IP Library › Granted Patent US 9,434,932
Granted Patent B2
US 9,434,932 · App. 14/129,565 · Granted Sep 6, 2016

Alpha-amylase variants

Inventors: Svend Kaasgaard (Skovlunde, DK); Jens Oebro (Humlebaek, DK); Signe Eskildsen Larsen (Kgs. Lyngby, DK); Allan Svendsen (Hoersholm, DK); Annette Helle Johansen (Broenshoej, DK); Michael Skjoet (Jyllinge, DK); Carsten Andersen (Vaerloese, DK); Lars Beier (Soeborg, DK); Esben Peter Friis (Herlev, DK); Miguel Duarte Guilherme Pereira Toscano (Frederiksberg, DK); Mads Bjoernvad (Virum, DK); Frank Winther Rasmussen (Roskilde, DK); Liv Spaanger Christiansen (Gentofte, DK)
Assignee: NOVOZYMES A/S
C12N9/2417C12Y302/01001
View Patent ↗
Loading inventors, assignments & file history…
Monitor This Case
Get email alerts when status or documents change.
Order Certified Copies
Most orders are placed with the USPTO same day — all within 24 business hours.
Order via The Patent Place →
Pre-filled with this patent's details
Quick Facts
Patent No.
US 9,434,932
App. No.
14/129,565
Granted
Sep 6, 2016
Kind
B2
Abstract

The present invention relates to variants of a parent alpha-amylase. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

Claims (105)

1. An isolated variant of a parent alpha-amylase, comprising at least two substitutions at positions corresponding to 140 and 206 of the polypeptide of SEQ ID NO: 1, wherein the variant has at least 90% sequence identity with the polypeptide of SEQ ID NO: 1, and has alpha-amylase activity, and wherein the substitution at position 206 is V206Y.

2. The variant according to claim 1 , which comprises a substitution at three positions selected from the group consisting of G304, W189, D134, E260, F262, W284, W347, W439, W469, G476, and G477.

3. The variant according to claim 1 , which comprises a substitution at four positions selected from the group consisting of G304, W189, D134, E260, F262, W284, W347, W439, W469, G476, and G477.

4. The variant according to claim 1 , which further comprises a substitution at two, three or four positions selected from the group consisting of G304, E260 and G476.

5. The variant of claim 1 , which further comprises two or more substitutions selected from the group consisting of G304R, W140YF, W189EGT, D134E, E260GHIKNRTY, W284DFR, W439RG, G476EK, G477EKMR.

6. The variant according to claim 1 , which comprises substitutions at two, three or four positions selected from the group consisting of G304R, W140YF, E260GHIKNRTY and G476EK.

7. The variant according to claim 6 , wherein the substitutions at the two, three or four positions are selected from the group consisting of G304R, W140Y, E260G and G476K.

8. The variant of claim 1 , further comprising one or more substitutions selected from the group consisting of N195F, Y243F, G109A, G273DV, G337N, K72R, R181H, S303G and Y100I.

9. The variant according to claim 1 , wherein the number of alterations is 2-20.

10. The variant according to claim 1 , which has at least 95% sequence identity with the polypeptide of SEQ ID NO: 1.

11. The variant according to claim 1 , which comprises substitutions in the positions, corresponding to the positions of the polypeptide of SEQ ID NO: 1, selected from the group consisting of:

W140Y+N195F+V206Y+Y243F+E260G+G477E,

W140Y+N195F+V206Y+Y243F+E260T+W284D,

W140Y+N195F+V206Y+Y243F+W284D,

G109A+W140Y+N195F+V206Y+Y243F+E260G,

W140Y+N195F+V206Y+Y243F+E260G,

W140Y+N195F+V206Y+Y243F+E260G+G476E,

W140Y+W189G+N195F+V206Y+Y243F+E260G,

W140Y+N195F+V206Y+Y243F+E260G+S303G,

W140Y+W189T+N195F+V206Y+Y243F+E260G,

W140Y+N195F+V206Y+Y243F+E260G+W284D,

Y100I+W140Y+N195F+V206Y+Y243F+E260G,

W140Y+N195F+V206Y+Y243F+E260G+G337N,

W140Y+N195F+V206Y+Y243F+E260G+W439R,

G109A+W140Y+E194D+N195F+V206Y+Y243F+E260G,

G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E,

T51I+Y100I+G109A+W140Y+N195F+V206Y+Y243F+E260G,

T51I+G109A+W140Y+N195F+V206Y+Y243F+E260G+W439R,

T51I+S52Q+N54K+G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E,

W140Y+N195F+V206Y+Y243F+E260G+G304R+G476K,

W140Y+N195F+V206Y+Y243F+E260G+W284R+G477K, and

W140Y+N195F+V206Y+Y243F+E260G+W284F+G477R.

