# Patent

## Title
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.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is 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 which are hereby incorporated by reference in their entirety.


STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
This invention was made with government support under Condition Award No: De-Fc36-08go18078 awarded by the Department of Energy. The government has certain rights in this invention.


SEQUENCE LISTING
The sequence listing submitted via EFS, in compliance with 37 C.F.R. §1.52(e), is incorporated herein by reference. The sequence listing text file submitted via EFS contains the file “31435US2 SequenceListing”, created on May 17, 2012, which is 315,033 bytes in size.
FIELD OF THE DISCLOSURE
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
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., 17: 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).
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 glycosides (Freer, J. Biol. Chem., 268; 9337-9342, 1993).
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, suck as Saccharomyces cerevisiae, lack the ability to hydrolyze cellulose (see, e.g., Wood et al., Methods in Enzymology, 160: 87-116, 1988).
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 knows 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.
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-types cellulases synergistically interact to efficiently degrade cellulose.
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
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.
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 expansion (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.
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 variants is L266A, I567E, S283F, S283P, T258E, T258I, T258K, T258Q, P536T, P536W, I532Y, Y503T, P607D, Q406M, Q406S, V602T, G300M, A630S, A630T, T180H, T180M, A450M, I444E, I444F, I444N, I444W, I444Y, V500Q, A633, S428P, A667V, A485L, A485W, Y678R, V603G, L266C, I567F, S624P, P607L, G606I, G606K, G606L, G606M, G606Q, G606V, G605E, I444V, A633V, A655W, Y678H, V522Y, G544F, L266N, F556L, S550I, S550T, S550V, T258I, P536I, P536V, F392R, S624G, S624N, S624Q, S624T, A601M, A630V, N463S, A450F, A450T, A450V, A450W, I486V, S282I, 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.
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, N369Y, N146A, N146Q, P607K, N369T, A655N, P671K, 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.
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, F436Y, 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, D547C, 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, A6667F, A667L, A667R, A667Y, A485T, V466S, Y478A, 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.
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, F206I, P607E, G605R, G300C, A377C, A377D, S308C, N146F, 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, I167K, 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, S332Y, Q316T, K345E, G427C, P661P, P661L, P666Q, T666C, S683W, F260D, F260G, F260Q, P607G, N400S, P607F, Q406D, G605C, N263T, N461V, I671C, K206A, T568E, E170F, P260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, F260L, A633C, S312C, or N455D.
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 BGL1variant 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, I167C, K206A, T368E, A630Y, A655D, S507G, F260E, T568K, or F260L.
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, G606L, 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, P260G, F260Q, P607G, N400S, Q406D, G605C, N263T, S308E, A338D, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, F260L, A633C, S312C, or N455D.
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 I266F, I567Y, A270R, S384C, A630W, E128R, N146M, N145V, N146W, I181F, V043C, Y639P, S507F, Q245P, G662C, A630H, V466T, N146A, N146Q, P607K, N369T, S384E, L181M, V043A, V043G, V043N, Q060D, A655Y, T242S, S474D, P607F, A630Y, S308E, A635D, or L293F.
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 BGL1variant is N261E, N261K, N400A, V602K, L293I, N461S, D457A, V043Q, G203N, 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.
In other aspects, the invention provides BGL1 variants, as described above and throughout this specifications 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, Q261I, D564V, A468T, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A458T, 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.
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 S233D, A270D, N146Y, F260A, S474R, A565C, K206S, D564T, N461V, I167C, K206A, T563E, A338D, P260E, T568K, or P260L.
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, G347A, F611A, G662C, G662F, or F260L.
In is other aspects, the invention provides BGL1variants, 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.
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, S692I, F260D, F260G, F260Q, P607G, N400S, P607I, A450P, T242H, S308E, A630Y, A338D, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, F260L or L293F.
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, S550T, S550V, T258L, P536I, P536V, F392R, S624G, S624N, S624Q, S624T, A601M, A630V, N463S, A450F, A450T, A450V, A450W, I486V, S482I, Y678I, G427F, D564D, 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.
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, A665H, N264L, S384E, L181M, V043A, Y043G, V043N, Q060D, A655Y, T242S, S474D, Y639G, K206S, A655D, or S507G.
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, V446T, Y530S, Q684N, F260W, Q406D, G605C, N263T, S308E, A630Y, L293F, A633C, S312C, or N455D.
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, N461Y, N146Q, P607K, N369T, T436E, Y639G, V530S, Q684N, Y639V, F260W, P607F, Q406D, G605C, N263T, A630Y, A655D, E170F, S507G, L293F, A633C, S312C, or N455D.
Is 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.
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.
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, A468T, Y639V, P607F, Q206D, G605C, N263T, S308E, 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), (b), and (d) over wild type BGL1, wherein the BGL1 variant is L266C, I567F, S624P, P607L, G606I, G606K, G606L, G606M, G606Q, G606V, G605E, I114V, A633V, A655W, Y678V, V522Y, G554E, 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, A603Q, D215S, G372A, G547A, F611A, G662C, G662F, F260L, 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 (b), (d), and (f) over wild type BGL1, wherein the BGL1 variant is L226N, F556L, S550I, S550T, S550V, T258L, P536I, P536V, F392R, S624F, 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, V446S, Y678A, Y678C, Y678Q, A468C, Q266W, Q226Y, N263C, N263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S683A, F260W, Y530F, P607I, A450P, T242H, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, A633C, S312C, N455D, or L293F.
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, S474B, D564T, T568E, A338D, F260E, T568K, or F260L.
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.
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 N566G, A565G, A270C, T258S, T258V, P536D, P536E, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A630D, N463K, N463R, A450E, S482A, A667F, A667L, A677R, 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.
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, T211I, 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.
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.
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.
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.
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.
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.
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.
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.
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.
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, Y043G, V043N, Q060D, A655Y, T242S, S474D, or A655D.
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, L273F, 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 (b), (c), and (g) over wild type BGL1, wherein the BGL1 variant is Y639G, P607F, A655D, or S507G.
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, I167K, F260T, P607S, F260D, F260G, F260Q, P607G, N400S, P607F, T568E, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, or P260L.
In other aspects, the invention provides BGL1 variant as described above and throughout this specification, having improved activities selected from any two or all three of (a), (e) 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, Q634N, 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, F661A, G662C, G662F, 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 (a), (b), and (g) over wild type BGL1, wherein the BGL1 variant is Y639V.
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 S863W.
In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected front 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.
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.
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.
In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected front 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.
In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, having improved activities selected front any two, any three, or all four of (a), (d), (e) and (g) over wild type BGL1, wherein the BGL1 variant is S308E.
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.
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 (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.
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.
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.
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, P761L|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, K343E|N369E|G372A|S683W, N369E| S683W, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, K345E|N369E|P661E|S383W, 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 (b) and (f) and the substitutions are: L167W|D177M|D225Q|Q626F|Q684G, L167W|D177M|D564V|Q684G, D215S|S312Y, E107F|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|G622C, E170F|Q226Y|N369Y|G372A|P661F, and L167W|D177M|D564T|Q626F|Q684G, K345E|N369E|G372A|S683W, N369E|S683W, K343E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|N345E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, K263C|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 (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|R263P|D370W, R179V|N238W|N264M|D370W|K636R, N264M|R265P, R265P|D370W (optionally also G662F), R179V|N264M|R265P|G369I|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|P611E|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|G316T, 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.
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|Q313T|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|V322Y|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|K423N|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 (b) and (e) and the substitutions are: K345E|N369E|K428N|P661L, Q316T|K320Y|V522Y, N369T|G372A|P661L|S683W, P176L|Q226W|K320Y|R363E, P176L|K320S|V552Y|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|N345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372C|N263C|K345E|N369E|G372A|P661E, or P176|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 (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.
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|Q626R|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.
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|Q303E|A338I, Q316T|K320Y|G662F, L167W|D117M|D564T|Q626F|Q684N, L167W|Q626F|Q684D, L167W|D177M|D564T|Q684R, L167W|D177M|D225Q|Q684D, R179V|R265P|N369I, Q316T|K320Y|R363E|V552Y|G662F, N238F|N264M|R265M|N369I, N238W|R265P|K656R, N264M|R265P, (optionally also G662F), N264I|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.
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|V552Y, 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|G373A|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|V552Y, P176L|Q226W|K320Y|R363E|V552Y, 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|G666F, 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.
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|K320|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.
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, K343E|N369E|P661I, 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.
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, N263W, 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), (c), and (d) and the substitutions are: D176M|D225Q|D564V|Q684G, D178N|N264K|A338D|S474R|G662K, L167W|D177M|Q626F, L167W|D177M|D225Q|D564V|Q684G, D177M|D225Q|D564T|Q684N, D177M|Q626F|Q684R, N233W|R265P|K656R, N264M|R265P (optionally also G662F). N264F|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|K363E|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|Y603G, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, K345E|N369E|P661E|S683W, K345E|F661E|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 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, G372A| K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|Q226W|G347A|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|Y603G, 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: K228N|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|P611E|S683W, K320S|R363E, E170F|Q226Y| Y242S|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|V552Y|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|G672C|P611E, or P176L|Q226W|G547A|G662C.
In other aspects, the invention provides BGL1 variants, as described above and throughout wherein the improved activities over wild type BGL1 are selected from any two or all 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|G683W, 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 (a), (c), (d), and (g) and the substitutions are: N238W|R263P|K656R, N264M|R265P (optionally also G662F), N264L|A338I|S474R|G662DE170F|V603G, G372A|P661E|S683W, P176L|G316T|K320S|R363E|G662F, N263C|N369T|G372A|K428N|S633W, 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|G683W, P176L|Q316T|K320S|R363E|G662F, N263C|G369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|G369E|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|G684N, K345E|N369T|G372A|P661E|S683W, K320S|R363E, E170F|Q226Y|T242S|S312Y|G372A| V603G|P661F|T666C, T242S|T666C, Q226Y|T666C, Q216E|G312K, 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|G683W, N369E|S683W, K345E|N369E|P661E|S683W, K345E|P661E|G683W, N263C|K343E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|G683W, N263C|G372A, G263C|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 (d) and the substitutions are: K345E|N369E|G372A|P661E, N369T|P661L|S683W, K345E|N369T|G372A|P661E|S683W, K320S|R363E, E170F|Q226Y|T242S|G312Y|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|K363E, Q316T|K320Y|R363E|G662F, Q226W|Q316T|R363E|V522Y|G662F, P176L|K320S|E363E|G662C, R363E|G547A|G662C, Q226W|K320S|G662C, P176L|Q226W|Q316T|R320Y|R363E|G662F, P176L|Q226W|Q316T|K320S|G662F, P176L|Q316T|K320S|R363E|V522Y|G662C, K320Y|R363E|G662C, K343E|N369E|P661L, G372A|P661E|S683W, P176L|Q316T|K320S|R363E| G662F, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K343E|N369E, N263C|N369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|G683W, N263C|G372A, N263C|K343E|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), (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|R428N|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, N263|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 (e) and the substitutions are: N369T|G372A|P661L|S683W, P176L|Q226W|K320Y|R363E, K345E|N369E|P661L, K345E|N369E|G372A|S683W, N369E|G683W, G372A|P661E|G683W, 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|V552Y|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.
