Alpha-amylase and alpha-amylase variants

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 . A variant of a parent Termamyl-like α-amylase with α-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 α-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; 
 
     
     
         2 . The variant according to  claim 1 , comprising the following mutations: N190F/Q264S in SEQ ID NO: 4 or in corresponding positions in another parent α-amylase. 
     
     
         3 . The variant according to  claim 1 , comprising the following mutations: I181*/G182*/N193F in SEQ ID NO: 3 or in corresponding positions in another parent Termamyl like α-amylase. 
     
     
         4 . The variant according to  claim 3 , further comprising a substitution in position E214Q in SEQ ID NO: 3 or in a corresponding position in another parent Termamyl like α-amylase. 
     
     
         5 . The variant according to  claim 1 , wherein the parent α-amylase is a hybrid α-amylase of SEQ ID NO: 4 and SEQ ID NO: 5. 
     
     
         6 . The variant according to  claim 5 , wherein the parent hybrid α-amylase is a hybrid alpha-amylase comprising the 445 C-terminal amino acid residues of the  B. licheniformis  α-amylase shown in SEQ ID NO: 4 and the 37 N-terminal amino acid residues of the α-amylase derived from  B. amyloliquefaciens  shown in SEQ ID NO: 5. 
     
     
         7 . The variant according to  claim 6 , wherein the parent hybrid Termamyl-like α-amylase further has the following mutations: H156Y+A181T+N190F+A209V+Q264S (using the numbering in SEQ ID NO: 4). 
     
     
         8 . The variant according to  claim 1 , exhibiting increased stability at acidic pH and/or low Ca 2+  concentration: 
     
     
         9 . A DNA construct comprising a DNA sequence encoding an α-amylase variant according to  claim 1 . 
     
     
         10 . A recombinant expression vector which carries a DNA construct according to  claim 9 . 
     
     
         11 . A cell which is transformed with a DNA construct according to  claim 9  or a vector according to  claim 10 . 
     
     
         12 . A cell according to  claim 11 , which is a microorganism. 
     
     
         13 . A cell according to  claim 12 , which is a bacterium or a fungus. 
     
     
         14 . The cell according to  claim 13 , which is a grampositive bacterium such as  Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus  or  Bacillus thuringiensis.    
     
     
         15 . A detergent additive comprising an α-amylase variant according to  claim 1 , optionally in the form of a non-dusting granulate, stabilised liquid or protected enzyme. 
     
     
         16 . A detergent additive according to  claim 15  which contains 0.02-200 mg of enzyme protein/g of the additive. 
     
     
         17 . A detergent additive according to  claim 15 , which additionally comprises another enzyme such as a protease, a lipase, a peroxidase, another amylolytic enzyme and/or a cellulase. 
     
     
         18 . A detergent composition comprising an α-amylase variant according to  claim 1 . 
     
     
         19 . The detergent composition according to  claim 18  which additionally comprises another enzyme such as a protease, a lipase, a peroxidase, another amylolytic enzyme and/or a cellulase. 
     
     
         20 . A manual or automatic dishwashing detergent composition comprising an α-amylase variant according to  claim 1 . 
     
     
         21 . A dishwashing detergent composition according to  claim 20  which additionally comprises another enzyme such as a protease, a lipase, a peroxidase, another amylolytic enzyme and/or a cellulase. 
     
     
         22 . A manual or automatic laundry washing composition comprising an α-amylase variant according to  claim 1 . 
     
     
         23 . A laundry washing composition according to  claim 22 , which additionally comprises another enzyme such as a protease, a lipase, a peroxidase, an amylolytic enzyme and/or a cellulase. 
     
     
         24 . A composition comprising:
 (i) a mixture of the α-amylase from  B. licheniformis  having the sequence shown in SEQ ID NO: 4 with one or more variants according to  claim 1  derived from (as the parent Termamyl-like α-amylase) the  B. stearothermophilus  α-amylase having the sequence shown in SEQ ID NO: 3; or   (ii) a mixture of the α-amylase from  B. stearothermophilus  having the sequence shown in SEQ ID NO: 3 with one or more variants according to  claim 1  derived from one or more other parent Termamyl-like α-amylases; or   (iii) a mixture of one or more variants according  claim 1  derived from (as the parent Termamyl-like α-amylase) the  B. stearothermophilus  α-amylase having the sequence shown in SEQ ID NO: 3 with one or more variants according to the invention derived from one or more other parent Termamyl-like α-amylases.   
     
     
         25 . A composition comprising:
 a mixture of one or more variants according  claim 1  derived from (as the parent Termamyl-like α-amylase) the  B. stearothermophilus  α-amylase having the sequence shown in SEQ ID NO: 3 and a Termamyl-like alpha-amylase derived from the  B. licheniformis  α-amylase having the sequence shown in SEQ ID NO: 4.   
     
     
         26 . The composition comprising:
 a mixture of one or more variants according  claim 1  derived from (as the parent Termamyl-like α-amylase) the  B. stearothermophilus  α-amylase having the sequence shown in SEQ ID NO: 3 and a hybrid alpha-amylase comprising a part of the  B. amyloliquefaciens  α-amylase shown in SEQ ID NO: 5 and a part of the  B. licheniformis  α-amylase shown in SEQ ID NO: 4.   
     
     
         27 . The composition according to  claim 26 , wherein the hybrid α-amylase is a hybrid alpha-amylase comprising the 445 C-terminal amino acid residues of the  B. licheniformis  α-amylase shown in SEQ ID NO: 4 and the 37 N-terminal amino acid residues of the α-amylase derived from  B. amyloliquefaciens  shown in SEQ ID NO: 5. 
     
     
         28 . The composition according to  claim 27 , wherein the hybrid α-amylase further has the following mutations: H156Y+A181T+N190F+A209V+Q264S (using the numbering in SEQ ID NO: 4). 
     
     
         29 . The composition according to  claim 26 , comprising a mixture of TVB146 and LE174. 
     
     
         30 . A method for generating a variant of a parent Termamyl-like α-amylase, which variant exhibits increased stability at low pH and at low calcium concentration relative to the parent, the method comprising:
 (a) subjecting a DNA sequence encoding the parent Termamyl-like α-amylase to random mutagenesis, 
 (b) expressing the mutated DNA sequence obtained in step (a) in a host cell, and 
 (c) screening for host cells expressing a mutated α-amylase which has increased stability at low pH and low calcium concentration relative to the parent α-amylase.