leu Trp Pro Phe Gln Asp Ser Val 225 230 235 240 Arg Ala Gly Leu Gly Ser Ile Met Gly Ser Tyr Asn Arg Val Asn Asn 245 250 255 Ser Tyr Ala Cys Lys Asn Ser Lys Val Leu Asn Gly Leu Leu Lys Ser 260 265 270 Glu Leu Gly Phe Gln Gly Phe Val Val Ser Asp Trp Gly Gly Gln His 275 280 285 Thr Gly Ile Ala Ser Ala Asn Ala Gly Leu Asp Met Ala Met Pro Ser 290 295 300 Ser Thr Tyr Trp Glu Glu Gly Leu Ile Glu Ala Val Lys Asn Gly Thr 305 310 315 320 Val Asp Gln Ser Arg Leu Asp Asp Met Ala Thr Arg Ile Ile Ala Ala 325 330 335 Trp Tyr Lys Tyr Ala Arg Leu Asp Asp Pro Gly Phe Gly Met Pro Val 340 345 350 Ser Leu Ala Glu Asp His Glu Leu Val Asp Ala Arg Asp Pro Ala Ala 355 360 365 Ala Ser Thr Ile Phe Gln Gly Ala Val Glu Gly His Val Leu Val Lys 370 375 380 Asn Glu Asn Ala Leu Pro Leu Lys Lys Pro Lys Tyr Ile Ser Leu Phe 385 390 395 400 Gly Tyr Asp Gly Val Ser Thr Asp Val Asn Thr Val Gly Gly Gly Phe 405 410 415 Ser Phe Phe Ser Phe Asp Val Lys Ala Ile Glu Asn Lys Thr Leu Ile 420 425 430 Ser Gly Gly Gly Ser Gly Thr Asn Thr Pro Ser Tyr Val Asp Ala Pro 435 440 445 Phe Asn Ala Phe Val Ala Lys Ala Arg Glu Asp Asn Thr Phe Leu Ser 450 455 460 Trp Asp Phe Thr Ser Ala Glu Pro Val Ala Asn Pro Ala Ser Asp Ala 465 470 475 480 Cys Ile Asp Phe Ile Asn Ala Ala Ala Ser Glu Gly Tyr Asp Arg Pro 485 490 495 Asn Leu Ala Asp Lys Tyr Ser Asp Lys Leu Val Glu Ala Val Ala Ser 500 505 510 Gln Cys Ser Asn Thr Ile Val Val Ile His Asn Ala Gly Ile Arg Leu 515 520 525 Val Asp Asn Trp Ile Glu His Glu Asn Val Thr Gly Val Ile Leu Ala 530 535 540 His Leu Pro Gly Gln Asp Thr Gly Thr Ser Leu Ile Glu Val Leu Tyr 545 550 555 560 Gly Asn Gln Ser Pro Ser Gly Arg Leu Pro Tyr Thr Val Ala Lys Lys 565 570 575 Ala Ser Asp Tyr Gly Gly Leu Leu Trp Pro Thr Glu Pro Glu Gly Asp 580 585 590 Leu Asp Leu Tyr Phe Pro Gln Ser Asn Phe Thr Glu Gly Val Tyr Ile 595 600 605 Asp Tyr Lys Tyr Phe Ile Gln Lys Asn Ile Thr Pro Arg Tyr Glu Phe 610 615 620 Gly Tyr Gly Leu Thr Tyr Thr Thr Phe Asp Tyr Ser Glu Leu Glu Val 625 630 635 640 Asp Ala Ile Thr Asn Gln Ser Tyr Leu Pro Pro Asp Cys Thr Ile Glu 645 650 655 Glu Gly Gly Ala Lys Ser Leu Trp Asp Ile Val Ala Thr Val Lys Phe 660 665 670 Thr Val Thr Asn Thr Gly Asp Val Ala Ala Ala Glu Val Pro Gln Leu 675 680 685 Tyr Val Gly Ile Pro Asn Gly Pro Pro Lys Val Leu Arg Gly Phe Asp 690 695 700 Lys Lys Leu Ile His Pro Gly Gln Ser Glu Glu Phe Val Phe Glu Leu 705 710 715 720 Thr Arg Arg Asp Leu Ser Thr Trp Asp Val Val Ala Gln Asn Trp Gly 725 730 735 Leu Gln Ala Gly Thr Tyr Gln Phe Tyr Val Gly Arg Ser Val Phe Asp 740 745 750 Val Pro Leu Thr Ser Ala Leu Val Phe Thr Asn 755 760 32765PRTTrichoderma reesei 32Met Arg Leu Cys Asp Leu Ser Ser Leu Ala Ser Trp Val Leu Val Thr 1 5 10 15 Val Ala Leu Pro Ser Ser Gly Ala Ala Ala Lys Gly Val Ser Gln Ile 20 25 30 Pro Ser Thr His Ser Ser Gln Ser Lys Gly Asn Gly Pro Trp Ala His 35 40 45 Ala Tyr Arg Arg Ala Glu Lys Leu Val Arg Gln Met Thr Leu Glu Glu 50 55 60 Lys Ala Asn Ile Thr Arg Gly Phe Thr Gly Asp Asn Val Cys Ala Gly 65 70 75 80 Asn Thr Gly Ser Val Pro Arg Leu Gly Trp Pro Gly Met Cys Val His 85 90 95 Asp Ala Gly Asn Gly Val Arg Ala Thr Asp Leu Val Asn Ser Tyr Pro 100 105 110 Ser Gly Ile His Val Gly Ala Ser Trp Asp Arg Asn Leu Thr Tyr Glu 115 120 125 Arg Gly Leu His Met Gly Gly Glu Phe Lys Ala Lys Gly Val Asn Val 130 135 140 Pro Leu Gly Pro Asn Ala Gly Pro Leu Gly Arg Thr Pro Leu Gly Gly 145 150 155 160 Arg Asn Trp Glu Gly Phe Ser Ile Asp Pro Tyr Leu Ser Gly Gln Leu 165 170 175 Asn Ala Glu Thr Ile Thr Gly Met Gln Asp Ala Gly Val Ile Ala Asn 180 185 190 Ile Lys His Phe Ile Ala Asn Glu Gln Glu Thr Leu Arg Arg Pro Tyr 195 200 205 Phe Gly Val Glu Ala Val Ser Ala Asn Ile Asp Asp Arg Thr Leu His 210 215 220 Glu Tyr Tyr Leu Trp Pro Phe Met Asp Ser Val His Ala Gly Val Gly 225 230 235 240 Ser Val Met Cys Ser Tyr Asn Arg Ile Asn Asn Thr Tyr Gly Cys Met 245 250 255 Asn Asp Lys Leu Met Asn Gly Ile Leu Lys Ala Glu Leu Gly Phe Gln 260 265 270 Gly Phe Val Met Leu Asp Trp Asn Ala Gln His Asp Leu Gln Ser Ala 275 280 285 Asn Ala Gly Leu Asp Met Val Met Pro Leu Gly Gly Ser Trp Gly Lys 290 295 300 Asn Leu Thr Asp Ala Val Ala Asn Gly Thr Val Ser Glu Ser Arg Ile 305 310 315 320 Thr Asp Met Ala Thr Arg Ile Ile Ala Ala Trp Tyr Leu Val Gly Gln 325 330 335 Asp Gly Asn Asn Phe Pro Val Pro Gly Ile Gly Leu Lys Gln Leu Thr 340 345 350 Lys Pro His Glu Gln Val Asp Ala Arg Asp Pro Ala Ser Lys Pro Val

