Alpha-amylase variants with altered properties
The present invention relates to variants (mutants) of polypeptides, in particular Termamyl-like alpha-amylases, which variant has alpha-amylase activity and exhibits an alteration in at least one of the following properties relative to said parent alpha-amylase: substrate specificity, substrate binding, substrate cleavage pattern, thermal stability, pH/activity profile, pH/stability profile, stability towards oxidation, Ca2+ dependency, specific activity, in particular laundry and dish-wash applications.
1. An isolated variant of a parent Termamyl-like alpha-amylase, comprising an alteration at position 270
wherein
(a) the alteration is a substitution of the amino acid which occupies the position with a different amino acid,
(b) the variant has alpha-amylase activity,
(c) the position corresponds to a position of the amino acid sequence of the parent alpha-amylase having the amino acid sequence of the parent Termamyl-like alpha-amylase having the amino acid sequence of AA560 shown in SEQ ID NO: 12, wherein the variant has an amino acid sequence which has a degree of identity of at least 90% to SEQ ID NO: 12; and
(d) the variant has an additional mutation in one or more methionine residues.
2. The variant of claim 1 , wherein the variant further comprises alterations selected from the group consisting of:
R26S, D30N, N33D, R82H, K37T, N106D, R118K, N128Y, G133E,A, G149A,N, N150H,Q, Y160F, Y178F, T193S,N,D,E,Q, Y203L, V214I,T, D231N, G256K, T257I, G258D, N270F,Y,D, L272I,V,A, N283D, Y295F,N,D,Q,E, N296K,Q,E, Y304F,R,K, G305D, G315N,S,T, V318L, Q319E,K,S,T, A339S,T, Q361E, G378K, T419N, H421Y, F441L, R444E,Y, N445Q, K446R, A447Y, V450T, T461P, and W482Y.
3. The variant of claim 1 , wherein the methionine residues are:
M9, M10, M116, M202, M208, M261, M309, M323, M382, M410, M430, and M440.
4. The variant of claim 1 , wherein the mutations are:
M9L,I, M10L, M105L,I,F, M116N,D,L,I,F,W,R,K, M202I,L,V,T, M208F,Y,L,I, M261L,I, M309L,I, M323L,I,S,T,A,Q,E,N,D, M382L,I,Y,F,K, M410L,I,V, M430L,I, and M440L,I,F,Y.
5. The variant of claim 1 , wherein the variant further comprises one or more of the following mutations:
M9L+M202I,
M9L+M202I+M323T,
M9L+M202I+323T+M382Y,
M9L+M202I+Y295F+A339S,
M9L+M202I+Y295F,
M9L+M202I+A339S,
M9L+M202I+Y295F+A339S,
M9L+M202I+Y295F+A339S+E345R,
M9L+G149A+M202I+Y295F+A339S+E345R,
M9L+M202T,
M9L+M202L+M323T,
M9L+M202L+M232T+M382Y,
M9L+M202L+Y295F+A339S,
M9L+M202L+Y295F,
M9L+M202L+A339S,
M9L+M202L+Y295F+A339S,
M9L+M202L+Y295F+A339S, E345R,
M9L+G149A+M202L+Y295F+A339S+E345R,
M9L+M202T,
M9L+M202T+M323T,
M9L+M202T+M323T+M382Y,
M9L+M202T+Y295F+A339S,
M9L+M202T+Y295F,
M9L+M202T+A339S,
M9L+M202T+Y295F+A339S,
M9L+M202T+Y295F+A339S+E345R,
M9L+G149A+M202T+Y295F+A339S+E345R,
M9L+G149A+M202I+V214T+Y295F+N299Y+M323T+A339S+E345R,
M9L+G149A+M202L+V214I+Y295F+M323T+A339S+E345R+M382Y,
M9L+G149A+G182T+M202I+V214I+Y295F+N299Y+M323T+A339S,
M9L+G149A+G182T+M202L+T257I+Y295F+N299Y+M323T+A339S+E345R,
M9L+G149A+M202L+V214T+Y295F+N299Y+M323T+A339S+E345R,
M9L+G149A+M202I+V214I+Y295F+M323T+A339S+E345R+M382Y,
M9L+G149A+G182T+M202L+V214I+Y295F+N299Y+M323T+A339S,
M9L+G149A+G182T+M202I+T257I+Y295F+N299Y+M323T+A339S+E345R,
M9L+G149A+M202I+V214T+Y295F+N299Y+M323T+A339S+E345R+N471E,
M9L+G149A+M202L+V214I+Y295F+M323T+A339S+E345R+M382Y+N471E,
M9L+G149A+G182T+M202I+V214I+Y295F+N299Y+M323T+A339S+N471E,
M9L+G149A+G182T+M202L+T257I+Y295F+N299Y+M323T+A339S+E345R+N471E,
M202L+M105F+M208F,
G133E+M202L+Q361E,
G133E+M202L+R444E,
M202L+Y295F,
M202L+A339S,
M202L+M323T,
M202L+M323T+M309L,
M202L+M323T+M430I,
M202L+V214T+R444Y,
M202L+N283D+Q361E,
M202L+M382Y+K383R,
M202L+K446R,
M202I+Y295F,
M202I+A339S,
M202I+M105F+M208F,
G133E+M202I+Q361E,
G133E+M202I+R444E,
M202I+M202I+M323T,
M202I+M202I+M323T+M309L,
M202I+M323T+M430I,
M202I+V214T+R444Y,
M202I+N283D+Q361E,
M202I+M382Y+K383R,
M202I+K446R,
M202V+M105F+M208F,
G133E+M202V+Q361E,
G133E+M202V+R444E,
M202V+M323T,
M202V+M323T+M309L,
M202V+M323T+M430I,
M202V+M323T+M9L,
M202V+V214T+R444Y,
M202V+N283D+Q361E,
M202V+M382Y+K383R,
M202V+K446R,
M202T+M105F+M208F,
G133E+M202T+Q361E,
G133E+M202T+R444E,
M202T+Y295F,
M202T+A339S,
M202T+M323T,
M202T+M323T+M309L,
M202T+M323T+M430I,
M202T+M323T+M9L,
M202T+V214T+R444Y,
M202T+N283D+Q361E,
M202T+A339S,
M202T+Y295F
M202T+N299F,Y,
M202T+M382Y+K383R,
M202T+K446R.
