Alpha-amylase combinatorial variants

Abstract

Disclosed are compositions and methods relating to variant alpha-amylases. The variant alpha-amylases are useful, for example, for starch liquefaction and saccharification, for cleaning starchy stains in laundry, dishwashing, and other applications, for textile processing (e.g., desizing), in animal feed for improving digestibility, and for baking and brewing.

What is claimed is: 
     
         1 . A recombinant variant of a parent α-amylase comprising:
 a deletion of at least one amino acid residue in the X 1 G/S 1 X 2 G 2  motif adjacent to the calcium-binding loop of the parent α-amylase, and corresponding to the residues at positions 178-181 of SEQ ID NO: 1, wherein in at least one non-G residue remains in the motif following the deletion; and 
 a substitution of the amino acid residue corresponding to the residue at position 132 of SEQ ID NO: 1, and/or a substitution of the non-G amino acid residue remaining in the motif of the variant α-amylase, which substitution(s) allows a stabilizing interaction between the amino acid residue at position 132 and the non-G amino acid residue remaining in the motif of the variant α-amylase; 
 wherein the variant α-amylase has α-amylase activity and improved stability compared to a reference α-amylase differing from the variant α-amylase only by the absence of the substitution; and 
 wherein SEQ ID NO: 1 is used for numbering. 
 
     
     
         2 . The variant α-amylase of  claim 1 , wherein the deletion of at least one amino acid residue in the motif is the deletion of the residues at positions 178 and 179 or position 180 and 181, using SEQ ID NO: 1 for numbering. 
     
     
         3 . The variant α-amylase of any of the preceding claims, wherein the substitution changes the charge of the amino acid residue at position 132 or of the remaining amino acid residue in the motif at a predetermined pH. 
     
     
         4 . The variant α-amylase of any of the preceding claims, wherein the substitution is at position 132, using SEQ ID NO: 1 for numbering. 
     
     
         5 . The variant α-amylase of any of the preceding claims, wherein the substitution is at position 178 or 180, using SEQ ID NO: 1 for numbering. 
     
     
         6 . The variant α-amylase of any of the preceding claims, wherein the amino acid residue at position 132 is negatively charged and the non-G amino acid residue remaining in the calcium-binding loop is changed to positively charged amino acid residue. 
     
     
         7 . The variant α-amylase of any of the preceding claims, wherein the amino acid residue at position 132 is positively charged and the non-G amino acid residue remaining in the calcium-binding loop is changed to negatively charged amino acid residue. 
     
     
         8 . The variant α-amylase of any of the preceding claims, wherein the amino acid residue at position 132 is H, R, or K and the non-G amino acid residue remaining in the calcium-binding loop is D or E. 
     
     
         9 . The variant α-amylase of any of the preceding claims, wherein the amino acid residue at position 132 is D or E and the non-G amino acid residue remaining in the calcium-binding loop is H, R, or K. 
     
     
         10 . The variant of any of the preceding claims, further comprising a mutation at an amino acid residue corresponding to E187 or S241, using SEQ ID NO: 1 for numbering. 
     
     
         11 . The variant α-amylase of any of the preceding claims, further comprising at least one mutation at an amino acid residue corresponding to an amino acid residue selected from the group consisting of N126, Y150, F153, L171, T180, and, I203 using SEQ ID NO: 1 for numbering. 
     
     
         12 . The variant α-amylase of any of the preceding claims, further comprising a mutation at an amino acid residue corresponding to G476 and/or G477, using SEQ ID NO: 1 for numbering. 
     
     
         13 . The variant α-amylase of any of the preceding claims, further comprising a mutation in an amino acid residue corresponding to an amino acid residue selected from the group consisting of E132, Q167, and A277, using SEQ ID NO: 1 for numbering. 
     
     
         14 . The variant α-amylase of any of the preceding claims, further comprising a mutation in an amino acid residue corresponding to an amino acid residue selected from the group consisting of R458, T459, and D460, using SEQ ID NO: 1 for numbering. 
     
     
         15 . The variant α-amylase of any of the preceding claims, further comprising a mutation in an amino acid residue corresponding to N205, using SEQ ID NO: 3 for numbering. 
     
