Amylase variants

Abstract

The present invention relates to variants of a parent α-amylase, which parent α-amylase (i) has an amino acid sequence selected from the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 7, respectively; or (ii) displays at least 80% homology with one or more of these amino acid sequences; and/or displays immunological cross-reactivity with an antibody raised against an α-amylase having one of these amino acid sequences; and/or is encoded by a DNA sequence which hybridizes with the same probe as a DNA sequence encoding an α-amylase having one of these amino acid sequences; in which variant: (a) at least one amino acid residue of the parent α-amylase has been deleted; and/or (b) at least one amino acid residue of the parent α-amylase has been replaced by a different amino acid residue; and/or (c) at least one amino acid residue has been inserted relative to the parent α-amylase; the variant having α-amylase activity and exhibiting at least one of the following properties relative to the parent α-amylase: increased thermostability; increased stability towards oxidation; and reduced Ca 2+ dependency; with the proviso that the amino acid sequence of the variant is not identical to any of the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 7, respectively.

1 . A variant of a parent α-amylase, which parent α-amylase (i) has an amino acid sequence selected from the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 7, respectively, or (ii) displays at least 80% homology with one or more of said amino acid sequences: and/or displays immunological cross-reactivity with an antibody raised against an α-amylase having one of said amino acid sequences; and/or is encoded by a DNA sequence which hybridizes with the same probe as a DNA sequence encoding an α-amylase having one of said amino acid sequences; in which variant:
 (a) at least one amino acid residue of said parent α-amylase has been deleted; and/or   (b) at least one amino acid residue of said parent α-amylase has been replaced by a different amino acid residue; and/or   (c) at least one amino acid residue has been inserted relative to said parent α-amylase,   
       said variant having α-amylase activity and exhibiting at least one of the following properties relative to said parent α-amylase: increased thermostability; increased stability towards oxidation; and reduced Ca 2+  dependency; 
       provided that the amino acid sequence of said variant is not identical to any of the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2. SEQ ID No. 3 and SEQ ID No. 7, respectively. 
     
     
         2 . A variant according to  claim 1 , wherein at least one oxidizable amino acid residue of said parent α-amylase has been deleted or has been replaced by a different amino acid residue which is less susceptible to oxidation than said oxidizable amino acid residue. 
     
     
         3 . A variant according to  claim 2 , wherein said oxidizable amino acid residue is selected from the group consisting of methionine, tryptophan, cysteine and tyrosine. 
     
     
         4 . A variant according to  claim 2 , wherein said oxidizable amino acid residue is a methionine which is, or which is equivalent to, M9, M10, M105, M202, M208, M261, M309, M382, M430 or M440 of the amino acid sequence shown in SEQ ID No. 1. 
     
     
         5 . A variant according to  claim 4 , comprising a methionine substitution which is, or which is equivalent to, one of the following substitutions in the amino acid sequence shown in SEQ ID No. 1: M9L; M10L; M105L; M202L, T, F, I, V; M208L; M261L; M309L; M382L; M430L; M440L. 
     
     
         6 . A variant according to  claim 3 , wherein a said methionine residue has been replaced by threonine. 
     
     
         7 . A variant according to  claim 1 , wherein at least one amino acid which is, or which is equivalent to, F180, R181, G182, T183, G184 or K185 of the amino acid sequence shown in SEQ ID No. 1 as been deleted. 
     
     
         8 . A variant according to  claim 7 , wherein the deleted amino acids are, or are equivalent to, any two of said amino acid residues. 
     
     
         9 . A variant according to  claim 8 , wherein the deletions are, or are equivalent to, R181*+G182*; or T183*+G184*. 
     
     
         10 . A variant according to  claim 1 , comprising an amino acid substitution which is, or which is equivalent to, one of the following substitutions in the amino acid sequence shown in SEQ ID No. 1, K269R; P260E; R124P; M105F, I, L, V, M208F, W, Y; L2171, V206I, L, F. 
     
     
         11 . A variant according to  claim 1 , comprising an amino acid substitution which is, or which is equivalent to, one of the following substitutions in the amino acid sequence shown in SEQ ID No. 1: Y243F; K108R; K179R; K239R, K242R: K269R; D163N; D188N; D192N; D199N, D205N; D207N; D209N; E190Q; E194Q; N106D. 
     
     
         12 . A DNA construct comprising a DNA sequence encoding an α-amylase variant according to  claim 1 . 
     
     
         13 . A recombinant expression vector which carries a DNA construct according to  claim 12 . 
     
     
         14 . A cell which is transformed with a DNA construct according to  claim 12 . 
     
     
         15 . A cell according to  claim 14 , which is a microorganism. 
     
     
         16 . A cell according to  claim 15 , which is a bacterium or a fungus. 
     
