Alpha-amylase variants with altered properties

Abstract

Disclosed are compositions comprising variants of alpha-amylase that have alpha-amylase activity and that exhibit altered properties relative to a parent AmyS-like alpha-amylase from which they are derived. The compositions generally comprise at least one of an additional enzyme, a detergent,.a surfactant, a chelator, an oxidizing agent, an acidulant, an alkalizing agent, a source of peroxide, a source of hardness, a salt, a detergent complexing agent, a polymer, a stabilizing agent, or a fabric conditioner. Also disclosed are detergent formulations comprising the variants. Methods of using the compositions for desizing woven material and washing or cleaning items, such as dishes or laundry, are disclosed. Kits related thereto are also provided.

1 . A composition comprising:
 a) at least one variant alpha-amylase comprising an amino acid sequence at least 95% identical to that of a parent AmyS-like alpha-amylase, and having a substitution at an amino acid position corresponding to position 242 of a reference alpha-amylase, said variant alpha-amylase having detectable alpha-amylase activity, and   b) at least one of an additional enzyme, a detergent, a surfactant, a chelator, an oxidizing agent, an acidulant, an alkalizing agent, a source of peroxide, a source of hardness, a salt, a detergent complexing agent, a polymer, a stabilizing agent, or a fabric conditioner.   
     
     
         2 . The composition of  claim 1 , wherein the variant alpha-amylase is altered, as compared to a parent AmyS-like alpha-amylase or a reference alpha-amylase, in one or more characteristics of: (a) net charge, (b) substrate specificity, (c) substrate cleavage, (d) substrate binding, (e) thermal stability, (f) activity at one or more pH's, (g) stability at one or more pH's, (h) stability in oxidizing conditions, (i) Ca 2+  requirements, (j) specific activity, (k) catalytic rate, (l) catalytic efficiency, (m) activity in the presence of a chelator, (n) thermal or pH stability in the presence of a chelator, (o) utility for desizing, or utility for a cleaning process, or (p) amount of expression in a protein expression system. 
     
     
         3 . The composition of  claim 1 , wherein the reference alpha-amylase is SEQ ID NO: 1 or 2. 
     
     
         4 . The composition of  claim 1 , which is a component of a product for use in laundry, dish, or hard-surface cleaning, desizing, or fabric or stain treatment. 
     
     
         5 . The composition of  claim 1 , wherein the additional enzyme is a protease, a lipase, an amylase, a cellulase, a peroxidase, an oxidase, a pectinase, a lyase, a cutinase, a laccase, or a combination thereof. 
     
     
         6 . The composition of  claim 1 , wherein the surfactant is nonionic, anionic, cationic, or zwitterionic. 
     
     
         7 . The composition of  claim 1 , wherein the variant alpha-amylase is a S242A, S242D, S242E, S242F, S242G, S242H, S242L, S242M, S242N, S242Q, or S242T variant. 
     
     
         8 . The composition of  claim 7 , wherein the variant alpha-amylase has altered stability to oxidation and the variant alpha-amylase further includes deletion or substitution of one or more methionine residues including residues located at amino positions 8, 9, 96, 200, 206, 284, 307, 311, 316, and 438 of a parent AmyS-like alpha-amylase, where the reference alpha-amylase in SEQ ID NO: 2. 
     
     
         9 . The composition of  claim 7 , wherein the variant alpha-amylase further comprises a sequence modification at one or more amino acid positions corresponding to amino acid positions 97, 179, 180, 193, 319, 349, 358, 416, 428, or 443 of the reference alpha-amylase. 
     
     
         10 . The composition of  claim 9 , wherein the variant alpha-amylase comprises one or more of substitution at positions as follows: a cysteine at 349, a cysteine at 428, a glutamic acid at 97, an arginine at 97, a glutamic acid at 319, an arginine at 319, a glutamic acid at 358, an arginine at 358, a glutamic acid at 443, or an arginine at 443. 
     
     
         11 . The composition of  claim 9 , wherein the variant alpha-amylase comprises a substitution of an N193 or a V416 or both. 
     
