Alpha-amylase mutants

Abstract

The invention relates to a variant of a parent Termamyl-like a-amylase, which variant has a-amylase activity and exhibits an alteration in at least one of the following properties relative to said parent a-amylase: substrate specificity, substrate binding, substrate cleavage pattern, thermal stability, pH/activity profile, pH/stability profile, stability towards oxidation, Ca 2+ dependency and specific activity.

1 . A variant of a parent Termamyl-like α-amylase, which variant has α-amylase activity and exhibits an alteration in at least one of the following properties relative to said parent α-amylase: substrate specificity, substrate binding, substrate cleavage pattern, thermal stability, pH/activity profile, pH/stability profile, stability towards oxidation, Ca 2+  dependency and specific activity; said variant comprising one or more mutations corresponding to one or more of the following mutations in the amino acid sequence shown in SEQ ID No. 2: 
 the mutations: A181E,D,Q,N,V; I201(bulkier amino acid), including I201W,F,L; Y203Q; Q9K,L,E; F11R,K,E; E12Q; D100N,L; V101H,R,K,D,E,F; V102A,T; I103H,K; N104R,K,D; H105R,K,D,E,W,F; L196R,K,D,E,F,Y; I212R,K,D,E; L230H,K,I; A232G,H,F,S,V; V233D; K234L,E; I236R,K,N,H,D,E; L241R,K,D,E,F; A260S; W263H; Q264R,D,K,E; N265K,R,D; A269R,K,D,E; L270R,K,H,D,E; V283H,D; F284H; D285N,L; V286R,K,H,D,E; Y290R,E,K; V312R,K,D,E; F323H; D325N; N326K,H,D,L; H327Q,N,E,D,F; Q330L,E; G332D; Q333R,K,H,E,L; S334A,V,T,L,I,D; L335G,A,S,T,N; E336R+R375E; T337D,K; T338D,E; T339D; Q360K,R,E; D365N; or G371D,R;  
 substitutions at positions H68, H91, H247, R305, K306, H382, K389, H405, H406, H450 or R483;  
 the mutations: H140Y; H142Y; H159Y; H140D+H142R; H140K+H142D; or H142Y+H156Y;  
 deletion of three amino acids within the partial sequence from T369 to I377, including the deletions: K370*+G371*+D372* or D372*+S373*+Q374*;  
 replacement of the partial amino acid sequence from T369 to I377 with one of the following sequences (sequence number increasing from left to right): I-P-T-H-S-V; I-P-T-H-G-V; I-P-Q-Y-N-I;  
 substitutions at positions R169 or R173, including R169I,L,F,T or R173I,L,F,T;  
 the mutations H156D; I201F; I212F; A209L,T; or V208I;  
 substitutions at positions N172, A181, N188, N190, H205, D207, A209, A210, E211, Q264 or N265, including N172R,H,K; A181T; N188P; N190L,F; H205C; D207Y; A209L,T,V; A210S; E211Q; Q264A,E,L,K,S,T; N265A,S,T,Y; or Q264S+N265Y;  
 the mutations: 
 H156Y+A181T+A209V;  
 H156Y+A181T+N190F+A209V+Q264S;  
 A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I+H156Y+A181T+A2 09V;  
 A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I+H156Y+A181T+N1 90F+A209V; or  
 
 A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I+H156Y+A181T+N1 90F+A209V+Q264S.  
 
     
     
         2 . A variant according to  claim 1 , exhibiting increased stability at low pH and low Ca 2+  concentration relative to the parent Termamyl-like α-amylase, and comprising mutations selected from the following: 
 H156Y+A181T+A209V;  
 H156Y+A181T+N190F+A209V+Q264S;  
 A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I+H156Y+A181T+A2 09V;  
 A1*+N2*+L3V+M1ST+R23K+S29A+A30E+Y31H+A33S+E34D+H35I+H156Y+A181T+N1 90F+A209V;  
 A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I+H156Y+A181T+N1 90F+A209V+Q264S.  
 
     
     
         3 . A variant according to  claim 1  or  2 , wherein the parent Termamyl-like α-amylase is selected from: 
 the  B. licheniformis  α-amylase having the sequence shown in SEQ ID No. 2,  
 the  B. amyloliquefaciens  α-amylase having the sequence shown in SEQ ID No. 4,  
 the  B. stearothermophilus  α-amylase having the sequence shown in SEQ ID No. 6,  
 the Bacillus strain NCIB 12512 α-amylase having the sequence shown in FIGS. 1 and 2,  
 the Bacillus strain NCIB 12513 α-amylase having the sequence shown in FIG. 2, and  
 the Bacillus sp. #707 α-amylase having the sequence shown in FIG. 2.  
 
     
     
         4 . A DNA construct comprising a DNA sequence encoding an α-amylase variant according to any one of claims  1 - 3 .  
     
     
         5 . A recombinant expression vector which carries a DNA construct according to  claim 4 .  
     
     
         6 . A cell which is transformed with a DNA construct according to  claim 4  or a vector according to  claim 5 .  
     
     
         7 . A cell according to  claim 6 , which is a microorganism.  
     
     
         8 . A cell according to  claim 6 , which is a bacterium or a fungus.  
     
     
         9 . The cell according to  claim 8 , 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.    
     
     
         10 . Use of an α-amylase variant according to any one of claims  1 - 3  for washing and/or dishwashing.  
     
     
         11 . Use of an α-amylase variant according to any one of claims  1 - 3  for textile desizing.  
     
     
         12 . Use of an α-amylase variant according to any of claims  1 - 3  for starch liquefaction.  
     
     
         13 . A detergent additive comprising an α-amylase variant according to any one of claims  1 - 3 , optionally in the form of a non-dusting granulate, stabilised liquid or protected enzyme.  
     
     
         14 . A detergent additive according to  claim 13  which contains 0.02-200 mg of enzyme protein/g of the additive.  
     
     
         15 . A detergent additive according to  claim 13  or  14 , which additionally comprises another enzyme such as a protease, a lipase, a peroxidase, another amylolytic enzyme and/or a cellulase.  
     
     
         16 . A detergent composition comprising an α-amylase variant according to any of claims  1 - 3 .  
     
     
         17 . A detergent composition according to  claim 16  which additionally comprises another enzyme such as a protease, a lipase, a peroxidase, another amylolytic enzyme and/or a cellulase.  
     
     
         18 . A manual or automatic dishwashing detergent composition comprising an α-amylase variant according to any one of claims  1 - 3 .  
     
     
         19 . A dishwashing 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 laundry washing composition comprising an α-amylase variant according to any one of claims  1 - 3 .  
     
     
         21 . A laundry washing composition according to  claim 20 , which additionally comprises another enzyme such as a protease, a lipase, a peroxidase, an amylolytic enzyme and/or a cellulase.  
     
     
         22 . A composition comprising: 
 (i) a mixture of the α-amylase from  B. licheniformis  having the sequence shown in SEQ ID No. 2 with one or more variants according to the invention derived from (as the parent Termamyl-like α-amylase) the  B. stearothermophilus  α-amylase having the sequence shown in SEQ ID No. 6; or    (ii) a mixture of the α-amylase from  B. stearothermophilus  having the sequence shown in SEQ ID No. 6 with one or more variants according to the invention derived from one or more other parent Termamyl-like α-amylases; or    (iii) a mixture of one or more variants according to the invention derived from (as the parent Termamyl-like α-amylase) the  B. stearothermophilus  α-amylase having the sequence shown in SEQ ID No. 6 with one or more variants according to the invention derived from one or more other parent Termamyl-like α-amylases.    
     
     
         23 . 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.