Subtilase variants and polynucleotides encoding same
The present invention relates to novel subtilase variants exhibiting increased stability and preferably on par or improved wash performance. The variants of the invention are suitable for use in e.g. cleaning or detergent compositions, such as laundry detergent compositions and dish wash compositions, including automatic dish wash compositions. The present invention also relates to isolated DNA sequences encoding the variants, expression vectors, host cells, and methods for producing and using the variants of the invention.
1 . A subtilase variant having protease activity and having at least 25% improved stability compared to a subtilase with SEQ ID NO: 1, when measured as described in the accelerated storage stability assay in Example 3 herein, wherein the amino acid sequence of the subtilase variant has at least 85% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 and wherein the subtilase variant comprises substitutions in positions 206 and 209, wherein each position corresponds to the position of the polypeptide of SEQ ID NO: 2.
2 . The subtilase variant according to claim 1 , wherein said subtilase variant further comprises one or more of the substitutions selected from the group consisting of X9C, X9D, X9E, X9Q, X43A, X43C, X43L, X43R, X43W, X72A, X72V, X78D, X104F, X114V, X115T, X115W, X120I, X120T, X120V, X129D, X147W, X149C, X149N, X158E, X160D, X160P, X161C, X161E, X163A, X163D, X182C, X182E, X185C, X185E, X188C, X188D, X188E, X191N, X204D, X204V, X205I, X212A, X212D, X212G, X212N, X216I, X216T, X216V, X217C, X217M, X218T, X222C, X222R, X225A, X255C, X255E, X256A, X256C, X256D, X256V, X256Y, X259D, X260E, X260P, X261C, X261E, X261L, X261M, X261V, X261W, X261Y, X261F, X262C, X262E and X262Q, wherein each position corresponds to the position of the polypeptide of SEQ ID NO: 2.
3 . The subtilase variant of claim 1 , wherein the total number of alterations compared to SEQ ID NO: 1 is between 3 and 30.
4 . The subtilase variant of claim 1 , wherein the parent subtilase is selected from a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.
5 . The subtilase variant of claim 1 , further comprising one or more substitutions selected from the group consisting of S3T, V4I, A15T, S24G, S24R, V68A, S99D, S99E, S101R, S101K, S101N, S101M, S101E, S101D, S101L, S101I, S103A, V104I, S128A, S128L, S128S, P129N, P129Q S130A, V199M, L217Y, L217D, L217E, L217Q, N218D, N218E, Q245K and Q245R (numbering according to SEQ ID NO: 2).
6 . The variant of claim 1 , wherein the variant subtilase is a polypeptide that has at least 90% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1.
7 . The variant of claim 1 , wherein the variant subtilase is a polypeptide that has at least 95% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1.
8 . The variant of claim 1 , wherein the variant subtilase is a polypeptide that has at least 98% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1.
9 . The variant of claim 1 , wherein the substitution at position 206 is selected from X206C, X206E, X206I, X206K, X206L, X206T, X206V, and X206W and the substitution at position 209 is selected from X209W and X209L.
10 . The variant of claim 1 , wherein the substitution at position 206 is X206L and the substitution at position 209 is X209W.
11 . A detergent composition comprising the subtilase variant according to claim 1 and one or more detergent components.
12 . The detergent composition according to claim 11 , further comprising one or more additional enzymes selected from the group consisting of proteases, amylases, lipases, cutinases, cellulases, endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidases, haloperoxygenases, catalases and mannanases, and any mixture thereof.
13 . The detergent composition according to claim 11 in the form of a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid.
14 . A method for producing the subtilase variant according to claim 1 comprising:
a. introducing into a parent subtilase one or more alterations from the group consisting of S9C, S9D, S9E, S9Q, N43A, N43C, N43L, N43R, N43W, I72A, I72V, S78D, V104F, A114V, G115T, G115W, H120I, H120T, H120V, P129D, V147W, V149C, V149N, A158E, G160D, G160P, S161C, S161E, S163A, S163D, Q182C, Q182E, N185C, N185E, S188C, S188D, S188E, Q191N, N204D, N204V, V205I, Q206C, Q206E, Q206I, Q206K, Q206T, Q206V, Q206W, Q206L Y209W, S212A, S212D, S212G, S212N, S216I, S216T, S216V, L217C, L217M, N218T, M222C, M222R, P225A, T255C, T255E, S256A, S256C, S256D, S256V, S256Y, S259D, T260E, T260P, N261C, N261E, N261L, N261M, N261V, N261Y, N261W, N261F, L262C, L262E and L262Q, wherein each position corresponds to the position of SEQ ID NO: 2, and
b. recovering the subtilase variant,
wherein the amino acid sequence of the subtilase variant has at least 85% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1.