Compositions of high stability alpha amylase variants

Patent metadata

Field	Value
Publication number	US-9896673-B2
Application number	US-201113024770-A
Country	US
Kind code	B2
Filing date	Feb 10, 2011
Priority date	Feb 10, 2010
Publication date	Feb 20, 2018
Grant date	Feb 20, 2018

Abstract

The present invention relates to variants of an alpha-amylase having improved stability to chelating agents relative to its parent enzyme, compositions comprising the variants, nucleic acids encoding the variants, methods of producing the variants, and methods for using the variants.

First claim

The invention claimed is:

1. A composition comprising:
a variant of a parent alpha-amylase, wherein the variant comprises a substitution at positions corresponding to 195 and 243 of SEQ ID NO: 6, wherein the variant has alpha-amylase activity and at least 95% sequence identity with SEQ ID NO: 6; and at least one chelating agent, wherein said chelating agent at a concentration below 10 mM reduces the concentration of free calcium ions from 2.0 mM to 0.10 mM when measured at 21° C. and pH 8.0.

2. The composition of claim 1 , wherein the variant further comprises an alteration at one or more positions corresponding to 193, 197, 198, 200, 203, 206, 210, 212, 213, 243, 116, 118, 129, 133, 142, 146, 147, 149, 151, 152, 169, 174, 186, 235, 244, 303, 320, 339, 359, 418, 431, 434, 447 or 458 of SEQ ID NO: 6.

3. The composition of claim 1 , wherein the chelating agent at a concentration below 10 mM reduces the concentration of free calcium ions from 2.0 mM to 0.10 mM when measured in 80 mM potassium chloride and 49 mM EPPS at 21° C. and pH 8.0.

4. The composition of claim 1 , wherein the chelating agent reduces the concentration of free calcium ions from 2.0 mM to 0.10 mM at a chelator agent concentration below 8 mM, below 7 mM, below 6 mM, below 5 mM, or below 4 mM.

5. The composition of claim 1 , wherein the chelating agent reduces the free calcium ion concentration from 2.0 mM to 0.10 mM at a chelating agent concentration below 0.9 times the concentration of citrate which reduces the free calcium ion concentration from 2.0 mM to 0.10 mM, when measured at 21° C. and pH 8.

6. The composition of claim 1 , wherein the chelating agent reduces the free calcium ion concentration from 2.0 mM to 0.10 mM at a chelating agent concentration below 0.7 times, below 0.5 times, or below 0.3 times the concentration of citrate which reduces the free calcium ion concentration from 2.0 mM to 0.10 mM.

7. The composition of claim 1 , wherein the parent alpha-amylase sequence is modified by at least one of the following substitutions: position 195 is [F, W, Y, L, I or V], position 243 is [F, W, Y, L, I or V] or position 195 is [F, W, Y, L, I or V] and position 243 is [F, W, Y, L, I or V].

8. The composition of claim 1 , wherein the parent alpha-amylase sequence is modified by at least one of the following substitutions: position 195 is F or Y, position 243 is F or position 195 is F or Y and position 243 is F.

9. The composition of claim 1 , wherein the variant further comprises at least one, at least two, or at least three deletions in amino acid region of 181, 182, 183 or 184 corresponding to SEQ ID NO: 6.

10. The composition of claim 1 , wherein the variant has at least 60% residual activity after 18 hours at pH 8 and 31° C. in the presence of a chelating agent, and wherein said chelating agent at a concentration below 10 mM reduces the concentration of free calcium ions from 2.0 mM to 0.10 mM at 21° C. and pH 8.0.

11. The composition of claim 1 , wherein the variant has at least 70% residual activity after 18 hours at pH 8 and 31° C. in the presence of a chelating agent.

12. The composition of claim 1 , wherein the variant has improved wash performance compared to the parent alpha-amylase when measured in AMSA.

13. The composition of claim 1 , wherein the chelating agent is selected from the group consisting of: EDTA, MGDA, EGTA, DTPA, DTPMP and HEDP.

