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, wherein the amino acid sequence of the variant has at least 80% but less than 100% sequence identity to SEQ ID NO: 1, and wherein the variant comprises the substitution N76D and two or more substitutions selected from positions 259, 260, 261 and 262, where the substitution in position 259 is S259D, where the substitution in position 260 is T260A, T260E or T260P, where the substitution in position 261 is N261D, N261C, N261E, N261L, N261M, N261R, N261V, N261W, N261Y, or N261F and where the substitution in position 262 is L262Y, L262C, L262E, or L262Q, wherein each position corresponds to the position of the polypeptide of SEQ ID NO: 2.
2. The subtilase variant of claim 1 , wherein the variant further comprises one or more substitutions selected from the group consisting of Q2R, W6I, I8{C,H}, S9{C,D,E,Q}, N18S, N43{A,C,L,R,W}, S49T, G53{A,L}, G61D, I72{A, V}, S78{D,N},V104{F,P,T,Y}, A114V, G115{T,W}, H120{I,T,V}, P129D, V147W, V149{C,N,Q}, A158E, G160{D,P}, S161{C,Y,E}, I162L, S163{A, D}, Q182{C,E}, N184D, N185{C,E}, S188{C,D,E}, Q191N, A194{D,E}, N204{D,V}, V205{I,L}, Q206{C,E,I,K,T,V,W,L}, Y209{L,W}, S212{A,D,G,N}, S216{I,T,V}, L217{C,M}, N218T, M222{C,N,R}, P225{A,S}, T255{C,E,Q}, S256{A,C,D,V,Y}, S259D, T260{A,E,P}, N261{D,C,E,L,M,R,V,W,Y,F} and L262{Y,C,E,Q}.
3. The variant of claim 1 , wherein the variant further comprises one or more substitutions selected from the group consisting of S3T, V4I, A15T, S24{G,R}, V68A, S99{D,E}, S101{R,K,N,M,E,D,L,I}, S103A, V104I, S128{A,L,S}, P129{N,Q} S130A, V199M, L217{Y,D,E,Q}, N218{D, E} and Q245{K,R}.
4. The subtilase variant of claim 1 , wherein the total number of alterations compared to SEQ ID NO: 1 is between 3 and 30.
5. The subtilase variant of claim 1 , which has an improved storage stability compared to the polypeptide of SEQ ID NO: 3.
6. The subtilase variant of claim 1 , wherein the amino acid sequence of the variant has at least 85% but less than 100% sequence identity to SEQ ID NO: 1.
7. The subtilase variant of claim 1 , wherein the amino acid sequence of the variant has at least 90% but less than 100% sequence identity to SEQ ID NO: 1.
8. The subtilase variant of claim 1 , wherein the amino acid sequence of the variant has at least 95% but less than 100% sequence identity to SEQ ID NO: 1.
9. A detergent composition comprising the variant of claim 1 and one or more detergent components.
10. The detergent composition of claim 9 , wherein the composition is a liquid detergent composition.
11. A method for producing a subtilase variant, comprising (a) introducing into a parent subtilase the substitution N76D and two or more substitutions in positions 259, 260, 261 and 262, where the substitution in position 259 is S259D, where the substitutions in position 260 is T260A, T260E or T260P, where the substitution in position 261 is N261D, N261C, N261E, N261L, N261M, N261R, N261V, N261W, N261Y, or N261F and where the substitution in position 262 is L262Y, L262C, L262E, or L262Q, wherein each position corresponds to the position of the polypeptide of SEQ ID NO: 2, and wherein the amino acid sequence of the variant has at least 80% but less than 100% sequence identity to SEQ ID NO: 1, and
(b) recovering the variant.
12. The method of claim 11 , wherein the amino acid sequence of the variant has at least 85% but less than 100% sequence identity to SEQ ID NO: 1.
13. The method of claim 11 , wherein the amino acid sequence of the variant has at least 90% but less than 100% sequence identity to SEQ ID NO: 1.
14. The method of claim 11 , wherein the amino acid sequence of the variant has at least 95% but less than 100% sequence identity to SEQ ID NO: 1.