IP Library › Granted Patent US 9,096,837
Granted Patent B2
US 9,096,837 · App. 13/008,619 · Granted Aug 4, 2015

Alpha-amylase variants with altered properties

Inventors: Carsten Andersen (Vaerlose, DK); Henrik Ostdal (Virum, DK); Peter Skagerlind (Hoganas, SE)
Assignee: Novozymes A/S
C12N9/2417C07K2299/00
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Quick Facts
Patent No.
US 9,096,837
App. No.
13/008,619
Granted
Aug 4, 2015
Kind
B2
Abstract

The present disclosure 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.

Claims (161)

1. An isolated variant of a parent Termamyl-like alpha-amylase, comprising an alteration at one or more positions selected from the group of:

R26S, D30N, N33D, R82H, K37T, N106D, N128Y, G133E,A, G149A,N, N150H,Q, Y160F, Y178F, Y203L, V2141,T, D231N, G256K, T2571, G258D, N270F,Y,D, L2721,V,A, N283D, Y295F,N,D,Q,E, N296K,Q,E, Y304F,R,K, G305D, G315N,S,T, V318L, A339S,T, Q361E, G378K, T419N, H421Y, F441L, K446R, A447Y, V450T, T461P, and W482Y, wherein

the variant has alpha-amylase activity, and wherein

each 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.

2. The variant of claim 1 , wherein the variant has an additional mutation in one or more methionine residues.

3. The variant of claim 2 , wherein the methionine residues are:

M9, M10, M116, M202, M208, M261, M309, M323, M382, M410, M430, and M440.

4. The variant of claim 2 , wherein the mutations are:

M9L,I, M10L, M105L,I,F, M116N,D,L,I,F,W,R,K, M2021,L,V,T, M208F,Y,L,I, M261 L,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+M2021,

M9L+M2021+M323T,

M9L+M2021+323T+M382Y,

M9L+M2021+Y295F+A339S,

M9L+M2021+Y295F,

M9L+M2021+A339S,

M9L+M2021+Y295F+A339S,

M9L+M2021+Y295F+A339S+E345R,

M9L+G149A+M2021+Y295F+A339S+E345R,

M9L+M202L,

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,

M9 L+M202T+Y295F+A339S,

M9L+M202T+Y295F,

M9L+M202T+A339S,

M9 L+M202T+Y295F+A339S,

M9L+M202T+Y295F+A339S+E345R,

M9L+G149A+M202T+Y295F+A339S+E345R,

M9L+G149A+M2021+V214T+Y295F+N299Y+M323T+A339S+E345R,

M9L+G149A+M202L+V2141+Y295F+M323T+A339S+E345R+M382Y,

M9L+G149A+G182T+M202I+V2141+Y295F+N299Y+M323T+A339S,

M9L+G149A+G182T+M202L+T2571+Y295F+N299Y+M323T+A339S+E345R,

M9L+G149A+M202L+V214T+Y295F+N299Y+M323T+A339S+E345R,

M9L+G149A+M2021+V2141+Y295F+M323T+A339S+E345R+M382Y,

M9L+G149A+G182T+M202L+V2141+Y295F+N299Y+M323T+A339S,

M9L+G149A+G182T+M2021+T2571+Y295F+N299Y+M323T+A339S+E345R,

M9L+G149A+M202I+V214T+Y295F+N299Y+M323T+A339S+E345R+N471E,

M9L+G149A+M202L+V2141+Y295F+M323T+A339S+E345R+M382Y+N471E,

M9L+G149A+G182T+M202I+V2141+Y295F+N299Y+M323T+A339S+N471E,

M9L+G149A+G182T+M202L+T2571+Y295F+N299Y+M323T+A339S+E345R+N471E,

M202L+M105F+M208F,

G133E+M202L+Q361E,

G133E+M202L+R444E,

M202L+Y295F,

M202 L+A339S,

M202 L+M323T,

M202L+M323T+M309L,

M202L+M323T+M430I,

M202L+V214T+R444Y,

M202L+N283D+Q361E,

M202L+M382Y+K383R,

M202L+K446R,

M2021+Y295F,

M2021+A339S,

M2021+M105F+M208F,

G133E+M202I+Q361E,

G133E+M2021+R444E,

M2021+M2021+M323T,

M2021+M2021+M323T+M309L,

M202I+M323T+M4301,

M202I+V214T+R444Y,

M202I+N283D+Q361E,

M202I+M382Y+K383R,

M2021+K446R,

M202V+M105F+M208F,

G133E+M202V+Q361E,

G133E+M202V+R444E,

M202V+M323T,

M202V+M323T+M309L,

M202V+M323T+M4301,

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+M4301,

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,

K383R,

N445Q,

Y178F,

V318L,

W482Y,

N283D+Q361E,

M105F+M208F,

M202L+M323T+M4301,

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,

M2021,

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

M2021+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 depicted in SEQ ID NO: 12.

15. The variant of claim 1 , wherein the parent Termamyl-like alpha-amylase is encoded by a nucleic acid sequence, which hybridizes under high stringency conditions, with the nucleic acid sequence of SEQ ID NO: 11.

16. A detergent additive comprising an alpha-amylase variant according to claim 1 .

17. 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.

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 96% 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 97% 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 98% 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 99% to SEQ ID NO: 12.

Priority Claims (2)
DK 2004 01059 · Jul 5, 2004 · national
DK 2004 01325 · Sep 2, 2004 · national
Continuity (4)
Continuation 11571708
Provisional Application 60609065 · Sep 10, 2004
Provisional Application 60585763 · Jul 6, 2004
Related Publication 20110136205A1 · Jun 9, 2011