US2009181875A1PendingUtilityA1

Subtilase variants

Assignee: NOVOZYMES ASPriority: Nov 6, 2002Filed: Mar 13, 2009Published: Jul 16, 2009
Est. expiryNov 6, 2022(expired)· nominal 20-yr term from priority
C11D 3/386C12N 9/54
64
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Claims

Abstract

The present invention relates to novel subtilase variants exhibiting alterations relative to the parent subtilase in one or more properties including: wash performance, thermal stability, storage stability or catalytic activity. The variants of the invention are suitable for use in, e.g., cleaning or detergent compositions, such as laundry detergent compositions and dish wash compositions, including automatic dish wash compositions.

Claims

exact text as granted — not AI-modified
1 - 15 . (canceled) 
     
     
         16 . A variant of a parent subtilase comprising substitutions at positions 9 and 15, wherein the variant has protease activity and each position corresponds to a position of the amino acid sequence of SEQ ID NO: 1. 
     
     
         17 . The variant of  claim 16 , wherein the substitutions are S9R+A15T. 
     
     
         18 . The variant of  claim 16 , further comprising a substitution at position 68. 
     
     
         19 . The variant of  claim 18 , wherein the substitution at position 68 is V68A. 
     
     
         20 . The variant of  claim 17 , further comprising a substitution at position 68. 
     
     
         21 . The variant of  claim 20 , wherein the substitution at position 68 is V68A. 
     
     
         22 . The variant of  claim 16 , comprising a set of substitutions selected from the group consisting of: 
       
         
           
                 
                 
               
                     
                     
                 