     
         16 . The variant α-amylase of any of the preceding claims, further comprising a mutation in an amino acid residue corresponding to an amino acid residue selected from the group consisting of T333G, A335S, and Q337E, using SEQ ID NO: 3 for numbering. 
     
     
         17 . The variant α-amylase of any of the preceding claims, comprising a combinations of mutations selected from the group consisting of:
 N126Y+E132H+T180D+E187P+I203Y+Y303D+G476T+G477E, 
 N126Y+E132H+F153W+T180D+I203Y+S241Q+A277F, 
 N126Y+E132H+F153W+Q167E+T180D+I203Y+S241Q+A277F, 
 N126Y+E132H+F153W+Q167E+T180D+I203Y+S241Q+A277F+T400K, 
 T134E+T182H+E189P+G475R, 
 N128Y+T134E+T182H+E189P+G475R, 
 T134E+F155W+T182H+E189P+G475R, and 
 N128Y+T134E+F155W+T182H+E189P+G475R. 
 
     
     
         18 . A method for increasing the stability of a recombinant α-amylase, comprising:
 deleting at least one amino acid residue in the X 1 G/S 1 X 2 G 2  motif adjacent to the calcium-binding loop of the parent α-amylase, and corresponding to the residues at positions 178-181 of SEQ ID NO: 1, wherein in at least one non-G residue remains in the motif following the deletion; and 
 substituting the amino acid residue corresponding to the residue at position 132 of SEQ ID NO: 1, and/or a substituting the non-G amino acid residue remaining in the motif such that the residue at a position corresponding to position 132 and the non-G amino acid residue remaining in the motif are capable of participating in a stabilizing interaction; 
 wherein the resulting α-amylase has increased stability compared to a reference α-amylase that differs only in not having the substitution at the amino acid residue reresponding to position 132 and/or the substitution at the non-G amino acid residue remaining in the motif; and 
 wherein SEQ ID NO: 1 is used for numbering. 
 
     
     
         19 . A method for increasing the stability of a recombinant α-amylase having a deletion of at least one amino acid residue in the X 1 G/S 1 X 2 G 2  motif adjacent to the calcium-binding loop corresponding to the residues at positions 178-181 of SEQ ID NO: 1, wherein in at least one non-G residue remains in the motif, comprising:
 substituting the amino acid residue corresponding to the residue at position 132 of SEQ ID NO: 1, and/or a substituting the non-G amino acid residue remaining in the motif such that the residue at a position corresponding to position 132 and the non-G amino acid residue remaining in the motif are capable of participating in a stabilizing interaction; 
 wherein the resulting α-amylase has increased stability compared to a reference α-amylase that differs only in not having the substitution at the amino acid residue reresponding to position 132 and/or the substitution at the non-G amino acid residue remaining in the motif; and 
 wherein SEQ ID NO: 1 is used for numbering. 
 
     
     
         20 . The method of  claim 18  or  19 , wherein the deletion of at least one amino acid residue in the motif is the deletion of the residues at positions 178 and 179 or position 180 and 181, using SEQ ID NO: 1 for numbering. 
     
     
         21 . The method of any of  claims 18 - 20 , wherein the substitution changes the charge of the amino acid residue at position 132 or of the remaining amino acid residue in the motif at a predetermined pH. 
     
     
         22 . The method of any of  claims 18 - 21 , wherein the substitution (i) is at position 132 or (ii) is at position 178 or 180, using SEQ ID NO: 1 for numbering. 
     
     
         23 . The method of any of  claims 18 - 22 , wherein the amino acid residue at position 132 of the resulting α-amylase is negatively charged and the non-G amino acid residue remaining in the calcium-binding loop is positively charged or the amino acid residue at position 132 in the resulting α-amylase is positively charged and the non-G amino acid residue remaining in the calcium-binding loop is negatively charged. 
     
     
         24 . The method of any of  claims 18 - 23 , wherein the amino acid residue at position 132 of the resulting α-amylase is H, R, or K and the non-G amino acid residue remaining in the calcium-binding loop is D or E; or the amino acid residue at position 132 is D or E and the non-G amino acid residue remaining in the calcium-binding loop is H, R, or K. 
     