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|T242S|T666C, Q226Y|T666C, Q216E|S312K|S692K, P176L|G662F, P176L|Q226W|Q316T|K320Y|R363E, P176L|Q226W|K320S|R363E|G662F, P176L|Q226W|Q316T|K320Y|V552Y, P176L|Q226W|K320Y|R363E|V552Y, 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.
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|G177M|D564T|Q626F|Q684G, 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), (d), (d), (e), and (g) and the substitutions are: L177W|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, any three, any four, or all five of (a), (b), (c), (e), and (g) and the substitutions are: K345E|N369E|G372A|S683W, N369E|S683W, N263C|N369T, N369T|G372A|K428N|S648W, 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), (b), (c), (d), (e) and (g) 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, or all five or (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|G683W, 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 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.
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 insults in a beta-glucosidase variant with improvements in one or more of expression, activity and stability, in comparison to the reference BGL1.
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, 166, 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 patent beta-glucosidase selected from the group consisting of Hypocrea jecorina BGL1 (TrireBGL1), Hansenula anomala BGL (HananBglu), Piromyces sp BGL (PirspBglu), Coccidiodies immitis BGL (CocimBglu), Saccharomycopsis fibuligera BGL2 (SacfiBglu2), Saccharomycopsis fibuligera BGL1 (SacfiBglu1), Septoria lycopersici BGL (SeplyBgfu), 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 BGL1 (AsporBGL1), Aspergillus aculeatus BGL1 (AspacBGL1), Aspergillus niger BGL (AspniBGL), Talaromyces emersonni 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% of 99%) identical, so 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 comprise from one to fifty-nine of the conserved residues selected from the group consisting of A16, K28, G44, C58, D61, R67, E100, G105, L110, P124, G124, R125, E128, D133, P134, L136, G147, Q149, K158, H159, R169, S173, D178, P188, P189, 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.
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; S033 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, I052 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, D, 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, L, K, 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, V, 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, I, M, N, Q, S, V, or W; Q216 to A, C, D, E, 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, 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, L, K, M, 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, 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, M, N, S, V, W, or V; 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, MM, N, Q, R, T, V, W, or Y; R313 to A, C, D, E, G, K, T, 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 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, to 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, N, R, S, T, V, W, or Y; D370E, 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, D, 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; T441 to D, F, E, 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, to 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, to 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 so 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 S to A, E, F, G, I, L, M, N, Q, R, S, T, V, W, or Y; S692 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.
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, W037G, 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, E169K, 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, P236A, 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, T282G, T282N, T282R, R282V, 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, R335S, K335T, A338D, A338E, A338G, A338N, A338R, A347Y, R363A, R363G, R363K, R363M, R363V, D370E, D370Q, K371A, E371H, D374A, Y396A, D397N, I399L, S420A, S420D, G427E, G427S, K428A, E441A, E441C, E441D, E441G, V446A, E447A, E447N, G448A. G448D, G448E, 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, R560H, N583D, R586D, V603L, V603M, V603Q, V603S, Q612D, Q612G, Q612K, Q612V, A622L, A622W, A622Y, Q626I, Q626L, Q626T, Q626V, T538D, S642D, A643M, K649A, or R649W; 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).
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, L025V, L025Y, Q026C, Q026D, Q026E, L026G, 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, Y035H, Y035K, V035L, V035N, V035P, V035Q, V035R, V035T, V035Y, G036C, G036D, G036E, G036K, G036N, G036R, G036S, W037M, W037V, S050A, G050C, S050F, S050G, G050I, G050K, 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, E093H, R093K, R093L, R093M, R093Q, R093S, R093T, R093V, R093W, E093A, 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, E167E, L167F, L167G, L167M, L167N, L167Q, E167R, L167S, E167V, E167Y, N168A, N168D, N168E, N168G, N168H, N168Q, N168R, N168T, N168Y, R169F, R169H, R169Q, R169S, R169T, E170D, E170F, R170I, E170M, E170V, P176E, F176F, P176H, P176K, P176L, P176M, P176Q, P176R, P176S, F167T, P176V, P176W, P176Y, D177A, D177E, D177F, D177H, D177I, D177M, D177Q, D177R, D177V, D177W, D177Y, D178C, D178K, D178N, D178Q178R, D178S, D178Y, R179A, R179C, R179G, R179I, R179K, R179S, R179T, R179V, R179W, Q194C, Q194E, Q194F, Q194G, Q194H, Q194K, Q194I, Q194M, Q194R, Q194T, Q194W, N196E, N196G, N196H, N196L, N196M, N196P, N196Q, R196R, N196T, S199G, S199N, S199T, S199V, Y204F, Y204M, N208K, N208R, T209C, T209D, T209E, T209G, T207H, T209I, T209K, T209L, T209M, T209Q, Q209R, T209S, T209V, T209W, T209Y, E214A, E214C, E214D, E214G, E214H, E214K, E214L, E214M, E214N, E214P, E214Q, E214R, E214S, E214T, E214V, F214Y, D215A, D215C, D215E, D215F, D215G, D215H, D215L, P215M, 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, G225Y, 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, D278S, S287V, D287W, D287Y, Q301A, Q301E, Q301G, Q301L, Q301N, Q301R, Q301S, Q301T, Q301V, D302A, D302C, D302E, Q302F, 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, Y301K, Y306M, Y306N, Y306P, Y306Q, Y306R, Y306S, Y306T, Y306V, S312C, S312K, S312I, S312M, S312Q, S312T, S312V, S312W, S312Y, R313A, R313C, R313G, R313K, E313I, 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, R326M, 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, R335H, R335I, K335M, R335N, 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, R344E, 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, H361D, 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, N369N, 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, M3830I, M380N, M380Q, M380S, M380T, M380V, M380Y, W382F, W382N, W382Y, Y396C, Y396D, Y396E, Y396F, Y396G, Y396H, Y396I, Y396K, Y396I, Y396M, Y396N, Y396Q, Y396R, Y396S, Y396T, Y396V, Y396W, D397A, D397C, D397E, D397F, D397H, D391I, D397K, D339L, 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, I999S, 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, T441N, T411Q, T411R, T411S, T411V, T411Y, S420C, S420G, S420H, S420K, S420N, S420Q, S420T, S420Y, R426E, R426F, R426I, E426K, 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, R428F, K428G, K428H, K428I, K428L, K428M, K428N, K428P, K428Q, K428R, K428S, K428T, K428V, K428W, K428Y, T445A, T445C, T451D, T445E, K445F, T445G, T445I, T445K, T445L, T445M, T445N, T445P, T445Q, T445R, T445S, T445V, T445Y, V446C, V446K, V446Q, V446R, E447K, E447L, E447S, E447V, E447W, E447Y, G447C, G448E, G448H, G448K, G445L, G448Q, G448V, N449A, N449C, N449E, N449F, N449G, N449H, N449K, N449L, N449M, N449P, N449R, N449T, N449V, N449W, D452N, R453A, T453L, R453M, N454F, N454G, N454K, N454L, N454M, N454R, N454S, N454T, N454V, N455A, N455C, N455D, N455E, N455F, N455G, N455H, N455H, N455L, N455M, N455S, N455T, N455V, N455W, N455Y, H460A, H460C, G460D, H460E, H460F, H460G, G460I, H460K, H460L, H460M, H460N, H460Q, H460R, H460S, H460W, G460Y, 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, Q492P, Q496S, Q496T, Q496V, V497A, Y497C, 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, Y522W, V522Y, K534C, K534D, K534E, K534F, K534G, K534H, K534I, K534N, K534R, K534S, K534T, K534V, R542C, R542E, R542F, R542G, R542H, R542I, R542K, R542I, R542M, R542N, R542P, R542Q, R542S, R524T, R542V, R542W, R542Y, G574A, 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, G534F, G554H, G554K, G554L, G554M, G554Q, G554R, G554S, G554T, G554V, G554W, L555A, L555C, L555D, L555E, E555F, E555G, L555H, L555I, L555K, L555M, L555N, L555P, L555Q, L555T, L555V, L555W, L555Y, K560A, K560C, K560E, K560G, K560I, K560L, K560M, K560N, K560P, K560Q, K560R, R560S, K560T, K560V, K560W, K560Y, H561A, H561C, H561D, H561E, H561F, H561G, H561I, H564M, H561N, H561Q, H561S, H561T, H561V, H561W, D563A, D563C, D563E, D563F, D563I, D0563L, 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, M583S, N583T, N53V, N583W, N583Y, R586E, R586F, R586G, R586I, 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, F661I, F611K, F661L, 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, Q627D, 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, R645Q, R645T, R645V, R645W, R645Y, K649C, K649F, K649I, K649L, H649M, K649Q, K649S, R649T, 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, P662R, P661S, P661Y, P622V, 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, T672F, 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, D686S, D680V, D680W, D680Y, T681A, T681K, T681L, T681M, T681N, T681Q, T681R, T681S, T681V, 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, K702K, K702L, R702M, R702N, R702Q, R702S, R702T, R702V, R702W, R702C, R705F, R705H, R705I, R705L, R705M, R705P, R705S, R705T, R705V, and R705 W, 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).
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, R062Q, R067S, R067W, R091A, R091D, R091F, R091L, R091Q, R091V, R091W, E092C, E092K, E092L, E092N, E100A, E100G, E100I, E100M, E100S, E100T, E164S, Q165C, Q165E, Q165H, Q165I, Q165L, Q651M, Q165R, Q165S, Q165T, Q165V, Q165W, Q165Y, E166D, E166K, E661L, E661P, E166R, E666T, T167A, 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, E179W, Q194A, Q194K, Q194Y, N196E, Y204F, T209C, T709D, T209E, T209G, T209H, T209I, T209K, T209L, T209M, T209Q, T209R, T209S, T209V, T209W, T209Y, E214A, E214A, E214D, E214G, E214H, E214L, E214M, E214N, E214Q, E214R, E214S, E214T, E214Y, E215E, 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, T242Y, N248A, 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, Q302A, Q302C, D302W, Q303A, Q303C, Q303E, Q303H, Q303I, Q303K, Q303L, Q303M, Q303N, Q303R, Q303S, Q303T, Q303V, Q303Y, T306C, 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, A333P, A338R, A338V, A338W, K344D, R344E, K344F, K344G, K344I, K344L, K344M, K344N, K344R, K344S, K344T, K344V, K345A, K345E, K345F, K345H, K345P, K345Q, K345R, E345S, 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, G372E, 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, A448C, 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, K571A, K581D, K584E, K581F, K581G, K581H, K581I, K581L, K584M, K581N, K581R, K581S, K581T, K581V, K581W, K581Y, N583A, N583C, N583D, N583G, N583R, N583V, N586E, 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, G626T, 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, T660L, T660W, P661C, P661D, P661F, P661L, P661L, P661S, P664V, P661W, G662A, G662C, G662F, G662H, G662T, G662W, G662Y, Q663A, Q663C, Q663D, Q663E, Q663G, Q663I, Q663K, Q663S, Q663W, T666A, T666C, T666N, R672K, R673R, R673N, R673S, R673T, D675E, D675H, D675S, D675Y, D680A, D680C, D680E, D680M, D680Q, D680R, D680V, D680Y, T681G, T661M, T681S, T681S, T681W, S683G, S683V, S683W, Q684A, Q684C, Q684G, Q684N, Q684P, K685A, K685F, K685G, K685I, K685L, K665M, K685N, K685Q, K685R, K685S, K685T, K685V, K685W, K685Y, S692C, S692E, S692H, S692I, S692K, S692L, S692M, S692N, S692P, S692Q, S692T, S692V, S692W, R702G, R705L and R703V, 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).
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, R037Y, R091A, R091D, R091E, R091F, R091G, R091H, R091I, R091K, R094L, R091N, R091Q, R091S, R091T, R091V, R091W, E092K, E092T, R093A, R093C, R930D, R093E, R093F, R093G, R093H, R093K, R093L, R093M, R093Q, R093S, R093T, R093V, R093W, E099A, E099F, E099I, E099M, E999W, E099Y, E1001I, E100K, E103L, 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, D177A, 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, C226D, 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, S227A, 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, D278F, D287H, D278I, 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, Q315F, Q316G, Q316H, Q316I, Q316K, Q316N, Q316P, Q316R, Q316S, Q316T, Q316V, Q316W, Q316Y, K320E, K320G, K320M, K320N, K320T, R342C, 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, A346K, A347K, 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, D379Y, K371A, K371D, K371F, K371G, K371L, K371S, K371T, K371W, G374W, 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, A328H, 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, G449C, N447E, N449G, G449K, 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, R498T, 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, Q554S, 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, G563Y, D564A, D564C, D564E, D564F, D564G, D564N, D564Y, R570E, R570G, R570M, R570N, R570Q, R579T, 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, R645P, R645I, R64L, 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, G662F, 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, T681I, A682D, 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, E702G, E702I, R702K, R702L, R702M, R702Q, R702S, R702V, R702W, 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).
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, N042A, N024C, N042E, N042M, L025I, L025T, Q026H, Q026K, Q026R, Q026S, V035L, V035S, V035T, W037E, W037F, W037H, W037M, W073S, 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, 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, A369W, N369Y, G372A, I399L, R402A, V410L, T411F, T411H, T441L, 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, G542E, G347K, G347L, G547P, G547R, G547V, S548C, S548E, S548F, S548H, S481I, S481L, S548M, S548V, S548W, Q554Q, H561N, D563A, D563E, N583D, N583R, R586D, V603A, V603E, V603H, V603M, V603N, V603Q, V603S, F661A, 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, G622F, G662H, G662K, G662I, G662M, G662N, G662Q, G662R, G662S, G662T, G662W, G662Y, T666A, R673W, S663V, Q684F, Q684H, Q684K, Q684L, Q684M, Q684S, Q684T, K685A, K685I, K685S, S692H, S679K, 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).