355 360 365 Leu Leu Glu Gly Ala Ile Ala Gly His Val Leu Val Lys Asn Glu Asn 370 375 380 Asn Ala Leu Pro Phe Asn Lys Lys Leu Thr Met Ile Ser Val Phe Gly 385 390 395 400 Tyr Asp Ala Thr Ile Pro Arg Thr Lys Asn Thr Asp Ile Leu Phe Gln 405 410 415 Leu Gly Tyr Thr Ser Ser Pro Glu Met Ala Gln Ala Val Leu Gly Asn 420 425 430 Glu Ala His Phe Asp Gln Ala Ala Lys Gly Gly Thr Ile Met Thr Gly 435 440 445 Gly Arg Ala Gly Ala Asn Ala Pro Ser Tyr Ile Asp Asp Pro Leu Ala 450 455 460 Ala Ile Gln Arg Arg Ala Arg Lys Asp Asp Thr Trp Val Asn Trp Asp 465 470 475 480 Leu Asp Ser Phe Asn Pro Glu Val Asn Ala Ala Ser Asp Ala Cys Leu 485 490 495 Val Phe Ile Asn Ala Ile Ala Thr Glu Gly Trp Asp Arg Asp Gly Leu 500 505 510 His Asp Asp Phe Ser Asp Gly Leu Val Leu Asn Val Ala Ala Asn Cys 515 520 525 Ser Asn Thr Ile Val Val Val His Ala Ala Gly Thr Arg Leu Val Asp 530 535 540 Gln Trp Ile Glu His Pro Asn Val Thr Ala Ala Val Ile Ala His Leu 545 550 555 560 Pro Gly Gln Asp Ser Gly Arg Ala Leu Val Lys Leu Leu Tyr Gly Glu 565 570 575 Ala Asn Phe Ser Gly Lys Leu Pro Tyr Thr Ile Ala Lys Asn Glu Ser 580 585 590 Asp Tyr Ser Val Tyr Thr Pro Cys Gln Arg Arg Ser Pro Glu Asp Thr 595 600 605 Asp Pro Gln Cys Asp Phe Thr Glu Gly Val Tyr Leu Asp Tyr Arg Ala 610 615 620 Phe Asp Ala Asn Asn Met Thr Pro Arg Phe Glu Phe Gly Tyr Gly Leu 625 630 635 640 Ser Tyr Thr Ser Phe Asn Tyr Ser Ala Leu Ser Ile Lys Lys Ala Lys 645 650 655 Gly Leu Arg Gln Ser Arg Cys Thr Asp Asp Leu Trp Gln Ala Ala Ala 660 665 670 Gln Val Thr Ala Ser Ile Thr Asn Ser Gly Gly Met Ser Gly Ser Glu 675 680 685 Val Ala Gln Leu Tyr Leu Ala Ile Pro Asn Ser Pro Pro Lys Gln Leu 690 695 700 Arg Gly Phe Asn Lys Leu Leu Leu Arg Pro His Glu Ser Gly Thr Val 705 710 715 720 His Phe Gly Leu Thr Lys Arg Asp Leu Ser Val Trp Asp Val Val Ser 725 730 735 Gln Ser Trp Val Ile Gln Glu Gly Glu Tyr Lys Val Phe Val Gly Ala 740 745 750 Ser Ser Arg Asp Ile Arg Leu Ser Gly Lys Leu His Ile 755 760 765 33843PRTUromyces fabae 33Met Lys Thr Pro Leu Gly Ile Gly Ser Thr Ala Ala Val Leu Tyr Ile 1 5 10 15 Leu Ser Asn Ile Ser His Val Gln Leu Ala Thr Thr Ser Pro Ser Glu 20 25 30 Asn Gln Asn Gln Ser Tyr Asn Pro Gln Ile Glu Gly Leu Thr Val Gln 35 40 45 Pro Ser Thr Val Ala Asn Gly Leu Arg Ile Asn Ser Asn Ser Leu Ile 50 55 60 Ser Asn Phe Asp Phe Glu Ile Ile Gln Pro Pro Pro Gly Tyr Glu Glu 65 70 75 80 Trp Thr Ser Pro Val Val Leu Pro Ala Pro Val Gln Ser Gly Leu Ser 85 90 95 Pro Trp Ser Glu Ser Ile Val Arg Ala Arg Ala Phe Val Ala Gln Leu 100 105 110 Thr Ile Glu Glu Lys Val Asn Leu Thr Thr Gly Ala Gly Thr Gln Gly 115 120 125 Arg Cys Val Gly Glu Thr Gly Thr Val Pro Arg Leu Gly Phe Asn Gln 130 135 140 Pro Ile Cys Leu Gln Asp Gly Pro Val Gly Ile Arg Tyr Thr Asp Phe 145 150 155 160 Asn Ser Val Phe Pro Ala Ala Ile Asn Val Ala Ala Thr Phe Asp Lys 165 170 175 Gln Leu Met Phe Lys Arg Ala Gln Ala Met Ala Glu Glu Phe Arg Gly 180 185 190 Lys Gly Ala Asn Val Val Leu Ala Pro Met Thr Asn Leu Met Arg Thr 195 200 205 Pro Gln Ala Gly Arg Ala Trp Glu Gly Tyr Gly Ser Asp Pro Tyr Leu 210 215 220 Ser Gly Val Ala Thr Val Gln Ser Val Leu Gly Ile Gln Ser Thr Arg 225 230 235 240 Ala Ser Ala Cys Val Lys His Tyr Ile Gly Asn Glu Gln Glu His Tyr 245 250 255 Arg Gly Gly Ser Gly Ala Thr Ala Ser Ser Ser Asn Ile Asp Asp Arg 260 265 270 Thr Leu Arg Glu Leu Tyr Glu Trp Pro Phe Ala Glu Ala Ile His Ala 275 280 285 Gly Val Asp Tyr Ile Met Cys Ser Tyr Asn Arg Val Asn Gln Thr Tyr 290 295 300 Ala Cys Glu Asn Ser Lys Leu Ile Asn Gly Ile Ala Lys Gly Glu His 305 310 315 320 Lys Phe Gln Gly Val Met Val Thr Asp Trp Ala Ala Ala Glu Ser Gly 325 330 335 Val Arg Thr Ala Leu Ala Gly Thr Asp Met Asn Met Pro Gly Phe Met 340 345 350 Ala Tyr Gly Gln Pro Ser Glu Pro Asn Pro Ser Thr Ala Asn Gly Ser 355 360 365 Tyr Trp Gly Leu Arg Met Ile Glu Ala Val Lys Asn Gly Thr Val Pro 370 375 380 Met Glu Arg Leu Asp Asp Met Val Thr Arg Val Ile Ser Thr Tyr Tyr 385 390 395 400 Lys Gln Gly Gln Asp Lys Ser Asp Tyr Pro Lys Leu Asn Phe Met Ser 405 410 415 Met Gly Gln Gly Thr Pro Ala Glu Gln Ala Val Ser Asn His His Val 420 425 430 Asn Val Gln Lys Asp His Tyr Leu Ile Ile Arg Gln Ile Ala Thr Ala 435 440 445 Ser Thr Ile Leu Leu Lys Asn Val Asn His Thr Leu Pro Leu Lys Ser 450 455 460 Pro Asp Lys Met Arg Ser Val Val Val Val Gly Ser Asp Ala Gly Asp 465 470 475 480 Asn Pro Gln Gly Pro Asn Ser Cys Val Asp Arg Gly Cys Asn Arg Gly 485 490 495 Ile Leu Ala Ile Gly Trp Gly Ser Gly Thr Ala Asn Phe Ala His Leu 500 505 510 Thr Ala Pro Ala Thr Ser Ile Gln Asn Tyr Leu Leu Gln Ser Asn Pro 515 520 525 Thr Ile Thr Tyr Arg Ser Ile Phe Asp Asp Tyr Ala Tyr Asp Glu Ile 530 535 540 Ala Lys Ala Ala Ser Thr Ala Asp Val Ser Ile Val His Val Ser Ser 545 550 555 560 Asp Ser Gly Glu Gly Tyr Leu Thr Val Glu Gly Asn Gln Gly Asp Arg 565 570 575 Ser Asn Thr Ser Leu Trp Asn Lys Gly Asp Glu Leu Ile Leu Lys Ala 580 585 590 Ala Glu Ala Cys Asn Asn Val Val Val Val Ile His Ser Val Gly Pro 595 600 605 Val Asp Met Glu Ala Trp Ile Asn His Pro Asn Val Thr Ala Val Leu 610 615 620 Leu Ala Gly Leu Pro Gly Gln Glu Ala Gly Ser Ala Glu Val Asp Val 625 630 635 640 Leu Trp Gly Ser Thr Asn Pro Ser Gly Arg Leu Pro Tyr Thr Ile Ala 645 650 655 Lys Lys Pro Ser Asp Tyr Pro Ala Glu Leu Leu Tyr Glu Ser Asn Met 660 665 670 Thr Val Pro Gln Ile Asn Tyr Ser Glu Arg Leu Asn Ile Asp Tyr Arg 675 680 685 His Phe Asp Thr Tyr Asn Ile Glu Pro Arg Phe Glu Phe Gly Phe Gly 690 695 700 Leu Ser Tyr Thr Thr Phe Ala Trp Asn Ser Leu Lys Phe Ser Ser Ser 705 710 715 720 Phe Gln Leu Gln Lys Thr Ser Pro Val Ile Val Pro Pro Asn Leu Asp 725 730 735 Leu Tyr Gln Asp Val Ile Glu Phe Glu Phe Gln Val Thr Asn Ser Gly 740 745 750 Pro Phe Asp Gly Ser Glu Val Ala Gln Leu Tyr Val Asp Phe Pro Asn 755 760 765 Gln Val Asn Glu Pro Pro Lys Val Leu Arg Gly Phe Glu Arg Ala Tyr 770 775 780 Ile Pro Ser Lys Gln Ser Lys Thr Ile Glu Ile Lys Leu Arg Val Lys 785 790 795 800 Asp Leu Ser Phe Trp Asp Val Ile Thr Gln Ser Trp Gln Ile Pro Asp 805 810 815 Gly Lys Phe Asn Phe Met Ile Gly Ser Ser Ser Arg Lys Ile Ile Phe 820 825 830 Thr Gln Glu Ile Ser Leu Gln His Ser His Met 835 840 34736PRTAspergillus terreus 34Met Asn Tyr Arg Val Pro Ser Leu Lys Ala Thr Ala Leu Ala Met Ala 1 5 10 15 Ala Leu Thr Gln Ala Leu Thr Thr Trp Asp Ala Ala Tyr Glu Lys Ala 20 25 30 Leu Ala Asp Leu Ala Ser Leu Thr Gln Ser Glu Lys Val Gly Val Val 35 40 45 Ser Gly Ile Thr Trp Glu Gly Gly Pro Cys Val Gly Asn Thr Tyr Ala 50 55 60 Pro Glu Ser Ile Ala Tyr Pro Ser Leu Cys Leu Gln Asp Gly Pro Leu 65 70 75 80 Gly Ile Arg Phe Ala Asn Pro Val Thr Ala Phe Pro Ala Gly Ile Asn 85 90 95 Ala Gly Ala Thr Trp Asp Arg Glu Leu Leu Arg Ala Arg Gly Ala Ala 100 105 110 Met Gly Glu Glu Ala Lys Gly Leu Gly Val His Val Gln Leu Ala Pro 115 120 125 Val Ala Gly Ala Leu Gly Lys Ile Pro Ser Ala Gly Arg Asn Trp Glu 130 135 140 Gly Phe Thr Ser Asp Pro Tyr Leu Ser Gly Ile Ala Met Ala Glu Thr 145 150 155 160 Ile His Gly Met Gln Gly Ser Gly Val Gln Ala Cys Ala Lys His Tyr 165 170 175 Ile Leu Asn Glu Gln Glu His Ser Arg Glu Thr Ile Ser Ser Asn Val 180 185 190 Asp Asp Arg Thr Met His Glu Val Tyr Leu Trp Pro Phe Tyr Asp Ala 195 200 205 Val Lys Ala Asn Val Ala Ser Val Met Cys Ser Tyr Asn Lys Ile Asn 210 215 220 Gly Thr Trp Ala Cys Glu Asn Glu Gly Ile Leu Asp Thr Leu Leu Lys 225 230 235 240 Gln Glu Leu Gly Phe Arg Gly Tyr Val Met Ser Asp Trp Asn Ala Gln 245 250 255 His Ser Thr Val Ala Ser Ala Asn Thr Gly Leu Asp Met Thr Met Pro 260 265 270 Gly Ser Asp Phe Ser Gln Pro Pro Gly Ser Ile Tyr Trp Asn Glu Asn 275 280 285 Leu Ala Glu Ala Val Ala Asn Gly Ser Val Pro Gln Ala Arg Val Asp 290 295 300 Asp Met Val Thr Arg Ile Leu Ala Ala Trp Tyr Leu Leu Glu Gln Asp 305 310 315 320 Gln Gly Tyr Pro Ala Val Ala Phe Asp Ser Arg Asn Gly Gly Lys Ala 325 330 335 Ser Val Asp Val Thr Ala Asp His Ala Asp Ile Ala Arg Thr Val Ala 340 345 350 Arg Asp Ser Ile Val Leu Leu Lys Asn Ser Asn Asn Thr Leu Pro Leu 355 360 365 Arg Asn Pro Ser Ser Ile Ala Val Val Gly Ser Asp Ala Ile Val Asn 370 375 380 Pro Asp Gly Pro Asn Ala Cys Thr Asp Arg Gly Cys Asn Val Gly Thr 385 390 395 400 Leu Ala Gln Gly Trp Gly Ser Gly Thr Ala Glu Phe Pro Tyr Leu Val 405 410 415 Ala Pro Leu Asp Ala Ile Gln Glu Arg Ser Ser Gly Asn Gly Thr Lys 420 425 430 Val Val Thr Ser Thr Thr Asp Asp Ala Thr Ala Gly Ala Asp Ala Ala 435 440 445 Ala Ser Ala Asp Ile Ala Ile Val Phe Ile Ser Ser Asp Ser Gly Glu 450 455 460 Gly Tyr Ile Thr Val Glu Gly His Gln Gly Asp Arg Asn Asn Leu Asp 465 470 475 480 Pro Trp His Gly Gly Asn Asp Leu Val Lys Ala Val Ala Ala Val Asn 485 490 495 Lys Lys Thr Ile Val Val Val His Ser Thr Gly Pro Val Val Leu Glu 500 505 510 Thr Ile Leu Ala Gln Pro Asn Val Val Ala Val Val Trp Ala Gly Ile 515 520 525 Pro Gly Gln Glu Ser Gly Asn Ala Leu Ala Asp Val Leu Tyr Gly Asp 530 535 540 Val Ser Pro Ser Gly Lys Leu Pro Tyr Thr Ile Gly Lys Ser Glu Ala 545 550 555 560 Asp Tyr Gly Thr Thr Trp Val Ala Asn Gly Ala Asp Asp Asp Phe Pro 565 570 575 Glu Gly Leu Phe Ile Asp Tyr Arg His Phe Asp Lys Asn Glu Ile Glu 580 585 590 Pro Arg Tyr Glu Phe Gly Phe Gly Leu Ser Tyr Thr Arg Phe Asn Phe 595 600 605 Ser Asn Leu Ala Ile Asn Ile Asp Ala Thr Ser Gly Pro Thr Ser Gly 610 615 620 Ala Val Asp Val Gly Gly Ala Ala Asp Leu Tyr Asp Ser Val Gly Thr 625 630 635 640 Ile Ser Ala Thr Val Thr Asn Val Gly Gly Val Ser Gly Ala Glu Val 645 650 655 Ala Gln Leu Tyr Ile Gly Phe Pro Ser Ser Ala Pro Glu Thr Pro Pro 660 665 670 Lys Gln Leu Arg Gly Phe Gln Lys Leu Pro Leu Ala Gly Gly Ala Asp 675 680 685 Gly Val Ala Glu Phe Glu Leu Thr Arg Arg Asp Ile Ser Tyr Trp Asp 690 695 700 Val Gly Gln Gln Lys Trp Val Val Pro Glu Gly Ser Phe Gln Val Tyr 705 710 715 720 Val Gly Ala Ser Ser Arg Asp Ile Arg Leu Asp Gly Ser Phe Thr Val 725 730 735 35726PRTChaetomium globosum 35Met Thr Thr Leu Arg Asn Phe Ala Leu Leu Ala Ala Ala Val Leu Ala 1 5 10 15 Arg Val Glu Ala Leu Glu Ala Ala Asp Trp Ala Ala Ala Glu Ala Ser 20 25 30 Ala Lys Thr Ala Leu Ala Lys Met Ser Gln Gln Asp Lys Ile Ser Ile 35 40 45 Val Thr Gly Ile Gly Trp Asp Lys Gly Pro Cys Val Gly Asn Thr Ala 50 55 60 Ala Ile Asn Ser Ile Asn Tyr Pro Gln Leu Cys Leu Gln Asp Gly Pro 65 70 75 80 Leu Gly Ile Arg Phe Gly Thr Gly Ser Thr Ala Phe Thr Pro Gly Val 85 90 95 Gln Ala Ala Ser Thr Trp Asp Thr Glu Leu Met Arg Gln Arg Gly Glu 100 105 110 Tyr Leu Gly Ala Glu Ala Lys Gly Cys Gly Ile His Val Leu Leu Gly 115 120 125 Pro Val Ala Gly Ala Leu Gly Lys Ile Pro His Gly Gly Arg Asn Trp 130 135 140 Glu Gly Phe Gly Thr Asp Pro Tyr Leu Ala Gly Ile Ala Met Ala Glu 145 150 155 160 Thr Ile Glu Gly Leu Gln Ser Ala Gly Val Gln Ala Cys Ala Lys His 165 170 175 Tyr Ile Val Asn Glu Gln Glu Leu Asn Arg Glu Thr Ile Ser Ser Asp 180 185 190 Val Asp Asp Arg Thr Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp 195 200 205 Ala Val His Ala Asn Val Ala Ser Val Met Cys Ser Tyr Asn Lys Ile 210 215 220 Asn Gly Ser Trp Gly Cys Glu Asn Asp His Ala Gln Asn Gly Leu Leu 225 230 235 240 Lys Lys Glu Leu Gly Phe Lys Gly Tyr Val Val Ser Asp Trp Asn Ala 245 250 255 Gln His Thr Thr Asp Gly Ala Ala Asn Asn Gly Met Asp Met Thr Met 260 265 270 Pro Gly Ser Asp Tyr Asn Gly Asn Asn Val Leu Trp Gly Pro Gln Leu 275 280 285 Ser Asn Ala Val Asn Ser Asn Arg Val Ser Arg Asp Arg Leu Asp Asp 290 295 300 Met Ala Lys Arg Ile Leu Thr Ser Trp Tyr Leu Leu Gly Gln Asn Ser 305

