Alpha-Amylase Mutants

Abstract

The invention relates to a novel Termamyl-like alpha-amylase, and Termamyl-like alpha-amylases comprising mutations in two, three, four, five or six regions/positions. The variants have increased thermostability at acidic pH and/or at low Ca 2+ concentrations (relative to the parent). The invention also relates to a DNA construct comprising a DNA sequence encoding an alpha-amylase variant of the invention, a recombinant expression vector which carries a DNA construct of the invention, a cell which is transformed with a DNA construct of the invention, the use of an alpha-amylase variant of the invention for washing and/or dishwashing, textile desizing, starch liquefaction, a detergent additive comprising an alpha-amylase variant of the invention, a manual or automatic dishwashing detergent composition comprising an alpha-amylase variant of the invention, a method for generating a variant of a parent Termamyl-like alpha-amylase, which variant exhibits increased thermostability at acidic pH and/or at low Ca 2+ concentrations (relative to the parent).

1 - 56 . (canceled) 
     
     
         57 . An isolated polypeptide having alpha-amylase activity, selected from the group consisting of:
 (a) a polypeptide having an amino acid sequence which has at least 96% identity with amino acids 1 to 485 of SEQ ID NO:24 or SEQ ID NO:26;   (b) a polypeptide which is encoded by a nucleic acid sequence which hybridizes under medium stringency conditions with (i) the nucleic acid sequence of SEQ ID NO: 23 or SEQ ID NO: 25, (ii) the cDNA sequence of SEQ ID NO: 23 or SEQ ID NO: 25, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii);   (c) an allelic variant of (a) or (b);   (d) a fragment of (a), (b), or (c) that has alpha-amylase activity;   (e) a pH optimum determined using the Phadebas method (37° C.) in the range between pH 8 and 9;   (f) a temperature optimum determined using the Phasebas method (pH 9.0) in the range between 55 and 65° C.;   (g) a pl between 7-8 determined by isoelectric focusing (Pharmacia, Ampholine, pH 3.5-9.3); and   (h) improved wash and/or dishwash performance between pH 9-11.   
     
     
         58 . A variant of a parent Termamyl-like alpha-amylase with alpha-amylase activity comprising mutations in two, three, four, five, or six of the following regions/positions or in corresponding positions in other parent Termamyl-like alpha-amylases:
 (relative to SEQ ID NO: 1):   1: R181*, G182*, T183*, G184*   2: N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;   3: V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;   4: E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;   5: E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;   6: K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;   (relative to SEQ ID NO: 2):   1: R181*,G182*,D183*,G184*   2: N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;   3: V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;   4: E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;   5: E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;   6: K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;   (Relative to SEQ ID NO: 3):   1: R179*,G180,I181*,G182*   2: N193A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;   3: L204A,R,D,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V;   4: E210A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;   5: E214A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;   6: S267A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V   relative to SEQ ID NO: 4):   1: N190A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;   2: I201A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;   3: D207A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;   4: E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;   5: Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;   (relative to SEQ ID NO: 5):   1: R176*,G177*,E178,G179*   2: N190A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;   3: V201A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;   4: D207A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;   5: E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;   6: Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;   (relative to SEQ ID NO: 6):   1: R181*,G182*,H183*,G184*   2: N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;   3: I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;   4: E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;   5: E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;   6: K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V; and   (relative to SEQ ID NO: 24):   1: R181*,G182*,H183*,G184*   2: N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;   3: I206 A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;   4: E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;   5: E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;   6: K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V.   
     
     
         59 . A detergent composition comprising an alpha-amylase variant of  claim 58  and a surfactant.