Variants and compositions comprising variants with high stability in presence of a chelating agent

Abstract

The present invention relates to variants of an alpha-amylase having improved stability to chelating agents relative to its parent enzyme, compositions comprising the variants, nucleic acids encoding the variants, methods of producing the variants, and methods for using the variants.

1 . A variant of a parent alpha-amylase comprising a substitution at a position corresponding to position 206 of SEQ ID NO: 6, wherein the variant has alpha-amylase activity and at least 60% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 26. 
     
     
         2 . The variant of  claim 1 , wherein the substitution is selected from the group consisting of F, W, Y, N, L, I, V, H, Q, D or E. 
     
     
         3 . The variant of  claim 1 , wherein the substitution is Y. 
     
     
         4 . The variant of  claim 1 , wherein the substitution is selected from the group consisting of F, Y, N, L, I, V, H, Q, A, C, D, E, F, or M. 
     
     
         5 . The variant of  claim 1 , wherein the variant further comprises a substitution at a position corresponding to a position of SEQ ID NO: 6 selected from the group consisting of 193, 195, 198, 203, 210, 212, 213 and 243. 
     
     
         6 . The variant of  claim 5 , wherein:
 (i) the substitution is at a position corresponding to position 193 of SEQ ID NO: 6 and the substitution is selected from the group consisting of S and P;   (ii) the substitution is at a position corresponding to position 195 of SEQ ID NO: 6 and the substitution is selected from the group consisting of S, M, N, R, F, H and Y;   (iii) the substitution is at a position corresponding to position 198 of SEQ ID NO: 6 and the substitution is D or Y;   (iv) the substitution is at a position corresponding to position 203 of SEQ ID NO: 6 and the substitution is selected from the group consisting of E, L and Y;   (v) the substitution is at a position corresponding to position 210 of SEQ ID NO: 6 and the substitution is selected from the group consisting of A, K, R, H, F, E, M, S, Y, and N;   (vi) the substitution is at a position corresponding to position 212 of SEQ ID NO: 6 and the substitution is C, I, D and G;   (vii) the substitution is at a position corresponding to position 213 of SEQ ID NO: 6 and the substitution is selected from the group consisting of S and V; or   (viii) the substitution is at a position corresponding to position 243 of SEQ ID NO: 6 and the substitution is selected from the group consisting of D and F.   
     
     
         7 . The variant of  claim 1 , wherein the variant further comprises a substitution at a position corresponding to a position of SEQ ID NO: 6 selected from the group consisting of 116, 118, 129, 133, 134, 142, 146, 147, 149, 151, 152, 169, 174, 186, 235, 244, 303, 320, 339, 418, 431, 434, 447, and 458. 
     
     
         8 . The variant of  claim 7 , wherein:
 (i) the substitution is at a position corresponding to position 116 of SEQ ID NO: 6 and the substitution is G;   (ii) the substitution is at a position corresponding to position 118 of SEQ ID NO: 6 and the substitution is selected from the group consisting of T, A, C, D, E, G, H, I, K, L, M, N, S, V and W;   (iii) the substitution is at a position corresponding to position 129 of SEQ ID NO: 6 and the substitution is selected from the group consisting of C, Y, Q, A, M, H, I, K, R, T and V;   (iv) the substitution is at a position corresponding to position 133 of SEQ ID NO: 6 and the substitution is selected from the group consisting of F, M, T, A, H, I, K, N, W, P and Q;   (v) the substitution is at a position corresponding to position 134 of SEQ ID NO: 6 and the substitution is selected from the group consisting of E, N, S, A, C, G, H, I, L, M, R, T, V, Y, P and Q;   (vi) the substitution is at a position corresponding to position 142 of SEQ ID NO: 6 and the substitution is selected from the group consisting of M, D, C, E, F, H, I, L, T, V, Y, S, K and N;   (vii) the substitution is at a position corresponding to position 146 of SEQ ID NO: 6 and the substitution is selected from the group consisting of Q, F, P, C, H, M, A, G, K, L, N, R, Y, I and T;   (viii) the substitution is at a position corresponding to position 147 of SEQ ID NO: 6 and the substitution is selected from the group consisting of L, W, Y, I, M, G, E,Q, T, A, H, K, N, R and S;   (ix) the substitution is at a position corresponding to position 149 of SEQ ID NO: 6 and the substitution is selected from the group consisting of G, C, L, V, E, K, N, Q and R;   (x) the substitution is at a position corresponding to position 151 of SEQ ID NO: 6 and the substitution is selected from the group consisting of T, A, C, D, E, H, I, K, M, L, R, S, W and Y;   (xi) the substitution is at a position corresponding to position 152 of SEQ ID NO: 6 and the substitution is selected from the group consisting of D, P, Q, G, M, Y, F, H, W and I;   (xii) the substitution is at a position corresponding to position 169 of SEQ ID NO: 6 and the substitution is selected from the group consisting of Q, A, C, G, H, K, N, R, S, T, V and Y;   (xiii) the substitution is at a position corresponding to position 174 of SEQ ID NO: 6 and the substitution is selected from the group consisting of L, F, G, I, M, R, V, W, Y, H, K, N, Q and S;   (xiv) the substitution is at a position corresponding to position 186 of SEQ ID NO: 6 and the substitution is selected from the group consisting of A, E, K, and R;   (xv) the substitution is at a position corresponding to position 235 of SEQ ID NO: 6 and the substitution is selected from the group consisting of L, R and V;   (xvi) the substitution is at a position corresponding to position 244 of SEQ ID NO: 6 and the substitution is selected from the group consisting of T, S, F, R, P, A, C, D, E, G, I, K, L, N, Q, V, W and Y;   (xvii) the substitution is at a position corresponding to position 303 of SEQ ID NO: 6 and the substitution is selected from the group consisting of Q, G, A, K, L, and R;   (xviii) the substitution is at a position corresponding to position 320 of SEQ ID NO: 6 and the substitution is selected from the group consisting of W, A, S, G, L, T, C, E, M, Q, Y, H and K;   (xix) the substitution is at a position corresponding to position 339 of SEQ ID NO: 6 and the substitution is selected from the group consisting of S, A, and D;   (xx) the substitution is at a position corresponding to position 418 of SEQ ID NO: 6 and the substitution is selected from the group consisting of D, A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, W and Y;   (xxi) the substitution is at a position corresponding to position 431 of SEQ ID NO: 6 and the substitution is selected from the group consisting of D, T and A;   (xxii) the substitution is at a position corresponding to position 434 of SEQ ID NO: 6 and the substitution is selected from the group consisting of M, P, R, D, Q and S;   (xxiii) the substitution is at a position corresponding to position 447 of SEQ ID NO: 6 and the substitution is selected from the group consisting of A, G, K, Q, R, S, T, and V; or   (xxiv) the substitution is at a position corresponding to position 458 of SEQ ID NO: 6 and the substitution is selected from the group consisting of R, D, E, F, S, W, A, C, I, M, and Y.   
     
