Glucoamylase variants and polynucleotides encoding same

Patent metadata

Field	Value
Publication number	US-9353362-B2
Application number	US-201214351469-A
Country	US
Kind code	B2
Filing date	Oct 11, 2012
Priority date	Oct 11, 2011
Publication date	May 31, 2016
Grant date	May 31, 2016

Abstract

The present invention relates to glucoamylase variants having improved thermostability. 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.

First claim

The invention claimed is:

1. A glucoamylase variant having improved thermostability, comprising a substitution at a positon corresponding to position 65 of the polypeptide of SEQ ID NO: 3,wherein the variant has glucoamylase activity and wherein the variant has at least 90% sequence identity to the polypeptide of SEQ ID NO: 3.

2. The variant of claims 1 , wherein the variant has at least at 95% sequence identity to SEQ ID NO: 3.

3. The variant of claim 1 , wherein the variant has at least 97% sequence identity to the polypeptide of SEQ ID NO: 3.

4. The variant of claim 1 , wherein the variant has at least 98% sequence identity to SEQ ID NO: 3.

5. The variant of claim 1 , wherein the variant has at least 99% sequence identity to SEQ ID NO: 3.

6. The variant of claim 1 , wherein the variant has a number of substitutions in the amount of 1-20.

7. The variant of claim 1 , further comprising one or more substitution or deletion in at least one position selected from position 327, 501, and 504 of SEQ ID NO: 3.

8. The variant of claim 1 , wherein the variant further comprises or consists of one or more substitutions selected from the group consisting of T65A, Q327F, E501V, Y504T, and Y504*.

9. The variant of claim 1 , wherein the variant comprises at least one of the following alterations or combinations of alterations:
T65A; or
T65A +Q327F; or
T65A +E501V; or
T65A +Y504T; or
T65A +Y504*; or
T65A +Q327F +E501V; or
T65A +Q327F +Y504T; or
T65A +E501V +Y504T; or
T65A +Q327F +Y504*; or
T65A +E501V +Y504*; or
T65A +Q327F +E501V +Y504T; or
T65A +Q327F +E501V +Y504*.