     
         12 . The composition of  claim 11 , comprising a substitution of N193F or V416G, or both. 
     
     
         13 . The composition of  claim 7 , further comprising deletion of one or more amino acids at positions F178, R179, G180, I181, G182 and K183. 
     
     
         14 . The composition of  claim 13 , wherein the variant alpha-amylase has altered metal ion dependence or altered stability or activity in an absence of added calcium or a presence of a chelator. 
     
     
         15 . The composition of  claim 1 , wherein the variant alpha-amylase has at least 95% homology to SEQ ID NO: 2 and comprises a substitution of amino acid 242 relative to numbering in a reference alpha-amylase comprising SEQ ID NO: 1, and wherein the variant alpha-amylase has alpha-amylase activity. 
     
     
         16 . The composition of  claim 1 , wherein the parent AmyS-like alpha-amylase is SEQ ID NO: 1, 2, 6, 7, 8, 9, 10, 11, 12, 15, or 16, and the reference alpha-amylase is SEQ ID NO: 1 or 2. 
     
     
         17 . The composition of  claim 1 , wherein the variant alpha-amylase has improved performance in a wash process at a pH≧about 8, relative to the parent AmyS-like alpha-amylase. 
     
     
         18 . The composition of  claim 10 , wherein the variant alpha-amylase comprises a set of substitutions of a) Q97E, Q319E, Q358E, Q443E; b) Q97E, Q319R, Q358E, Q443R; c) Q97E, Q319R, Q358E; d) Q97E, Q319R, Q443E; e) Q97E, Q319R, Q443R; f) Q97E, Q358R; g) Q97E, Q443E; h) Q319R, Q358E, Q443E; or i) Q319R, Q358R, Q443E. 
     
     
         19 . A detergent or cleaning formulation comprising at least one variant alpha-amylase comprising an amino acid sequence at least 95% identical to that of a parent AmyS-like alpha-amylase, and having a substitution at an amino acid position corresponding to position 242 of a reference alpha-amylase, said variant alpha-amylase having detectable alpha-amylase activity; wherein the reference amylase is SEQ ID NO: 1 or 2. 
     
     
         20 . The detergent or cleaning formulation of  claim 19 , wherein the variant alpha-amylase is an S242 variant alpha-amylase comprising at least a S242A, S242D, S242E, S242F, S242G, S242H, S242L, S242M, S242N, S242Q, or S242T substitution. 
     
     
         21 . A method of desizing a woven material subsequent to a weaving process comprising contacting the woven material with a variant alpha-amylase comprising an amino acid sequence at least 95% identical to that of a parent AmyS-like alpha-amylase, and having a substitution at an amino acid position corresponding to position 242 of a reference alpha-amylase, said variant alpha-amylase having detectable alpha-amylase activity, under conditions and for a time effective for at least partially removing sizing from the woven material. 
     
     
         22 . The method of  claim 21 , wherein the variant alpha-amylase is altered, as compared to a parent AmyS-like alpha-amylase or a reference alpha-amylase, in one or more of: (a) net charge, (b) substrate specificity, (c) substrate cleavage, (d) substrate binding, (e) thermal stability, (f) activity at one or more pH's, (g) stability at one or more pH's, (h) stability in oxidizing conditions, (i) Ca 2+  requirements, (j) specific activity, (k) catalytic rate, (l) catalytic efficiency, (m) activity in a presence of a chelator, (n) thermal or pH stability in the presence of a chelator, (o) effectiveness for desizing, or (p) amount of expression in a protein expression system. 
     
     
         23 . The method of  claim 21 , wherein the parent AmyS-like alpha-amylase is SEQ ID NO: 1, 2, 6, 7, 8, 9, 10, 11, 12, 15, or 16, and the reference alpha-amylase is SEQ ID NO: 1 or 2. 
     
     
         24 . The method of  claim 21 , wherein the variant alpha-amylase is a S242A, S242D, S242E, S242F, S242G, S242H, S242L, S242M, S242N, S242Q, or S242T variant. 
     