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, K024F, K024P, K024Q, N024C, N024F, N024Q, N024R, N024Y, L025A, L025D, L025F, L025G, L025K, L025N, L025Q, L025R, L025S, L025V, K025Y, 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, Y033Q, 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, E029N, 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, 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, D215I, 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, T424C, 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, T282I, 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, K344T, K344T, K544V, K345A, K345D, K345F, K345G, K345H, K345N, K345P, K345Q, K345Q, K345R, K345S, K345T, R345V, 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, Y395P, Y396G, Y396H, Y396I, Y396K, Y396L, Y396M, Y396N, Y396Q, Y396S, Y396T, Y396V, Y396W, D397A, D397C, D397E, D397P, D397Q, D397R, D397S, D397T, D397V, A398C, A398D, A398E, A393F, 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, Q402C, Q409D, Q409G, Q409H, Q409I, Q409V, V410A, V410C, V410F, V410G, V410H, V410L, V410N, V410S, V410T, V410W, V410Y, T411D, T411E, T411G, T411I, T411K, T411N, T411R, T441S, 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, K428A, K428D, K428E, K428F, K428G, K428H, R428I, K428L, K428M, K428N, K428P, K428Q, K428R, R428T, 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, N445T, N455V, N455W, N455Y, Q467A, Q467C, Q467D, Q467N, Q467S, N473A, N473C, G473E, N473G, N473H, N473K, N473L, N473M, N473P, N473Q, N473R, N473T, N473V, S474A, S474C, S474E, S474F, S474L, S474P, S474Q, N475I, G475L, N475M, N475P, N475Q, N475R, N475S, N475T, N475V, N475W, N475Y, E489N, Q490A, Q490C, Q490E, Q490F, Q490G, Q490H, Q490K, Q490L, Q490P, Q490R, Q490S, G490T, 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, R542I, G542M, R542N, R542P, R542Q, R542S, R542T, R542V, R542W, R542Y, G547C, G547E, 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, D564I, 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, R581R, 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, F661D, F611G, F611I, F611K, F611L, F611M, F611N, F611R, F611S, F611T, F611V, G611W, F611Y, Q612C, Q612F, Q612H, Q612I, Q612L, Q611M, Q611R, Q612S, Q612W, A622E, A622F, A922K, 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, R643T, R645V, R645W, R645Y, K649C, K649F, K649I, K649S, K649T, R649Y, Q650A, Q650C, Q650E, Q650F, Q650G, Q650H, Q550I, 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, Q661T, 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, Q684E, Q684E, Q684G, Q684I, Q684N, Q684P, K685E, K685F, K685G, K685I, K685M, K685N, K685Q, K685R, E685T, 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 improved thermostability (PI greater than 0.1 but less than 1).
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, G026H, Q026I, Q026K, Q026L, Q026P, Q026R, Q026S, Q026T, Q026V, Q026W, D027A, D027C, D027E, S033C, V035C, V035P, V035T, G036D, G036E, G036K, G036R, G036S, G036T, 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, D215I, 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, W226W, Q226Y, N238A, N238C, N238R, N238G, T242A, T242C, T242E, T242F, T242G, T242H, T242K, T422L, T242M, T242N, T242Q, T242R, T242S, T242V, T424W, T242Y, N248A, N248F, N248G, N248T, N248W, S249M, S249V, N263C, N263D, N263G, N263S, N263T, N264C, N276A, N276C, N276C, S277C, S277F, S277W, S277Y, N278C, N278F, N278G, N278V, Q279C, T228C, D287C, D278S, Q301G, Q301K, Q301L, D302A, D302C, D302E, D302F, D302G, D302K, D302M, D302T, Q303A, Q303C, Q303K, Q303M, Q302P, 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, K372W, 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, A393V, A398W, I399C, I399Y, R402A, R402E, R402G, R402L, R402Q, R402S, R402W, Q409G, T411D, D411E, T411F, T411G, T411H, T411I, T411K, T411L, T411N, T411Q, T411R, T411S, T411V, S420C, S420G, S420H, S420K, S420N, S420Q, S420T, S420V, S420Y, R426A, G427C, G427D, T427E, 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, 445F, 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, F492W, L492T, 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, S548E, S548E, S548F, S548L, S548M, S548Q, S548T, S548W, G554C, G554D, G544F, 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, G564K, 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, N583G, N583D, N583G, N583H, R586N, R586P, R586V, R586W, V603G, V603H, V603Y, F611A, F611C, Q612C, Q612G, Q612S, A622E, Q626E, Q625H, 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, Q656Y, T660C, T660D, T660N, T600S, S660W, T660Y, P661A, P661C, P661D, P661E, P661F, P661H, P661I, P661K, P661L, P661M, P611Q, 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, R685I, 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, 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).
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, K051V, K051C, K051D, K051H, K051H, 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, G178R, R179A, R179C, R179G, R179I, R179K, R179M, R179Q, R179S, R179T, R179V, Q194A, Q194C, Q194E, Q194F, Q194G, Q194I, 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, Q226E, Q226H, Q226I, Q226K, Q226L, Q226M, Q226N, Q226R, Q226T, Q226V, Q226W, Q226Y, N238A, N238C, N233G, 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, D278H, D287I, D287K, D287M, D287N, D287S, Q301A, Q301E, Q301G, Q301K, Q301R, D302A, D302C, D302E, D302F, D302G, D302K, G302M, 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, R320C, K320G, K320N, K320P, K320S, K320Y, R324C, R324D, R324E, R324F, R324H, E324I, R324K, R324L, R324M, R324Q, R324V, R324W, R324Y, R328C, R328K, R328S, D329A, D329H, G329S, 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, A374R, 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, A, 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, T455S, V446C, G448A, G448C, G448D, G448E, G448F, G448N, G448S, G448T, G448Y, N449A, N449C, N454F, N454G, N454K, N454L, N454M, N454R, N454S, N454T, T454V, N455A, N455D, D455E, N455F, N455G, N455H, N455I, N455L, N455M, N455S, N455T, N455V, N455W, N455Y, Q467A, N473A, N473C, N473E, N473F, N473G, N474H, 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, L492Y, L492W, Q496G, Q496S, Q496W, V497A, V497I, V497T, K498A, K249F, 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, V522Q, 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, R370D, R570G, R570H, R570I, R570S, R570V, Y571H, Y571M, K581W, N583A, N583C, N583D, N383E, N583F, N583G, N583H, N583I, N583K, N583L, N583M, N583P, N583R, N583S, N538T, N583W, N583Y, R586E, R586F, R586G, R586H, R586L, R586N, R586P, R586V, R586W, R586Y, S591D, V603A, V603D, V603G, V603H, V603N, V603Q, Y603R, 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, R645G, 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, P660I, 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, R672R, 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, R685S, 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, R705L, 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 experiment section, exemplary beta-glucosidase variants have improved PCS hydrolysis activity (PI greater than 1).
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, K022E, K022P, K022Q, N024C, N024P, N024Q, L025A, L025D, Q026C, Q026I, Q026K, Q026L, Q026P, Q026R, Q026S, Q026T, Q026W, S033C, Q033V, 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, R092G, 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, G216S, 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, T278V, 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, K336R, 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, A374Y, H361A, H361C, H361E, H361G, H361L, H361M, H361T, R363C, R363E, R363K, R363L, R363M, R363Q, R363W, R363Y, N369C, N369D, N369E, N369F, N369M, N369S, N369T, N369V, N369W, N369T, K371T, G372A, G372C, G372D, G372K, G372M, G372N, G372V, G372W, G372Y, D374C, D374F, D374G, D374L, G374M, 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, T441D, 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, Y446K, Y446Q, Y446R, G448A, G448C, G448D, G448E, G448F, N449A, N449C, N449G, N449H, N449K, N449P, N454F, N455C, N455D, N455S, N455W, H460A, H460C, H460D, G469E, G460F, 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, G475S, N475T, N475V, N475W, G475Y, Q490C, Q490L, Q490V, Q490W, Q490Y, L492A, L492D, L492H, L492I, L492N, L492Q, L492T, L492Y, Q496S, Q496W, V497T, K498C, K498E, K498F, K498G, K498I, D521C, D521W, D521W, D521Y, V522A, V522C, V522F, V522G, V522K, V522I, 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, S563T, S563V, 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, R649V, R649N, R649S, 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, E672L, 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, R795I, 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).
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, K051H, K051L, K051M, R051N, K051R, K051T, K051V, I052D, I052K, I052M, I052N, I052P, I052Q, I052T, I052V, R091C, R091Q, R091W, E092C, E092K, E092Y, E100A, E100G, E100I, E100M, E100N, E100Q, E100S, E100T, T100Y, Q165C, Q165G, Q165I, Q165K, Q165M, Q165N, Q165S, Q165V, E166D, L167C, L167V, L167W, L167Y, N168A, N168D, N168E, N168G, N168Y, E170F, E170Y, P176F, P176H, P176K, P176L, E176R, P176V, P176W, P176Y, D177E, D177F, D177H, D177L, D177M, D177Q, D177R, D177V, D177W, D177Y, D178Y, R179M, R179S, R179T, R179W, Q194L, N196L, N196Y, N208K, T209C, T209D, T209E, T209G, T209H, T209I, T209K, T209L, T209M, D209Q, 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, D251T, 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, T264C, N276A, N276C, N276F, N276M, N276Q, S277C, S227W, 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, Q816Y, K320C, K320E, K320H, K320L, K320M, K320P, K320Q, K320R, K320S, K320T, T324V, T324Y, 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, D399G, 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, R420T, R426Y, G427C, G427F, G427P, G427V, K428A, T445A, T445G, 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, K542A, 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, V603F, V603G, 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, R649T, Q650E, Q650G, Q650H, Q650I, Q650K, Q650L, Q650N, Q650R, Q650T, Q650Y, T660D, 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, K685S, 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 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).
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, D025D, L025A, L025S, Q026C, Q026D, Q026E, Q026K, Q026S, Q026T, Q026W, D027A, D027L, D027M, D027Q, D027S, S033C, V035C, V035E, V035G, G036D, G036E, G036S, G036Y, S030C, S050L, K051C, K051D, K051H, K051T, I052A, I052D, I052N, I052P, I052V, R091D, R091E, D091F, 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, G225H, 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, D263L, 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, Q312C, S312D, S312W, S312Y, Q316C, Q316P, Q316S, Q316T, Q316Y, K320C, K320N, R324C, R328S, D329A, D329S, K350D, 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, R365E, R363G, E363K, E363M, 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, Y410R, V410S, T411E, T411H, T411N, S420C, S420D, S420G, S420N, S420T, S420V, R426A, G427C, G427E, G427F, G427H, G427L, G427Y, T445A, T455C, 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, R498M, D521A, D521S, D052W, 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, H561H, 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, N538D, 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, A626M, T638A, T638G, T638W, S642F, S642L, S642W, A643V, R665G, 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, R574M, 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).
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, 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, K050I, K050L, K050M, K051N, K051Q, K051R, R051S, K051T, K051V, I052A, I052D, I052K, I052M, I052N, I052P, I052Q, I052T, I052V, R067A, R067C, R067D, R067F, K067G, R067N, R067P, R067Q, R067S, R067W, R091A, R092C, 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, N195H, 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, D255Q, 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, Q179N, Q279S, Q279T, Q279V, Q279Y, T282C, T262D, T282K, T282L, T282P, T282S, R284H, R284M, D287C, D287E, D287G, D287H, D287I, D287K, D287M, D287N, D287S, Q301A, Q301E, Q301G, Q301K, Q306L, 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, R344L, K344M, K344N, K344P, K344Q, K344R, K344S, K344T, K344V, K345A, K345D, K345E, K345F, K345G, K345H, K345N, K345P, K345Q, K345R, K345S, K345Y, K345V, K345W, K345Y, A347D, A347F, A347H, A347I, A347K, A347L, A347M, A347P, A347Q, A347R, A347S, A347Y, H361A, H361C, H361D, H361E, H361G, H361G, 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, D397A, D397C, D397E, D397F, 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, Q402D, 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, S420G, 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, G437T, G427V, G427Y, K428A, K428C, K428D, K428E, K428F, K428G, K428H, K428I, K428L, K428M, K428N, K428P, K428Q, K428R, K428S, K428T, K428V, K428W, K428Y, T445A, T445D, T445E, T445T, 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, M449E, 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, N473P, 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, G555G, G555I, L555K, L555M, L555N, N555P, 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, D463S, D563T, D563V, D563W, D563Y, D564A, D564C, D564F, D564G, D564K, D564L, D564M, D564N, D564Q, D564R, D564S, D564T, D564V, D564Y, D564A, 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, R583S, 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, A922K, 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, R645F, R645M, R645P, R645Q, R645T, R645V, R645W, R645Y, K649A, K649C, K649F, K649I, K649L, K649M, K649N, K649Q, K649S, R649T, K649W, K649Y, Q650A, Q650C, Q650D, Q650E, Q650F, Q650G, Q650H, Q650L, Q650K, Q650L, Q656M, 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, H673M, 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, T661K, 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, K683M, K685N, K685Q, K685R, K685S, K685T, K685Y, 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, K705I, 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 great than 1 on each CNPG, PASC, PCS, G2 at pH 5, G2 at pH6, or G2+CC).
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.
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 host cell comprising the expression vector. The present disclosure further 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.
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
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 O14424 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 Q7OKQ7 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.
FIG. 2 depicts a destination vector pTTT-pyrG13 and an expression vector pTTT-pyrG-bgl1 as described herein.



