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 protein comprising a subtiliase variant comprising the substitutions N76D, X9{C,D,E,Q}, and X43{A,C,L,R,W}
wherein each position corresponds to the position of the polypeptide of SEQ ID NO: 2, and
wherein the variant has protease activity and is a polypeptide that has at least 85% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1.
2. The variant of claim 1 , wherein the total number of alterations compared to a parent subtilase is between 3 and 30 alterations.
3. The variant of claim 2 , wherein the parent subtilase is amino acids 1 to 269 of SEQ ID NO: 1 or amino acids 1 to 275 of SEQ ID NO: 2.
4. The variant of claim 1 , wherein the variant comprises one or more of the substitutions selected from the group consisting of:
N76D+X2R
N76D+X115W
N76D+X194E
N76D+X255C
N76D+X6I
N76D+X120I
N76D+X204D
N76D+X255E
N76D+X8C
N76D+X120T
N76D+X204V
N76D+X255Q
N76D+X8H
N76D+X120V
N76D+X205I
N76D+X256A
N76D+X9C
N76D+X129D
N76D+X205L
N76D+X256C
N76D+X9D
N76D+X147W
N76D+X206C
N76D+X256D
N76D+X9E
N76D+X149C
N76D+X206E
N76D+X256V
N76D+X9Q
N76D+X149N
N76D+X206I
N76D+X256Y
N76D+X18S
N76D+X149Q
N76D+X206K
N76D+X259D
N76D+X43A
N76D+X158E
N76D+X206T
N76D+X260A
N76D+X43C
N76D+X160D
N76D+X206V
N76D+X260E
N76D+X43L
N76D+X160P
N76D+X206W
N76D+X260P
N76D+X43R
N76D+X161C
N76D+X209L
N76D+X261D
N76D+X43W
N76D+X161Y
N76D+X209W
N76D+X261C
N76D+X49T
N76D+X161E
N76D+X212A
N76D+X261E
N76D+X53A
N76D+X162L
N76D+X212D
N76D+X261L
N76D+X53L
N76D+X163A
N76D+X212G
N76D+X261M
N76D+X61D
N76D+X163D
N76D+X212N
N76D+X261R
N76D+X72A
N76D+X182C
N76D+X216I
N76D+X261V
N76D+X72V
N76D+X182E
N76D+X216T
N76D+X261W
N76D+X78D
N76D+X184D
N76D+X217C
N76D+X261Y
N76D+X78N
N76D+X185C
N76D+X217M
N76D+X261F
N76D+X104F
N76D+X185E
N76D+X218T
N76D+X262Y
N76D+X104P
N76D+X188C
N76D+X222C
N76D+X262C
N76D+X104T
N76D+X188D
N76D+X222N
N76D+X262E
N76D+X104Y
N76D+X188E
N76D+X222R
N76D+X262Q
N76D+X114V
N76D+X191N
N76D+X225A
N76D+X115T
N76D+X194D
N76D+X225S
N76D+X216V and
N76D+X206L.
5. The variant of claim 1 , further comprising one or more substitutions selected from the group consisting of X3T, X4I, X15T, X24{G,R}, X68A, X99{D,E}, X101{R,K,N,M,E,D,L,I}, X103A, X1041, X128{A,L,S}, X129{N,Q} X130A, X199M, L217{Y,D,E,Q}, X218{D,E} and X245{K,R} (numbering according to SEQ ID NO: 2).
6. The variant of claim 1 , which has an improved stability compared to SEQ ID NO: 3.
7. The variant of claim 6 , which has an improved wash performance compared to SEQ ID NO: 3.
8. A detergent composition comprising the variant according to claim 1 and one or more detergent components.
9. The detergent composition according to claim 8 , 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, or any mixture thereof.
10. The detergent composition according to claim 8 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.
11. The variant of claim 5 , comprising N76D and at least one substitution selected from X205I, X206L, X209W, X212G and X216V.
12. The variant of claim 1 , which is (i) a polypeptide that has at least 90% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1.
13. The protein of claim 1 wherein the variant further comprises one or more substitutions selected from the group consisting of: X2R, X6I, X8{C,H}, X18S, X49T, X53{A,L}, X61 D, X72{A,V}, X78{D,N}, X104{F,P,T,Y}, X114V, X115{T,W}, X120{I,T,V}, X129D, X147W, X149{C,N,Q}, X158E, X160{D,P}, X161{C,Y,E}, X162L, X163{A, D}, X182{C,E}, X184D, X185{C,E}, X188{C, D,E}, X191N, X194{D,E}, X204{D,V}, X205{I,L}, X206{C,E,I,K,T,V,W,L}, X209{L,W}, X212{A,D,G,N}, X216{I,T,V}, X217{C,M}, X218T, X222{C,N,R}, X225{A,S}, X255{C,E,Q}, X256{A,C,D,V,Y}, X259D, X260{A,E,P}, X261{D,C,E,L,M,R,V,W,Y,F} and X262{Y,C,E,Q}.