                     
                   S3T + S9R + P14H + A15M + R19L + S99SD + V139I 
                 
                     
                   V4A + S9R + A15T + G97GV + H120D 
                 
                     
                   S9L + A15T + T22TV + V139L + Q245F 
                 
                     
                   S9L + A15T + T22TV + V139L + Q245F + L262S 
                 
                     
                   S9R + A13V + A15T + I35V + N62D + Q245F 
                 
                     
                   S9R + A15M + A16P + T22S + S99AD 
                 
                     
                   S9R + A15T 
                 
                     
                   S9R + A15T + A16P + G97GA + P131S + Q137D + N204S 
                 
                     
                   S9R + A15T + G20A + G97GV + H120D + P131H 
                 
                     
                   S9R + A15T + G20* + L21F + P52T + N62D + Q245R 
                 
                     
                   S9R + A15T + G20* + L21F + G61E + *61aP + Q245R 
                 
                     
                   S9R + A15T + G20* + L21F + *61aA + V68A + Q245R 
                 
                     
                   S9R + A15T + G20* + L21F + *61aS + V68A + G160D + Q245R 
                 
                     
                   S9R + A15T + G20* + L21F + N62D + Q245N 
                 
                     
                   S9R + A15T + G20* + L21F + N62D + Q245R 
                 
                     
                   S9R + A15T + G20* + L21F + N62D + Q245R + S259G 
                 
                     
                   S9R + A15T + G20* + L21F + N62E + Q245R 
                 
                     
                   S9R + A15T + G20* + L21F + *62aS + N218D + Q245R 
                 
                     
                   S9R + A15T + G20* + L21F + *63aG + Q245R + N272V 
                 
                     
                   S9R + A15T + L21LC + V139L + R186H + Q245M 
                 
                     
                   S9R + A15T + L21LG + T22TV + V139L + N204D + Q245N 
                 
                     
                   S9R + A15T + L21LP + T22TV + M119I + N218D + Q245I 
                 
                     
                   S9R + A15T + L21LP + T22TY + V139L + G160D + Q245L 
                 
                     
                   S9R + A15T + L21LW + G100S + V139L + Q245V 
                 
                     
                   S9R + A15T + T22A + N62D 
                 
                     
                   S9R + A15T + T22A + V139L + Q245E 
                 
                     
                   S9R + A15T + T22G + V139L + Q245L 
                 
                     
                   S9R + A15T + T22L + V139L + Q245V + A254S 
                 
                     
                   S9R + A15T + T22TG + N62D + V139L + Q245G 
                 
                     
                   S9R + A15T + T22TG + N62D + V139L + Q245S 
                 
                     
                   S9R + A15T + T22TG + N62D + V139L + Q245V 
                 
                     
                   S9R + A15T + T22TL + G61E + L96LG + Q137D + Q245R 
                 
                     
                   S9R + A15T + T22TL + N62D + I107V + V139L + Q245W 
                 
                     
                   S9R + A15T + T22TL + N62D + Q245W 
                 
                     
                   S9R + A15T + T22TQ + S101P 
                 
                     
                   S9R + A15T + T22TW + N204D + Q245I 
                 
                     
                   S9R + A15T + S24P + G61E + A85T + P239S + Q245A 
                 
                     
                   S9R + A15T + V28I + V68A + Q245R + N252A 
                 
                     
                   S9R + A15T + T38S + A98R + S99C + G100S + 
                 
                     
                   H120N + P131S + Q137H 
                 
                     
                   S9R + A15T + G46D + V68A + N218D + Q245R 
                 
                     
                   S9R + A15T + F50S + H120D + P131H 
                 
                     
                   S9R + A15T + D60DG 
                 
                     
                   S9R + A15T + G61E + V68A + A98S + S99G + Q245R 
                 
                     
                   S9R + A15T + G61E + A85T + Q137H + Y209C + Q245G 
                 
                     
                   S9R + A15T + G61E + A85T + N218D + P239S + Q245L 
                 
                     
                   S9R + A15T + G61E + A85T + P239L + Q245C 
                 
                     
                   S9R + A15T + G61E + A85T + P239S + Q245C 
                 
                     
                   S9R + A15T + G61E + A85T + P239S + Q245V 
                 
                     
                   S9R + A15T + G61E + A98S + S99M + Q245R 
                 
                     
                   S9R + A15T + G61E + H120S + Q137D + V139L + N218D 
                 
                     
                   S9R + A15T + G61GP + Q245L 
                 
                     
                   S9R + A15T + N62D + H120N + P131T 
                 
                     
                   S9R + A15T + N62D + Q245R + N252M 
                 
                     
                   S9R + A15T + N62D + Q245W + N252S 
                 
                     
                   S9R + A15T + N62D + Q245W + N252V 
                 
                     
                   S9R + A15T + N62ND + V139L + Q245E 
                 
                     
                   S9R + A15T + N62ND + V139L + N261D 
                 
                     