310 315 320 Gly Tyr Pro Asn Ile Asn Ile Asn Ala Asn Val Gln Gly Asn His Lys 325 330 335 Glu Asn Val Arg Ala Val Ala Arg Asp Gly Ile Val Leu Leu Lys Asn 340 345 350 Asp Glu Gly Val Leu Pro Leu Lys Lys Pro Gly Lys Val Ala Leu Val 355 360 365 Gly Ser Ala Ala Ser Val Asn Ser Ala Gly Pro Asn Ala Cys Val Asp 370 375 380 Lys Gly Cys Asn Thr Gly Ala Leu Gly Met Gly Trp Gly Ser Gly Ser 385 390 395 400 Val Asn Tyr Pro Tyr Phe Val Ala Pro Tyr Asp Ala Leu Lys Thr Arg 405 410 415 Ala Gln Ala Asp Gly Thr Thr Leu Ser Leu His Asn Ser Asp Ser Thr 420 425 430 Asn Gly Val Ser Gly Val Val Ser Gly Ala Asp Val Ala Ile Val Val 435 440 445 Ile Thr Ala Asp Ser Gly Glu Gly Tyr Ile Thr Val Glu Gly His Ala 450 455 460 Gly Asp Arg Asn His Leu Asp Pro Trp His Asp Gly Asn Ala Leu Val 465 470 475 480 Lys Ala Val Ala Ala Ala Asn Lys Asn Thr Ile Val Val Val His Ser 485 490 495 Thr Gly Pro Ile Ile Leu Glu Thr Ile Leu Ala Thr Glu Gly Val Lys 500 505 510 Ala Val Val Trp Ala Gly Leu Pro Ser Gln Glu Asn Gly Asn Ala Leu 515 520 525 Val Asp Val Leu Tyr Gly Leu Thr Ser Pro Ser Gly Lys Leu Val Tyr 530 535 540 Ser Ile Ala Lys Arg Pro Glu Asp Tyr Gly Thr Ala Pro Ser Lys Gly 545 550 555 560 Ser Asn Asp Lys Phe Thr Glu Gly Leu Phe Val Asp Tyr Arg His Phe 565 570 575 Asp Asn Ala Lys Ile Glu Pro Arg Tyr Glu Phe Gly Phe Gly Leu Ser 580 585 590 Tyr Thr Glu Phe Thr Tyr Ala Asp Leu Ser Val Thr Ser Thr Val Thr 595 600 605 Ala Gly Pro Ala Ser Gly Glu Thr Ile Pro Gly Gly Ala Ala Asp Leu 610 615 620 Trp Glu Thr Val Ala Thr Val Thr Ala Ser Ile Thr Asn Ser Gly Glu 625 630 635 640 Val Glu Gly Ala Glu Val Ala Gln Leu Tyr Ile Thr Leu Pro Ser Ala 645 650 655 Ala Pro Ser Thr Pro Pro Lys Gln Leu Arg Gly Phe Ala Lys Leu Lys 660 665 670 Leu Glu Pro Gly Ala Ser Gly Val Ala Thr Phe Asn Leu Arg Arg Arg 675 680 685 Asp Leu Ser Tyr Trp Asp Ala Gly Arg Gly Gln Trp Val Val Pro Ala 690 695 700 Gly Glu Phe Thr Val Ser Val Gly Ala Ser Ser Arg Asp Val Arg Leu 705 710 715 720 Thr Gly Ser Leu Thr Ala 725 36874PRTTrichoderma reesei 36Met Lys Thr Leu Ser Val Phe Ala Ala Ala Leu Leu Ala Ala Val Ala 1 5 10 15 Glu Ala Asn Pro Tyr Pro Pro Pro His Ser Asn Gln Ala Tyr Ser Pro 20 25 30 Pro Phe Tyr Pro Ser Pro Trp Met Asp Pro Ser Ala Pro Gly Trp Glu 35 40 45 Gln Ala Tyr Ala Gln Ala Lys Glu Phe Val Ser Gly Leu Thr Leu Leu 50 55 60 Glu Lys Val Asn Leu Thr Thr Gly Val Gly Trp Met Gly Glu Lys Cys 65 70 75 80 Val Gly Asn Val Gly Thr Val Pro Arg Leu Gly Met Arg Ser Leu Cys 85 90 95 Met Gln Asp Gly Pro Leu Gly Leu Arg Phe Asn Thr Tyr Asn Ser Ala 100 105 110 Phe Ser Val Gly Leu Thr Ala Ala Ala Ser Trp Ser Arg His Leu Trp 115 120 125 Val Asp Arg Gly Thr Ala Leu Gly Ser Glu Ala Lys Gly Lys Gly Val 130 135 140 Asp Val Leu Leu Gly Pro Val Ala Gly Pro Leu Gly Arg Asn Pro Asn 145 150 155 160 Gly Gly Arg Asn Val Glu Gly Phe Gly Ser Asp Pro Tyr Leu Ala Gly 165 170 175 Leu Ala Leu Ala Asp Thr Val Thr Gly Ile Gln Asn Ala Gly Thr Ile 180 185 190 Ala Cys Ala Lys His Phe Leu Leu Asn Glu Gln Glu His Phe Arg Gln 195 200 205 Val Gly Glu Ala Asn Gly Tyr Gly Tyr Pro Ile Thr Glu Ala Leu Ser 210 215 220 Ser Asn Val Asp Asp Lys Thr Ile His Glu Val Tyr Gly Trp Pro Phe 225 230 235 240 Gln Asp Ala Val Lys Ala Gly Val Gly Ser Phe Met Cys Ser Tyr Asn 245 250 255 Gln Val Asn Asn Ser Tyr Ala Cys Gln Asn Ser Lys Leu Ile Asn Gly 260 265 270 Leu Leu Lys Glu Glu Tyr Gly Phe Gln Gly Phe Val Met Ser Asp Trp 275 280 285 Gln Ala Gln His Thr Gly Val Ala Ser Ala Val Ala Gly Leu Asp Met 290 295 300 Thr Met Pro Gly Asp Thr Ala Phe Asn Thr Gly Ala Ser Tyr Phe Gly 305 310 315 320 Ser Asn Leu Thr Leu Ala Val Leu Asn Gly Thr Val Pro Glu Trp Arg 325 330 335 Ile Asp Asp Met Val Met Arg Ile Met Ala Pro Phe Phe Lys Val Gly 340 345 350 Lys Thr Val Asp Ser Leu Ile Asp Thr Asn Phe Asp Ser Trp Thr Asn 355 360 365 Gly Glu Tyr Gly Tyr Val Gln Ala Ala Val Asn Glu Asn Trp Glu Lys 370 375 380 Val Asn Tyr Gly Val Asp Val Arg Ala Asn His Ala Asn His Ile Arg 385 390 395 400 Glu Val Gly Ala Lys Gly Thr Val Ile Phe Lys Asn Asn Gly Ile Leu 405 410 415 Pro Leu Lys Lys Pro Lys Phe Leu Thr Val Ile Gly Glu Asp Ala Gly 420 425 430 Gly Asn Pro Ala Gly Pro Asn Gly Cys Gly Asp Arg Gly Cys Asp Asp 435 440 445 Gly Thr Leu Ala Met Glu Trp Gly Ser Gly Thr Thr Asn Phe Pro Tyr 450 455 460 Leu Val Thr Pro Asp Ala Ala Leu Gln Ser Gln Ala Leu Gln Asp Gly 465 470 475 480 Thr Arg Tyr Glu Ser Ile Leu Ser Asn Tyr Ala Ile Ser Gln Thr Gln 485 490 495 Ala Leu Val Ser Gln Pro Asp Ala Ile Ala Ile Val Phe Ala Asn Ser 500 505 510 Asp Ser Gly Glu Gly Tyr Ile Asn Val Asp Gly Asn Glu Gly Asp Arg 515 520 525 Lys Asn Leu Thr Leu Trp Lys Asn Gly Asp Asp Leu Ile Lys Thr Val 530 535 540 Ala Ala Val Asn Pro Lys Thr Ile Val Val Ile His Ser Thr Gly Pro 545 550 555 560 Val Ile Leu Lys Asp Tyr Ala Asn His Pro Asn Ile Ser Ala Ile Leu 565 570 575 Trp Ala Gly Ala Pro Gly Gln Glu Ser Gly Asn Ser Leu Val Asp Ile 580 585 590 Leu Tyr Gly Lys Gln Ser Pro Gly Arg Thr Pro Phe Thr Trp Gly Pro 595 600 605 Ser Leu Glu Ser Tyr Gly Val Ser Val Met Thr Thr Pro Asn Asn Gly 610 615 620 Asn Gly Ala Pro Gln Asp Asn Phe Asn Glu Gly Ala Phe Ile Asp Tyr 625 630 635 640 Arg Tyr Phe Asp Lys Val Ala Pro Gly Lys Pro Arg Ser Ser Asp Lys 645 650 655 Ala Pro Thr Tyr Glu Phe Gly Phe Gly Leu Ser Trp Ser Thr Phe Lys 660 665 670 Phe Ser Asn Leu His Ile Gln Lys Asn Asn Val Gly Pro Met Ser Pro 675 680 685 Pro Asn Gly Lys Thr Ile Ala Ala Pro Ser Leu Gly Ser Phe Ser Lys 690 695 700 Asn Leu Lys Asp Tyr Gly Phe Pro Lys Asn Val Arg Arg Ile Lys Glu 705 710 715 720 Phe Ile Tyr Pro Tyr Leu Ser Thr Thr Thr Ser Gly Lys Glu Ala Ser 725 730 735 Gly Asp Ala His Tyr Gly Gln Thr Ala Lys Glu Phe Leu Pro Ala Gly 740 745 750 Ala Leu Asp Gly Ser Pro Gln Pro Arg Ser Ala Ala Ser Gly Glu Pro 755 760 765 Gly Gly Asn Arg Gln Leu Tyr Asp Ile Leu Tyr Thr Val Thr Ala Thr 770 775 780 Ile Thr Asn Thr Gly Ser Val Met Asp Asp Ala Val Pro Gln Leu Tyr 785 790 795 800 Leu Ser His Gly Gly Pro Asn Glu Pro Pro Lys Val Leu Arg Gly Phe 805 810 815 Asp Arg Ile Glu Arg Ile Ala Pro Gly Gln Ser Val Thr Phe Lys Ala 820 825 830 Asp Leu Thr Arg Arg Asp Leu Ser Asn Trp Asp Thr Lys Lys Gln Gln 835 840 845 Trp Val Ile Thr Asp Tyr Pro Lys Thr Val Tyr Val Gly Ser Ser Ser 850 855 860 Arg Asp Leu Pro Leu Ser Ala Arg Leu Pro 865 870 37878PRTPenicillium brasilianum 37Met Gln Gly Ser Thr Ile Phe Leu Ala Phe Ala Ser Trp Ala Ser Gln 1 5 10 15 Val Ala Ala Ile Ala Gln Pro Ile Gln Lys His Glu Pro Gly Phe Leu 20 25 30 His Gly Pro Gln Ala Ile Glu Ser Phe Ser Glu Pro Phe Tyr Pro Ser 35 40 45 Pro Trp Met Asn Pro His Ala Glu Gly Trp Glu Ala Ala Tyr Gln Lys 50 55 60 Ala Gln Asp Phe Val Ser Gln Leu Thr Ile Leu Glu Lys Ile Asn Leu 65 70 75 80 Thr Thr Gly Val Gly Trp Glu Asn Gly Pro Cys Val Gly Asn Thr Gly 85 90 95 Ser Ile Pro Arg Leu Gly Phe Lys Gly Phe Cys Thr Gln Asp Ser Pro 100 105 110 Gln Gly Val Arg Phe Ala Asp Tyr Ser Ser Ala Phe Thr Ser Ser Gln 115 120 125 Met Ala Ala Ala Thr Phe Asp Arg Ser Ile Leu Tyr Gln Arg Gly Gln 130 135 140 Ala Met Ala Gln Glu His Lys Ala Lys Gly Ile Thr Ile Gln Leu Gly 145 150 155 160 Pro Val Ala Gly Pro Leu Gly Arg Ile Pro Glu Gly Gly Arg Asn Trp 165 170 175 Glu Gly Phe Ser Pro Asp Pro Val Leu Thr Gly Ile Ala Met Ala Glu 180 185 190 Thr Ile Lys Gly Met Gln Asp Thr Gly Val Ile Ala Cys Ala Lys His 195 200 205 Tyr Ile Gly Asn Glu Gln Glu His Phe Arg Gln Val Gly Glu Ala Ala 210 215 220 Gly His Gly Tyr Thr Ile Ser Asp Thr Ile Ser Ser Asn Ile Asp Asp 225 230 235 240 Arg Ala Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg 245 250 255 Ala Gly Val Gly Ser Phe Met Cys Ser Tyr Ser Gln Ile Asn Asn Ser 260 265 270 Tyr Gly Cys Gln Asn Ser Gln Thr Leu Asn Lys Leu Leu Lys Ser Glu 275 280 285 Leu Gly Phe Gln Gly Phe Val Met Ser Asp Trp Gly Ala His His Ser 290 295 300 Gly Val Ser Ser Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp 305 310 315 320 Thr Glu Phe Asp Ser Gly Leu Ser Phe Trp Gly Ser Asn Leu Thr Ile 325 330 335 Ala Ile Leu Asn Gly Thr Val Pro Glu Trp Arg Leu Asp Asp Met Ala 340 345 350 Met Arg Ile Met Ala Ala Tyr Phe Lys Val Gly Leu Thr Ile Glu Asp 355 360 365 Gln Pro Asp Val Asn Phe Asn Ala Trp Thr His Asp Thr Tyr Gly Tyr 370 375 380 Lys Tyr Ala Tyr Ser Lys Glu Asp Tyr Glu Gln Val Asn Trp His Val 385 390 395 400 Asp Val Arg Ser Asp His Asn Lys Leu Ile Arg Glu Thr Ala Ala Lys 405 410 415 Gly Thr Val Leu Leu Lys Asn Asn Phe His Ala Leu Pro Leu Lys Gln 420 425 430 Pro Arg Phe Val Ala Val Val Gly Gln Asp Ala Gly Pro Asn Pro Lys 435 440 445 Gly Pro Asn Gly Cys Ala Asp Arg Gly Cys Asp Gln Gly Thr Leu Ala 450 455 460 Met Gly Trp Gly Ser Gly Ser Thr Glu Phe Pro Tyr Leu Val Thr Pro 465 470 475 480 Asp Thr Ala Ile Gln Ser Lys Val Leu Glu Tyr Gly Gly Arg Tyr Glu 485 490 495 Ser Ile Phe Asp Asn Tyr Asp Asp Asn Ala Ile Leu Ser Leu Val Ser 500 505 510 Gln Pro Asp Ala Thr Cys Ile Val Phe Ala Asn Ala Asp Ser Gly Glu 515 520 525 Gly Tyr Ile Thr Val Asp Asn Asn Trp Gly Asp Arg Asn Asn Leu Thr 530 535 540 Leu Trp Gln Asn Ala Asp Gln Val Ile Ser Thr Val Ser Ser Arg Cys 545 550 555 560 Asn Asn Thr Ile Val Val Leu His Ser Val Gly Pro Val Leu Leu Asn 565 570 575 Gly Ile Tyr Glu His Pro Asn Ile Thr Ala Ile Val Trp Ala Gly Met 580 585 590 Pro Gly Glu Glu Ser Gly Asn Ala Leu Val Asp Ile Leu Trp Gly Asn 595 600 605 Val Asn Pro Ala Gly Arg Thr Pro Phe Thr Trp Ala Lys Ser Arg Glu 610 615 620 Asp Tyr Gly Thr Asp Ile Met Tyr Glu Pro Asn Asn Gly Gln Arg Ala 625 630 635 640 Pro Gln Gln Asp Phe Thr Glu Ser Ile Tyr Leu Asp Tyr Arg His Phe 645 650 655 Asp Lys Ala Gly Ile Glu Pro Ile Tyr Glu Phe Gly Phe Gly Leu Ser 660 665 670 Tyr Thr Thr Phe Glu Tyr Ser Asp Leu Arg Val Val Lys Lys Tyr Val 675 680 685 Gln Pro Tyr Ser Pro Thr Thr Gly Thr Gly Ala Gln Ala Pro Ser Ile 690 695 700 Gly Gln Pro Pro Ser Gln Asn Leu Asp Thr Tyr Lys Phe Pro Ala Thr 705 710 715 720 Tyr Lys Tyr Ile Lys Thr Phe Ile Tyr Pro Tyr Leu Asn Ser Thr Val 725 730 735 Ser Leu Arg Ala Ala Ser Lys Asp Pro Glu Tyr Gly Arg Thr Asp Phe 740 745 750 Ile Pro Pro His Ala Arg Asp Gly Ser Pro Gln Pro Leu Asn Pro Ala 755 760 765 Gly Asp Pro Val Ala Ser Gly Gly Asn Asn Met Leu Tyr Asp Glu Leu 770 775 780 Tyr Glu Val Thr Ala Gln Ile Lys Asn Thr Gly Asp Val Ala Gly Asp 785 790 795 800 Glu Val Val Gln Leu Tyr Val Asp Leu Gly Gly Asp Asn Pro Pro Arg 805 810 815 Gln Leu Arg Asn Phe Asp Arg Phe Tyr Leu Leu Pro Gly Gln Ser Ser 820 825 830 Thr Phe Arg Ala Thr Leu Thr Arg Arg Asp Leu Ser Asn Trp Asp Ile 835 840 845 Glu Ala Gln Asn Trp Arg Val Thr Glu Ser Pro Lys Arg Val Tyr Val 850 855 860 Gly Arg Ser Ser Arg Asp Leu Pro Leu Ser Ser Gln Leu Glu 865 870 875 38866PRTPericonia sp. 38Met Ala Ser Trp Leu Ala Pro Ala Leu Leu Ala Val Gly Leu Ala Ser 1 5 10 15 Ala Gln Ala Pro Phe Pro Asn Gly Ser Ser Pro Leu Asn Asp Ile Thr 20 25 30 Ser Pro Pro Phe Tyr Pro Ser Pro Trp Met Asp Pro Ser Ala Ala Gly 35 40 45 Trp Ala Glu Ala Tyr Thr Lys Ala Gln Ala Phe Val Arg Gln Leu Thr 50 55 60 Leu Leu Glu Lys Val Asn Leu Thr Thr Gly Val Gly Trp Glu Gly Glu 65 70 75 80 Ala Cys Val Gly Asn Thr Gly Ser Ile Pro Arg Leu Gly Phe Pro Gly 85 90 95 Phe Cys Thr Gln Asp Ser Pro Leu Gly Val Arg Phe Ala Asp Tyr Val 100 105 110 Ser Ala Phe Thr Ala Gly Gly Thr Ile Ala Ala Ser Trp Asp Arg Ser 115 120 125 Glu Phe Tyr Arg