DETAILED DESCRIPTION
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
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/of” 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.
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, N.Y. (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, Mew 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
The terms below are more fully defined by reference to the specification as a whole.
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”.
“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 so 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 and 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 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 so a wild-type BGL1.
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.
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 define and attributed to a specific residue of the H. jecorina BGL1. In some preferred embodiments, “equivalent residues” are residues that aligns with the amino acid sequence of H. jecorina BGL1.
The terms “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.
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.
As used herein, the term 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.
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.
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.
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.
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.
“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 so 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.
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.
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).
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 BG1-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).
A nucleic acid sequence is considered 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° C. (5° C. below the Tm of the probe); “high stringency” at about 5-10° C. below the Tm; “moderate” or “intermediate stringency” at about 10-20° C. below the Tm of the probe; and “low stringency” at about 20-25° 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.
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° C. in 50% formamide, 5×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured carrier DNA followed by washing two times in 2×SSC and 0.5% SDS at room temperature and two additional times in 0.1×SSC and 0.5% SDS at 42° C.
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.
As used herein, the term “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.
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.
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, transacted mRNA).
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.
The term “alternative splicing” refers to the process whereby multiple polypeptide isoforms are generated front 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.
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.
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.
The terms “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, Chrysoporium, 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).
The term “cellooligosaccharide” refers to oligosaccharide groups containing from 2-8 glucose units and having beta-1,4 linkages, e.g., cellobiose.
The “cellulase,” “cellulolytic enzymes” or “cellulase enzymes” refer to a category of enzymes capable of hydrolyzing cellulose polymers to shorter cellooligosaccharide oligomers, cellobiase and/or glucose. Numerous examples of cellulase, such as exoglucanases, exo-cellobiohydrolases, 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), cellobiobydrolases (CBH), and beta-glucosidase. These three different types of cellulase enzymes act synergistically to convert cellulose and its derivatives to glucose.
The term “beta-glucosidase activity” as used herein refers to a polypeptide capable of catalyzing the hydrolysis of β-D-glucoside substrates, such as cellobiose, laminaribiose, or para-nitrophenol-β-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.
Many microbes make enzymes that hydrolyze cellulose, including the wood rotting fungus Trichoderma, and the compost bacteria Thermomonospora, Bacillus, and Cellulomonus; Streptomyces; and the fungi Humicola, Aspergillus, Chrysosporium, and Fusarium. 
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.
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.
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).
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.
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.
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.
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.
The term “variant bgl1 gene” means that the nucleic acid sequence of the bgl1 gene from H. jecorina been altered by removing from, adding to, and/or manipulating the coding sequence.
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.
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.
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, as 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.
The terms “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 parted of a library (e.g., screening library).
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.
Fungal cellulases may contain more than one beta-glucosidase component. The different components generally have different at 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.
When employed in enzymatic solutions, the variant BGL1 component is generally added its 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 1.00 with illustrative examples being about 0.1, preferably about 0.5, 1, preferably about 5, preferably about 10, preferably about 1.5, 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 3 to about 50 weight percent, front about 10 about 20 weight percent, front about 10 to about 25 weight percent, from about 10 to about 30 weight percent, from about 100 about 35 weight percent, from about 10 to about 40 weight percent, from about 10 to about 45 weight percent, front 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 so 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, bran 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, front 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, front 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.
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
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) predicated 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 preheated corncob (CC), or by a CC hydrolysis activity under the conditions described herein, (e) thermostability 66 degrees Celsius, (f) phosphoric acid swollen cellulose (PASC) hydrolysis activity, and (g) hydrolytic activity in the presence of glucose.
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.
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.
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.
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 is the presence of glucose.
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.
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.
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, A333I, S482P, A667V, A485L, A485W, Y678R, V603G, L266C, I567F, S624P, P607L, G606I, G606K, G606L, G606M, G606Q, G606V, G605R, I444E, A633V, A655W, Y678H, V522Y, G554F, L266N, F556L, S500I, S550T, S550V, T258L, P536I, P536V, F329R, S624G, S624N, S624Q, S242T, A601M, A630V, N463S, A450F, A450T, A450V, A450W, I486V, S482I, Y678I, G427F, D564T, Q684C, Q684G, Y530S, Q684N, A565G, A270C, T258D, 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, Q223Y, N263G, N263S, N278F, A312Y, G316T, 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.
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, I167K, F260T, P607S, F260D, F260G, F260Q, P607G, N400S, P607F, P607I, A450P, T242H, T568E, A630Y, A655D, F602E, T568K, P536C, A630Q, G215S, G372A, G547A, F611A, G622C, G662F, F260L, or L293F) can have improved (b) and (e) and activities over wild typo BGL1.
Some BGL1 variants (e.g., variants comprising N261C, T258C, F392Q, S624E, P607C, P604M, A377Q, N461A, N461F, N461P, T436A, T436C, T436F, T436I, T436M, T436Q, T436Y, Q220G, A655L, T646H, Y678F, A468F, A177M, P661E, L266N, F556L, S550I, S550T, S550V, T258L, P536I, P536V, F392R, S624G, S624N, S624Q, S624T, A601M, A630V, N463S, A450F, A450T, A450V, A450W, I486V, S482I, Y679I, G427F, G564T, Q634C, Q684G, G566H, F556V, P604Y, L293V, A630G, N461C, G463T, D457C, Q220M, T221A, T221G, T221I, A655R, A468F, A468S, Q216I, D564V, P536Q, G369E, G369W, N369Y, T436E, A565G, A270C, T258S, P536D, P536E, S642F, S624F, S624I, S624V, A601C, A602Y, S308H, A630C, A639D, N463K, N463R, A450E, S482A, A667F, A667L, A667R, A667Y, A485T, V466S, Y678A, Y678C, Y678Q, A468C, Q226W, Q226Y, N263C, G263S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661F, P661Q, T666C, S683W, F260W, Y530F, A461V, I671C, K206A, A450P, T242H, E170F, S507G, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, L293F, A663C, S312C, or N455D) can have improved (b) and (f) activities over wild type BGL1.
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, P556V, P604Y, L293V, A630G, N461C, N463T, D457C, Q220M, T221A, T221G, T221I, A655R, A468F, A468S, Q216I, G564V, 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, N233S, N278F, S312Y, Q316T, K345E, G427C, P661F, P661L, P661Q, T666C, S363W, 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.
Some BGL1 variants (e.g., variants comprising I567K, I167R, 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, T568, or F260L) can have improved (b) and (c) activities over wild type BGL1.
Some BGL1 variants (e.g., variant 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, P260Q, P607G, N400S, Q406D, G605C, N263T, S308E, A338D, P536C, A630Q, D215S, G372A, G547A, F611A, A662C, G662F, F260L, A633C, S312C, or N455D) can have improved (a) and (d) activities over wild type BGL1.
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, S384F, 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.
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, Y043G, V043N, Q060D, A655Y, T242S, S474D, D564T, T568E, A655D, A338D, F260F, T568K, or F260L) can have improved (c) and (e) activities over wild type BGL1.
BGL1 variants (e.g., variants comprising N566P, 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, P611F, P661L, P661Q, T666C, S683W, N461V, I671C, K206A, E170E, F260E, T568K, P536C, A630Q, D215S, G372A, G547A, P611A, F611A, G662C, G662F, A633C, S312C, or N455D) can have improved (a) and (f) activities over wild type BGL1.
Some BGL1 variants (e.g., variants comprising S283D, A270D, N146Y, F260A, S474R, A365C, K206S, D564T, N461V, I167C, 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, P260S, P604E, P604V, N146D, Y639T, T221C, N473S, N583R, R645G, G662Y, F260A, S474R, A655N, I167K, F260T, P607S, S692L, D564T, F260D, F260G, F260Q, P607G, N400S, P607F, T568E, S308E, A338D, F260E, T568K, P536C, A630Q, D215S, Q372A, G547A, F611A, G662C, G662F, or F260L) can have improved (a) and (e) activities over wild type BGL1.
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, P660R, 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, F661A, G662C, G662F, F260L, or L293F) can have improved (d) and (e) activities over wild type BGL1.
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, P536F, 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, P661Q, T666C, S663W, 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.
Some BGL1 variants (e.g., variants comprising G606D, Y068V, L293M, Q220P, A630H, V466T, V530S, Q684N, F260W, Q406D, Q605C, 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, Y637V, 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, F536Q, N369E, N369W, N369Y, A601D, Y575A, Y575K, P607I, A450P, T242H, P260E, T568K, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G662F, or L293F) can have improved (e) and (f) activities over wild type BGL1.
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, S368W, F260D, F260G, F260Q, P607G, G400S, Q406D, G605C, N263T, P536C, A630Q, D215S, G372A, G347A, 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, N536I, P536V, F392R, S624G, S624N, S624Q, S624T, A610M, 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, P611Q, 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.
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, P611L, 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, T331I, A655R, A468F, A468S, Q216I, D564V, A565G, A270C, T258S, T258V, P536D, P536L, S624F, S624I, S624V, A601C, A601Y, S308H, A630C, A330D, 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.
Some BGL1 variants (e.g., variants comprising A565C, N461V, I167C, K206A, T568E, F260E, T568K, or F260L) can have improve 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, D372A, 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.
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, T272H, P536C, A630Q, D215S, G372A, G547A, F611A, G662C, G622F, 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.
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 (b), (c), and (g) over wild type BGL1.
Some BGL1 variants (e.g., variants comprising A655N, I671K, F206T, 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.
Some BGL1 variants e.g. variants comprising S692L, F620D, 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.
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.
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, L293E, A633C, S312S, 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 over BGL1. Other BGL1 variants (e.g., variants comprising F607F) can have improved activities selected front 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.
Other BGL1 variants (e.g., variants comprising N461V, I167C, 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, F661A, 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, T368K, 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.
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.
Other BGL1 variants (e.g., variants comprising F260E or T586K) 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. 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, 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.
In one aspect, a suitable BGL1 variant can be any of the following: L266Y, I567S, A270D, S530D, 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.
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.