12. The variant according to claim 1 , which consists of substitutions in the positions, corresponding to the positions of the polypeptide of SEQ ID NO: 1, selected from the group consisting of:

W140Y+N195F+V206Y+Y243F+E260G+G477E,

W140Y+N195F+V206Y+Y243F+E260T+W284D,

W140Y+N195F+V206Y+Y243F+W284D,

G109A+W140Y+N195F+V206Y+Y243F+E260G,

W140Y+N195F+V206Y+Y243F+E260G,

W140Y+N195F+V206Y+Y243F+E260G+G476E,

W140Y+W189G+N195F+V206Y+Y243F+E260G,

W140Y+N195F+V206Y+Y243F+E260G+S303G,

W140Y+W189T+N195F+V206Y+Y243F+E260G,

W140Y+N195F+V206Y+Y243F+E260G+W284D,

Y100I+W140Y+N195F+V206Y+Y243F+E260G,

W140Y+N195F+V206Y+Y243F+E260G+G337N,

W140Y+N195F+V206Y+Y243F+E260G+W439R,

G109A+W140Y+E194D+N195F+V206Y+Y243F+E260G,

G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E,

T51I+Y100I+G109A+W140Y+N195F+V206Y+Y243F+E260G,

T51I+G109A+W140Y+N195F+V206Y+Y243F+E260G+W439R,

T51I+S52Q+N54K+G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E,

W140Y+N195F+V206Y+Y243F+E260G+G304R+G476K,

W140Y+N195F+V206Y+Y243F+E260G+W284R+G477K,

W140Y+N195F+V206Y+Y243F+E260G+W284F+G477K.

13. The variant according to claim 1 , which further comprises deletions at positions corresponding to positions G182*+D183* or D183*+G184* of SEQ ID NO:1.

14. The variant according to claim 1 , which comprises alterations in the positions, corresponding to the positions of the polypeptide of SEQ ID NO: 1, selected from the group consisting of:

D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G477E,

D183*+G184*+W140Y+N195F+V206Y+Y243F+E260T+W284D,

D183*+G184*+W140Y+N195F+V206Y+Y243F+W284D,

D183*+G184*+G109A+W140Y+N195F+V206Y+Y243F+E260G,

D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G,

D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G476E,

D183*+G184*+W140Y+W189G+N195F+V206Y+Y243F+E260G,

D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+S303G,

D183*+G184*+W140Y+W189T+N195F+V206Y+Y243F+E260G,

D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W284D,

D183*+G184*+Y100I+W140Y+N195F+V206Y+Y243F+E260G,

D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G337N,

D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W439R,

D183*+G184*+G109A+W140Y+E194D+N195F+V206Y+Y243F+E260G,

D183*+G184*+G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E,

D183*+G184*+T51I+Y100I+G109A+W140Y+N195F+V206Y+Y243F+E260G,

D183*+G184*+T51I+G109A+W140Y+N195F+V206Y+Y243F+E260G+W439R,

D183*+G184*+T51I+S52Q+N54K+G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E,

D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G304R+G476K,

D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W284R+G477K, and

D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W284F+G477R.

15. The variant according to claim 1 , which consists of alterations in the positions, corresponding to the positions of the polypeptide of SEQ ID NO: 1, selected from the group consisting of:

D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G477E,

D183*+G184*+W140Y+N195F+V206Y+Y243F+E260T+W284D,

D183*+G184*+W140Y+N195F+V206Y+Y243F+W284D,

D183*+G184*+G109A+W140Y+N195F+V206Y+Y243F+E260G,

D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G,

D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G476E,

D183*+G184*+W140Y+W189G+N195F+V206Y+Y243F+E260G,

D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+S303G,

D183*+G184*+W140Y+W189T+N195F+V206Y+Y243F+E260G,

D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W284D,

D183*+G184*+Y100I+W140Y+N195F+V206Y+Y243F+E260G,

D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G337N,

D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W439R,

D183*+G184*+G109A+W140Y+E194D+N195F+V206Y+Y243F+E260G,

D183*+G184*+G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E,

D183*+G184*+T51I+Y100I+G109A+W140Y+N195F+V206Y+Y243F+E260G,

D183*+G184*+T51I+G109A+W140Y+N195F+V206Y+Y243F+E260G+W439R,

D183*+G184*+T51I+S52Q+N54K+G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E,

D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G304R+G476K,

D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W284R+G477K, and

D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W284F+G477R.

16. An isolated polynucleotide encoding the variant of claim 1 .

17. A nucleic acid construct comprising the polynucleotide of claim 16 .

18. An expression vector comprising the polynucleotide of claim 16 .

19. A host cell comprising the polynucleotide of claim 16 .

20. A method of producing a variant of a parent alpha-amylase, comprising cultivating the host cell of claim 19 under conditions suitable for the expression of the variant; and recovering the variant.

21. An isolated variant of a parent alpha-amylase, comprising substitutions at positions corresponding to 140 and 206 of the polypeptide of SEQ ID NO: 1, wherein the variant has at least 99% sequence identity with the polypeptide of SEQ ID NO: 1, wherein the substitution at position 206 is characterized as V206Y, and wherein the variant has alpha-amylase activity.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 4, 2014
From: KAASGAARD, SVEND; OEBRO, JENS; LARSEN, SIGNE ESKILDSEN; SVENDSEN, ALLAN; JOHANSEN, ANNETTE HELLE; SKJOET, MICHAEL; ANDERSEN, CARSTEN; BEIER, LARS; FRIIS, ESBEN PETER; TOSCANO, MIGUEL DUARTE GUILLERME PEREIRA; BJOERNVAD, MADS; RASMUSSEN, FRANT WINTHER
To: NOVOZYMES A/S
Reel/Frame 032137/0685 →
Continuity (2)
Provisional Application 61503768 · Jul 1, 2011
Related Publication 20140141489A1 · May 22, 2014