Arg Gly Tyr Gln Met Gly Val Glu His Arg Gly Lys 130 135 140 Gly Val Asp Val Gln Leu Gly Pro Val Val Gly Pro Ile Gly Arg His 145 150 155 160 Pro Lys Gly Gly Arg Asn Trp Glu Gly Phe Ser Pro Asp Pro Val Leu 165 170 175 Ser Gly Ile Ala Val Ala Glu Thr Val Lys Gly Ile Gln Asp Ala Gly 180 185 190 Val Ile Ala Cys Thr Lys His Phe Ile Leu Asn Glu Gln Glu His Phe 195 200 205 Arg Gln Pro Gly Asn Val Gly Asp Phe Gly Phe Val Asp Ala Val Ser 210 215 220 Ala Asn Leu Ala Asp Lys Thr Leu His Glu Leu Tyr Leu Trp Pro Phe 225 230 235 240 Ala Asp Ala Val Arg Ala Gly Thr Gly Ser Ile Met Cys Ser Tyr Asn 245 250 255 Lys Ala Asn Asn Ser Gln Val Cys Gln Asn Ser Tyr Leu Gln Asn Tyr 260 265 270 Ile Leu Lys Gly Glu Leu Gly Phe Gln Gly Phe Thr Met Ser Asp Trp 275 280 285 Asp Ala Gln His Ser Gly Val Ala Ser Thr Leu Ala Gly Leu Asp Met 290 295 300 Asn Met Pro Gly Asp Thr Asp Phe Asp Ser Gly Phe Ser Phe Trp Gly 305 310 315 320 Pro Asn Met Thr Leu Ser Ile Ile Asn Gly Thr Val Pro Glu Trp Arg 325 330 335 Leu Asp Asp Ala Ala Thr Arg Ile Met Ala Ala Tyr Tyr Leu Val Gly 340 345 350 Arg Asp Arg His Ala Val Pro Val Asn Phe Asn Ser Trp Ser Lys Asp 355 360 365 Thr Tyr Gly Tyr Gln His Ala Tyr Ala Lys Val Gly Tyr Gly Leu Ile 370 375 380 Asn Gln His Val Asp Val Arg Ala Asp His Phe Lys Ser Ile Arg Thr 385 390 395 400 Ala Ala Ala Lys Ser Thr Val Leu Leu Lys Asn Asn Gly Val Leu Pro 405 410 415 Leu Lys Gly Thr Glu Lys Tyr Thr Ala Val Phe Gly Asn Asp Ala Gly 420 425 430 Glu Ala Gln Tyr Gly Pro Asn Gly Cys Ala Asp His Gly Cys Asp Asn 435 440 445 Gly Thr Leu Ala Met Gly Trp Gly Ser Gly Thr Ala Asp Tyr Pro Tyr 450 455 460 Leu Val Thr Pro Leu Glu Ala Ile Lys Arg Thr Val Gly Asp His Gly 465 470 475 480 Gly Val Ile Ala Ser Val Thr Asp Asn Tyr Ala Phe Ser Gln Ile Met 485 490 495 Ala Leu Ala Lys Gln Ala Thr His Ala Ile Val Phe Val Asn Ala Asp 500 505 510 Ser Gly Glu Gly Tyr Ile Thr Val Asp Gly Asn Glu Gly Asp Arg Asn 515 520 525 Asn Leu Thr Leu Trp Gln Asn Gly Glu Glu Leu Val Arg Asn Val Ser 530 535 540 Gly Tyr Cys Asn Asn Thr Ile Val Val Ile His Ser Val Gly Pro Val 545 550 555 560 Leu Val Asp Ser Phe Asn Asn Ser Pro Asn Val Ser Ala Ile Leu Trp 565 570 575 Ala Gly Leu Pro Gly Gln Glu Ser Gly Asn Ala Ile Thr Asp Val Leu 580 585 590 Tyr Gly Arg Val Asn Pro Gly Gly Lys Leu Pro Phe Thr Ile Gly Lys 595 600 605 Ser Ala Glu Glu Tyr Gly Pro Asp Ile Ile Tyr Glu Pro Thr Ala Gly 610 615 620 His Gly Ser Pro Gln Ala Asn Phe Glu Glu Gly Val Phe Ile Asp Tyr 625 630 635 640 Arg Ser Phe Asp Lys Lys Asn Ile Thr Pro Val Tyr Glu Phe Gly Phe 645 650 655 Gly Leu Ser Tyr Thr Asn Phe Ser Tyr Ser Asn Leu Val Val Thr Arg 660 665 670 Val Asn Ala Pro Ala Tyr Val Pro Thr Thr Gly Asn Thr Thr Ala Ala 675 680 685 Pro Thr Leu Gly Asn Ser Ser Lys Asp Ala Ser Asp Tyr Gln Trp Pro 690 695 700 Ala Asn Leu Thr Tyr Val Asn Lys Tyr Ile Tyr Pro Tyr Leu Asn Ser 705 710 715 720 Thr Asp Leu Lys Glu Ala Ser Asn Asp Pro Glu Tyr Gly Ile Glu His 725 730 735 Glu Tyr Pro Glu Gly Ala Thr Asp Gly Ser Pro Gln Pro Arg Ile Ala 740 745 750 Ala Gly Gly Gly Pro Gly Gly Asn Pro Gln Leu Trp Asp Val Leu Tyr 755 760 765 Lys Val Thr Ala Thr Val Thr Asn Asn Gly Ala Val Ala Gly Asp Glu 770 775 780 Val Ala Gln Leu Tyr Val Ser Leu Gly Gly Pro Glu Asp Pro Pro Val 785 790 795 800 Val Leu Arg Asn Phe Asp Arg Leu Thr Ile Ala Pro Gly Gln Ser Val 805 810 815 Glu Phe Thr Ala Asp Ile Thr Arg Arg Asp Val Ser Asn Trp Asp Thr 820 825 830 Val Ser Gln Asn Trp Val Ile Ser Asn Ser Thr Lys Thr Val Tyr Val 835 840 845 Gly Ala Ser Ser Arg Lys Leu Pro Leu Lys Ala Thr Leu Pro Ser Ser 850 855 860 Ser Tyr 865 39871PRTPhaeosphaeria avenaria 39Met Ala Leu Ala Val Ala Phe Phe Val Thr Gln Val Leu Ala Gln Gln 1 5 10 15 Tyr Pro Thr Ser Asn Thr Ser Ser Pro Ala Ala Asn Ser Ser Ser Pro 20 25 30 Leu Asp Asn Ala Val Ser Pro Pro Phe Tyr Pro Ser Pro Trp Ile Glu 35 40 45 Gly Leu Gly Asp Trp Glu Ala Ala Tyr Gln Lys Ala Gln Ala Phe Val 50 55 60 Ser Gln Leu Thr Leu Leu Glu Lys Val Asn Leu Thr Thr Gly Thr Gly 65 70 75 80 Trp Gln Ser Asp His Cys Val Gly Asn Thr Gly Gly Val Pro Arg Leu 85 90 95 Asn Phe Thr Gly Ile Cys Asn Gln Asp Ala Pro Leu Gly Val Arg Phe 100 105 110 Ala Asp Tyr Val Ser Ala Phe Pro Ser Gly Gly Thr Ile Ala Ala Ala 115 120 125 Trp Asp Arg Gly Glu Trp Tyr Leu Arg Gly Tyr Gln Met Gly Ser Glu 130 135 140 His Arg Ser Lys Gly Val Asp Val Gln Leu Gly Pro Val Val Gly Pro 145 150 155 160 Leu Gly Arg Asn Pro Lys Gly Gly Arg Asn Trp Glu Gly Phe Ser Pro 165 170 175 Asp Pro Tyr Leu Ser Gly Ile Ala Ser Ala Glu Ser Val Arg Gly Ile 180 185 190 Gln Asp Ala Gly Val Ile Ala Cys Thr Lys His Tyr Ile Met Asn Glu 195 200 205 Gln Glu His Phe Arg Gln Pro Gly Asn Phe Glu Asp Gln Gly Phe Val 210 215 220 Asp Ala Leu Ser Ser Asn Leu Asp Asp Lys Thr Leu His Glu Leu Tyr 225 230 235 240 Leu Trp Pro Phe Ala Asp Ala Val Arg Ala Gly Thr Gly Ser Ile Met 245 250 255 Cys Ser Tyr Asn Lys Val Asn Asn Ser Gln Ala Cys Gln Asn Ser Tyr 260 265 270 Leu Gln Asn Tyr Ile Leu Lys Gly Glu Leu Gly Phe Gln Gly Phe Ile 275 280 285 Met Ser Asp Trp Asp Ala Gln His Ser Gly Val Ala Ser Thr Phe Ala 290 295 300 Gly Leu Asp Met Thr Met Pro Gly Asp Thr Asp Phe Asn Ser Gly Lys 305 310 315 320 Thr Phe Trp Gly Thr Asn Phe Thr Thr Ser Ile Leu Asn Gly Thr Val 325 330 335 Pro Gln Trp Arg Leu Asp Asp Ala Val Thr Arg Ile Met Ala Ala Phe 340 345 350 Tyr Tyr Val Gly Arg Asp Lys Ala Arg Ile Pro Val Asn Phe Asp Ser 355 360 365 Trp Ser Arg Asp Thr Tyr Gly Phe Asp His Tyr Tyr Gly Lys Ala Gly 370 375 380 Tyr Ser Gln Ile Asn Ser His Val Asp Val Arg Ala Asp His Phe Arg 385 390 395 400 Ser Ile Arg Arg Thr Ala Ala Met Ser Thr Val Leu Leu Lys Asn Glu 405 410 415 Gly Ala Leu Pro Leu Thr Gly Ser Glu Lys Trp Thr Ala Val Phe Gly 420 425 430 Asp Asp Ala Gly Glu Gly Gln Leu Gly Pro Asn Gly Phe Pro Asp His 435 440 445 Gly Gly Asn Asn Gly Thr Leu Ala Met Gly Trp Gly Ser Gly Thr Ser 450 455 460 Asp Tyr Pro Tyr Leu Val Thr Pro Leu Glu Ser Ile Lys Ala Thr Val 465 470 475 480 Ala Gln Asn Gly Gly Ile Val Thr Ser Val Thr Asp Asn Trp Ala Tyr 485 490 495 Thr Gln Ile Gln Thr Leu Ala Lys Gln Ala Ser Val Ala Ile Val Phe 500 505 510 Val Asn Ala Asp Ser Gly Glu Gly Tyr Ile Thr Val Asp Gly Asn Ala 515 520 525 Gly Asp Arg Asn Asn Leu Thr Leu Trp Gln Asp Gly Asp Thr Leu Ile 530 535 540 Lys Asn Val Ser Ser Leu Cys Asn Asn Thr Ile Val Val Ile His Ser 545 550 555 560 Val Gly Pro Val Leu Val Asn Ser Phe Tyr Asp Ser Glu Asn Val Thr 565 570 575 Ala Ile Leu Trp Ala Gly Leu Pro Gly Gln Glu Ser Gly Asn Ala Ile 580 585 590 Ala Asp Ile Leu Tyr Gly Arg His Asn Pro Gly Gly Lys Leu Pro Phe 595 600 605 Thr Ile Gly Ser Asp Ala Ala Glu Tyr Gly Pro Asp Leu Ile Tyr Glu 610 615 620 Pro Thr Asn Asn Ser Ser Ser Pro Gln Asp Asn Phe Glu Glu Gly Val 625 630 635 640 Phe Ile Asp Tyr Arg Ala Phe Asp Lys Gln Asn Val Thr Pro Ile Tyr 645 650 655 Glu Phe Gly Phe Gly Leu Ser Tyr Thr Lys Phe Ser Tyr Ser Asn Leu 660 665 670 Thr Val Lys Lys Ala Asn Ala Gly Ala Tyr Thr Pro Ala Thr Gly Gln 675 680 685 Ser Lys Ala Ala Pro Thr Leu Gly Asn Phe Ser Thr Asp Ala Ser Gln 690 695 700 Tyr Gln Trp Pro Ser Asp Phe Thr Tyr Ile Asp Thr Phe Ile Tyr Pro 705 710 715 720 Tyr Leu Asn Ser Thr Asp Leu Lys Thr Ala Ser Gln Asp Pro Glu Tyr 725 730 735 Gly Leu Asn Tyr Thr Trp Pro Ala Gly Ala Thr Asp Gly Thr Pro Gln 740 745 750 Ala Arg Ile Pro Ala Gly Gly Ala Pro Gly Gly Asn Pro Gln Leu Trp 755 760 765 Asp Val Leu Phe Ser Val Glu Ala Thr Ile Thr Asn Asn Gly Thr Val 770 775 780 Pro Gly Asp Glu Val Val Gln Leu Tyr Val Ser Leu Gly Asn Pro Asp 785 790 795 800 Asp Pro Lys Ile Val Leu Arg Gly Phe Asp Arg Leu Ser Ile Gln Pro 805 810 815 Gly Lys Thr Ala Thr Phe His Ala Asp Ile Thr Arg Arg Asp Val Ser 820 825 830 Asn Trp Asp Val Ala Ser Gln Asn Trp Val Ile Thr Ser Ala Pro Lys 835 840 845 Thr Val Tyr Val Gly Ala Ser Ser Arg Lys Leu Pro Leu Thr Ala Thr 850 855 860 Leu Asp Thr Ser Asp Phe Gln 865 870 40873PRTAspergillus fumigatus 40Met Arg Phe Gly Trp Leu Glu Val Ala Ala Leu Thr Ala Ala Ser Val 1 5 10 15 Ala Asn Ala Gln Val Phe Asp Asn Ser His Gly Asn Asn Gln Glu Leu 20 25 30 Ala Phe Ser Pro Pro Phe Tyr Pro Ser Pro Trp Ala Asp Gly Gln Gly 35 40 45 Glu Trp Ala Asp Ala His Arg Arg Ala Val Glu Ile Val Ser Gln Met 50 55 60 Thr Leu Ala Glu Lys Val Asn Leu Thr Thr Gly Thr Gly Trp Glu Met 65 70 75 80 Asp Arg Cys Val Gly Gln Thr Gly Ser Val Pro Arg Leu Gly Ile Asn 85 90 95 Trp Gly Leu Cys Gly Gln Asp Ser Pro Leu Gly Ile Arg Phe Ser Asp 100 105 110 Leu Asn Ser Ala Phe Pro Ala Gly Thr Asn Val Ala Ala Thr Trp Asp 115 120 125 Lys Thr Leu Ala Tyr Leu Arg Gly Lys Ala Met Gly Glu Glu Phe Asn 130 135 140 Asp Lys Gly Val Asp Ile Leu Leu Gly Pro Ala Ala Gly Pro Leu Gly 145 150 155 160 Lys Tyr Pro Asp Gly Gly Arg Ile Trp Glu Gly Phe Ser Pro Asp Pro 165 170 175 Ala Leu Thr Gly Val Leu Phe Ala Glu Thr Ile Lys Gly Ile Gln Asp 180 185 190 Ala Gly Val Ile Ala Thr Ala Lys His Tyr Ile Leu Asn Glu Gln Glu 195 200 205 His Phe Arg Gln Val Gly Glu Ala Gln Gly Tyr Gly Tyr Asn Ile Thr 210 215 220 Glu Thr Ile Ser Ser Asn Val Asp Asp Lys Thr Met His Glu Leu Tyr 225 230 235 240 Leu Trp Pro Phe Ala Asp Ala Val Arg Ala Gly Val Gly Ala Val Met 245 250 255 Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr Gly Cys Gln Asn Ser Gln 260 265 270 Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu Gly Phe Gln Gly Phe Val 275 280 285 Met Ser Asp Trp Ser Ala His His Ser Gly Val Gly Ala Ala Leu Ala 290 295 300 Gly Leu Asp Met Ser Met Pro Gly Asp Ile Ser Phe Asp Asp Gly Leu 305 310 315 320 Ser Phe Trp Gly Thr Asn Leu Thr Val Ser Val Leu Asn Gly Thr Val 325 330 335 Pro Ala Trp Arg Val Asp Asp Met Ala Val Arg Ile Met Thr Ala Tyr 340 345 350 Tyr Lys Val Gly Arg Asp Arg Leu Arg Ile Pro Pro Asn Phe Ser Ser 355 360 365 Trp Thr Arg Asp Glu Tyr Gly Trp Glu His Ser Ala Val Ser Glu Gly 370 375 380 Ala Trp Thr Lys Val Asn Asp Phe Val Asn Val Gln Arg Ser His Ser 385 390 395 400 Gln Ile Ile Arg Glu Ile Gly Ala Ala Ser Thr Val Leu Leu Lys Asn 405 410 415 Thr Gly Ala Leu Pro Leu Thr Gly Lys Glu Val Lys Val Gly Val Leu 420 425 430 Gly Glu Asp Ala Gly Ser Asn Pro Trp Gly Ala Asn Gly Cys Pro Asp 435 440 445 Arg Gly Cys Asp Asn Gly Thr Leu Ala Met Ala Trp Gly Ser Gly Thr 450 455 460 Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu Gln Ala Ile Gln Arg Glu 465 470 475 480 Val Ile Ser Asn Gly Gly Asn Val Phe Ala Val Thr Asp Asn Gly Ala 485 490 495 Leu Ser Gln Met Ala Asp Val Ala Ser Gln Ser Ser Val Ser Leu Val 500 505 510 Phe Val Asn Ala Asp Ser Gly Glu Gly Phe Ile Ser Val Asp Gly Asn 515 520 525 Glu Gly Asp Arg Lys Asn Leu Thr Leu Trp Lys Asn Gly Glu Ala Val 530 535 540 Ile Asp Thr Val Val Ser His Cys Asn Asn Thr Ile Val Val Ile His 545 550 555 560 Ser Val Gly Pro Val Leu Ile Asp Arg Trp Tyr Asp Asn Pro Asn Val 565 570 575 Thr Ala Ile Ile Trp Ala Gly Leu Pro Gly Gln Glu Ser Gly Asn Ser 580 585 590 Leu Val Asp Val Leu Tyr Gly Arg Val Asn Pro Ser Ala Lys Thr Pro 595 600 605 Phe Thr Trp Gly Lys Thr Arg Glu Ser Tyr Gly Ala Pro Leu Leu Thr 610 615 620 Glu Pro Asn Asn Gly Asn Gly Ala Pro Gln Asp Asp Phe Asn Glu Gly 625 630 635 640 Val Phe Ile Asp Tyr Arg His Phe Asp Lys Arg Asn Glu Thr Pro Ile 645 650 655 Tyr Glu Phe Gly His Gly Leu Ser Tyr Thr Thr Phe Gly Tyr Ser His 660 665 670 Leu Arg Val Gln Ala Leu Asn Ser Ser Ser Ser Ala Tyr Val Pro Thr 675 680 685 Ser Gly Glu Thr Lys Pro Ala Pro