     
         25 . The method of  claim 24 , wherein the variant alpha-amylase further comprises one or more of substitution at positions as follows: a cysteine at 349, a cysteine at 428, a glutamic acid at 97, an arginine at 97, a glutamic acid at 319, an arginine at 319, a glutamic acid at 358, an arginine at 358, a glutamic acid at 443, or an arginine at 443, wherein the reference alpha-amylase is SEQ ID NO: 1 or 2. 
     
     
         26 . A method of washing or cleaning comprising contacting one or more items to be washed or cleaned with a composition comprising a variant alpha-amylase comprising an amino acid sequence at least 95% identical to that of a parent AmyS-like alpha-amylase, and having a substitution at an amino acid position corresponding to position 242 of a reference alpha-amylase, said variant alpha-amylase having detectable alpha-amylase activity, under conditions and for a time effective for at least partially washing or cleaning the one or more items. 
     
     
         27 . The method of  claim 26 , wherein at least one item is soiled with at least one starch-containing material, removal of said starch soil is aided by the variant alpha-amylase. 
     
     
         28 . The method of  claim 26 , wherein the composition further comprises one or more of an additional enzyme, a detergent, a surfactant, a chelator, an oxidizing agent, an acidulant, an alkalizing agent, a source of peroxide, a source of hardness, a salt, a detergent complexing agent, a polymer, a stabilizing agent, or a fabric conditioner. 
     
     
         29 . The method of  claim 26 , wherein the parent AmyS-like alpha-amylase is SEQ ID NO: 1, 2, 6, 7, 8, 9, 10, 11, 12, 15, or 16, and the reference alpha-amylase is SEQ ID NO: 1 or 2. 
     
     
         30 . The method of  claim 29 , wherein the variant alpha-amylase is a S242A, S242D, S242E, S242F, S242G, S242H, S242L, S242M, S242N, S242Q, or S242T variant. 
     
     
         31 . The method of  claim 30 , wherein the variant alpha-amylase has improved performance in a wash process at a pH≧about 8, relative to the parent AmyS-like alpha-amylase. 
     
     
         32 . The method of  claim 30 , wherein the variant alpha-amylase comprises one or more of substitution at positions as follows: a cysteine at 349, a cysteine at 428, a glutamic acid at 97, an arginine at 97, a glutamic acid at 319, an arginine at 319, a glutamic acid at 358, an arginine at 358, a glutamic acid at 443, or an arginine at 443. 
     
     
         33 . The method of  claim 32 , wherein the variant alpha-amylase comprises a set of substitutions of a) Q97E, Q319E, Q358E, Q443E; b) Q97E, Q319R, Q358E, Q443R; c) Q97E, Q319R, Q358E; d) Q97E, Q319R, Q443E; e) Q97E, Q319R, Q443R; f) Q97E, Q358R; g) Q97E, Q443E; h) Q319R, Q358E, Q443E; or i) Q319R, Q358R, Q443E. 
     
     
         34 . The method of  claim 30 , wherein the variant alpha-amylase comprises deletion of one or more amino acids at positions F178, R179, G180, I181, G182, or K183. 
     
     
         35 . The method of  claim 34 , wherein the variant alpha-amylase has altered metal ion dependence or altered stability, or activity in an absence of added calcium or the presence of a chelator. 
     
     
         36 . A kit comprising:
 a) one or more variant alpha-amylases comprising an amino acid sequence at least 95% identical to that of a parent AmyS-like alpha-amylase, and having a substitution at an amino acid position corresponding to position 242 of a reference alpha-amylase, said variant alpha-amylase having detectable alpha-amylase activity; and   b) at least one of an additional enzyme, a detergent, a surfactant, a chelator, an oxidizing agent, an acidulant, an alkalizing agent, a source of peroxide, a source of hardness, a salt, a detergent complexing agent, a polymer, a stabilizing agent, or a fabric conditioner.   
     
     
         37 . The kit of  claim 36  further comprising instructions for using the kit in a process for desizing a woven material or washing or cleaning one or more items soiled with a starch-containing substance.