Thermostable asparaginase variants and polynucleotides encoding same

Patent metadata

Field	Value
Publication number	US-9828595-B2
Application number	US-201314420399-A
Country	US
Kind code	B2
Filing date	Aug 15, 2013
Priority date	Aug 17, 2012
Publication date	Nov 28, 2017
Grant date	Nov 28, 2017

Abstract

The present invention relates to asparaginase variants. 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. The invention further relates to a process of producing a fermentation product, comprising: liquefying a starch-containing material to dextrins with an alpha-amylase in the presence of an asparaginase of the invention; saccharifying the dextrins to a sugar with a glucoamylase; and fermenting the sugar using a fermenting organism.

First claim

What is claimed is:

1. An asparaginase variant comprising a substitution at one or more positions corresponding to positions 140 or 241 of SEQ ID NO: 2, or a deletion at a position corresponding to position 27 of SEQ ID NO: 2, wherein the variant has at least 80% sequence identity to the mature polypeptide of either of SEQ ID NOs: 2 or 4, and wherein the variant has asparaginase activity.

2. The variant of claim 1 , in which the amino acid at a position corresponding to position 140 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val.

3. The variant of claim 1 , in which the amino acid at a position corresponding to position 140 is substituted with Asp.

4. The variant of claim 1 , in which the amino acid at a position corresponding to position 241 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val.

5. The variant of claim 1 , in which the amino acid at a position corresponding to position 241 is substituted with Glu.

6. The variant of claim 1 , which further comprises one or more of the following substitutions: K122A, K122R, D197E, S238C, N239C, K240R, K253R, I258V, I258Y, R259C, R259V, S297V or E373H.

7. The variant of claim 1 , which further comprises a substitution at one or more positions corresponding to positions 71, 74, 139, 194, 228, 299, 314, 333, 334, 337, 338, 356 or 363 of SEQ ID NO: 2.

8. The variant of claim 1 , which further comprises one or more of the following substitutions: T71C, T74A, T74C, V139G, K194L, I228M, S299A, T314A, P333L, S334P, S334W, E337S, S338G, S338W, G356D or K363R.

9. The variant of claim 1 , which further comprises a substitution at one or more positions corresponding to positions 70, 307, 323, 327, 349, 351 or 353 of SEQ ID NO: 2.

10. The variant of claim 1 , which further comprises one or more of the following substitutions: N70K, S307A, A323R, T327V, A349Q, S351A or V353I.

11. A method for producing a food product from a food material, comprising:
a) adding the variant of claim 1 to the food material; and
b) heating the enzyme treated food material.

12. The variant of claim 1 , which has at least 85% sequence identity to the mature polypeptide of any of SEQ ID NOs: 2 or 4.

13. The variant of claim 1 , which has at least 90% sequence identity to the mature polypeptide of any of SEQ ID NOs: 2 or 4.

14. The variant of claim 1 , which has at least 95% sequence identity to the mature polypeptide of any of SEQ ID NOs: 2 or 4.

Assignees

• Novozymes As

Inventors

• Matsui Tomoko
• Tomiki Aki
• Svendsen Allan
• Hendriksen Hanne Vang
• Stringer Mary Ann
• Ayabe Keiichi

Classifications

• C12N9/82 Primary

  Asparaginase {(3.5.1.1)} · CPC title

• A23L5/25

  using enzymes · CPC title

• C12Y305/01001

  Asparaginase (3.5.1.1) · CPC title