Thr Tyr Gly Glu Ile Gly Ser Ala 690 695 700 Ala Asp Tyr Leu Tyr Pro Glu Gly Leu Lys Arg Ile Thr Lys Phe Ile 705 710 715 720 Tyr Pro Trp Leu Asn Ser Thr Asp Leu Glu Asp Ser Ser Asp Asp Pro 725 730 735 Asn Tyr Gly Trp Glu Asp Ser Glu Tyr Ile Pro Glu Gly Ala Arg Asp 740 745 750 Gly Ser Pro Gln Pro Leu Leu Lys Ala Gly Gly Ala Pro Gly Gly Asn 755 760 765 Pro Thr Leu Tyr Gln Asp Leu Val Arg Val Ser Ala Thr Ile Thr Asn 770 775 780 Thr Gly Asn Val Ala Gly Tyr Glu Val Pro Gln Leu Tyr Val Ser Leu 785 790 795 800 Gly Gly Pro Asn Glu Pro Arg Val Val Leu Arg Lys Phe Asp Arg Ile 805 810 815 Phe Leu Ala Pro Gly Glu Gln Lys Val Trp Thr Thr Thr Leu Asn Arg 820 825 830 Arg Asp Leu Ala Asn Trp Asp Val Glu Ala Gln Asp Trp Val Ile Thr 835 840 845 Lys Tyr Pro Lys Lys Val His Val Gly Ser Ser Ser Arg Lys Leu Pro 850 855 860 Leu Arg Ala Pro Leu Pro Arg Val Tyr 865 870 41861PRTAspergillus oryzae 41Met Lys Leu Gly Trp Ile Glu Val Ala Ala Leu Ala Ala Ala Ser Val 1 5 10 15 Val Ser Ala Lys Asp Asp Leu Ala Tyr Ser Pro Pro Phe Tyr Pro Ser 20 25 30 Pro Trp Ala Asp Gly Gln Gly Glu Trp Ala Glu Val Tyr Lys Arg Ala 35 40 45 Val Asp Ile Val Ser Gln Met Thr Leu Thr Glu Lys Val Asn Leu Thr 50 55 60 Thr Gly Thr Gly Trp Gln Leu Glu Arg Cys Val Gly Gln Thr Gly Ser 65 70 75 80 Val Pro Arg Leu Asn Ile Pro Ser Leu Cys Leu Gln Asp Ser Pro Leu 85 90 95 Gly Ile Arg Phe Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn 100 105 110 Val Ala Ala Thr Trp Asp Lys Thr Leu Ala Tyr Leu Arg Gly Gln Ala 115 120 125 Met Gly Glu Glu Phe Ser Asp Lys Gly Ile Asp Val Gln Leu Gly Pro 130 135 140 Ala Ala Gly Pro Leu Gly Ala His Pro Asp Gly Gly Arg Asn Trp Glu 145 150 155 160 Gly Phe Ser Pro Asp Pro Ala Leu Thr Gly Val Leu Phe Ala Glu Thr 165 170 175 Ile Lys Gly Ile Gln Asp Ala Gly Val Ile Ala Thr Ala Lys His Tyr 180 185 190 Ile Met Asn Glu Gln Glu His Phe Arg Gln Gln Pro Glu Ala Ala Gly 195 200 205 Tyr Gly Phe Asn Val Ser Asp Ser Leu Ser Ser Asn Val Asp Asp Lys 210 215 220 Thr Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala 225 230 235 240 Gly Val Gly Ala Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr 245 250 255 Gly Cys Glu Asn Ser Glu Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu 260 265 270 Gly Phe Gln Gly Phe Val Met Ser Asp Trp Thr Ala His His Ser Gly 275 280 285 Val Gly Ala Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Val 290 295 300 Thr Phe Asp Ser Gly Thr Ser Phe Trp Gly Ala Asn Leu Thr Val Gly 305 310 315 320 Val Leu Asn Gly Thr Ile Pro Gln Trp Arg Val Asp Asp Met Ala Val 325 330 335 Arg Ile Met Ala Ala Tyr Tyr Lys Val Gly Arg Asp Thr Lys Tyr Thr 340 345 350 Pro Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly Phe Ala His 355 360 365 Asn His Val Ser Glu Gly Ala Tyr Glu Arg Val Asn Glu Phe Val Asp 370 375 380 Val Gln Arg Asp His Ala Asp Leu Ile Arg Arg Ile Gly Ala Gln Ser 385 390 395 400 Thr Val Leu Leu Lys Asn Lys Gly Ala Leu Pro Leu Ser Arg Lys Glu 405 410 415 Lys Leu Val Ala Leu Leu Gly Glu Asp Ala Gly Ser Asn Ser Trp Gly 420 425 430 Ala Asn Gly Cys Asp Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met 435 440 445 Ala Trp Gly Ser Gly Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu 450 455 460 Gln Ala Ile Gln Asn Glu Val Leu Gln Gly Arg Gly Asn Val Phe Ala 465 470 475 480 Val Thr Asp Ser Trp Ala Leu Asp Lys Ile Ala Ala Ala Ala Arg Gln 485 490 495 Ala Ser Val Ser Leu Val Phe Val Asn Ser Asp Ser Gly Glu Gly Tyr 500 505 510 Leu Ser Val Asp Gly Asn Glu Gly Asp Arg Asn Asn Ile Thr Leu Trp 515 520 525 Lys Asn Gly Asp Asn Val Val Lys Thr Ala Ala Asn Asn Cys Asn Asn 530 535 540 Thr Val Val Ile Ile His Ser Val Gly Pro Val Leu Ile Asp Glu Trp 545 550 555 560 Tyr Asp His Pro Asn Val Thr Gly Ile Leu Trp Ala Gly Leu Pro Gly 565 570 575 Gln Glu Ser Gly Asn Ser Ile Ala Asp Val Leu Tyr Gly Arg Val Asn 580 585 590 Pro Gly Ala Lys Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ser Tyr 595 600 605 Gly Ser Pro Leu Val Lys Asp Ala Asn Asn Gly Asn Gly Ala Pro Gln 610 615 620 Ser Asp Phe Thr Gln Gly Val Phe Ile Asp Tyr Arg His Phe Asp Lys 625 630 635 640 Phe Asn Glu Thr Pro Ile Tyr Glu Phe Gly Tyr Gly Leu Ser Tyr Thr 645 650 655 Thr Phe Glu Leu Ser Asp Leu His Val Gln Pro Leu Asn Ala Ser Arg 660 665 670 Tyr Thr Pro Thr Ser Gly Met Thr Glu Ala Ala Lys Asn Phe Gly Glu 675 680 685 Ile Gly Asp Ala Ser Glu Tyr Val Tyr Pro Glu Gly Leu Glu Arg Ile 690 695 700 His Glu Phe Ile Tyr Pro Trp Ile Asn Ser Thr Asp Leu Lys Ala Ser 705 710 715 720 Ser Asp Asp Ser Asn Tyr Gly Trp Glu Asp Ser Lys Tyr Ile Pro Glu 725 730 735 Gly Ala Thr Asp Gly Ser Ala Gln Pro Arg Leu Pro Ala Ser Gly Gly 740 745 750 Ala Gly Gly Asn Pro Gly Leu Tyr Glu Asp Leu Phe Arg Val Ser Val 755 760 765 Lys Val Lys Asn Thr Gly Asn Val Ala Gly Asp Glu Val Pro Gln Leu 770 775 780 Tyr Val Ser Leu Gly Gly Pro Asn Glu Pro Lys Val Val Leu Arg Lys 785 790 795 800 Phe Glu Arg Ile His Leu Ala Pro Ser Gln Glu Ala Val Trp Thr Thr 805 810 815 Thr Leu Thr Arg Arg Asp Leu Ala Asn Trp Asp Val Ser Ala Gln Asp 820 825 830 Trp Thr Val Thr Pro Tyr Pro Lys Thr Ile Tyr Val Gly Asn Ser Ser 835 840 845 Arg Lys Leu Pro Leu Gln Ala Ser Leu Pro Lys Ala Gln 850 855 860 42860PRTAspergillus aculeatus 42Met Lys Leu Ser Trp Leu Glu Ala Ala Ala Leu Thr Ala Ala Ser Val 1 5 10 15 Val Ser Ala Asp Glu Leu Ala Phe Ser Pro Pro Phe Tyr Pro Ser Pro 20 25 30 Trp Ala Asn Gly Gln Gly Glu Trp Ala Glu Ala Tyr Gln Arg Ala Val 35 40 45 Ala Ile Val Ser Gln Met Thr Leu Asp Glu Lys Val Asn Leu Thr Thr 50 55 60 Gly Thr Gly Trp Glu Leu Glu Lys Cys Val Gly Gln Thr Gly Gly Val 65 70 75 80 Pro Arg Leu Asn Ile Gly Gly Met Cys Leu Gln Asp Ser Pro Leu Gly 85 90 95 Ile Arg Asp Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn Val 100 105 110 Ala Ala Thr Trp Asp Lys Asn Leu Ala Tyr Leu Arg Gly Gln Ala Met 115 120 125 Gly Gln Glu Phe Ser Asp Lys Gly Ile Asp Val Gln Leu Gly Pro Ala 130 135 140 Ala Gly Pro Leu Gly Arg Ser Pro Asp Gly Gly Arg Asn Trp Glu Gly 145 150 155 160 Phe Ser Pro Asp Pro Ala Leu Thr Gly Val Leu Phe Ala Glu Thr Ile 165 170 175 Lys Gly Ile Gln Asp Ala Gly Val Val Ala Thr Ala Lys His Tyr Ile 180 185 190 Leu Asn Glu Gln Glu His Phe Arg Gln Val Ala Glu Ala Ala Gly Tyr 195 200 205 Gly Phe Asn Ile Ser Asp Thr Ile Ser Ser Asn Val Asp Asp Lys Thr 210 215 220 Ile His Glu Met Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala Gly 225 230 235 240 Val Gly Ala Ile Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr Gly 245 250 255 Cys Gln Asn Ser Tyr Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu Gly 260 265 270 Phe Gln Gly Phe Val Met Ser Asp Trp Gly Ala His His Ser Gly Val 275 280 285 Gly Ser Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Ile Thr 290 295 300 Phe Asp Ser Ala Thr Ser Phe Trp Gly Thr Asn Leu Thr Ile Ala Val 305 310 315 320 Leu Asn Gly Thr Val Pro Gln Trp Arg Val Asp Asp Met Ala Val Arg 325 330 335 Ile Met Ala Ala Tyr Tyr Lys Val Gly Arg Asp Arg Leu Tyr Gln Pro 340 345 350 Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly Phe Lys Tyr Phe 355 360 365 Tyr Pro Gln Glu Gly Pro Tyr Glu Lys Val Asn His Phe Val Asn Val 370 375 380 Gln Arg Asn His Ser Glu Val Ile Arg Lys Leu Gly Ala Asp Ser Thr 385 390 395 400 Val Leu Leu Lys Asn Asn Asn Ala Leu Pro Leu Thr Gly Lys Glu Arg 405 410 415 Lys Val Ala Ile Leu Gly Glu Asp Ala Gly Ser Asn Ser Tyr Gly Ala 420 425 430 Asn Gly Cys Ser Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met Ala 435 440 445 Trp Gly Ser Gly Thr Ala Glu Phe Pro Tyr Leu Val Thr Pro Glu Gln 450 455 460 Ala Ile Gln Ala Glu Val Leu Lys His Lys Gly Ser Val Tyr Ala Ile 465 470 475 480 Thr Asp Asn Trp Ala Leu Ser Gln Val Glu Thr Leu Ala Lys Gln Ala 485 490 495 Ser Val Ser Leu Val Phe Val Asn Ser Asp Ala Gly Glu Gly Tyr Ile 500 505 510 Ser Val Asp Gly Asn Glu Gly Asp Arg Asn Asn Leu Thr Leu Trp Lys 515 520 525 Asn Gly Asp Asn Leu Ile Lys Ala Ala Ala Asn Asn Cys Asn Asn Thr 530 535 540 Ile Val Val Ile His Ser Val Gly Pro Val Leu Val Asp Glu Trp Tyr 545 550 555 560 Asp His Pro Asn Val Thr Ala Ile Leu Trp Ala Gly Leu Pro Gly Gln 565 570 575 Glu Ser Gly Asn Ser Leu Ala Asp Val Leu Tyr Gly Arg Val Asn Pro 580 585 590 Gly Ala Lys Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ala Tyr Gly 595 600 605 Asp Tyr Leu Val Arg Glu Leu Asn Asn Gly Asn Gly Ala Pro Gln Asp 610 615 620 Asp Phe Ser Glu Gly Val Phe Ile Asp Tyr Arg Gly Phe Asp Lys Arg 625 630 635 640 Asn Glu Thr Pro Ile Tyr Glu Phe Gly His Gly Leu Ser Tyr Thr Thr 645 650 655 Phe Asn Tyr Ser Gly Leu His Ile Gln Val Leu Asn Ala Ser Ser Asn 660 665 670 Ala Gln Val Ala Thr Glu Thr Gly Ala Ala Pro Thr Phe Gly Gln Val 675 680 685 Gly Asn Ala Ser Asp Tyr Val Tyr Pro Glu Gly Leu Thr Arg Ile Ser 690 695 700 Lys Phe Ile Tyr Pro Trp Leu Asn Ser Thr Asp Leu Lys Ala Ser Ser 705 710 715 720 Gly Asp Pro Tyr Tyr Gly Val Asp Thr Ala Glu His Val Pro Glu Gly 725 730 735 Ala Thr Asp Gly Ser Pro Gln Pro Val Leu Pro Ala Gly Gly Gly Ser 740 745 750 Gly Gly Asn Pro Arg Leu Tyr Asp Glu Leu Ile Arg Val Ser Val Thr 755 760 765 Val Lys Asn Thr Gly Arg Val Ala Gly Asp Ala Val Pro Gln Leu Tyr 770 775 780 Val Ser Leu Gly Gly Pro Asn Glu Pro Lys Val Val Leu Arg Lys Phe 785 790 795 800 Asp Arg Leu Thr Leu Lys Pro Ser Glu Glu Thr Val Trp Thr Thr Thr 805 810 815 Leu Thr Arg Arg Asp Leu Ser Asn Trp Asp Val Ala Ala Gln Asp Trp 820 825 830 Val Ile Thr Ser Tyr Pro Lys Lys Val His Val Gly Ser Ser Ser Arg 835 840 845 Gln Leu Pro Leu His Ala Ala Leu Pro Lys Val Gln 850 855 860 43860PRTAspergillus niger 43Met Arg Phe Thr Leu Ile Glu Ala Val Ala Leu Thr Ala Val Ser Leu 1 5 10 15 Ala Ser Ala Asp Glu Leu Ala Tyr Ser Pro Pro Tyr Tyr Pro Ser Pro 20 25 30 Trp Ala Asn Gly Gln Gly Asp Trp Ala Gln Ala Tyr Gln Arg Ala Val 35 40 45 Asp Ile Val Ser Gln Met Thr Leu Asp Glu Lys Val Asn Leu Thr Thr 50 55 60 Gly Thr Gly Trp Glu Leu Glu Leu Cys Val Gly Gln Thr Gly Gly Val 65 70 75 80 Pro Arg Leu Gly Val Pro Gly Met Cys Leu Gln Asp Ser Pro Leu Gly 85 90 95 Val Arg Asp Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Met Asn Val 100 105 110 Ala Ala Thr Trp Asp Lys Asn Leu Ala Tyr Leu Arg Gly Lys Ala Met 115 120 125 Gly Gln Glu Phe Ser Asp Lys Gly Ala Asp Ile Gln Leu Gly Pro Ala 130 135 140 Ala Gly Pro Leu Gly Arg Ser Pro Asp Gly Gly Arg Asn Trp Glu Gly 145 150 155 160 Phe Ser Pro Asp Pro Ala Leu Ser Gly Val Leu Phe Ala Glu Thr Ile 165 170 175 Lys Gly Ile Gln Asp Ala Gly Val Val Ala Thr Ala Lys His Tyr Ile 180 185 190 Ala Tyr Glu Gln Glu His Phe Arg Gln Ala Pro Glu Ala Gln Gly Phe 195 200 205 Gly Phe Asn Ile Ser Glu Ser Gly Ser Ala Asn Leu Asp Asp Lys Thr 210 215 220 Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Ile Arg Ala Gly 225 230 235 240 Ala Gly Ala Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr Gly 245 250 255 Cys Gln Asn Ser Tyr Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu Gly 260 265 270 Phe Gln Gly Phe Val Met Ser Asp Trp Ala Ala His His Ala Gly Val 275 280 285 Ser Gly Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Val Asp 290 295 300 Tyr Asp Ser Gly Thr Ser Tyr Trp Gly Thr Asn Leu Thr Ile Ser Val 305 310 315 320 Leu Asn Gly Thr Val Pro Gln Trp Arg Val Asp Asp Met Ala Val Arg 325 330 335 Ile Met Ala Ala Tyr Tyr Lys Val Gly Arg Asp Arg Leu Trp Thr Pro 340 345 350 Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly Tyr Lys Tyr Tyr 355 360 365 Tyr Val Ser Glu Gly Pro Tyr Glu Lys Val Asn Gln Tyr Val Asn Val 370 375 380 Gln Arg Asn His Ser Glu Leu Ile Arg Arg Ile Gly Ala Asp Ser Thr 385