                   S9R + A15T + N62NG + Q245T 
                 
                     
                   S9R + A15T + N62S + H120N + P131T + N218D 
                 
                     
                   S9R + A15T + V68A 
                 
                     
                   S9R + A15T + V68A + A98G + S99I + K237R + Q245R 
                 
                     
                   S9R + A15T + V68A + A98G + S99V + Q245R 
                 
                     
                   S9R + A15T + V68A + A98L + S99G + Q245R 
                 
                     
                   S9R + A15T + V68A + A98L + Q245R 
                 
                     
                   S9R + A15T + V68A + A98M + S99G + Q245R + T274A 
                 
                     
                   S9R + A15T + V68A + A98M + Q245R + N248D 
                 
                     
                   S9R + A15T + V68A + S99G + Q245R + N261D 
                 
                     
                   S9R + A15T + V68A + H120D + P131S + Q137H + Q245R 
                 
                     
                   S9R + A15T + V68A + H120N + P131S + Q137H + Q245M 
                 
                     
                   S9R + A15T + V68A + H120N + P131S + Q137H + Q245R 
                 
                     
                   S9R + A15T + V68A + H120N + N218D + Q245R 
                 
                     
                   S9R + A15T + V68A + Q137D 
                 
                     
                   S9R + A15T + V68A + A174V + Q245R 
                 
                     
                   S9R + A15T + V68A + A194T + Q245R + N252E 
                 
                     
                   S9R + A15T + V68A + N218D + Q245R 
                 
                     
                   S9R + A15T + V68A + Q245R 
                 
                     
                   S9R + A15T + V68A + Q245R + N252S 
                 
                     
                   S9R + A15T + N76S + L111V + P131H + Q137D 
                 
                     
                   S9R + A15T + N77S + L96LG + H120D + P131Q 
                 
                     
                   S9R + A15T + A88V + A98R + S99G + G100C + 
                 
                     
                   H120N + P131S + Q137H 
                 
                     
                   S9R + A15T + K94N + H120N + P131H 
                 
                     
                   S9R + A15T + L96LG + H120D + P131H + R186L 
                 
                     
                   S9R + A15T + L96LG + H120D + P131S + Q137E 
                 
                     
                   S9R + A15T + L96LG + H120D + G160D 
                 
                     
                   S9R + A15T + L96LG + H120N + P131H + Q137E 
                 
                     
                   S9R + A15T + L96LG + H120N + P131S 
                 
                     
                   S9R + A15T + L96LG + H120N + P131S + Q137H 
                 
                     
                   S9R + A15T + L96LG + H120N + P131S + Q137H + M222S 
                 
                     
                   S9R + A15T + L96LG + H120Q + Q137E 
                 
                     
                   S9R + A15T + L96LG + S130* 
                 
                     
                   S9R + A15T + L96LG + P131H + Q137D 
                 
                     
                   S9R + A15T + L96LG + P131Q + Q137D 
                 
                     
                   S9R + A15T + L96LG + P131T + Q137H 
                 
                     
                   S9R + A15T + L96LG + Q137E + Y209H 
                 
                     
                   S9R + A15T + G97GA + H120D + P131H + Q137E 
                 
                     
                   S9R + A15T + G97GA + H120D + P131S + Q137E 
                 
                     
                   S9R + A15T + G97GA + H120D + Q137D 
                 
                     
                   S9R + A15T + G97GA + H120D + Q137H + M222V 
                 
                     
                   S9R + A15T + G97GA + H120N + Q137D + N248D 
                 
                     
                   S9R + A15T + G97GA + H120N + Q137E 
                 
                     
                   S9R + A15T + G97GA + H120N + S212L 
                 
                     
                   S9R + A15T + G97GA + H120Q + P131H + Q137E 
                 
                     
                   S9R + A15T + G97GA + H120Y + Q137H 
                 
                     
                   S9R + A15T + G97GA + Q137D 
                 
                     
                   S9R + A15T + G97GA + Q137E 
                 
                     
                   S9R + A15T + G97GA + Q137E + L262V 
                 
                     
                   S9G + A15T + G97GA + Q137H 
                 
                     
                   S9R + A15T + G97GA + Q137H + N218S 
                 
                     
                   S9R + A15T + G97GA + R186C 
                 
                     
                   S9R + A15T + G97GG + L111I + P131T + Q137H 
                 
                     
                   S9R + A15T + G97GG + H120D + P131H + Q137H 
                 
                     
                   S9R + A15T + G97GG + H120D + Q137E 
                 
                     
                   S9R + A15T + G97GG + H120N + Q137D 
                 
                     
                   S9R + A15T + G97GG + P131S + Q137H 
                 
                     
                   S9R + A15T + G97GV + S106A + P131H 
                 
                     