10. The variant of claim 1 , wherein the variant comprises one of the following combinations of substitutions:
T65A +K161S;
T65A +Q405T;
T65A +Q327W;
T65A +Q327F;
T65A +Q327Y;
P11F +T65A +Q327F;
R1 K +D3W +K5Q +G7V +N8S +T1OK +P11S +T65A +Q327F;
P2N +P4S +P11F +T65A +Q327F;
P11F +D26C +K33C +T65A +Q327F;
P2N +P4S +P11F +T65A +Q327W +E501V +Y504T;
R1E +D3N +P4G +G6R +G7A +N8A +T10D+P11D +T65A +Q327F;
P11F +T65A +Q327W;
P2N +P4S +P11F +T65A +Q327F +E501V +Y504T;
P11F +T65A +Q327W +E501V +Y504T;
T65A +Q327F +E501V +Y504T;
T65A +S105P +Q327W;
T65A +S105P +Q327F;
T65A +Q327W +S364P;
T65A +Q327F +S364P;
T65A +S103N +Q327F;
P2N +P4S +P11F +K34Y +T65A +Q327F;
P2N +P4S +P11F +T65A +Q327F +D445N +V447S;
P2N +P4S +P11F +T65A +I172V +Q327F;
P2N +P4S +P11F +T65A +Q327F +N502*;
P2N +P4S +P11F +T65A +Q327F +N502T +P563S +K571E;
P2N +P4S +P11F +R31S +K33V +T65A +Q327F +N564D +K571S;
P2N +P4S +P11F +T65A +Q327F +S377T;
P2N +P4S +P11 F +T65A +V325T+Q327W;
P2N +P4S +P11 F +T65A +Q327F +D445N +V447S +E501V +Y504T;
P2N +P4S +P11F +T65A +1172V +Q327F +E501V +Y504T;
P2N +P4S +P11F +T65A +Q327F +S377T +E501V +Y504T;
P2N +P4S +P11F +D26N +K34Y +T65A +Q327F;
P2N +P4S +P11F +T65A +Q327F +1375A +E501V +Y504T;
P2N +P4S +P11F +T65A +K218A +K221D +Q327F +E501V +Y504T;
P2N +P4S +P11F +T65A +S103N +Q327F +E501V +Y504T;
P2N +P4S +T1 OD +T65A +Q327F +E501V +Y504T;
P2N +P4S +F12Y +T65A +Q327F +E501V +Y504T;
K5A +P11F +T65A +Q327F +E501V +Y504T;
P2N +P4S +T1OE +E18N +T65A +Q327F +E501V +Y504T;
P2N +T1OE +E18N +T65A +Q327F +E501V +Y504T;
P2N +P4S +P11F +T65A +Q327F +E501V +Y504T +T568N;
P2N +P4S +P11 F +T65A +Q327F +E501V +Y504T +K524T +G526A;
P2N +P4S +P11 F +K34Y +T65A +Q327F +D445N +V447S +E501V +Y504T;
P2N +P4S +P11F +R31S +K33V +T65A +Q327F +D445N +V447S +E501V +Y504T;
P2N +P4S +P11F +D26N +K34Y +T65A +Q327F +E501V +Y504T;
P2N +P4S +P11F +T65A +F80* +Q327F +E501V +Y504T;
P2N +P4S +P11F +T65A +K112S +Q327F +E501V +Y504T;
P2N +P4S +P11F +T65A +Q327F +E501V +Y504T +T516P +K524T +G526A;
P2N +P4S +P11F +T65A +Q327F +E501V +N502T +Y504*;
P2N +P4S +P11F +T65A +Q327F +E501V +Y504T;
P2N +P4S +P11F +T65A +S103N +Q327F +E501V +Y504T;
K5A +P11F +T65A +Q327F +E501V +Y504T;
P2N +P4S +P11F +T65A +Q327F +E501V +Y504T +T516P +K524T +G526A;
P2N +P4S +P11F +T65A +V79A +Q327F +E501V +Y504T;
P2N +P4S +P11F +T65A +V79G +Q327F +E501V +Y504T;
P2N +P4S +P11 F +T65A +V791 +Q327F +E501V +Y504T;
P2N +P4S +P11 F +T65A +V79L +Q327F +E501V +Y504T;
P2N +P4S +P11F +T65A +V79S +Q327F +E501V +Y504T;
P2N +P4S +P11 F +T65A +L72V +Q327F +E501V +Y504T;
P2N +P4S +P11 F +T65A +E74N +V79K +Q327F +E501V +Y504T;
P2N +P4S +P11F +T65A +G220N +Q327F +E501V +Y504T;
P2N +P4S +P11F +T65A +Y245N +Q327F +E501V +Y504T;
P2N +P4S +P11F +T65A +Q253N +Q327F +E501V +Y504T;
P2N +P4S +P11F +T65A +D279N +Q327F +E501V +Y504T;
P2N +P4S +P11F +T65A +Q327F +S359N +E501V +Y504T;
P2N +P4S +P11F +T65A +Q327F +D370N +E501V +Y504T;
P2N +P4S +P11F +T65A +Q327F +V460S +E501V +Y504T;
P2N +P4S +P11 F +T65A +Q327F +V460T +P468T +E501V +Y504T;
P2N +P4S +P11F +T65A +Q327F +T463N +E501V +Y504T;
P2N +P4S +P11F +T65A +Q327F +S465N +E501V +Y504T; or P2N +P4S +P11F +T65A +Q327F +T477N +E501V +Y504T.

11. The variant of claim 1 , which has an improved property relative to a parent, wherein the improved property is reduced sensitivity to protease degradation.

12. The variant of claim 1 , which has an improved property relative to a parent, wherein the improved property is improved thermostability.

13. A variant glucoamylase catalytic domain comprising a substitution at a position corresponding to position 65 of the polypeptide of SEQ ID NO: 3, wherein the variant has glucoamylase activity.

14. A composition comprising the polypeptide of claim 1 .

15. A composition according to claim 14 , comprising an alpha-amylase.

16. A process of producing a fermentation product from starch-containing material comprising the steps of:
(a) liquefying starch-containing material in the presence of an alpha amylase;
(b) saccharifying the liquefied material; and
(c) fermenting with a fermenting organism;

wherein step (a) and/or step (b) is carried out using at least a glucoamylase of claim 1 .

17. A process of producing a fermentation product from starch-containing material, comprising the steps of:
(a) saccharifying starch-containing material at a temperature below the initial gelatinization temperature of said starch-containing material; and
(b) fermenting with a fermenting organism,

wherein step (a) is carried out using at least a glucoamylase of claim 1 .

Assignees

• Novozymes As

Inventors

• Friis Esben Peter
• De Maria Leonardo
• Vind Jesper
• Poulsen Thomas A
• Svendsen Allan
• Danielsen Steffen
• Lenhard Rolf T
• Friis-Madsen Henrik
• Skov Lars K

Classifications

• Y02E50/10

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

• C12P19/20

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

• C12N9/2428 Primary

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

• C12Y302/01003

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

• C12P37/00

  Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin · CPC title