390 395 400 Val Leu Leu Lys Asn Asp Gly Ala Leu Pro Leu Thr Gly Lys Glu Arg 405 410 415 Leu Val Ala Leu Ile Gly Glu Asp Ala Gly Ser Asn Pro Tyr Gly Ala 420 425 430 Asn Gly Cys Ser Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met Gly 435 440 445 Trp Gly Ser Gly Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu Gln 450 455 460 Ala Ile Ser Asn Glu Val Leu Lys His Lys Asn Gly Val Phe Thr Ala 465 470 475 480 Thr Asp Asn Trp Ala Ile Asp Gln Ile Glu Ala Leu Ala Lys Thr Ala 485 490 495 Ser Val Ser Leu Val Phe Val Asn Ala Asp Ser Gly Glu Gly Tyr Ile 500 505 510 Asn Val Asp Gly Asn Leu Gly Asp Arg Arg Asn Leu Thr Leu Trp Arg 515 520 525 Asn Gly Asp Asn Val Ile Lys Ala Ala Ala Ser Asn Cys Asn Asn Thr 530 535 540 Ile Val Val Ile His Ser Val Gly Pro Val Leu Val Asn Glu Trp Tyr 545 550 555 560 Asp Asn Pro Asn Val Thr Ala Ile Leu Trp Gly Gly Leu Pro Gly Gln 565 570 575 Glu Ser Gly Asn Ser Leu Ala Asp Val Leu Tyr Gly Arg Val Asn Pro 580 585 590 Gly Ala Lys Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ala Tyr Gln 595 600 605 Asp Tyr Leu Val Thr Glu Pro Asn Asn Gly Asn Gly Ala Pro Gln Glu 610 615 620 Asp Phe Val Glu Gly Val Phe Ile Asp Tyr Arg Gly Phe Asp Lys Arg 625 630 635 640 Asn Glu Thr Pro Ile Tyr Glu Phe Gly Tyr Gly Leu Ser Tyr Thr Thr 645 650 655 Phe Asn Tyr Ser Asn Leu Glu Val Gln Val Leu Ser Ala Pro Ala Tyr 660 665 670 Glu Pro Ala Ser Gly Glu Thr Glu Ala Ala Pro Thr Phe Gly Glu Val 675 680 685 Gly Asn Ala Ser Asp Tyr Leu Tyr Pro Ser Gly Leu Gln Arg Ile Thr 690 695 700 Lys Phe Ile Tyr Pro Trp Leu Asn Gly Thr Asp Leu Glu Ala Ser Ser 705 710 715 720 Gly Asp Ala Ser Tyr Gly Gln Asp Ser Ser Asp Tyr Leu Pro Glu Gly 725 730 735 Ala Thr Asp Gly Ser Ala Gln Pro Ile Leu Pro Ala Gly Gly Gly Pro 740 745 750 Gly Gly Asn Pro Arg Leu Tyr Asp Glu Leu Ile Arg Val Ser Val Thr 755 760 765 Ile Lys Asn Thr Gly Lys Val Ala Gly Asp Glu Val Pro Gln Leu Tyr 770 775 780 Val Ser Leu Gly Gly Pro Asn Glu Pro Lys Ile Val Leu Arg Gln Phe 785 790 795 800 Glu Arg Ile Thr Leu Gln Pro Ser Glu Glu Thr Lys Trp Ser Thr Thr 805 810 815 Leu Thr Arg Arg Asp Leu Ala Asn Trp Asn Val Glu Lys Gln Asp Trp 820 825 830 Glu Ile Thr Ser Tyr Pro Lys Met Val Phe Val Gly Ser Ser Ser Arg 835 840 845 Lys Leu Pro Leu Arg Ala Ser Leu Pro Thr Val His 850 855 860 44857PRTTalaromyces emersonii 44Met Arg Asn Gly Leu Leu Lys Val Ala Ala Leu Ala Ala Ala Ser Ala 1 5 10 15 Val Asn Gly Glu Asn Leu Ala Tyr Ser Pro Pro Phe Tyr Pro Ser Pro 20 25 30 Trp Ala Asn Gly Gln Gly Asp Trp Ala Glu Ala Tyr Gln Lys Ala Val 35 40 45 Gln Phe Val Ser Gln Leu Thr Leu Ala Glu Lys Val Asn Leu Thr Thr 50 55 60 Gly Thr Gly Trp Glu Gln Asp Arg Cys Val Gly Gln Val Gly Ser Ile 65 70 75 80 Pro Arg Leu Gly Phe Pro Gly Leu Cys Met Gln Asp Ser Pro Leu Gly 85 90 95 Val Arg Asp Thr Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn Val 100 105 110 Ala Ala Thr Trp Asp Arg Asn Leu Ala Tyr Arg Arg Gly Val Ala Met 115 120 125 Gly Glu Glu His Arg Gly Lys Gly Val Asp Val Gln Leu Gly Pro Val 130 135 140 Ala Gly Pro Leu Gly Arg Ser Pro Asp Ala Gly Arg Asn Trp Glu Gly 145 150 155 160 Phe Ala Pro Asp Pro Val Leu Thr Gly Asn Met Met Ala Ser Thr Ile 165 170 175 Gln Gly Ile Gln Asp Ala Gly Val Ile Ala Cys Ala Lys His Phe Ile 180 185 190 Leu Tyr Glu Gln Glu His Phe Arg Gln Gly Ala Gln Asp Gly Tyr Asp 195 200 205 Ile Ser Asp Ser Ile Ser Ala Asn Ala Asp Asp Lys Thr Met His Glu 210 215 220 Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala Gly Val Gly Ser 225 230 235 240 Val Met Cys Ser Tyr Asn Gln Val Asn Asn Ser Tyr Ala Cys Ser Asn 245 250 255 Ser Tyr Thr Met Asn Lys Leu Leu Lys Ser Glu Leu Gly Phe Gln Gly 260 265 270 Phe Val Met Thr Asp Trp Gly Gly His His Ser Gly Val Gly Ser Ala 275 280 285 Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Ile Ala Phe Asp Ser 290 295 300 Gly Thr Ser Phe Trp Gly Thr Asn Leu Thr Val Ala Val Leu Asn Gly 305 310 315 320 Ser Ile Pro Glu Trp Arg Val Asp Asp Met Ala Val Arg Ile Met Ser 325 330 335 Ala Tyr Tyr Lys Val Gly Arg Asp Arg Tyr Ser Val Pro Ile Asn Phe 340 345 350 Asp Ser Trp Thr Leu Asp Thr Tyr Gly Pro Glu His Tyr Ala Val Gly 355 360 365 Gln Gly Gln Thr Lys Ile Asn Glu His Val Asp Val Arg Gly Asn His 370 375 380 Ala Glu Ile Ile His Glu Ile Gly Ala Ala Ser Ala Val Leu Leu Lys 385 390 395 400 Asn Lys Gly Gly Leu Pro Leu Thr Gly Thr Glu Arg Phe Val Gly Val 405 410 415 Phe Gly Lys Asp Ala Gly Ser Asn Pro Trp Gly Val Asn Gly Cys Ser 420 425 430 Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met Gly Trp Gly Ser Gly 435 440 445 Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu Gln Ala Ile Gln Arg 450 455 460 Glu Val Leu Ser Arg Asn Gly Thr Phe Thr Gly Ile Thr Asp Asn Gly 465 470 475 480 Ala Leu Ala Glu Met Ala Ala Ala Ala Ser Gln Ala Asp Thr Cys Leu 485 490 495 Val Phe Ala Asn Ala Asp Ser Gly Glu Gly Tyr Ile Thr Val Asp Gly 500 505 510 Asn Glu Gly Asp Arg Lys Asn Leu Thr Leu Trp Gln Gly Ala Asp Gln 515 520 525 Val Ile His Asn Val Ser Ala Asn Cys Asn Asn Thr Val Val Val Leu 530 535 540 His Thr Val Gly Pro Val Leu Ile Asp Asp Trp Tyr Asp His Pro Asn 545 550 555 560 Val Thr Ala Ile Leu Trp Ala Gly Leu Pro Gly Gln Glu Ser Gly Asn 565 570 575 Ser Leu Val Asp Val Leu Tyr Gly Arg Val Asn Pro Gly Lys Thr Pro 580 585 590 Phe Thr Trp Gly Arg Ala Arg Asp Asp Tyr Gly Ala Pro Leu Ile Val 595 600 605 Lys Pro Asn Asn Gly Lys Gly Ala Pro Gln Gln Asp Phe Thr Glu Gly 610 615 620 Ile Phe Ile Asp Tyr Arg Arg Phe Asp Lys Tyr Asn Ile Thr Pro Ile 625 630 635 640 Tyr Glu Phe Gly Phe Gly Leu Ser Tyr Thr Thr Phe Glu Phe Ser Gln 645 650 655 Leu Asn Val Gln Pro Ile Asn Ala Pro Pro Tyr Thr Pro Ala Ser Gly 660 665 670 Phe Thr Lys Ala Ala Gln Ser Phe Gly Gln Pro Ser Asn Ala Ser Asp 675 680 685 Asn Leu Tyr Pro Ser Asp Ile Glu Arg Val Pro Leu Tyr Ile Tyr Pro 690 695 700 Trp Leu Asn Ser Thr Asp Leu Lys Ala Ser Ala Asn Asp Pro Asp Tyr 705 710 715 720 Gly Leu Pro Thr Glu Lys Tyr Val Pro Pro Asn Ala Thr Asn Gly Asp 725 730 735 Pro Gln Pro Ile Asp Pro Ala Gly Gly Ala Pro Gly Gly Asn Pro Ser 740 745 750 Leu Tyr Glu Pro Val Ala Arg Val Thr Thr Ile Ile Thr Asn Thr Gly 755 760 765 Lys Val Thr Gly Asp Glu Val Pro Gln Leu Tyr Val Ser Leu Gly Gly 770 775 780 Pro Asp Asp Ala Pro Lys Val Leu Arg Gly Phe Asp Arg Ile Thr Leu 785 790 795 800 Ala Pro Gly Gln Gln Tyr Leu Trp Thr Thr Thr Leu Thr Arg Arg Asp 805 810 815 Ile Ser Asn Trp Asp Pro Val Thr Gln Asn Trp Val Val Thr Asn Tyr 820 825 830 Thr Lys Thr Ile Tyr Val Gly Asn Ser Ser Arg Asn Leu Pro Leu Gln 835 840 845 Ala Pro Leu Lys Pro Tyr Pro Gly Ile 850 855 45843PRTThermoascus aurentiacus 45Met Arg Leu Gly Trp Leu Glu Leu Ala Val Ala Ala Ala Ala Thr Val 1 5 10 15 Ala Ser Ala Lys Asp Asp Leu Ala Tyr Ser Pro Pro Phe Tyr Pro Ser 20 25 30 Pro Trp Met Asn Gly Asn Gly Glu Trp Ala Glu Ala Tyr Arg Arg Ala 35 40 45 Val Asp Phe Val Ser Gln Leu Thr Leu Ala Glu Lys Val Asn Leu Thr 50 55 60 Thr Gly Val Gly Trp Met Gln Glu Lys Cys Val Gly Glu Thr Gly Ser 65 70 75 80 Ile Pro Arg Leu Gly Phe Arg Gly Leu Cys Leu Gln Asp Ser Pro Leu 85 90 95 Gly Val Arg Phe Ala Asp Tyr Val Ser Ala Phe Pro Ala Gly Val Asn 100 105 110 Val Ala Ala Thr Trp Asp Lys Asn Leu Ala Tyr Leu Arg Gly Lys Ala 115 120 125 Met Gly Glu Glu His Arg Gly Lys Gly Val Asp Val Gln Leu Gly Pro 130 135 140 Val Ala Gly Pro Leu Gly Arg His Pro Asp Gly Gly Arg Asn Trp Glu 145 150 155 160 Gly Phe Ser Pro Asp Pro Val Leu Thr Gly Val Leu Met Ala Glu Thr 165 170 175 Ile Lys Gly Ile Gln Asp Ala Gly Val Ile Ala Cys Ala Lys His Phe 180 185 190 Ile Gly Asn Glu Met Glu His Phe Arg Gln Ala Gly Glu Ala Val Gly 195 200 205 Tyr Gly Phe Asp Ile Thr Glu Ser Val Ser Ser Asn Ile Asp Asp Lys 210 215 220 Thr Leu His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala 225 230 235 240 Gly Val Gly Ser Phe Met Cys Ser Tyr Asn Gln Val Asn Asn Ser Tyr 245 250 255 Ser Cys Ser Asn Ser Tyr Leu Leu Asn Lys Leu Leu Lys Ser Glu Leu 260 265 270 Asp Phe Gln Gly Phe Val Met Ser Asp Trp Gly Ala His His Ser Gly 275 280 285 Val Gly Ala Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Thr 290 295 300 Ala Phe Gly Thr Gly Lys Ser Phe Trp Gly Thr Asn Leu Thr Ile Ala 305 310 315 320 Val Leu Asn Gly Thr Val Pro Glu Trp Arg Val Asp Asp Met Ala Val 325 330 335 Arg Ile Met Ala Ala Phe Tyr Lys Val Gly Arg Asp Arg Tyr Gln Val 340 345 350 Pro Val Asn Phe Asp Ser Trp Thr Lys Asp Glu Tyr Gly Tyr Glu His 355 360 365 Ala Leu Val Gly Gln Asn Tyr Val Lys Val Asn Asp Lys Val Asp Val 370 375 380 Arg Ala Asp His Ala Asp Ile Ile Arg Gln Ile Gly Ser Ala Ser Val 385 390 395 400 Val Leu Leu Lys Asn Asp Gly Gly Leu Pro Leu Thr Gly Tyr Glu Lys 405 410 415 Phe Thr Gly Val Phe Gly Glu Asp Ala Gly Ser Asn Arg Trp Gly Ala 420 425 430 Asp Gly Cys Ser Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met Gly 435 440 445 Trp Gly Ser Gly Thr Ala Asp Phe Pro Tyr Leu Val Thr Pro Glu Gln 450 455 460 Ala Ile Gln Asn Glu Ile Leu Ser Lys Gly Lys Gly Leu Asp Ser Val 465 470 475 480 Ser Ile Val Phe Val Asn Ala Asp Ser Gly Glu Gly Tyr Ile Asn Val 485 490 495 Asp Gly Asn Glu Gly Asp Arg Lys Asn Leu Thr Leu Trp Lys Gly Gly 500 505 510 Glu Glu Val Ile Lys Thr Val Ala Ala Asn Cys Asn Asn Thr Ile Val 515 520 525 Val Met His Thr Val Gly Pro Val Leu Ile Asp Glu Trp Tyr Asp Asn 530 535 540 Pro Asn Val Thr Ala Ile Val Trp Ala Gly Leu Pro Gly Gln Glu Ser 545 550 555 560 Gly Asn Ser Leu Val Asp Val Leu Tyr Gly Arg Val Ser Pro Gly Gly 565 570 575 Lys Thr Pro Phe Thr Trp Gly Lys Thr Arg Glu Ser Tyr Gly Ala Pro 580 585 590 Leu Leu Thr Lys Pro Asn Asn Gly Lys Gly Ala Pro Gln Asp Asp Phe 595 600 605 Thr Glu Gly Val Phe Ile Asp Tyr Arg Arg Phe Asp Lys Tyr Asn Glu 610 615 620 Thr Pro Ile Tyr Glu Phe Gly Phe Gly Leu Ser Tyr Thr Thr Phe Glu 625 630 635 640 Tyr Ser Asn Ile Tyr Val Gln Pro Leu Asn Ala Arg Pro Tyr Thr Pro 645 650 655 Ala Ser Gly Ser Thr Lys Ala Ala Pro Thr Phe Gly Asn Ile Ser Thr 660 665 670 Asp Tyr Ala Asp Tyr Leu Tyr Pro Glu Asp Ile His Lys Val Pro Leu 675 680 685 Tyr Ile Tyr Pro Trp Leu Asn Thr Thr Asp Pro Glu Glu Val Leu Arg 690 695 700 Arg Ser Arg Leu Thr Glu Met Lys Ala Glu Asp Tyr Ile Pro Ser Gly 705 710 715 720 Ala Thr Asp Gly Ser Pro Gln Pro Ile Leu Pro Ala Gly Gly Ala Pro 725 730 735 Gly Gly Asn Pro Gly Leu Tyr Asp Glu Met Tyr Arg Val Ser Ala Ile 740 745 750 Ile Thr Asn Thr Gly Asn Val Val Gly Asp Glu Val Pro Gln Leu Tyr 755 760 765 Val Ser Leu Gly Gly Pro Asp Asp Pro Lys Val Val Leu Arg Asn Phe 770 775 780 Asp Arg Ile Thr Leu His Pro Gly Gln Gln Thr Met Trp Thr Thr Thr 785 790 795 800 Leu Thr Arg Arg Asp Ile Ser Asn Trp Asp Pro Ala Ser Gln Asn Trp 805 810 815 Val Val Thr Lys Tyr Pro Lys Thr Val Tyr Ile Gly Ser Ser Ser Arg 820 825 830 Lys Leu His Leu Gln Ala Pro Leu Pro Pro Tyr 835 840