     
         25 . The method of any of  claims 18 - 24 , wherein the resulting α-amylase further comprises:
 (i) a mutation at an amino acid residue corresponding to E187 or S241; 
 (ii) a mutation at an amino acid residue corresponding to corresponding to an amino acid residue selected from the group consisting of N126, Y150, F153, L171, T180, and, I203; 
 (iii) a mutation at an amino acid residue corresponding to G476 and/or G477; 
 (iv) a mutation in an amino acid residue corresponding to an amino acid residue selected from the group consisting of E132, Q167, and A277; 
 (v) a mutation in an amino acid residue corresponding to an amino acid residue selected from the group consisting of R458, T459, and D460; 
 (vi) a mutation in an amino acid residue corresponding to N205, using SEQ ID NO: 3 for numbering; and/or 
 (vii) a mutation in an amino acid residue corresponding to an amino acid residue selected from the group consisting of T333G, A335S, and Q337E; 
 wherein for (i)-(v) SEQ ID NO: 1 is used for numbering and for (vi) and (vii) for (i)-(v) SEQ ID NO: 3 is used for numbering. 
 
     
     
         26 . A variant α-amylase produced by the method of any of  claims 18 - 25 . 
     
     
         27 . The variant amylase of any of  claim 1 - 7  or  26 , wherein the parental α-amylase is from a  Cytophaga  species. 
     
     
         28 . The variant amylase of any of  claim 1 - 7  or  26 , wherein the parental α-amylase is from a  Paenibacillus  species. 
     
     
         29 . The variant amylase of any of  claim 1 - 7  or  26 , wherein the parental α-amylase is not from a  Bacillus  species. 
     
     
         30 . The variant amylase of any of  claim 1 - 7  or  26 , wherein the parental α-amylase or the variant α-amylase has at least 70% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. 
     
     
         31 . The variant amylase of any of  claim 1 - 7  or  26 , wherein the parental α-amylase or the variant α-amylase has at least 70% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. 
     
     
         32 . The variant amylase of any of  claim 1 - 7  or  26 , wherein the parental α-amylase or the variant α-amylase has at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. 
     
     
         33 . The variant amylase of any of  claim 1 - 7  or  26 , wherein the parental α-amylase or the variant α-amylase has at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. 
     
     
         34 . A composition comprising the variant α-amylase of any of the preceding claims. 
     
     
         35 . The composition of  claim 34 , wherein the composition is effective for removing starchy stains from laundry, dishes, or textiles. 
     
     
         36 . The composition of  claim 34  or  35 , further comprising a surfactant. 
     
     
         37 . The composition of any of  claims 34 - 36 , wherein the composition is a detergent composition. 
     
     
         38 . The composition of any of  claims 34 - 36 , wherein the composition is a laundry detergent or a laundry detergent additive. 
     
     
         39 . The composition of any of  claims 34 - 36 , wherein the composition is a manual or automatic dishwashing detergent. 
     
     
         40 . The composition of any of  claims 34 - 36 , further comprising one or more additional enzymes selected from the group consisting of protease, hemicellulase, cellulase, peroxidase, lipolytic enzyme, metallolipolytic enzyme, xylanase, lipase, phospholipase, esterase, perhydrolase, cutinase, pectinase, pectate lyase, mannanase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, pullulanase, tannase, pentosanase, malanase, β-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, metalloproteinase, amadoriase, glucoamylase, arabinofuranosidase, phytase, isomerase, transferase, and an amylase other than the amylase of any one of  claim 1 - 17  or  26 . 
     
     
         41 . The composition of  claim 34 , wherein the composition is for liquifying starch. 
     
     
         42 . The composition of  claim 34 , wherein the composition is for saccharifying a composition comprising starch, for SSF post liquefaction, or for direct SSF without prior liquefaction. 
     
     
         43 . The composition of  claim 34 , wherein the composition is for producing a fermented beverage. 
     
     
         44 . The composition of  claim 34 , wherein the composition is for producing a baked food product. 
     
     
         45 . The composition of  claim 34 , wherein the composition is for textile desizing. 
     
     
         46 . A method for removing a starchy stain or soil from a surface, comprising:
 contacting the surface in the presence of a composition comprising an effective amount of the variant amylase of any of the  claim 1 - 17  or  26 , and   allowing the polypeptide to hydrolyze starch components present in the starchy stain to produce smaller starch-derived molecules that dissolve in the aqueous composition,   thereby removing the starchy stain from the surface.   
     
