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