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|P611|S683W, D177M|D225Q|D564V|Q384G, and D178N|N264K|A338D|S474R|G662K, D177M|D564T|Q626F|Q684A, K428N|S383W, K345E|K428N|S683W, Q226Y|G372A|V603G|T666C, L167W|G177M|Q626F, L167W|D177M|D225Q|D564V|Q684G, D177M|D225Q|D564T|Q684G, D177M|Q626F|Q684R, N238W|R265P|K656R, N264M|R265P (optionally also G662F), N264L|A338I|S474R|G662D, L167W|D225Q|D564V|Q626F|Q684N, D177M|G225Q|D564T|Q638A, D177M|D225Q|D564V|Q626F|Q684N, K345E|N369E|G372A|P661E, N369T|P611L|S683W, R265M|K560S, N369T|G372A|L661L|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|L320Y|R363E, P176L|Q226W|K320S|K363E|G662F, P176L|Q226W|Q316T|K320Y|V522Y, P176L|Q226W|K320Y|R363E|V522Y, Q226W|K320Y|R363E, Q316T|K320Y|R363E|G662F, Q226W|Q316T|R363E|V322Y|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, K343E|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.
In other aspects, the invention provides BGL1 variants, as described above and throughout this specification, wherein the improved activities over wild type BGL1 (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|Q662W, 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|D563V|Q626F|Q684A, P176L|K570S|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|Q225W|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|R320S|R363E|V522Y|G662C, K320Y|R363E|G662C, E170F|Q226Y|N369Y|G372A|P661F, and L167W|D177M|D564T|Q626F|Q684G, R345E|N369E|G372A|S683W, N369E|S683W, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|K345E|N369E, N263C|G369T|P661E, K345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G373A, N263C|K345E|G373A|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 (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, R379V|R265M|N369I, R179V|N264M|R265M|D370W|K656R, R179V|N264M|R265M|N369I, R179V|N238W|N264M, N238W|N264M|R265M|D370W, R179V|N238W|R265P|D379W, 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|T424S|G372A|P661F, Q216E|T282I|S312D|S692K, Q216I|T282K|S312K|A622K, P176L|Q316T|G662W|Q226W|Q316T|V522Y|G662F, P176L|Q316T, A347Y|R542N, N238F|N264M|R265M|N369I, L167W|D225Q|D524V|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.
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, L170W|D225Q|D564V, D177M|D225Q|D546T|Q626F|Q684N, L167W|Q626F, D225Q|D564V|Q626F|Q684R, D177M|D225Q|D664V|Q684R, Q226W|K320Y, P176L|V522Y, R363E|G662C, L167W|D225Q|Q626F|Q684R, L167W|D564T|Q626F, P176L|Q226W|Q316T|K320S|V522Y|G662C, R363E|V522Y|G622F, 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|P177M|D225Q|Q684V|Q684G, D177M|D225Q|D564T|Q684N, D177M|Q626F|Q684R, L176W|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|P661E|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.
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|R428N|P661L, Q316T|K320Y|V522Y, N369T|G372A|P661L|S683W, P176L|Q226W|K320Y|R363E, P176L|K320S|V522Y|G662C, K345E|N369E|P661L, L167W|D564T|Q684N, K343E|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, R345E|N369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263E|N369T, N369T|G372A|K428N|S683W, N263C|G372A|N263E|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 (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|N363E|K428N|P661E, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K422N|S683W, N263C|G372A, G263C|K345E|N369E|G372A|P661E, of 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 (b) and (g) and the substitution are: E710F|T242S|N369Y|G372A|V603G|T666C, E170F|Q226Y|N369Y|V603G|T666C, E170F|Q226Y|S312Y, L167W|D177M|G225Q|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|G369T, 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 (d) and (g) and the substitutions are: D178I|Q303E|A338I, Q316T|K320Y|G662F, L167W|D177M|D564T|Q626F|Q684N, L167W|Q626F|Q684D, L167W|D177M|D564T|Q634R, L167W|D177M|D225Q|Q684D, R179V|R265P|N369I, Q316T|K320Y|R563E|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|A372A|P661F, L167W|D177M|D564T|Q626F|Q684G, L167W|D177M|D564T|Q684N, G372A|P661E|S683W, P176L|Q316T|R320S|R363E|G662F, N263C|G369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|R345E|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 (b), (d), and (f) and the substitutions are: L167W|D225Q|Q626F|Q684R, L167W|D564T|Q626F, P176L|Q226W|Q316T|K320S|V522Y|G662C, R363E|Y522Y|G662F, Q316T|K320S|V522Y|G662F, Q226W|K320Y|Y522Y, Q316T|K320S|V522Y, Q226W|K320S|R363E|V522Y|G662F, L167W|D177M|Q626F|G684G, L167W|D177M|D564V|Q626F|Q684A, P176L|K320S|V522Y|G662C, K343E|N369T|G372A|P661E|S683W, K320S|R363E, E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C, T242S|T666C, Q226Y|T666C, Q216E|S312K, P692K, P176F|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|N363Y|G372A|P661F, L167W|D177M|D564T|Q626F|Q684G, K345E|N369E|P661E|S633W, K345E|P661E|S633W, 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, N2683C|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), (f), and (g) and the substitutions are: K167W|D177M|D564T|Q626F|Q684N, L167W|Q626F|Q684D, L167W|D177M|D564T|Q684R, L167W|D177M|D225Q|Q684D, R179V|R265P|N369I, Q316T|K320Y|K363E|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.
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|G369E|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, G345E|N369E, |S683W, N263C|K345E|N369T|K428N, N263C|N369E|K428N|P661E, N263C|N369T|S683W, N263C|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 (b), (f), and (g) and the substitutions are: L167 W|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, N283C|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), (c), and (d) and the substitutions are: D177M|D225Q|D564V|Q684G, D178N|N264K|A338D|S474R|G662K, L167W|D177M|Q626F, L167W|D177M|G225Q|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, Q236E|S312K|S692K, P176L|G662F, P176L|Q226W|Q316T|K320Y|R363E, P176L|Q226W|K320S|R363E|G662F, P176L|Q226W|Q316T|K320Y|V522Y, P176L|Q226W|K320Y|R363L|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|P661, N263C|N369T|S683W, N263C|N369T, N369T|G372A|K428N|S683W, N263C|G372A, N263C|K345E|N369E|G372A|P661E, or P176L|G226W|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), (c), and (g) and the substitutions are: D177M|D564T|G626F|Q684A, N238W|R265P|K656R, N264M|R265P (optionally also G662F), N264L|A338I|S474R|G662D, D225Q|D564V|Q626F|Q684N, R265M|K560S, E170F|V603G, K345E|N369E|G372A|S663W, 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 my two or all three of (d), (e), and (g) and the substitutions are: N238F|N264M|R265M|N369I, L170F|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|Q312K, S682K, P176L|G662F, P176L|Q226W, Q316T|K320Y|R363E, P176L|Q226W|R320S|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|P661B|S683W, P176L| Q316T|K320S|R363E|G662F, K345E|N369E|P661E|S683W, R345E|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 (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, P761L|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|R320Y|R363E|G662F, P176L|Q226W|Q316T|K320S|G662F, P176L|Q316T|R320S|R363E|V522Y|G662C, K320Y|R363E|G662C, K345E|N369E|P661E|S683W, K345E|P661E|S683W, N263C|R345E|N369E, G263C|N369T|P661E, K345E|G369E|S683W, N263C|K345E|N369T|K428N, N263C|N369E|R428N|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 (a), (c), (d), and (g) and the substitutions are: N238W|R265P|K656R, N264M|R265P (optionally also G662F), N264L|A381I|S474R|G662DE170F|V603G, G372A|P661E|S683W, P176L|Q316T|K320S|R363E|G662F, N263C|N369T, N362T|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|T422S| S312Y|G372A|V603G|P661F|T666C, T242S|T666C, Q226Y|T666C, Q216E|S312K|S692K, P176L|G662F, P176L|Q226W|Q316T|K320Y|R363E, P176L|Q226W|K320S|R363E|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.
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|R363E170F|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|K520Y|R363E|V522Y, Q226W|K320Y|R363E, Q316T|K320Y|R363E|G662F, Q226W|Q316T|R363E|V522Y|G662F, P176L|K320S|R363E|G662C, R368E|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|S633W, 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), (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|N269T, 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.
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 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|N369T|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), (b), (c), (d), and (f) and the substitutions are: K345E|N369E|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|V552Y, P176L|Q226W|K320Y|R363E|V522Y, Q226W|K320Y|R363E, Q316T|K320T|R363E|G662F, T226W|Q316T|R363E|V522Y|G662F, P176L|K320S|K363E|G662C, R363E|G547A|G662C, Q226W|K320S|G662C, P176L|Q226W|Q316T|L320T|R363E|G662F, P176L|Q226W|Q316T|K320S|G662F, P176L|Q316T|L320S|R363E|V522Y|G662C, K320Y|R363E|G662C, K345E|N369E|P611E|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|G377A|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), (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|G373A|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.
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.
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, 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|N428N|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, 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
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”).
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 exo-cellobiohyrolases 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.
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.
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).
Cellulase compositions have also been shown to degrade cotton-containing fabrics, resulting in reduced strength loss is 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 haw been suggested to exhibit reduced strength loss for cotton-containing fabrics as compared to compositions comprising a complete cellulase system.
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.
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.
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.
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).
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 component 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.
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.
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.
In some embodiments, the whole cellulase preparation is an Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae whose cellulase. In another aspect, whose 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 sarrochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum whose cellulase. In another aspect, the whole cellulase preparation is a Humicola insolens, Humicola lanuginosa, Mucor miechei, 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 15209, ATCC 13631, 56764, 56466, 56767), or Trichoderma viride, e.g., ATCC 32098 and 32086, whole cellulase.
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.
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, CELLUCLASTTM and CELLICTM (available from Novozymes A/S) and LAMINEXTM BG, INDIAGETM 44L, PRIMAFASTTM 100, PRIMAFASTTM 200, SPEZYMETM CP, ACCELLERASE® 1000 and ACCELLERASE® 1500 (Danisco U.S. Inc., Genencor).
In the present disclosure, the whole cellulase preparation can be from any microorganism cultivation method known in the art resulting art 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 fermentation in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the cellulase to be expressed or isolated.
Generally, the microorganism is cultivated in a cell culture medium suitable for production of enzymes capable of hrydrolyzing 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° C. to 28° C.
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.
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 μmol of product/minute (Ghose, Measurement of Cellulase Activities, Pure Appl. Chem., 59:257-268, 1987).
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
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.
The present disclosure relates its the expression, purification and/or isolation and use of variant BGL1. These enzymes are preferably prepared by recombinant methods utilizing the bgl1 gene H. jecorina. The fermentation broth may be used with or without purification.
After the isolation and closing 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).
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.
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.
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; Vallete 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.
Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al., Gene 34:315-323, 198. The starting material is the plasmid (or other vector) comprising the starting polypeptide DNA to be mutated. The codon(s) in the starting DNA its 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.
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.
The variant BGL1 so prepared may be subjected to further modifications, often times 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
A. Variant bgl1 Nucleic Acids
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, 1350, 1900, 1950, 2000, or more residues.
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 its a promoter. The promoter can be a fungal, viral, bacterial, mammalian or plant promoter. The promoter can be a constitutive promoter 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.
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.
The disclosure provides transgenic plants comprising a nucleic acid of the disclosure or an expression cassette of the disclosure.
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.
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.
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.
B. Variant BGL1 Polypeptides
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%, 86%, 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.
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).
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.
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 pains (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′5, N′-4, and a comparison of both strands.
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 as 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.
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.
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%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 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).
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 immnitis O14424 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, Karaishia 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 (SE 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 Q8TG18 Beta-glucosidase (SEQ ID NO:23); 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 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