     
         47 . The method of  claim 46 , wherein the aqueous composition further comprises a surfactant. 
     
     
         48 . The method of any of  claim 46  or  47 , wherein the surface is a textile surface or a surface on dishes. 
     
     
         49 . The method of any of  claims 46 - 48 , wherein the composition further comprises at least one additional enzymes selected from the group consisting of protease, hemicellulase, cellulase, peroxidase, lipolytic enzyme, metallolipolytic enzyme, xylanase, lipase, phospholipase, esterase, perhydrolase, cutinase, pectinase, pectate lyase, mannanase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, pullulanase, tannase, pentosanase, malanase, β-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, metalloproteinase, amadoriase, glucoamylase, arabinofuranosidase, phytase, isomerase, transferase, and an amylase other than the amylase of any one of  claim 1 - 17  or  26 . 
     
     
         50 . A method for saccharifying a composition comprising starch to produce a composition comprising glucose, wherein the method comprises:
 (i) contacting the solution comprising starch with effective amount of the variant amylase of any of the  claim 1 - 17  or  26 ; and   (ii) saccharifying the solution comprising starch to produce the composition comprising glucose; wherein the variant amylase catalyzes the saccharification of the starch solution to glucose or other enriched carbohydrate syrups.   
     
     
         51 . The method of  claim 50 , wherein the composition comprising starch comprises liquefied starch, gelatinized starch, granular starch, or starch heat-treated below its gelatinization temperature. 
     
     
         52 . The method of  claim 50  or  51 , wherein the fermentation is a simultaneous saccharification and fermentation (SSF) reaction. 
     
     
         53 . The method of any of  claims 50 - 52 , wherein the method further comprises contacting a mash and/or a wort with an amylase. 
     
     
         54 . The method of any one of  claims 50 - 53 , further comprising adding glucoamylase, hexokinase, xylanase, glucose isomerase, xylose isomerase, phosphatase, phytase, pullulanase, β-amylase, α-amylase that is not the variant α-amylase, protease, cellulase, hemicellulase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, pectinase, alpha-glucosidase, beta-glucosidase, or a combination thereof, to the starch solution. 
     
     
         55 . The method of any one of  claims 50 - 54 , wherein the amylase is expressed and secreted by a host cell. 
     
     
         56 . The method of  claim 55 , wherein the composition comprising starch is contacted with the host cell. 
     
     
         57 . The method of  claim 55  or  56 , wherein the host cell further expresses and secretes one or more enzymes selected from the group consisting of glucoamylase, hexokinase, xylanase, glucose isomerase, xylose isomerase, phosphatase, phytase, pullulanase, β-amylase, α-amylase that is not the variant α-amylase, protease, cellulase, hemicellulase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, pectinase, alpha-glucosidase, and beta-glucosidase. 
     
     
         58 . The method of any one of  claims 55 - 57 , wherein the host cell further expresses and secretes a glucoamylase. 
     
     
         59 . The method of any one of  claims 55 - 58 , wherein the host cell is capable of fermenting the composition. 
     
     
         60 . A composition comprising glucose produced by the method of any one of  claims 50 - 59 . 
     
     
         61 . A liquefied starch produced by the method of any one of  claims 50 - 59 . 
     
     
         62 . A fermented beverage produced by the method of any one of  claims 50 - 59 . 
     
     
         63 . Use of an amylase of any of  claim 1 - 17  or  26  in the production of a composition comprising glucose, in the production of a liquefied starch, in the production of a fermented beverage, in cleaning starchy stains, or in textile desizing. 
     
     
         64 . A method of desizing a textile comprising contacting a desizing composition with a sized textile for a time sufficient to desize the textile, wherein the desizing composition comprises a variant α-amylase of any one of  claim 1 - 17  or  26 . 
     
     
         65 . An isolated polynucleotide encoding a polypeptide of any of  claim 1 - 17  or  26 . 
     
     
         66 . An expression vector comprising the polynucleotide of  claim 65 . 
     
     
         67 . A host cell comprising the expression vector of  claim 66 . 
     
     
         68 . A polypeptide according to any one of  claim 1 - 17  or  26  encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide complementary to the full-length of the polynucleotide of SEQ ID NO: 7, SEQ ID NO: 33, or SEQ ID NO: 38.