Alpha-Amylase Variants With Altered Properties

Abstract

The present invention relates to variants (mutants) of polypeptides, in particular Termamyl-like alpha-amylases, which variant has alpha-amylase activity and exhibits an alteration in at least one of the following properties relative to said parent alpha-amylase: substrate specificity, substrate binding, substrate cleavage pattern, thermal stability, pH/activity profile, pH/stability profile, stability towards oxidation, Ca2+ dependency, specific activity, in particular laundry and dish-wash applications.

1 - 37 . (canceled) 
     
     
         38 . A variant of a parent Termamyl-like alpha-amylase, comprising an alteration at one or more positions selected from the group of:
 26, 30, 33, 82, 37, 106, 118, 128, 133, 149, 150, 160, 178, 182, 186, 193, 203, 214, 231, 256, 257, 258, 269, 270, 272, 283, 295, 296, 298, 299, 303, 304, 305, 311, 314, 315, 318, 319, 339, 345, 361, 378, 383, 419, 421, 437, 441, 444, 445, 446, 447, 450, 461, 471, 482, 484,   
       wherein
 (a) the alteration(s) are independently
 (i) an insertion of an amino acid downstream of the amino acid which occupies the position, 
 (ii) a deletion of the amino acid which occupies the position, or 
 (iii) a substitution of the amino acid which occupies the position with a different amino acid, 
 
 (b) the variant has alpha-amylase activity, and 
 (c) each position corresponds to a position of the amino acid sequence of the parent alpha-amylase having the amino acid sequence of the parent Termamyl-like alpha-amylase having the amino acid sequence of AA560 shown in SEQ ID NO: 12. 
 
     
     
         39 . The variant of  claim 38 , wherein the mutations are:
 R26S, D30N, N33D, R82H, K37T, N106D, K118Q, N128Y, G133E,A, G149A,N, N150H, Q, Y160F, Y178F, G182T, G186A, T193S,N,D,E,Q, Y203L, V214I,T, D231N, G256K, T257I, G258D, K269S,Q, N270F,Y,D, L272I,V,A, N283D, Y295F,N,D,Q,E, N296K,Q,E, Y298F,H, N299F,Y,Q,T, S303Q,K, Y304F,R,K, G305D, Q311N,Q,K,R,T,S,Y,F, N314D,S,T,Q, G315N,S,T, V318L, Q319E,K,S,T, A339S,T, E345N,R, Q361E, G378K, K383R, T419N, H421Y, N437H, F441L, R444E,Y, N445Q, K446R, A447Y, V450T, T461P, N471E, W482Y, N484Q.   
     
     
         40 . The variant of  claim 38 , wherein the variant has an additional mutation in one or more methionine residues. 
     
     
         41 . The variant of  claim 40 , wherein the methionine residues are:
 M9, M10, M116, M202, M208, M261, M309, M323, M382, M410, M410, and M440.   
     
     
         42 . The variant of  claim 40 , wherein the mutations are:
 M9L,I, M10L, M105L,I,F, M116N,D,L,I,F,W,R,K, M202I,L,V,T, M208F,Y,L,I, M261L,I, M309L,I, M323L,I,S,T,A,Q,E,N,D, M382L,I,Y,F,K, M410L,I,V, M430L,I, and M440L,I,F,Y.   
     
     
         43 . The variant of  claim 38 , wherein the variant has one or more of the following mutations:
 M9L+M202I,   M9L+M202I+M323T,   M9L+M202I+323T+M382Y,   M9L+M202I+Y295F+A339S,   M9L+M202I+Y295F,   M9L+M202I+A339S,   M9L+M202I+Y295F+A339S,   M9L+M202I+Y295F+A339S+E345R,   M9L+G149A+M202I+Y295F+A339S+E345R,   M9L+M202L,   M9L+M202L+M323T,   M9L+M202L+M232T+M382Y,   M9L+M202L+Y295F+A339S,   M9L+M202L+Y295F,   M9L+M202L+A339S,   M9L+M202L+Y295F+A339S,   M9L+M202L+Y295F+A339S, E345R,   M9L+G149A+M202L+Y295F+A339S+E345R,   M9L+M202T,   M9L+M202T+M323T,   M9L+M202T+M323T+M382Y,   M9L+M202T+Y295F+A339S,   M9L+M202T+Y295F,   M9L+M202T+A339S,   M9L+M202T+Y295F+A339S,   M9L+M202T+Y295F+A339S+E345R,   M9L+G149A+M202T+Y295F+A339S+E345R,   M9L+G149A+M202I+V214T+Y295F+N299Y+M323T+A339S+E345R,   M9L+G149A+M202L+V214I+Y295F+M323T+A339S+E345R+M382Y,   M9L+G149A+G182T+G186A+M202I+V214I+Y295F+N299Y+M323T+A339S,   M9L+G149A+G182T+G186A+M202L+T257I+Y295F+N299Y+M323T+A339S+E345R,   M9L+G149A+M202L+V214T+Y295F+N299Y+M323T+A339S+E345R,   M9L+G149A+M202I+V214I+Y295F+M323T+A339S+E345R+M382Y,   M9L+G149A+G182T+G186A+M202L+V214I+Y295F+N299Y+M323T+A339S,   M9L+G149A+G182T+G186A+M202I+T257I+Y295F+N299Y+M323T+A339S+E345R,   M9L+G149A+M202I+V214T+Y295F+N299Y+M323T+A339S+E345R+N471E,   M9L+G149A+M202L+V214I+Y295F+M323T+A339S+E345R+M382Y+N471E,   M9L+G149A+G182T+G186A+M202I+V214I+Y295F+N299Y+M323T+A339S+N471E,   M9L+G149A+G182T+G186A+M202L+T257I+Y295F+N299Y+M323T+A339S+E345R+N471E,   M202L+M105F+M208F,   G133E+M202L+Q361E,   G133E+M202L+R444Er   M202L+Y295F,   M202L+A339S,   M202L+M323T,   M202L+M323T+M309L,   M202L+M323T+M430I,   M202L+V214T+R444Y,   M202L+N283D+Q361E,   M202L+M382Y+K383R,   M202L+K446R+N484Q,   M202I+Y295F,   M202I+A339S,   M202I+M105F+M208S,   G133E+M202I+Q361E,   G133E+M202I+R444E,   M202I+M202I+M323T,   M202I+M202I+M323T+M309L,   M202I+M323T+M430I,   M202I+V214T+R444Y,   M202I+N283D+Q361E,   M202I+M382Y+K383R,   M202I+K446R+N484O,   M202V+M105F+M208F,   G133E+M202V+Q361E,   G133E+M202V+R444E,   M202V+M323T   M202V+M323T+M309L,   M202V+M323T+M430I,   M202V+M323T+M9L,   M202V+V214T+R444Y,   M202V+N283D+Q361E,   M202V+M382Y+K383R,   M202V+K446R+N484Q,   M202T+M105F+M208F,   G133E+M202T+Q361E,   G133E+M202T+R444E,   M202T+Y295F,   M202T+A339S,   M202T+M323T,   M202T+M323T+M309L,   M202T+M323T+M430I,   M202T+M323T+M9L,   M202T+V214T+R444Y,   M202T+N283D+Q361,   M202T+A339S,   M202T+Y295F,   M202T+N299F,Y,   M202T+M382Y+K383R,   M202T+K446R+N484Q   
     
