Trichoderma reesei glucoamylase variants resistant to oxidation-related activity loss and the use thereof

Patent metadata

Field	Value
Publication number	US-9540669-B2
Application number	US-201314419906-A
Country	US
Kind code	B2
Filing date	Aug 12, 2013
Priority date	Aug 14, 2012
Publication date	Jan 10, 2017
Grant date	Jan 10, 2017

Abstract

Embodiments of the present disclosure relate to Trichoderma reesei glucoamylase (TrGA) variants having improved properties (e.g., improved thermostability, improved specific activity, and/or resistant to oxidation-related activity loss). Also provided are compositions comprising variant glucoamylases. These compositions are useful in various starch process applications.

First claim

What is claimed is:

1. A glucoamylase variant comprising amino acid substitutions corresponding to positions: 50, 417, 430, 511, 539, and 563 of SEQ ID NO: 2, wherein the glucoamylase variant has at least 90% sequence identity with SEQ ID NO: 2, and wherein the amino acid substitution at position 50 is M50Y, G, F, or K.

2. The glucoamylase variant of claim 1 , wherein the amino acid substitution at position 50 is M50Y.

3. The glucoamylase variant of claim 1 , wherein the amino acid substitutions at positions 417, 430, 511, 539, and 563 are: L417V, T430A, Q511H, A539R, and N563I, respectively.

4. The glucoamylase variant of claim 1 , wherein the glucoamylase variant has at least 95%, 97%, or 99% sequence identity with SEQ ID NO: 2.

5. The glucoamylase variant of claim 1 , wherein the glucoamylase variant comprises the amino acid sequence of SEQ ID NO: 6.

6. The glucoamylase variant of claim 1 , wherein the glucoamylase variant consists of the amino acid sequence of SEQ ID NO: 6.

7. The glucoamylase variant of claim 1 further comprising one or more additional amino acid substitutions corresponding to positions: 43, 44, 61, 73, 294, 431, 503, or 535 of SEQ ID NO: 2.

8. The glucoamylase variant of claim 7 , wherein the amino acid substitutions are: I4Q/R3, D44C/R, N61I, G73F, G294C, A431L/Q, E503A/V, or A535R of SEQ ID NO: 2.

9. The glucoamylase variant of claim 1 , wherein the glucoamylase variant exhibits increased thermostability or increased specific activity as compared to a glucoamylase comprising the amino acid sequence of SEQ ID NO: 2.

10. The glucoamylase variant of claim 1 , wherein the glucoamylase variant loses less activity upon oxidation, when compared to a second glucoamylase variant comprising the amino acid sequence of SEQ ID NO: 5 under the same conditions.

11. An enzyme composition comprising the glucoamylase variant of claim 1 .

12. The enzyme composition of claim 11 , further comprising a hexokinase, a xylanase, a glucose isomerase, a xylose isomerase, a phosphatase, a phytase, a pullulanase, a β-amylase, an α-amylase, a protease, a cellulase, a hemicellulase, a lipase, a cutinase, a trehalase, an isoamylase, a redox enzyme, an esterase, a transferase, a pectinase, a lyase, an α-glucosidase, a β-glucosidase, or a combination thereof.

13. A method of processing starch comprising contacting a starch substrate with the glucoamylase variant of claim 1 to produce a composition comprising glucose.

14. The method of claim 13 , further comprising adding a hexokinase, a xylanase, a glucose isomerase, a xylose isomerase, a phosphatase, a phytase, a pullulanase, a β-amylase, an α-amylase, a protease, a cellulase, a hemicellulase, a lipase, a cutinase, a trehalase, an isoamylase, a redox enzyme, an esterase, a transferase, a pectinase, a hydrolase, an alpha-glucosidase, an beta-glucosidase, or a combination thereof to the starch substrate.

15. The method of claim 13 , wherein processing starch comprises saccharifying the starch substrate resulting in a high glucose syrup.

16. The method of claim 13 , further comprising fermenting the composition comprising glucose to an end product.

17. The method of claim 15 , wherein saccharifying and fermenting are carried out as a simultaneous saccharification and fermentation (SSF) process.

18. The method of claim 16 , wherein the end product is an alcohol.

19. The method of claim 18 , wherein the end product is ethanol.

20. The method of claim 16 , wherein the end product is citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, glucono delta-lactone, sodium erythorbate, omega 3 fatty acid, butanol, lysine, itaconic acid, 1,3-propanediol, biodiesel, or isoprene.

21. The method of claim 13 , wherein the starch substrate is about 15% to 50% dry solid (DS).

22. The method of claim 13 , wherein the glucoamylase is dosed at a range of about 0.2 to about 1.0 glucoamylase unit (GAU) per gram of dry solid starch (dss).

23. The method of claim 13 , wherein the starch substrate is wheat, barley, corn, rye, rice, sorghum, bran, cassava, milo, millet, potato, sweet potato, tapioca, or any combination thereof.

24. The method of claim 13 , wherein the starch substrate comprises liquefied starch, gelatinized starch, or granular starch.

Assignees

• Danisco Us Inc

Inventors

• Chow Su Yin Marina
• Graycar Thomas P
• Huitink Jacquelyn A
• Vroemen Casper
• Wong David L

Classifications

• C12P19/14 Primary

  produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase} · CPC title

• C12Y302/01003

  Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase · CPC title

• C12P2203/00

  Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source · CPC title

• C12P19/20 Primary

  produced by the action of an exo-1,4 alpha-glucosidase, e.g. dextrose · CPC title

• C12N9/2428

  Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase · CPC title