Variants of glycoside hydrolases

Abstract

The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

1 . An isolated variant of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variant has glycoside hydrolase activity.  
     
     
         2 - 135 . (Canceled)  
     
     
         136 . The variant of  claim 1 , which comprises one or more substitutions selected from the group consisting of S21P, G94S, G94A, G94R, G94Q, K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G, T246I, Y247C, D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L, T455A, G467S, and C486W of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprises one or more substitutions selected from the group consisting of S8P, G22D, T41I, N49S, S57N, S113N, S196T, T226A, P227A, T255P, T356I, Y371C, S411F, and T462A of amino acids 1 to 513 of SEQ ID NO: 2.  
     
     
         137 - 145 . (Canceled).  
     
     
         146 . The variant of  claim 1 , which is encoded by the nucleotide sequence contained in NRRL B-30658, NRRL B-30659, NRRL B-30661, NRRL B-30662, NRRL B-30663, NRRL B-30664, NRRL B-30665, NRRL B-30666, NRRL B-30674, NRRL B-30675, NRRL B-30676, NRRL B-30677, NRRL B-30678, NRRL B-30679, NRRL B-30680, NRRL B-30681, NRRL B-30682, or NRRL B-30762.  
     
     
         147 . An isolated nucleotide sequence encoding the variant of  claim 1 .  
     
     
         148 . (Canceled).  
     
     
         149 . An expression vector comprising the nucleotide sequence of  claim 147 .  
     
     
         150 . A host cell comprising the nucleotide sequence of  claim 147 .  
     
     
         151 . A method for producing a variant, comprising (a) cultivating the host cell of  claim 150  under conditions suitable for production of the variant; and (b) recovering the variant from the cultivation medium.  
     
     
         152 . An isolated polypeptide having glycoside hydrolase activity, wherein the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprises a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2.  
     
     
         153 - 272 . (Canceled)  
     
     
         273 . The polypeptide of  claim 152 , which comprises one or more differences selected from the group consisting of S21P, G94S, G94A, G94R, G94Q, K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G, T246I, Y247C, D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L, T455A, G467S, and C486W of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further differ by one or more differences selected from the group consisting of S8P, G22D, T41I, N49S, S57N, S113N, S196T, T226A, P227A, T255P, T356I, Y371C, S411F, and T462A of amino acids 1 to 513 of SEQ ID NO: 2.  
     
     
         274 - 277 . (Canceled)  
     
     
         278 . An isolated nucleotide sequence encoding the polypeptide of claim  152 - 277 .  
     
     
         279 . (Canceled).  
     
     
         280 . An expression vector comprising the nucleotide sequence of  claim 278 .  
     
     
         281 . A host cell comprising the nucleotide sequence of  claim 278 .  
     
     
         282 . A method for producing a polypeptide having glycoside hydrolase activity, comprising (a) cultivating the host cell of  claim 281  under conditions suitable for production of the variant; and (b) recovering the variant from the cultivation medium.  
     
     
         283 . A method for obtaining a variant of a parent glycoside hydrolase, comprising: 
 (a) introducing into the parent glycoside hydrolase a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further introducing a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variant has glycoside hydrolase activity; and    (b) recovering the variant.    
     
     
         284 - 417 . (Canceled)  
     
     
         418 . The method of  claim 283 , wherein the variant comprises one or more substitutions selected from the group consisting of S21P, G94S, G94A, G94R, G94Q, K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G, T2461, Y247C, D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L, T455A, G467S, and C486W of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprises one or more substitutions selected from the group consisting of S8P, G22D, T41I, N495, S57N, S113N, S196T, T226A, P227A, T255P, T356I, Y371C, S411F, and T462A of amino acids 1 to 513 of SEQ ID NO: 2.  
     
     
         419 - 428 . (Canceled)  
     
     
         429 . An isolated polypeptide having cellobiohydrolase activity comprising amino acids 1 to 513 of SEQ ID NO: 2.  
     
     
         430 . An isolated nucleotide sequence encoding the polypeptide of  claim 429 .  
     
     
         431 . The nucleotide sequence of  claim 430 , which is contained in pAJO52 that is contained in  E. coli  NRRL B-30683.  
     
     
         432 . An expression vector comprising the nucleotide sequence of  claim 430 .  
     
     
         433 . A host cell comprising the nucleotide sequence of  claim 430 .  
     
     
         434 . A method for producing a polypeptide having cellobiohydrolase activity, comprising: 
 (a) cultivating the host cell of  claim 433  under conditions suitable for the expression of the polypeptide; and    (b) recovering the polypeptide from the cultivation medium of amino acids 1 to 513 of SEQ ID NO: 2.    
     
     
         435 . (Canceled).  
     
     
         436 . A detergent composition comprising a variant of  claim 1  and a surfactant.  
     
     
         437 . A method for degrading cellulose- and hemicellulose-containing biomass, comprising treating the biomass with an effective amount of a variant of  claim 1  and recovering the degraded biomass.  
     
     
         438 . (Cancelled).  
     
     
         439 . A method for degrading cellulose- and hemicellulose-containing biomass, comprising treating the biomass with a host cell of  claim 150  and recovering the degraded biomass.  
     
     
         440 . (Cancelled).  
     
     
         441 . (Canceled).