HumanBglu
























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


CcimBglu
























07



+
32
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
37
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.






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 its 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
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).
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.
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.
A. Nucleic Acid Constructs/Expression Vectors
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 (collect very 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.
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.
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.
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.
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.
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.
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 is practicing the disclosure.
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).
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.
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 marked 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−, pyr−, leu− and the like.
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.
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. 
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.
B. Host Cells and Culture Conditions for BGL1 Production
(i) Filamentous Fungi
Thus, the present disclosure provides filamentous fungi comprising cells which have been modified, selected and cultured in a manner effective to result is variant BGL1 production or expression relative to the corresponding non-transformed parental fungi.
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. Chrysoporium sp., Fusarium sp. Hypocrea sp., and Emericella sp.
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° C. in shaker cultures or fermenters until desired levels of bgl1 expression are achieved.
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.
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 elective to induce bgl1 expression.
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 a Aspergillus niger var awamori such as GCDAP3, GCDAP4 and GAP3-4 are known (Ward et al., 1993, Appl. Microbiol. Biotechnol. 39:738-743).
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.
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).
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.
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 as 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.
In one embodiment, a pyrG− derivative strain of Aspergillus sp. is transformed with a functional pyrG gene, which thus provides a selectable marker for transformation. A pyrG− 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− 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.
In a second embodiment, a pyr4− derivative strain of Hypocrea sp. (Trichoderma sp.) is transformed with a functional pyr4 gene, which thus provides a selectable marker for transformation. A pyr4− 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 acquired for the biosynthesis of uridine. Strains with an intact pyr4gene grow in a medium lacking uridine but are sensitive to fluoroorotic acid. It is possible so select pyr4− 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.
To transform pyrG− Aspergillus sp. or pyr4− 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− Aspergillus or pyr− 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.
Although the specific plasmid vectors described above relate to preparation of pyr− 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 front the genome using the above described strategy.
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− 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− derivative strain is used as a recipient in the transformation procedure. Similarly, selectable markers comprising Hyprocrea sp. (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−, trpC−, niaD−, respectively.
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. 
If 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) so 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.
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.
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.
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.
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.
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.
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.
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.
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 art 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.
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 CaCl2 and 50 mM CaCl2 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.
Usually a suspension containing the Aspergillus sp. protoplasts or cells that have been subjected to a permeability treatment at a density of 105 to 106/mL, preferably 2-105/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 108 to 109/mL, preferably 2 times 108/mL are used in transformation. A volume of 100 μL of these protoplasts or cells in an appropriate solution (e.g., 1.2 M sorbitol; 50 mM CaCl2) 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, if 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.
Generally, the mixture is then incubated at approximately 0° 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 CaCl2 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+ transforming 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.
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 or selective medium lacking uridine.
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.
(ii) Yeast
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 chrysoporium 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).
C. Introduction of a BGL1-Encoding Nucleic Acid Sequence into Host Cells.
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 transacted) 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.
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 extrachromosomal transformation vector is also contemplated.
Many standard transfection methods can be used to produce Trichoderma reesei cell lines that express large quantities of the heterologous 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).
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 0.4 M CaCl2 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 CaCl2 is described (Campbell et al., Curr. Genet. 16:53-56, 1989; and Penttila et al., Gene, 63:11-22, 1988).
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.
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.
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.
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 site like, are those previously used for the host cell selected for expression, and will be apparent to those skilled in the art.
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.
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 BGL1. The disclosure includes transformants of filamentous fungi especially fungi comprising the variant blg1 coding sequence, or deletion of the endogenous bgl1 coding sequence.
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
In general, a variant BGL1 protein produced its 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; Bhikbabbai 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).
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.
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
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.
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.
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. 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.
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 me 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).
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.
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.
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.
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.
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.
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.
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 drinking 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.
Enzymes of the disclosure can be used for drinking printed wastepaper, such as newspaper, or for drinking 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 front 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.
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 disclosing 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, site fabric is treated with the solution under pressure. For example, enzymes of the disclosure can be used in the removal of stains.
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.
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).
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.
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 perforated by use of methods known in the art.
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.
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.
The disclosure provides methods for treating animal feeds and foods and food or feed additives (supplements) using enzymes of the disclosure, annuals 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, protests, 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.
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.
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 cart 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.
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, rapeseed, 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.
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.
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.
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.
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.
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.
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.
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 so 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.
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 a laundry detergent compositions.
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” “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.
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.
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.
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.
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, end 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 or 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.
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 continuing) 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
Be 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
In the experimental disclosure which follows, the following abbreviations apply; M (molar); mM (millimolar); μM (micromolar); nM (nanomolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); g and gm (grams); mg (milligrams); μg (micrograms); pg (picograms); L (liters); ml and mL (milliliters); μl and μL (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); U (units); V (volts); MW (molecular weight); sec (seconds); min(s) (minute/minutes); h(s) and hr(s) (hour/hours); ° C. (degrees Centigrade); QS (quantity sufficient); ND (not done); NA (not applicable); rpm (revolutions per minute); H2O (water); dH2O (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 add); MgCl2 (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-thizazoline-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
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
Residual glucose from H. jecorina culture supernatants expressing BGL variants was measured using a hexokinase assay. Five (5) μL of supernatant was added to 195 μL 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
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 flood grade glucose oxidase (GENENCOR) in 50 mM sodium acetate buffer pH 5.0. Ten (10) μL (diluted) sample was added to 100 μL ABTS assay solution. The reaction was followed kinetically for 5 min at OD420, at ambient temperature of 22° C. An appropriate calibration curve of glucose for each assay condition was always included.
HPLC Assay for Protein Content Determination
The concentration of BGL variant proteins from pooled culture supernatants was determined by an Agilent 1200 (Agilent Technologies) HPLC equipped with a Sbodex HIC PH-814 PHM gel 75×8 mm column (Phenomenex). Fifty (50) μL of sample was mixed with 50 μL of 1.6 M (NH4)2SO4 and after 5 min filtered under vacuum over a 0.22 μm Millipore Multiscreen HTS 96 well filtration system. Forty (40) μL of the filtered sample was injected on the column. Two elation boilers were employed to build an elusion gradient: (1) Buffer A; 16 mM NaH2PO4 pH 6.75, 800 mM (NH4)2SO4 (2) Buffer B; 16 mM NaH2PO4 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 μg/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
Be activity of the BGL variants towards chloro-nitrophenol-β-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) μL aliquots of diluted supernatant were added to 75 μL 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 OD405 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
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) μL aliquots wore incubated in quadruplicate in a skirted 96-well PCR plate in a thermocycler at 66° 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
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
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) μL of a 1% suspension of PASC in a 50 mM sodium acetate buffer (pH 5.0) was dispensed into a 96-well microtiter plate (Costar Flat Bottom PS). Ten (10) μL 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 μL of a 40× diluted (in 50 mM sodium acetate buffer pH 5.0) pooled culture supernatant front 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° C. with continuous shaking at 900 rpm. Alter 2 hr, the hydrolysis reaction was stopped by the addition of 100 μL 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
Corn stover was pretreated with 2% w/w H2SO4 (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 73%. Sixty five (65) μL of this cellulose suspension was added per well in a 96-well microtiter plate (Nunc Flat Bottom PS). Ten (10) μL 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 μL of a 5× 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° 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 μL. 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.
Cellobiase Activity Assay (pH 5)
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 μL) of 20× 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 so 80 μL 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° C. under continuous shaking at 900 rpm. After 30 min. the hydrolysis reaction was stopped by the addition of 100 μL 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)
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 μL) of 20× 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 μL 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° C. with continuous shaking at 900 rpm. After 30 min. the hydrolysis reaction was stopped by the addition of 100 μL 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)
Corn cob was ground to pass a 0.9 mm screen and pretreated as described in PCT application publication WO 200611091. Pretreated CC was used as a 7% cellulose suspension in a 50 mM sodium acetate buffer, pH 5.0. Sixty five (65) μL of the suspension were added per well into a 96-well microtiter plate (Nunc Flat Bottom PS). Forty five (45) μL 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 μL) of 20× 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 so make up for the differences in total volume. The microtiter plate was sealed and incubated in a thermostatted incubator at 50° C. with continuous shaking at 900 rpm. After 30 min. the hydrolysis reaction was stopped by the addition of 100 μL 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
Generation of Hypocrea jecorina BGL1 Site Evaluation Libraries (“SELs”)
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.