     
         17 . A cell according to  claim 16 , which is a gram positive bacterium such as  Bacillus subtilis Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus thuringiensis  or  Streptomyces lividans  or  Streptomyces murinus , or a gram negative bacterium such as  E. coli.    
     
     
         18 . A method of producing an α-amylase variant, comprising culturing a cell according to  claim 14  under conditions conducive to the production of the α-amylase variant, and recovering the α-amylase variant from the culture. 
     
     
         19 . (canceled) 
     
     
         20 . A detergent additive comprising an α-amylase variant according to  claim 1 , optionally in the form of a non-dusting granulate, stabilized liquid or protected enzyme. 
     
     
         21 . A detergent additive according to  claim 20 , comprising 0.02-200 mg of enzyme protein per gram of the additive. 
     
     
         22 . A detergent additive according to  claim 20 , which additionally comprises another enzyme such as a protease, a lipase, a peroxidase, another amylolytic enzyme and/or a cellulase. 
     
     
         23 . A detergent composition comprising an α-amylase variant according to  claim 1  and a surfactant. 
     
     
         24 . A detergent composition according to  claim 23 , which additionally comprises another enzyme such as a protease, a lipase, a peroxidase, another amylolytic enzyme and/or a cellulase. 
     
     
         25 . A manual or automatic dishwashing detergent composition comprising an α-amylase variant according to  claim 1  and a surfactant. 
     
     
         26 . A dishwashing detergent composition according to  claim 25 , which additionally comprises another enzyme such as a protease, a lipase, a peroxidase, another amylolytic enzyme and/or a cellulase. 
     
     
         27 . A manual or automatic laundry washing composition comprising an α-amylase variant according to  claim 1  and a surfactant. 
     
     
         28 . A laundry washing composition according to  claim 27 , which additionally comprises another enzyme such as a protease, a lipase, a peroxidase, an amylolytic enzyme and/or a cellulase. 
     
     
         29 . (canceled) 
     
     
         30 . A variant according to  claim 1 , wherein at least three amino acids selected among F180, R181, G182, T183, G184 and KS185 (using SEQ ID NO:1 for numbering) have been deleted. 
     
     
         31 . A variant according to  claim 1 , comprising a deletion or substitution of a methionine residue in a position corresponding to M323 of the amino acid sequence shown in SEQ ID NO:2. 
     
     
         32 . A variant according to  claim 1 , comprising a deletion or substitution of a methionine residue in a position corresponding to deletion or substitution of one or more of the methionine residues in positions M9, M10, M105, M202, M208, M261, M309, M382, M430 and M440 of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:7. 
     
     
         33 . A variant according to  claim 1 , comprising a deletion or substitution of a methionine residue in a position corresponding to MS, M9, M96, M200, M206, M284, M307, M311, M316 and M438 of the amino acid sequence shown in SEQ ID NO:3. 
     
     
         34 . A variant according to  claim 1 , comprising a mutation corresponding to A354C+V479C; L351C+M430C; N457D, E+K385R; L355D, E+M430R, K; L355D, E+I411R, K; and N457D, E (using SEQ ID NO:1 for numbering). 
     
     
         35 . A variant according to  claim 1 , comprising one or more substitutions corresponding to K107R, K177R, K237R, K240R, D162N, D186N, D190N, D197N, D203N, D205N, D207N, E188Q and E192Q (using SEQ ID NO:1 for numbering). 
     
     
         36 . A variant according to  claim 1 , comprising a substitution of one or more of the amino acid residues from P260 to I275 (using SEQ ID NO:1 for numbering). 
     
     
         37 . A variant according to  claim 1 , comprising one or more substitutions corresponding to K107R, K177R, K237R, K240R, D162N, D186N, D190N, D197N, D203N, D205N, D207N, E188Q and E192Q (using SEQ ID NO:3 for numbering). 
     
     
         38 . A variant according to  claim 1  comprising one or more substitutions corresponding to pairwise substitutions of the amino acid residues present at positions 113 and 151 and positions 351 and 430 in the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 7; and at positions 112 and 150 and positions 349 and 428 in the amino acid sequence shown in SEQ ID No. 3. 
     
     
         39 . A variant according to  claim 1 , comprising one or more of the following G304W, F, Y, R, I, L, V, Q, N, G305A, S, N, D, Q, E, R, k; and H408Q, E, (using SEQ ID NO:1 for numbering). 
     
     
         40 . A variant according to  claim 1 , comprising one or more substitutions corresponding to the following substitutions, Q86E, R124P, S154D, T183D, V222E, P260E, R310A, Q346E, Q391E, N437E, K444Q and R452H (using SEQ ID NO:1 for numbering). 
     