14. The composition of claim 1 which is not for air care, car care, dishwashing, fabric conditioning, laundry detergent, laundry and rinse additive and/or care, hard surface cleaning and/or treatment, and other cleaning for consumer or institutional use.

15. A variant of a parent alpha-amylase comprising a substitution at positions corresponding to 195 and 243 of SEQ ID NO: 6, the variant having alpha-amylase activity and at least 99% sequence identity with SEQ ID NO: 6.

16. The variant of claim 15 , further comprising an alteration at one or more positions corresponding to 193, 197, 198, 200, 203, 206, 210, 212, 213, 243, 116, 118, 129, 133, 142, 146, 147, 149, 151, 152, 169, 174, 186, 235, 244, 303, 320, 339, 359, 418, 431, 434, 447 or 458 of SEQ ID NO: 6, wherein (a) the alteration(s) are independently
(i) an insertion of an amino acid immediately downstream and adjacent of the position,
(ii) a deletion of the amino acid which occupies the position, and/or
(iii) a substitution of the amino acid which occupies the position.

17. The variant of claim 15 , wherein the variant has improved stability relative to the parent alpha-amylase or relative to SEQ ID NO: 6 in a composition comprising a chelating agent, wherein said chelating agent at a concentration below 10 mM reduces the concentration of free calcium ions from 2.0 mM to 0.10 mM at 21° C. and pH 8.0.

18. The variant of claim 15 , wherein the variant has at least 60% residual activity after 18 hours at pH 8 in the presence of a chelating agent, and wherein said chelating agent at a concentration below 10 mM reduces the concentration of free calcium ions from 2.0 mM to 0.10 mM at 21° C. and pH 8.0.

19. The composition of claim 2 , wherein the parent alpha-amylase sequence is modified by at least one of the following substitutions: position 193 is [G, A, S, T or M]; position 197 is [F, W, Y, L, I or V]; position 198 is [Q or N]; position 200 is [F, W, Y, L, I or V]; position 203 is [F, W, Y, L, I or V]; position 206 is [F, W, Y, N, L, I, V, H, Q, D or E]; position 210 is [F, W, Y, L, I or V]; position 212 is [F, W, Y, L, I or V]; or position 213 is [G, A, S, T or M].

20. The variant of claim 15 , further comprising at least one, at least two or at least three amino acid deletions in amino acid region of 181, 182, 183 or 184 corresponding to SEQ ID NO: 6.

21. The variant of claim 15 , wherein the parent alpha-amylase sequence is modified by at least one of the following substitutions: position 193 is [G, A, S, T or M]; position 195 is [F, W, Y, L, I or V]; position 197 is [F, W, Y, L, I or V]; position 198 is [Q or N]; position 200 is [F, W, Y, L, I or V]; position 203 is [F, W, Y, L, I or V]; position 206 is [F, W, Y, N, L, I, V, H, Q, D or E]; position 210 is [F, W, Y, L, I or V]; position 212 is [F, W, Y, L, I or V]; position 213 is [G, A, S, T or M] or position 243 is [F, W, Y, L, I or V].

Assignees

• Svendsen Allan
• Johansen Annette Helle
• Bjoernvad Mads
• Rasmussen Frank W
• Skjoet Michael
• Larsen Signe Eskildsen
• Oebro Jens
• Kaasgaard Svend
• Beier Lars
• Novozymes As

Inventors

• Svendsen Allan
• Johansen Annette Helle
• Bjoernvad Mads
• Rasmussen Frank W
• Skjoet Michael
• Larsen Signe Eskildsen
• Oebro Jens
• Kaasgaard Svend
• Beier Lars

Classifications

• C12N9/2417 Primary

  from microbiological source · CPC title

• C11D3/38618

  Protease or amylase in liquid compositions only · CPC title

• C12Y302/01001

  Alpha-amylase (3.2.1.1) · CPC title

• Y02E50/10

  Biofuels, e.g. bio-diesel · CPC title

• Y02E50/30

  Fuel from waste, e.g. synthetic alcohol or diesel · CPC title