Claims

1. A beta-glucosidase 1 (BGL1) variant having at least two improved activities over wild type BGL1 selected from the group consisting of: (a) pre-treated corn stover (PCS) hydrolysis activity, (b) cellobiase activity, (c) protein expression, (d) beta-glucosidase activity measured by a cellobiase activity in the presence of ammonia pretreated corncob (CC) or by a CC hydrolysis activity, (e) thermostability, (f) phosphoric acid swollen cellulose (PASO) hydrolysis activity, and (g) hydrolytic activity in the presence of glucose, wherein the BGL1 variant is any variant as shown in Tables 4-8, 3-2, 4-2 and 4-3.

2. A BGL1 variant according to claim 1 having improved (b) and (d) activities over wild type BGL1, wherein the BGL1 variant is L266A, I567E, S283F, S283P, T258E, T258I, T258K, T258Q, P536T, P536W, I532Y, Y530T, P607D, Q406M, Q406S, V602T, G300M, A630S, A630T, T180H, T180M, A450M, I444E, I444F, I444N, I444W, I444Y, V500Q, A633I, S482P, A667V, A485L, A485W, Y678R, V603G, L266C, I567F, S624P, P607L, G606I, G606K, G606L, G606M, G606Q, G606V, G605E, I444V, A633V, A655W, Y678H, V522Y, G554F, L266N, F556L, S550I, S550T, S550V, T258L, P536I, P536V, F392R, S624G, S624N, S624Q, S624T, A601M, A630V, N463S, A450F, A450T, A450V, A450W, I486V, S482I, Y678I, G427F, D564T, Q684C, Q684G, Y530S, Q684N, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, F260D, F260G, F260Q, P607G, N400S, F260W, Y530F, Q406D, G605C, N263T, P607I, A450P, T242H, A630Y, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, F260L, L293F, A633C, S312C, or N455D; or having improved (b) and (e) activities over wild type BGL1, wherein the BGL1 variant is P607H, T011E, T011Y, N146E, I567V, N566G, A630K, Y639K, Q245N, K320Y, A347Y, P536Q, N369E, N369W, N369Y, N146A, N146Q, P607K, N369T, A655N, I671K, F260T, P607S, F260D, F260G, F260Q, P607G, N400S, P607F, P607I, A450P, T242H, T568E, A630Y, A655D, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, F260L, or L293F; or having improved (b) and (f) activities over wild type BGL1, wherein the BGL1 variant is N261C, T258C, F392Q, S624E, P607C, P604M, A377Q, N461A, N461F, N461P, T436A, T436C, T436F, T436I, T436M, T436Q, T436Y, Q220C, A655L, T646H, Y678F, A468I, D177M, P661E, L266N, F556L, S550I, S550T, S550V, T258L, P536I, P536V, F392R, S624G, S624N, S624Q, S624T, A601M, A630V, N463S, A450F, A450T, A450V, A450W, I486V, S482I, Y678I, G427F, D564T, Q684C, Q684G, N566H, F556V, P604Y, L293V, A630G, N461C, N463T, D457C, Q220M, T221A, T221G, T221I, A655R, A468F, A468S, Q216I, D564V, P536Q, N369E, N369W, N369Y, T436E, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, F260W, Y530F, N461V, I671C, K206A, A450P, T242H, E170F, S507G, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, L293F, A633C, S312C or N455D; or having improved (a) and (b) activities over wild type BGL1, wherein the BGL1 variant is I567Q, A565F, A565K, A565Q, A565V, F556E, F260I, P607E, G605R, G300C, A377C, A377D, S308C, N146H, N146S, A655C, A655G, P176L, T209I, L266C, I567F, S624P, P607L, G606I, G606K, G606L, G606M, G606Q, G606V, G605E, I444V, A633V, A655W, Y678H, V522Y, G554F, N566H, F556V, P604Y, L293V, A630G, N461C, N463T, D457C, Q220M, T221A, T221G, T221I, A655R, A468F, A468S, Q216I, D564V, A565C, A655N, I671K, F260T, P607S, Y639V, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, F260D, F260G, F260Q, P607G, N400S, P607F, Q406D, G605C, N263T, N461V I671C, K206A, T568E, E170F, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, F260L, A633C, S312C, or N455D; or having improved (b) and (c) activities over wild type BGL1, wherein the BGL1 variant is I567K, I567R, A565E, A565S, A565Y, F392Y, Q406H, Q406T, P604C, N038F, T568A, N461G, Y639L, Y639M, T243A, T243C, Q245H, Q245M, Q245T, T646A, T646C, I671F, I671L, I567V, N566G, A630K, Y639K, Q245N, K320Y, A347Y, A565C, Y639G, Y530F, N461V, I671C, K206A, T568E, A630Y, A655D, S507G, F260E, T568K, or F260L.