     
         9 . The variant of  claim 8 , wherein the variant comprises a Y substitution at the position corresponding to position 206 of SEQ ID NO: 6 and the variant comprises N substation at the position corresponding to position 458 of SEQ ID NO: 6. 
     
     
         10 . The variant of  claim 9 , further comprising at least one, at least two, or at least three deletions in amino acid region of 181, 182, 183 or 184. 
     
     
         11 . The variant of  claim 10 , comprising deletions in the amino acid region of 182 and 183. 
     
     
         12 . The variant of  claim 1 , further comprising at least one, at least two, or at least three deletions in amino acid region of 181, 182, 183 or 184. 
     
     
         13 . A composition comprising a variant of a parent alpha-amylase, wherein the variant comprises a substitution at a position corresponding to position 206 of SEQ ID NO: 6, and further comprising at least one chelating agent, wherein said chelating agent at a concentration below 10 mM reduces the concentration of free calcium ions from 2.0 mM to 0.10 mM when measured at 21° C. and pH 8.0, wherein the variant has at least 60% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:
 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 26. 
 
     
     
         14 . The composition of  claim 13 , wherein the chelating agent at a concentration below 10 mM reduces the concentration of free calcium ions from 2.0 mM to 0.10 mM when measured in 80 mM potassium chloride and 49 mM EPPS at 21° C. and pH 8.0. 
     
     
         15 . The composition of  claim 13 , wherein the chelating agent at a concentration below 10 mM reduces the concentration of free calcium ions from 2.0 mM to 0.10 mM. 
     
     
         16 . The composition of  claim 13 , wherein the chelating agent reduces the concentration of free calcium ions from 2.0 mM to 0.10 mM at a chelator agent concentration below 8 mM. 
     
     
         17 . The composition of  claim 13 , wherein the variant comprises a substitution at position 206, using the numbering according to SEQ ID NO: 6, and further comprising at least one chelating agent, wherein said chelating agent reduces the free calcium ion concentration from 2.0 mM to 0.10 mM at a chelating agent concentration below 0.9 times the concentration of citrate capable of reducing the free calcium ion concentration from 2.0 mM to 0.10 mM, when measured at 21° C. and pH 8. 
     
     
         18 . The composition of  claim 13 , wherein the chelating agent reduces the free calcium ion concentration from 2.0 mM to 0.10 mM at a chelating agent concentration below 0.7 times the concentration of citrate that reduces the free calcium ion concentration from 2.0 mM to 0.10 mM. 
     
     
         19 . The composition of  claim 13 , wherein the parent alpha-amylase sequence comprises a substitution at position 206 selected from the group consisting of F, W, Y, N, L, I, V, H, Q, D or E. 
     
     
         20 . The composition of  claim 13 , wherein the variant comprises a 206Y substitution. 
     
     
         21 . The composition of  claim 13 , wherein the parent alpha-amylase sequence is modified by a substitution at position 206 of Y. 
     
     
         22 . The composition of  claim 13 , wherein the variant further comprises at least one, at least two, or at least three deletions in amino acid region of 181, 182, 183 or 184, using SEQ ID NO: 6 for numbering. 
     
     
         23 . The composition of  claim 13 , wherein the variant comprises a substitution at a position corresponding to position 206 of SEQ ID NO: 6 selected from the group consisting of F, Y, N, L, I, V, H, Q, A, C, D, E, F, or M. 
     
     
         24 . The composition of  claim 23 , wherein the variant comprises a substitution at a position corresponding to position 458 of SEQ ID NO: 6. 
     
     
         25 . The composition of  claim 13 , wherein the variant has improved wash performance compared to the parent alpha-amylase when measured in an Automated Mechanical Wash Assay. 
     
     
         26 . The composition of  claim 13 , wherein the chelating agent is selected from the group consisting of: ethylenediaminetetraacetic acid (EDTA), methylgycinediacetic acid (MGDA), ethylene glucol-bis(beta-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), diethylenetriamine penta(methylene phosphonic acid) (DTPMP) and 1-hydroxyethane 1,1-diphosphic acid (HEDP).