6. The variant of claim 1 , wherein the variant further comprises the mutation D183*+G184*.
7. The variant of claim 1 , wherein the variant further comprises a mutation in R118.
8. The variant of claim 1 , wherein the variant further comprises a mutation in N195.
9. The variant of claim 1 , wherein the variant further comprises a mutation in R320.
10. The variant of claim 1 , wherein the variant further comprises a mutation in R458.
11. The variant of claim 1 , wherein the variant further comprises the mutation D183*+G184*+R118K+N195F+R320K+R458K in combination with one or more of the following mutations:
K37T,
H421Y,
V450T,
N445Q,
Y178F,
V318L,
W482Y,
N283D+Q361E,
M105F+M208F,
M202L+M323T+M430I,
K446R,
R444Y,
N106D,
Y203L,
G133E+Q361E,
M323E,
V214T,
M202L+M323T+M309L,
M202L,
M202L+M323T,
M202L+M323T+M9L+M382Y+K383R,
M202L+M323T+M9L+M382Y,
M202L+M323T+M9L.
12. The variant of claim 1 , wherein the variant further comprises the mutation D183*+G184*+R118K+N195F+R320K+R458K+M202L+M323T+M9L.
13. The variant of claim 1 , wherein the variant further comprises one or more of the following mutations:
T461P,
Y298H,
G133E+R444E,
Y298F,
M202T,
M202I,
M202V,
V214T+M323E+M382Y+K383R+N471E
Y178F+G258D+T419N+N437H
G149N+N150Q+M382Y+K383R
Y160F+V214T+M382Y
N128Y+G149A+V214T+D231N+M382Y+F441L
R82H+N128Y+G149A+V214T+M382Y
N150H+V214T
V214T+E345N
V214T+G305D+M382Y+R444E
V214T+M382Y+A447Y
M202I+V214T+M382Y+K383R+R444Y
V214T+G378K
V214T+A256K
R26S+D30N+N33D+V214T+M382Y.
14. The variant of claim 1 , wherein the parent Termamyl-like alpha-amylase is SEQ ID NO: 12.
15. The variant of claim 1 , wherein the parent Termamyl-like alpha-amylase has an amino acid sequence which has a degree of identity to SEQ ID NO: 12 of at least 95%.
16. A DNA construct comprising a DNA sequence encoding an alpha-amylase variant according to claim 1 .
17. A detergent additive comprising an alpha-amylase variant according to claim 1 .
18. The isolated variant of a parent Termamyl-like alpha-amylase of claim 1 , wherein the variant has an amino acid sequence which has a degree of identity of at least 95% to SEQ ID NO: 12.
19. The isolated variant of a parent Termamyl-like alpha-amylase of claim 1 , wherein the variant has an amino acid sequence which has a degree of identity of at least 96% to SEQ ID NO: 12.
20. The isolated variant of a parent Termamyl-like alpha-amylase of claim 1 , wherein the variant has an amino acid sequence which has a degree of identity of at least 97% to SEQ ID NO: 12.
21. The isolated variant of a parent Termamyl-like alpha-amylase of claim 1 , wherein the variant has an amino acid sequence which has a degree of identity of at least 98% to SEQ ID NO: 12.
22. The isolated variant of a parent Termamyl-like alpha-amylase of claim 1 , wherein the variant has an amino acid sequence which has a degree of identity of at least 99% to SEQ ID NO: 12.
23. An isolated variant of a parent Termamyl-like alpha-amylase, comprising an alteration at position 270 wherein:
(a) the alteration is a substitution of the amino acid which occupies the position with a different amino acid,
(b) the variant has alpha-amylase activity, and
(c) the position corresponds to a position of the amino acid sequence of the parent alpha-amylase having the amino acid sequence of the parent Termamyl-like alpha-amylase having the amino acid sequence of AA560 shown in SEQ ID NO: 12, wherein the variant has an amino acid sequence which has a degree of identity of at least 95% to SEQ ID NO: 12.
24. The isolated variant of claim 23 , wherein the substitution is one of N270F,Y or D.
25. The variant of claim 7 , wherein the mutation is R118K.
26. The variant of claim 8 , wherein the mutation is N195F.
27. The variant of claim 9 , wherein the mutation is R320K.
28. The variant of claim 10 , wherein the mutation is R458K.