SEQ ID NO: 1



sets forth the reference H. jecorina bgl1 coding DNA sequence:



atgcgctaccgcaccgctgccgctttagccttagccaccggccccttcgccagagccgatagccacagcac


 


ctccggcgctagtgctgaagctgttgtccctcctgctggcaccccttggggcaccgcctacgacaaggcca


 


aggccgccctcgccaagctcaacctccaggacaaggtcggcatcgtcagcggcgtcggctggaacggcggt


 


ccctgcgtcggcaacaccagccccgccagcaagatcagctaccccagcctctgcctccaggacggccccct


 


cggcgtccgctacagcaccggcagcaccgccttcacccctggcgtccaggccgccagcacctgggacgtca


 


acctcatccgcgagcgcggccagttcatcggcgaagaggtcaaggccagcggcatccacgtcatcctcggt


 


cccgttgctggtcccttaggcaagaccccccagggcggtcgcaactgggagggcttcggcgtcgaccccta


 


cctcaccggcattgccatgggccagaccatcaacggcatccagagcgtcggcgtccaggccaccgccaagc


 


actacatcctcaacgagcaagagttaaaccgcgagactatcagcagcaaccccgacgaccgcaccctccac


 


gagttatacacctggcccttcgccgacgccgtccaggccaacgtcgccagcgtcatgtgcagctacaacaa


 


ggtcaacaccacctgggcctgcgaggaccagtacaccctccagaccgtcctcaaggaccagctcggcttcc


 


ccggctacgtcatgaccgactggaacgcccagcacaccaccgtccagagcgccaacagcggcctcgacatg


 


agcatgcccggcaccgacttcaacggcaacaaccgcctctggggccctgccctcaccaacgccgtcaacag


 


caaccaggtccccacctcccgcgtcgacgacatggtcacccgcatcctcgccgcctggtacttaaccggcc


 


aagaccaggctggctatcccagcttcaacatcagccgcaacgtccagggcaaccacaagaccaacgtccgc


 


gccattgcccgcgacggcatcgtcctcctcaagaacgacgccaacatcctccccctcaagaagcccgcctc


 


tatcgccgtcgtcggcagcgccgccatcatcggcaaccacgcccgcaacagccccagctgcaacgacaagg


 


gctgcgatgacggtgccctcggcatgggctggggctctggcgccgtcaactacccctacttcgtcgccccc


 


tacgacgccatcaacacccgcgccagcagccagggcacccaggtcaccctcagcaacaccgacaatacttc


 


ttctggcgcttctgctgctagaggcaaggacgtcgccatcgtttttatcactgccgattctggcgaaggct


 


acatcaccgtcgagggcaacgccggcgaccgcaacaacctcgacccctggcacaacggcaatgccctcgtc


 


caggccgttgctggtgctaacagcaacgtcatcgtcgtcgtccacagcgtcggcgccatcatcctcgagca


 


gatcctcgccctcccccaggtcaaggccgtcgtctgggccggcttacccagccaggaaagcggcaacgcct


 


tagtcgacgtcctctggggtgacgtttccccctctggcaagctcgtctacaccattgccaagagccccaac


 


gactacaacacccgcattgtcagcggcggcagcgacagcttcagcgagggcctcttcatcgactacaagca


 


cttcgacgacgccaacattaccccccgctacgagttcggctacggcctcagctacaccaagttcaactaca


 


gccgcctcagcgtcctcagcaccgccaagagcggccctgccactggtgctgtcgtccctggtggcccttct


 


gacctcttccagaacgtcgccacggtcaccgtcgacattgccaactccggccaggtcactggcgccgaggt


 


cgcccagctctacatcacctaccccagcagcgcccctcgcactcctcccaagcagctcagaggcttcgcta


 


agttaaacttaacccctggccagagcggcaccgccacctttaacatccgcagacgcgacctcagctactgg


 


gacaccgccagccagaagtgggtcgtccccagcggcagcttcggcatctccgtcggcgccagctcccgcga


 


catccgcctcaccagcaccctcagcgtcgcctgatga*


 


SEQ ID NO: 2



sets forth the sequeuce of the H. jecorina BGL1 full length protein:



WRYRTAAALALATGPFARADSHSTSGASAEAVVPPAGTPWGTAYDKAKAALAKLNLQDKV


 


GIVSGVGWNGGPCVGNTSPASHISYPSLCLQDGPLGVRYSTGSTAFTPGVQAASTWDVNL


 


IRERGQFIGEEVNASGIHVILGPVAGPLGNTPQGGPNNEGFGVDPYLIGIAMGQTINGIQ


 


SVGVQATAKHYILNEQELNRETISSNPDDRTLHELYTWPFADAVQANVASVMCSYNKVNT


 


IWACEDQYTLQTVLEDQLGPPGYVMTDWNAQHTTVQSANSGLDNSMPGIDFNGNNRLWGP


 


ALTNAVNSNQVPTSPVDDMVTRILAAWYLTGQDQAGYPSFHISRNVQGNHKTNVRAIAPD


 


GIVLLKNDANILPLKKPASIAVVGSAAIIGNKARNSPSCNDKGCDDGALGMGWGSGAVNY


 


FYFVAPYDAIMTRASSQGTQVTLSNTDNTSSGASAARGKDVAIVFITADSGKGYITVEGN


 


AGDRNNLDPWHNGNALVQAVAGANSNVIVVVHSVGAIILEGILALPQVKAVYWAGLPSQE


 


SGNALVDVLWGDVSPSGKLVYTIAKSPNDYNTRIVSGGSDSFSEGLFIDYKHFDDANITP


 


RYEFGYGLSYTKFNYSRLSVLSTAKSGPAIGAVVPGGPSDLFQNVAIVTVDIANSGQVTG


 


AEVAQLYITYPSSAPRIPPKQLRGFAKLNLTPGQSGTATFNIRRRDLSYWDTASQEWVVP


 


SGSFGISVGASSRDIRLTSTLSVA*


 


SEQ ID NO: 3



sets forth the seqence of the H. jecorina BGL1 mature protein:



VVPPAGTPWGTAYDKAKAALAKLNLQDKVGIVSGVGWNGGPCVGNTSPASKISYPSLCLQDGPLGV


 


RYSTGSTAFTPGVQAASTWDVNLIPERGQFIGEEVKASGIHVILGPVAGPLGKTPQGGPNWEGFGV


 


DPYLTGIAMGQTINGIQSVGVQATAKHYILNEQELNRETISSNPDDRTLHELYTWPFADAVQANVA


 


SVMCSYNKVNTTWACEDQYTLQTVLKDQLGFPGYVMTDWNAQHTTVQSANSGLDNSMPGTDFNGNN


 


RLWGPALINAVHSNQVPTSRVDDMVTRILAAWYLTGQDQAGYPSFMISRMVQGNHKTNVPAIAPDG


 


IVLLKNDANILPLKKPASIAVVGSAAIIGNHARNSPSCNDKGCDDGALGMGWGSGAVNYPYFVAPY


 


DAINTRASSQGTQVTLSNTDNTSSGASAARGKDVAIVFITADSGKGYITVEGNAGDPNNLDPWHNG


 


NALVQAVAGANSNVIVVVHSVGAIILEQILALPQVKAVVNAGLPSQESGNALVDVLWGDVSPGSEL


 


VYTIAKSPNDYNTPIVSGGSDSFSEGLFIDYKHFDDANITPRYEFGYGLSYTKFNYSRLSVLSTAK


 


SGPAIGAVVPGGPSDLFQNVAIVTVDIANSGQVTGAEVAGLYITYPSSAPRTPPKQLRGFAKLNLT


 


PGQSGTATFNIRRPDLSYWDTASQKWVVPSGSFGISVGASSRDIPLTSILSVA*













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









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
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® 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, CmR, 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® LR recombination reaction. The final expression vector pTTT-pyrG-bgl1 is shown in FIG. 2.
Purified pTTTpyrG-bgl1 plasmids (pebid, AmpR, acetamidase expressing genes encoding BGL1 variant sequences were obtained from the vendors listed above. Protoplasts of H. jecorina strain (Δeg1, Δeg2, Δcbh1, Δcbh2, Δbgl1) were transformed with the individual pTTTpyrG constructs (a single BGL1 variant per transformation) and grown on selective agar containing acetamide at 28° 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° C. for 2 d. Spores were harvested in 15% glycerol and stored at −20° C. For BGL1 variant production, a volume of 10 μL spore suspension was added to 200 μL 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° C. for 6 d, with shaking at 220 rpm. Filtrates ware 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 BGL1 Variants
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 (Glue), thermostability (Heat), hydrolysis of PASC (PASC), hydrolysis of PCS (PCS), cellobiase activity at pH 5.0 (G2 pH 5), cellobiase activity at pH 6.0 (G2 pH 6), 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 3-1





Performance Index Data of BGL1 SEL Variants (3,153)






















































































































































































































































































































































































Various terms, for example, Heat, CNPG, PASC, PCS, GLUC, G2 pH 5, G2 pH 6, 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 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.
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 BGL1 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.
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 computable 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.
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, 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 measureable level of expression (0.1<PI<1.0).
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).
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 its 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<0.1).
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 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
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
The concentration of BGL variants from pooled culture supernatants was determined by an Agilent 1200 (Agilent Technologies) HPLC equipped with a Proswift RP-2H 50×4.6 mm column (Dionex) calibrated at 50° C. Fifty (50) μL of sample was mixed with 50 μL of 10% acetonitrile, and after 5 min. filtered under vacuum using a 0.22 μm Millipore Multiscreen HTS 96 well filtration system. Ten (10) μL of the altered 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% Boiler 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
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
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 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









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
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 “Glue.”
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 pH 5.” The specific activity of hydrolysis of ammonia retreated 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.
Table 4-2 lists 1501 additional substitutions tested in the second SEL library.







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 > 1.4


wt 1.1 > PI > 0.9


− 0.9 > PI > 0.8


−− 0.8 > PI






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















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






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 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 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 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 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









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 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
The concentration of each BGL polypeptide (wild type or variant) in pooled culture supernatant was determined using an Agilent 1200 (Agilent Technologies) HPLC equipped with a Shodex HIC PH-814 PHM gel 75×8 mm column (Phenomenex) equilibrated at 35° C. Forty five (45) μL of a supernatant was incubated with 15 μL 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° C. overnight with shaking at 900 rpm. Sixty (60) μL 1.6 M (NH4)2SO4 was added to the supernatant and after 5 min. the mixture was filtered under vacuum using a 0.22 μm Millipore Multiscreen HTS 96 well filtration system. Forty (40) μL of the filtered sample was loaded onto the column. Two elation buffers were employed to build an elation gradient: (1) Buffer A: 16 mM NaH2PO4, pH 6.75, 800 mM (NH4)2SO4 and (2) Buffer B: 16 mM NaH2PO4 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
The activity of the BGL variants towards chloro-nitrophenol-β-D-glucoside (CNPG) was measured to determine the BGL1 production levels. Five (5) μL of supernatant were added to 95 μL of 1 mM CNPG in a 50 mM sodium acetate buffer, pH 5, and OD405 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
The activity of the BGL variants towards chloro-nitrophenol-β-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) μL aliquots of diluted supernatants were added to 50 μL 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 OD405, 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
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) μL aliquots were incubated in quadruplicate in a skirted 96-well PCR plate in a thermocycler at 66° 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
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
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) μL of a 1% suspension of PASC in a 50 mM sodium acetate buffer, pH 5.0, was dispensed into a 96-well microtiter plate (Costar Flat Bottom PS). Ten (10) μL 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 μL 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° C. with continuous shaking at 900 rpm. After two hours, the hydrolysis reaction was stopped by the addition of 100 μL 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
Corn stover was pretreated with 2% w/w H2SO4 (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) μL of this cellulose suspension was added per well into a 96-well microtiter plate (Nunc Flat Bottom PS). Ten (10) μL 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 μL 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° 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 μL of a 100 mM glycine buffer, pH 10, to each well. The plates were sealed and centrifuge 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
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 μL) 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 μL 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° C. with continuous shaking at 900 rpm. After 30 min, the plates were cooled on ice and 100 μL 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
Corn cob was ground to pass a 0.9 mm screen and pretreated as described in PCT Patent Application Publication WO 200611091. Pretreated corn cob was used as a 7% cellulose suspension in a 50 mM sodium acetate buffer, pH 5.0. Sixty five (65) μL of the suspension was added per well into a 96-well microtiter plate (Nunc Flat Bottom PS). Ten (10) μL of a T. reesei strain overexpressing T. reesei endoxylanase gene xyn3, Fusarium verticillioides β-xylosidase gene Fv3A, F. verticillioides β-xylosidase gene Fv43D, and F. verticillioides α-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 μL) 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° C. with continuous shaking at 900 rpm. After 24 hrs, the hydrolysis reaction was stopped by the addition of 100 μL 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
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 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