     
         44 . The variant of  claim 38 , wherein the variant further comprises the mutation D183*+G84*. 
     
     
         45 . The variant of  claim 38 , wherein the variant further comprises a mutation in R118, in particular R118K. 
     
     
         46 . The variant of  claim 38 , wherein the variant further comprises a mutation in N195, in particular N195F. 
     
     
         47 . The variant of  claim 38 , wherein the variant further comprises a mutation in R320, in particular R320K. 
     
     
         48 . The variant of  claim 38 , wherein the variant further comprises a mutation in R458, in particular R458K. 
     
     
         49 . The variant of  claim 38 , wherein the variant comprises the mutation D183*+G184*+R118K+N195F+R320K+R458K in combination with one or more of the following mutations:
 K118Q,   K37T,   H421Y,   V450T,   K383R,   N445Q,   Y178F,   V318L,   W482Y,   N283D+Q361E,   M105F+M208F,   M202L+M323T+M430I,   K446R+N484Q,   R444Y,   N106D,   Y203L   G133E+Q361E,   M323E,   V214T,   M202L+M323T+M309L,   M202L,   M202L+M323T,   M202L+M323T+M9L+M382Y+K383R   M202L+M323T+M9L+M382Y,   M202L+M323T+M9L.   
     
     
         50 . The variant of  claim 38 , wherein the variant comprises the mutation D183*+G184*+R118K+N195F+R320K+R458K+M202L+M323T+M9L. 
     
     
         51 . The variant of  claim 38  wherein the variant further comprises one or more of the following mutations:
 T461P,   Y298H,   G133E+R444E,   Y298F,   M202T,   M202I,   M202V,   V214T+M323E+M382Y+K383R+N471E   Y178F+G258D+T419N+N437H   G149N+N150Q+M382Y+K383R   Y160F+V214T+M382Y   N128Y+G149A+V214T+D231N+M382Y+F441L   R82H+N128Y+G149A+V214T+M382Y   N150H+V214T   V214T+E345N   V214T+G305D+M382Y+R444E   V214T+M382Y+A447Y   M202I+V214T+M382Y+K383R+R444Y   V214T+G378K   V214T+A256K   R26S+D30N+N33D+V214T+M382Y   
     
     
         52 . The variant of  claim 38 , wherein the parent Termamyl-like alpha-amylase is derived from a strain of  B. licheniformis, B. amyloliquefaciens, B. stearothermophilus, Bacillus  sp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, or DSMZ no. 12649, KSM AP1378, or KSM K36 or KSM K38. 
     
     
         53 . The variant of  claim 38 , wherein the parent Termamyl-like alpha-amylase is any of the alpha-amylases selected from the group depicted in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 13, 14, 15 16, 17, and 18. 
     
     
         54 . The variant of  claim 38 , wherein the parent Termamyl-like alpha-amylase has an amino acid sequence which has a degree of identity to SEQ ID NO: 4 of at least 60%, preferably 70%, more preferably at least 80%, even more preferably at least about 90%, even more preferably at least 95%, even more preferably at least 97%, and even more preferably at least 99%. 
     
     
         55 . The variant of any of  claim 38 , wherein the parent Termamyl-like alpha-amylase is encoded by a nucleic acid sequence, which hybridizes under low, preferably medium, preferred high stringency conditions, with the nucleic acid sequence of SEQ ID NO: 11. 
     
     
         56 . A DNA construct comprising a DNA sequence encoding an alpha-amylase variant according to  claim 38 . 
     
     
         57 . A detergent additive comprising an alpha-amylase variant according to  claim 38 .