Amylase variants

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, Ca 2+ dependency, specific activity, and solubility, in particular under production conditions.

1 . A variant of a parent Termamyl-like alpha-amylase, comprising an alteration at one or more of the following positions:
 R28, R118, N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, and N484,   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 is numbered according to the amino acid sequence of SEQ ID NO: 12. 
   
     
     
         2 . The variant of  claim 1 , which further comprises one or more of the following mutations: Delta G184; Delta (R181-G182); Delta (D183-G184); R28N,K; S94K; R118K; N125A,R,K; N174D; R181Q,E,K; G186R; W189R,K; N195F; M202L; Y298H,F; N299A; K302R, S303Q, N306G,D,R,K; R310A,K,Q,E,H,D,N; N314D; R320K; H324K; E345R,D,K,N; Y396F; R400T,K; W439R; R444K; N445K,Q; K446N; Q449E; R458K; N471E; N484Q. 
     
     
         3 . The variant of  claim 1 , which further comprises a mutation in M202. 
     
     
         4 . The variant of  claim 3 , wherein the mutation is M202L,T. 
     
     
         5 . The variant of  claim 1 , which further comprises a mutation in N195. 
     
     
         6 . The variant of  claim 5 , wherein the mutation is N195F. 
     
     
         7 . The variant of  claim 1 , which further comprises a mutation in G186. 
     
     
         8 . The variant of  claim 7 , wherein the mutation is G186R. 
     
     
         9 . The variant of  claim 1 , which further comprises a mutation in R181. 
     
     
         10 . The variant of  claim 9 , wherein the mutation is R181Q. 
     
     
         11 . The variant of  claim 1 , 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. 
     
     
         12 . The variant of  claim 1 , wherein the parent Termamyl-like alpha-amylase has an amino acid sequence of SEQ ID NOS: 2, 4, 6, 8, 10, 12, or 13. 
     
     
         13 . The variant of  claim 1 , 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%. 
     
     
         14 . The variant of  claim 1 , which variant has altered solubility, preferably increased solubility, in particular under washing, dish washing or hard surface cleaning conditions. 
     
     
         15 . A DNA construct comprising a DNA sequence encoding a variant of  claim 1 . 
     
     
         16 . A recombinant expression vector which carries a DNA construct of  claim 15 . 
     
     
         17 . A cell which is transformed with a DNA construct of  claim 16 . 
     
     
         18 . A cell of  claim 17 , which is a microorganism, preferably a bacterium or a fungus, in particular a gram-positive bacterium, such as  Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, Bacillus subtilis , or  Bacillus thuringiensis.    
     
     
         19 . A composition comprising a variant of  claim 1 . 
     
     
         20 . A detergent composition comprising a variant of  claim 1  and a surfactant.