     
         41 . A variant according to  claim 1 , comprising one or more substitutions corresponding to the following substitutions. K107R, K177R, KQ37R, K240R, D162N, D186N, D190N, D197N, D203N, D205N, D207N, E188Q, and E192Q (using SEQ ID NO:3 for numbering). 
     
     
         42 . A variant according to  claim 1 , comprising one or more substitutions corresponding to the following substitutions:
 G113+N151 (using SEQ ID No. 1 for numbering),   A113+T151 (using SEQ ID No. 2 and SEQ ID No. 7 for numbering);   G112+T150 (using SEQ ID No. 3 for numbering); and   L351+M430 (using SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 7 for numbering);   L349+I428 (using SEQ ID No. 3 for numbering).   
     
     
         43 . A detergent comprising a surfactant and an alpha-amylase, wherein the alpha-amylase is selected from the group consisting of:
 an alpha-amylase comprising an amino acid sequence having at least 80% homology to SEQ ID NO:1 and which alpha-amylase is modified by having an amino acid deletion of two amino acids selected from the group of amino acids equivalent to positions 180, 181, 182, 183, 184 and 185 in SEQ ID NO:1;   an alpha-amylase comprising an amino acid sequence having at least 80% homology to SEQ ID NO:2 and which alpha-amylase is modified by having an amino acid deletion of two amino acids selected from the group of amino acids equivalent to positions 180, 181, 182, 183, 184 and 185 in SEQ ID NO:2:   an alpha-amylase comprising an amino acid sequence having at least 80% homology to SEQ ID NO:3 and which alpha-amylase is modified by having an amino acid deletion of two amino acids selected from the group of amino acids equivalent to positions 178, 179, 180, 181, 182 and 183 in SEQ ID NO:3; and   an alpha-amylase comprising an amino acid sequence having at least 80% homology to SEQ ID NO:7 and which alpha-amylase is modified by having an amino acid deletion of two amino acids selected from the group of amino acids equivalent to positions 180, 181, 182, 183, 184 and 185 in SEQ ID NO:7.   
     
     
         44 . A method for producing ethanol from starch, comprising treating starch with an alpha-amylase of  claim 1  and preparing ethanol from the treated starch. 
     
     
         45 . The method of  claim 44 , wherein the alpha-amylase is selected from the group consisting of:
 an alpha-amylase comprising an amino acid sequence having at least 80% homology to SEQ ID NO:1 and which alpha-amylase is modified by having an amino acid deletion of two amino acids selected from the group of amino acids equivalent to positions 180, 181, 182, 183, 185 and 185 in SEQ ID NO:1;   an alpha-amylase comprising an amino acid sequence having at least 80% homology to SEQ ID NO:2 and which alpha-amylase is modified by having an amino acid deletion of two amino acids selected from the group of amino acids equivalent to positions 180, 181, 182, 183, 185 and 185 in SEQ ID NO:2,   an alpha-amylase comprising an amino acid sequence having at least 80% homology to SEQ ID NO:3 and which alpha-amylase is modified by having an amino acid deletion of two amino acids selected from the group of amino acids equivalent to positions 178, 179, 180, 181, 182 and 183 in SEQ ID NO:3, and   an alpha-amylase comprising an amino acid sequence having at least 80% homology to SEQ ID NO:7 and which alpha-amylase is modified by having an amino acid deletion of two amino acids selected from the group of amino acids equivalent to positions 180, 181, 182, 183, 184 and 185 in SEQ ID NO:7.   
     
     
         46 . A method for producing a sweetener from starch, comprising treating starch with an alpha-amylase of  claim 1  and preparing the sweetener from the treated starch. 
     
     
         47 . The method of  claim 46 , wherein the alpha-amylase is selected from the group consisting of:
 an alpha-amylase comprising an amino acid sequence having at least 80% homology to SEQ ID NO:1 and which alpha-amylase is modified by having a deletion of two amino acids selected from the group of amino acids equivalent to positions 180, 181, 182, 183, 185 and 185 in SEQ ID NO:1;   an alpha-amylase comprising an amino acid sequence having at least 80% homology to SEQ ID NO:2 and which alpha-amylase is modified by having a deletion of two amino acids selected from the group of amino acids equivalent to positions 180, 181, 182, 183, 185 and 185 in SEQ ID NO:2;   an alpha-amylase comprising an amino acid sequence having at least 80% homology to SEQ ID NO:3 and which alpha-amylase is modified by having a deletion of two amino acids selected from the group of amino acids equivalent to positions 178, 179, 180, 181, 182 and 183 in SEQ ID NO:3; and   an alpha-amylase comprising an amino acid sequence having at least 80% homology to SEQ ID NO:7 and which alpha-amylase is modified by having a deletion of two amino acids selected of an amino acid selected from the group of amino acids equivalent to positions 180, 181, 182, 183, 184 and 185 in SEQ ID NO:7.