3-6. (canceled)

7. A BGL1 variant according to claim 1 having improved (a) and (d) activities over wild type BGL1, wherein the BGL1 variant is I567S, G606E, G606H, G606N, G606S, L293A, S308R, I444C, M201D, R542N, L266C, I567F, S624P, P607L, G606I, G606K, G606L, G606M, G606Q, G606V, G605E, I444V, A633V, A655W, Y678H, V522Y, G554F, N566F, L293M, Q220P, S692L, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, F260D, F260G, F260Q, P607G, N400S, Q406D, G605C, N263T, S308E, A338D, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, F260L, A633C, S312C, or N455D; or having improved (e) and (q) activities over wild type BGL1, wherein the BGL1 variant is L266F, I567Y, A270R, S384C, A630W, E128R, N146M, N146V, N146W, L181F, V043C, Y639P, S507F, Q245P, G662C, A630H, V466T, N146A, N146Q, P607K, N369T, S384E, L181M, V043A, V043G, V043N, Q060D, A655Y, T242S, S474D, P607F, A630Y, S308E, A655D, or L293F; or having improved (c) and (e) activities over wild type BGL1, wherein the BGL1 variant is selected from the group consisting of: N261E, N261K, N400A, V602K, L293I, N461S, D457A, V043Q, Q303N, K320S, G662D, F260A, S474R, I567V, N566G, A630K, Y639K, Q245N, K320Y, A347Y, A601D, S384E, L181M, V043A, V043G, V043N, Q060D, A655Y, T242S, S474D, D564T, T568E, A655D, A338D, F260E, T568K, or F260L; or having improved (a) and (f) activities over wild type BGL1, wherein the BGL1 variant is N566L, N566P, N566W, A270K, A270N, F556H, F556K, P604N, N461D, N463E, K206G, A468Q, A468Y, N566F, N566H, F556V, P604Y, L293V, A630G, N461C, N463T, D457C, Q220M, T221A, T221G, T221I, A655R, A468F, A468S, Q216I, D564V, A468T, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, N461V I671C, K206A, E170F, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, A633C, S312C, or N455D.

8-10. (canceled)

11. A BGL1 variant according to claim 1 having improved (a) and (c) activities over wild type BGL1, wherein the BGL1 variant is S283D, A270D, N146Y, F260A, S474R, A565C, K206S, D564T, N461V|I671C, K206A, T568E, A338D, F260E, T568K, or F260L; or having improved (a) and (e) activities over wild type BGL1, wherein the BGL1 variant is F556G, F260S, P604E, P604V, N146D, Y639T, T221C, N473S, N583R, R645G, G662Y, F260A, S474R, A655N, I671K, F260T, P607S, S692L, D564T, F260D, F260G, F260Q, P607G, N400S, P607F, T568E, S308E, A338D, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, or F260L; or having improved (c) and (d) activities over wild type BGL1, wherein the BGL1 variant is D259S, T243V, Y530F, A338D, or F260L; or having improved (d) and (e) activities over wild type BGL1, wherein the BGL1 variant is S550Q, P607R, N400Q, V602F, A601G, A601L, L293K, Y575C, Y575R, A450Q, I486C, I486Y, A655S, Q245F, D329A, P536G, P607Q, A655Q, Y575A, Y575K, A630H, V466T, S692L, F260D, F260G, F260Q, P607G, N400S, P607I, A450P, T242H, S308E, A630Y, A338D, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, F260L, or L293F; or having improved (d) and (f) activities over wild type BGL1, wherein the BGL1 variant is P536F, F392C, S624L, S624R, S624W, I486F, I486W, A667G, A667S, L266N, F556L, S550I, S550T, S550V, T258L, P536I, P536V, F392R, S624G, S624N, S624Q, S624T, A601M, A630V, N463S, A450F, A450T, A450V, A450W, I486V, S482I, Y678I, G427F, D564T, Q684C, Q684G, N566F, Y575A, Y575K, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, F260W, P607I, A450P, T242H, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, L293F, A633C, S312C, or N455D.

12-15. (canceled)

16. A BGL1 variant according to claim 1 having improved (c) and (g) activities over wild type BGL1, wherein the BGL1 variant is S384G, S384W, N038E, N038M, N038P, V043H, V043W, Y068E, Y068G, Y068M, L110C, L110G, L110Q, L110W, A655H, N264L, S384E, L181M, V043A, V043G, V043N, Q060D, A655Y, T242S, S474D, Y639G, K206S, A655D, or S507G; or having improved (d) and (q) activities over wild type BGL1, wherein the BGL1 variant is G606D, Y068V, L293M, Q220P, A630H, V466T, Y530S, Q684N, F260W, Q406D, G605C, N263T, S308E, A630Y, L293F, A633C, S312C, or N455D; or having improved (b) and (g) activities over wild type BGL1, wherein the BGL1 variant is A377I, N461Y, N146A, N146Q, P607K, N369T, T436E, Y639G, Y530S, Q684N, Y639V, F260W, P607F, Q406D, G605C, N263T, A630Y, A655D, E170F, S507G, L293F, A633C, S312C, or N455D; or having improved (c) and (f) activities over wild type BGL1, wherein the BGL1 variant is K206D, A601D, Y530F, N461V I671C, K206A, S507G, F260E, or T568K; or having improved (e) and (f) activities over wild type BGL1, wherein the BGL1 variant is A468G, P536Q, N369E, N369W, N369Y, A601D, Y575A, Y575K, P607I, A450P, T242H, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, or L293F; or having improved (a) and (a) activities over wild type BGL1, wherein the BGL1 variant is R179V, L293M, Q220P, K206S, A468T, Y639V, P607F, Q406D, G605C, N263T, S308E, E170F, A633C, S312C, or N455D.

17-21. (canceled)

22. A BGL1 variant according to claim 1 having improved activities selected from any two or all three of (a), (b), and (d) over wild type BGL1, wherein the BGL1 variant is L266C, I567F, S624P, P607L, G606I, G606K, G606L, G606M, G606Q, G606V, G605E, I444V, A633V, A655W, Y678H, V522Y, G554F, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468G, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, F260D, F260G, F260Q, P607G, N400S, Q406D, G605C, N263T, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, F260L, A633C, S312C, or N455D; or having improved activities selected from any two or all three of (b), (d), and (f) over wild type BGL1, wherein the BGL1 variant is L266N, F556L, S550I, S550T, S550V, T258L, P536I, P536V, F392R, S624G, S624N, S624Q, S624T, A601M, A630V, N463S, A450F, A450T, A450V, A450W, I486V, S482I, Y678I, G427F, D564T, Q684C, Q684G, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468G, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, F260W, Y530F, P607I, A450P, T242H, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, A633C, S312C, N455D, or L293F; or having improved activities selected from any two or all three of (a), (c), and (e) over wild type BGL1, wherein the BGL1 variant is F260A, S474R, D564T, T568E, A338D, F260E, T568K, or F260L; or having improved activities selected from any two or all three of (b), (c), and (e) over wild type BGL1, wherein the BGL1 variant is I567V, N566G, A630K, Y639K, Q245N, K320Y, A347Y, T568E, A655D, F260E, T568K, or F260L; or having improved activities selected from any two or all three of (a), (d), and (f) over wild type BGL1, wherein the BGL1 variant is N566F, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, A633C, S312C, or N455D.

23-26. (canceled)

27. A BGL1 variant according to claim 1 having improved activities selected from any two or all three of (a), (b), and (f) over wild type BGL1, wherein the BGL1 variant is N566H, F556V, P604Y, L293V, A630G, N461C, N463T, D457C, Q220M, T221A, T221G, T221I, A655R, A468F, A468S, Q216I, D564V, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683W, N461V, I671C, K206A, E170F, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, A633C, S312C, or N455D; or having improved activities selected from any two or all three of (a), (b), and (c) over wild type BGL1, wherein the BGL1 variant is A565C, N461V, I671C, K206A, T568E, F260E, T568K, or F260L; or having improved activities selected from any two or all three of (b), (d), and (e) over wild type BGL1, wherein the BGL1 variant is P536G, P607Q, A655Q, F260D, F260G, F260Q, P607G, N400S, P607I, A450P, T242H, A630Y, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, F260L, or L293F; or having improved activities selected from any two or all three of (b), (e), and (f) over wild type BGL1, wherein the BGL1 variant is P536Q, N369E, N369W, N369Y, P607I, A450P, T242H, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, or L293F; or having improved activities selected from any two or all three of (c), (e), and (f) over wild type BGL1, wherein the BGL1 variant is A601D, F260E or T568K.

28-31. (canceled)

32. A BGL1 variant according to claim 1 having improved activities selected from any two or all three of (a), (d), and (g) over wild type BGL1, wherein the BGL1 variant is L293M, Q220P, Q406D, G605C, N263T, S308E, A633C, S312C, or N455D; or having improved activities selected from any two or all three of (d), (e), and (f) over wild type BGL1, wherein the BGL1 variant is Y575A, Y575K, P607I, A450P, T242H, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, or L293F; or having improved activities selected from any two or all three of (d), (e), and (g) over wild type BGL1, wherein the BGL1 variant is A630H, V466T, S308E, A630Y, or L293F; or having improved activities selected from any two or all three of (b), (e), and (g) over wild type BGL1, wherein the BGL1 variant is N146A, N146Q, P607K, N369T, P607F, A630Y, A655D, or L293F; or having improved activities selected from any two or all three of (c), (e), and (g) over wild type BGL1, wherein the BGL1 variant is S384E, L181M, V043A, V043G, V043N, Q060D, A655Y, T242S, S474D, or A655D; or having improved activities selected from any two or all three of (b), (f), and (g) over wild type BGL1, wherein the BGL1 variant is T436E, F260W, E170F, S507G, L293F, A633C, S312C, or N455D; or having improved activities selected from any two or all three of (b), (c), and (g) over wild type BGL1, wherein the BGL1 variant is Y639G, P607F, A655D, or S507G.

33-38. (canceled)

39. A BGL1 variant according to claim 1 having improved activities selected from any two or all three of (a), (b), and (e) over wild type BGL1, wherein the BGL1 variant is A655N, I671K, F260T, P607S, F260D, F260G, F260Q, P607G, N400S, P607F, T568E, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, or F260L; or having improved activities selected from any two or all three of (a), (c), and (g) over wild type BGL1, wherein the BGL1 variant is K206S or P607F; or having improved activities selected from any two or all three of (b), (d), and (g) over wild type BGL1, wherein the BGL1 variant is Y530S, Q684N, F260W, Q406D, G605C, N263T, A630Y, L293F, A633C, S312C, or N455D; or having improved activities selected from any two or all three of (a), (f), and (g) over wild type BGL1, wherein the BGL1 variant is A468T, E170F, A633C, S312C, or N455D; or having improved activities selected from any two or all three of (a), (d), and (e) over wild type BGL1, wherein the BGL1 variant is S692L, F260D, F260G, F260Q, P607G, N400S, S308E, A338D, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, or F260L; or having improved activities selected from any two or all three of (a), (b), and (q) over wild type BGL1, wherein the BGL1 variant is Y639V; or having improved activities selected from any two, any three, or all four of (a), (b), (d) and (f) over wild type BGL1, wherein the BGL1 variant is A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, or S683W; or having improved activities selected from any two, any three, or all four of (a), (b), (d) and (e) over wild type BGL1, wherein the BGL1 variant is F260D, F260G, F260Q, P607G, or N400S.

40-46. (canceled)

47. A BGL1 variant according to claim 1 having improved activities selected from any two, any three, or all four of (b), (d), (f) and (g) over wild type BGL1, wherein the BGL1 variant is F260W, L293F, A633C, S312C, or N455D; or having improved activities selected from any two, any three, or all four of (b), (c), (d) and (f) over wild type BGL1, wherein the BGL1 variant is Y530F; or having improved activities selected from any two, any three, or all four of (a), (b), (e) and (q) over wild type BGL1, wherein the BGL1 variant is P607F; or having improved activities selected from any two, any three, or all four of (a), (b), (d) and (q) over wild type BGL1, wherein the BGL1 variant is Q406D, G605C, N263T, A633C, S312C, or N455D; or having improved activities selected from any two, any three, or all four of (a), (b), (c) and (f) over wild type BGL1, wherein the BGL1 variant is N461V, I671C, K206A, F260E or T568K.

48-51. (canceled)

52. A BGL1 variant according to claim 1 having improved activities selected from any two, any three, or all four of (b), (d), (e) and (f) over wild type BGL1, wherein the BGL1 variant is P607I, A450P, T242H, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, or L293F; or having improved activities selected from any two, any three, or all four of (a), (b), (c) and (e) over wild type BGL1, wherein the BGL1 variant is T568E, F260E, T568K, or F260L; or having improved activities selected from any two, any three, or all four of (a), (d), (e) and (a) over wild type BGL1, wherein the BGL1 variant is S308E; or having improved activities selected from any two, any three, or all four of (b), (d), (e) and (q) over wild type BGL1, wherein the BGL1 variant is A630Y or L293F; or having improved activities selected from any two, any three, or all four of (b), (c), (e) and (q) over wild type BGL1, wherein the BGL1 variant is A655D; or having improved activities selected from any two, any three, or all four of (a), (b), (f) and (g) over wild type BGL1, wherein the BGL1 variant is E170F, A633C, S312C, or N455D.

53-57. (canceled)

58. A BGL1 variant according to claim 1 having improved activities selected from any two, any three, or all four of (a), (c), (d) and (e) over wild type BGL1, wherein the BGL1 variant is A338D or F260L; or having improved activities selected from any two, any three, or all four of (b), (c), (f) and (a) over wild type BGL1, wherein the BGL1 variant is S507G; or having improved activities selected from any two, any three, any four, or all five of (a), (b), (c), (e) and (f) over wild type BGL1, wherein the BGL1 variant is F260E or T568K; or having improved activities selected from any two, any three, any four, or all five of (a), (b), (d), (e) and (f) over wild type BGL1, wherein the BGL1 variant is P536C, A630Q, D215S, G372A, G547A, F611A, G662C, or G662F.

59-61. (canceled)

62. A BGL1 variant according to claim 1 having improved activities selected from any two, any three, any four, or all five of (a), (b), (c), (d) and (e) over wild type BGL1, wherein the BGL1 variant is F260L; or having improved activities selected from any two, any three, any four, or all five of (b), (d), (e), (f) and (g) over wild type BGL1, wherein the BGL1 variant is L293F; or having improved activities selected from any two, any three, any four, or all five of (a), (b), (d), (f) and (g) over wild type BGL1, wherein the BGL1 variant is A633C, S312C, or N455D.

65-100. (canceled)

101. A composition comprising a BGL1 variant of claim 1.

102. The composition of claim 101 wherein the composition is enriched in the BGL1 variant.

103-105. (canceled)

106. A host cell comprising a nucleic acid encoding the BGL1 variant of claim 1.

107. A method for producing a BGL1 variant, comprising culturing the host cell of claim 106 in a culture medium under suitable conditions to produce the variant.

108. A method of converting biomass to sugars comprising contacting the biomass with the BGL1 variant of claim 1.