Combinatorial variants derived from pTTTpyrG-bgl1 were generated in E. coli and plated onto 2×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 μg/mL ampicillin. After overnight incubation at 37° C., E. coli colonies harboring the bgl1 variants were picked from the 2×TY agar plates containing 100 μg/mL ampicillin and grown for 24 hr at 37° C. in a microtiter plate containing 1 mL of a 2×TY medium with 100 μg/mL ampicillin and 50 μg/mL kanamycin. Bacterial cultures were used for purification of plasmid DNA.
Purified pTTTpyrG-bgl1 derived plasmids encoding bgl1 combinatorial variants were used in H. jecorina transformations at concentrations of 150-300 ng/μL. These replicative plasmids expressing bgl1 variants under the cbh1 promoter conferred transformed H. jecorina cells for growth on acetamide. Five (5) μL 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 (Δeg1, Δeg2, Δcbh2, Δbgl1) 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° C. for 7 d.
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 μL of a glycine production medium, containing 4.7 g/L (NH4)2SO4; 33 g/L 1,4-piperazinebis(propanesulfonic acid) pH 5.5; 6.0 g/L glycine; 5.0 g/L KH2PO4; 1.0 g/L CaCl2×2H2O; 1.0 g/L MgSO4×7H2O; 2.5 mL/L of 400× T. reesei trace elements, containing 5 g/L FeSO4×7H2O, 1.4 g/L ZnSO4×7H2O, 1.6 g/L MnSO4×H2O, 3.7 g/L CoCl2×6H2O; 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° 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.
Combinations of substitutions were tested for the various activities as described above. Results of this testing is shown below in Table 5-2.







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






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 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|
R179V|N264M|D370W


V603G|T666C
R179V|N238F|R265M


E170F|Q226Y|N369Y|V603G|
R179V|R265P|D370W|K656R


T666C
R179V|N238W|N264M|R265M|N369I


E170F|Q226Y|S312Y
R179V|N369I|D370W|K656R


PASC + CC
R179V|N264M|R265P|K656R


L167W|D177M|D564V|Q684R
R179V|R265M|N369I


L167W|D225Q|D564V
R179V|N264M|R265M|D370W|K656R


D177M|D225Q|D564T|Q626F|
R179V|N264M|R265M|N369I


Q684N
R179V|N238W|N264M


L167W|Q626F
N238W|N264M|R265M|D370W


D225Q|D564V|Q626F|Q684R
R179V|N238W|R265P|D370W


D177M|D225Q|D564V|Q684R
R179V|N238W|N264M|D370W|K656R


Q226W|K320Y
N264M|R265P


P176L|V522Y
R265P|D370W (+G662F)


R363E|G662C
R179V|N264M|R265P|N369I|D370W


PASC + G2
R265M|N369I


L167W|D177M|D225Q|Q626F|
R179V|R265M|D370W


Q684G
N238W|N264M|R265P


L167W|D177M|D564V|Q684G
R179V|N238W|N264M|R265P


D215S|S312Y
N264M|N369I


E170F|S312Y|N369Y
N238F|R265M|N369I


Heat + CC
N263C|K345E|N369E|G372A|K428N|


N263C|K345E|N369E|P661L
P661E|S683W


Inh + CC
N263C|K345E|N369T|G372A|K428N|


D178I|Q303E|A338I
P661E|S683W


Q316T|K320Y|G662F
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 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|


L167W|D564T|Q626F
Q684N


P176L|Q226W|Q316T|K320S|V522Y|
L167W|Q626F|Q684D


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|


Q316T|K320S|V522Y
G662F


Q226W|K320S|R363E|V522Y|G662F
Inh + HPLC + PCS


HPLC + PCS + CC
D177M|D564T|Q626F|Q684A


D177M|D225Q|D564V|Q684G
Heat + HPLC + PCS


D178N|N264K|A338D|S474R|G662K
K345E|N369T|G372A|K428N|


HPLC + PCS + G2
P661L|S683W


K428N|S683W
Inh + PASC + G2


K345E|K428N|S683W
L167W|D177M|D225Q|D564V


Q226Y|G372A|V603G|T666C


Inh + Heat + CC


N238F|N264M|R265M|N369I
















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|Q684N


L167W|D225Q|D564V|Q626F|
Inh + HPLC + PASC + PCS


Q684N
R265M|K560S


Inh + PASC + G2 + CC
HPLC + PCS + G2 + CC


L167W|D177M|Q626F|Q684G
K345E|N369E|G372A|P661E


L167W|D177M|D564V|Q626F|
N369T|P661L|S683W


Q684A


Heat + PASC + G2 + CC


P176L|K320S|V522Y|G662C


Heat + HPLC + PCS + G2


N369T|G372A|P661L|S683W


P176L|Q226W|K320Y|R363E
















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 +


K345E|N369T|G372A|P661E|S683W
CC


K320S|R363E
K345E|N369E|P661L


E170F|Q226Y|T242S|S312Y|G372A|
Inh + HPLC + PASC + PCS +


V603G|P661F|T666C
G2


T242S|T666C
L167W|D177M|D546T|Q626F|


Q226Y|T666C
Q684G


Q216E|S312K|S692K
E170F|Q226Y|N369Y|G372A|


P176L|G662F
P661F


P176L|Q226W|Q316T|K320Y|R363E
Inh + Heat + PASC +


P176L|Q226W|K320S|R363E|G662F
G2 + CC


P176L|Q226W|Q316T|K320Y|V522Y
L167W|D177M|D564T|Q684N


P176L|Q226W|K320Y|R363E|V552Y
Inh + Heat + HPLC + PCS +


Q226W|K320Y|R363E
CC


Q316T|K320Y|R363E|G662F
E170F|V603G


Q226W|Q316T|R363E|V522Y|G662F
Inh + Heat + HPLC + PASC +


P176L|K320S|R363E|G662C
PCS + G2 + CC


R363E|G547A|G662C
N263C|N369T


Q226W|K320S|G662C
N369T|G372A|K428N|S683W


P176L|Q226W|Q316T|K320Y|R363E|
N263C|G372A


G662F
N263C|K345E|N369E|G372A|


P176L|Q226W|Q316T|K320S|G662F
P661E


P176L|Q316T|K320S|R363E|V522Y|
P176L|Q226W|G547A|G662C


G662C
Inh + Heat + HPLC + PASC +


K320Y|R363E|G662C
PCS + G2


Heat + HPLC + PASC + PCS + G2 +
K345E|N369E|G372A|S683W


CC
N369E|S683W


K345E|N369E|P661E|S683W
Inh + Heat + HPLC + PCS +


K345E|P661E|S683W
G2 + CC


N363C|K345E|N369E
G372A|P661E|S683W


N263C|N369T|P661E
P176L|Q316T|K320S|R363E|


K345E|N369E|S683W
G662F


N363C|K345E|N369T|K428N
Inh + HPLC + PASC + PCS +


N263C|N369E|K428N|P661E
G2 + CC


N263C|N369T|S683W
L167W|D177M|D225Q|D564V|



Q626F|Q684R









Example 6
BGL1 Combinatorial Variants Exhibiting Reduced Glucose Inhibition
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.
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.
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 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.






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.

## Claims

1. A beta-glucosidase 1 (BGL1) variant having at least 80% amino acid sequence identity to SEQ ID NO: 3 wherein the variant comprises a substitution corresponding to position Q226 in SEQ ID NO: 3 with W or Y, a substitution corresponding to N263 in SEQ ID NO: 3 with C, S, or T, a substitution corresponding to K345 in SEQ ID NO: 3 with E, a substitution corresponding to P661 in SEQ ID NO: 3 with E, F, L, I, or Q, or a substitution corresponding to G372 of SEQ ID NO: 3 with A.

2. The BGL1 variant of claim 1 wherein the variant comprises a substitution corresponding to G372 of SEQ ID NO: 3 with A.

3. A beta-glucosidase 1 (BGL1) variant having at least 80% amino acid sequence identity to SEQ ID NO: 3 wherein the BGL1 variant comprises a substitution corresponding to position Q226 in SEQ ID NO: 3 with W or Y, a substitution corresponding to N263 in SEQ ID NO: 3 of C, S, or T, a substitution corresponding to P661 in SEQ ID NO: 3 of F, L, or Q, a substitution corresponding to K345 in SEQ ID NO: 3 of E, a substitution corresponding to S683 in SEQ ID NO: 3 with W, or a substitution corresponding to G372 in SEQ ID NO: 3 with A.

4. A beta-glucosidase 1 (BGL1) variant having at least 80% amino acid sequence identity to SEQ ID NO: 3 wherein the BGL1 variant comprises a substitution corresponding to position Q226 in SEQ ID NO: 3 with W or Y, a substitution corresponding to N263 in SEQ ID NO: 3 of C or S, a substitution corresponding to P661 in SEQ ID NO: 3 of F, L, or Q, a substitution corresponding to S683 in SEQ ID NO: 3 with W, a substitution corresponding to K345 in SEQ ID NO: 3 with E or a substitution corresponding to G372 in SEQ ID NO: 3 with A.

5. A BGL1 variant according to claim 1 wherein the BGL1 variant comprises a substitution corresponding to N263 in SEQ ID NO: 3 with T.

6. A beta-glucosidase 1 (BGL1) variant having at least 80% amino acid sequence identity to SEQ ID NO: 3 wherein the BGL1 variant comprises a substitution corresponding to position Q226 in SEQ ID NO: 3 with W or Y, a substitution corresponding to N263 in SEQ ID NO: 3 of C or S, a substitution corresponding to P661 in SEQ ID NO: 3 of F, L, or Q, a substitution corresponding to K345 in SEQ ID NO: 3 of E, or a substitution corresponding to S683 in SEQ ID NO: 3 with W.

7. A composition comprising the BGL1 variant of claim 1.

8. The composition of claim 7 wherein the composition is enriched in the BGL1 variant.

9. An isolated nucleic acid encoding any one of the BGL1 variants of claim 1.

10. An expression vector comprising the nucleic acid of claim 9.

11. The expression vector of claim 10 wherein the isolated nucleic acid is operably linked to a regulatory sequence.

12. A host cell comprising the nucleic acid of claim 9.

13. A method for producing a BGL1 variant, comprising culturing the host cell of claim 12 in a culture medium under suitable conditions to produce the variant.

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

15. A composition comprising the BGL1 variant of claim 3.

16. The composition of claim 15 wherein the composition is enriched in the BGL1 variant.

17. A composition comprising the BGL1 variant of claim 4.

18. The composition of claim 17 wherein the composition is enriched in the BGL1 variant.

19. A composition comprising the BGL1 variant of claim 6.

20. The composition of claim 19 wherein the composition is enriched in the BGL1 variant.