                   S9R + A15T + G97GV + H120D + Q137H 
                 
                     
                   S9R + A15T + G97GV + H120E + Q137H 
                 
                     
                   S9R + A15T + G97GV + H120N 
                 
                     
                   S9R + A15T + G97GV + H120Q 
                 
                     
                   S9R + A15T + G97GV + H120Q + Y263F 
                 
                     
                   S9R + A15T + G97GV + P131H + Q137H 
                 
                     
                   S9R + A15T + G97GV + P131S 
                 
                     
                   S9R + A15T + G97GV + P131T + Q137H 
                 
                     
                   S9R + A15T + G97GV + Q137H 
                 
                     
                   S9R + A15T + A98C + G100S + H120N + P131S + Q137H 
                 
                     
                   S9R + A15T + A98G + S99C + H120N + P131S + Q137H 
                 
                     
                   S9R + A15T + A98S + S99G + G100S + 
                 
                     
                   H120N + P131S + Q137H 
                 
                     
                   S9R + A15T + A98S + G100S + H120N + P131S + Q137H 
                 
                     
                   S9R + A13T + S99A + S99SD + P131F 
                 
                     
                   S9R + A15T + S99C + H120N + P131S + Q137H + M222S 
                 
                     
                   S9R + A15T + S99G + G100S + H120N + P131S + Q137H 
                 
                     
                   S9R + A15T + G102S + M175T + Q245R + N252D 
                 
                     
                   S9R + A15T + L111I + H120N + Q137E 
                 
                     
                   S9R + A15T + L111I + Q137E 
                 
                     
                   S9R + A15T + L111V + Q137E + G211D 
                 
                     
                   S9R + A15T + H120D + Q137D 
                 
                     
                   S9R + A15T + H120G + Q137E + N218D 
                 
                     
                   S9R + A15T + H120N + P131T + N218D 
                 
                     
                   S9R + A15T + H120N + P131T + N218D + N269T 
                 
                     
                   S9R + A15T + H120Q + P131C + Q137H 
                 
                     
                   S9R + A15T + H120R + Q137D + N173S 
                 
                     
                   S9R + A15T + G127E + P131R + Q137H 
                 
                     
                   S9R + A15T + S130P 
                 
                     
                   S9R + A15T + P131H + S144P 
                 
                     
                   S9R + A15T + Y167I + R170L 
                 
                     
                   S9R + A15T + N218D 
                 
                     
                   S9R + A15T + T224A 
                 
                     
                   S9R + A15T + Q245F 
                 
                     
                   S9R + A15T + Q245L 
                 
                     
                   S9R + A15T + Q245S 
                 
                     
                   S9R + A15T + Q245V and 
                 
                     
                   S9R + A15T + T260M. 
                 
                     
                     
                 
             
                
               
               
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         23 . The variant of  claim 16 , wherein the parent subtilase belongs to the sub-group I-S1. 
     
     
         24 . The variant of  claim 16 , wherein the parent subtilase belongs to the sub-group I-S2. 
     
     
         25 . The variant of  claim 16 , wherein the parent subtilase is subtilisin 309. 
     
     
         26 . The variant of  claim 16 , further comprising one or more of the modifications K27R, *36D, S56P, N62D, V68A, N76D, S87N, G97N, S99SE, S101G, S103A, V104A, V104I, V104N, V104Y, S106A, H120D, H120N, N123S, G159D, Y167A, R170S, R170L, A194P, N204D, V205I, Q206E, L217D, N218S, N218D, M222S, M222A, T224S, A232V, K235L, Q236H, Q245R, N248D, N252K, T274A, S101G+V104N, S87N+S101G+V104N, K27R+V104Y+N123S+T274A, N76D+S103A+V104I, S99D+S101R+S103A+V1041+G160S, S3T+V4I+S99D+S100R+S103A+V104I+G160S+V199M+V205I+L217D, S3T+V4I+S99D+S100R+S103A+V104I+G160S+A194P+V199M+V205I+L217D, S3T+V4I+S99D+S100R+S103A+V104I+G160S+V2051 and N76D+V104A. 
     
     
         27 . The variant of  claim 16 , further comprising the following substitutions: S101G+S103A+V104I+G159D+A232V+Q236H+Q245R+N248D+N252K. 
     
     
         28 . A cleaning or detergent composition, comprising a variant of  claim 16  and a surfactant. 
     
     
         29 . A composition of  claim 28 , which additionally comprises one or more of an amylase, cellulase, cutinase, esterase, beta-galactosidase, glycoamylase, hemicellulase, lactase, ligninase, lipase, polygalacturonase, and protease. 
     
     
         30 . An isolated DNA sequence encoding a protease variant of  claim 16 . 
     
     
         31 . An expression vector comprising the isolated DNA sequence of  claim 30 . 
     
     
         32 . A microbial host cell transformed with the expression vector of  claim 31 . 
     
     
         33 . A microbial host cell of  claim 32 , which is a  Bacillus.    
     
     
         34 . A method for producing a protease variant, comprising
 (a) culturing a host of  claim 32  under conditions conducive to the expression and secretion of the variant, and   (b) recovering the protease variant.

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