Synthesis of glucan comprising alpha-1,3 glycosidic linkages with phosphorylase enzymes

Patent metadata

Field	Value
Publication number	US-10808269-B2
Application number	US-201916281225-A
Country	US
Kind code	B2
Filing date	Feb 21, 2019
Priority date	Feb 23, 2018
Publication date	Oct 20, 2020
Grant date	Oct 20, 2020

Abstract

Reaction compositions are disclosed herein comprising at least water, beta-glucose-1-phosphate (beta-G1P), an acceptor molecule, and an alpha-1,3-glucan phosphorylase enzyme. These reactions can synthesize oligosaccharides and polysaccharides with alpha-1,3 glycosidic linkages. Further disclosed are alpha-1,3-glucan phosphorylase enzymes and methods of use thereof.

First claim

What is claimed is:

1. A reaction composition comprising at least water, beta-glucose-1-phosphate (beta-G1P), an acceptor molecule, and a phosphorylase enzyme that synthesizes alpha-1,3-glucan, wherein the phosphorylase enzyme comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:2 or SEQ ID NO:5.

2. The reaction composition of claim 1 , wherein the alpha-1,3-glucan has at least about 50% alpha-1,3 glycosidic linkages.

3. The reaction composition of claim 1 , wherein the alpha-1,3-glucan has at least about 90% alpha-1,3 glycosidic linkages.

4. The reaction composition of claim 1 , wherein the degree of polymerization (DP) of the alpha-1,3-glucan is at least 3.

5. The reaction composition of claim 4 , wherein the DP of the alpha-1,3-glucan is at least about 10.

6. The reaction composition of claim 1 , wherein the acceptor molecule comprises a disaccharide or oligosaccharide.

7. The reaction composition of claim 6 , wherein the acceptor molecule comprises nigerose.

8. The reaction composition of claim 1 , wherein the acceptor molecule comprises a polysaccharide.

9. The reaction composition of claim 8 , wherein the polysaccharide comprises alpha-glucan.

10. The reaction composition of claim 9 , wherein the alpha-glucan comprises dextran.

11. The reaction composition of claim 1 , wherein the phosphorylase enzyme comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:2 or SEQ ID NO:5.

12. A method for producing alpha-1,3-glucan, said method comprising:
contacting at least water, beta-glucose-1-phosphate (beta-G1P), an acceptor molecule, and a phosphorylase enzyme that synthesizes alpha-1,3-glucan, wherein the phosphorylase enzyme comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:2 or SEQ ID NO:5, and wherein alpha-1,3-glucan is produced.

13. The method of claim 12 , wherein the phosphorylase enzyme comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:2 or SEQ ID NO:5.

14. The method of claim 12 , wherein said beta-G1P is provided in step (a) by providing a second reaction, wherein the products of the second reaction comprise beta-G1P.

15. The method of claim 14 , wherein the second reaction is provided in the same vessel in which step (a) is performed, and wherein the second reaction is performed before and/or continuously with step (a).

16. The method of claim 14 , wherein the second reaction produces beta-G1P by contacting (i) water, (ii) inorganic phosphate, (iii) a glucose-comprising disaccharide, oligosaccharide, or polysaccharide, and (iv) a phosphorylase that phosphorolyzes the disaccharide, oligosaccharide, or polysaccharide.

17. The method of claim 16 , wherein the second reaction comprises:
(i) water, inorganic phosphate, maltose, and a maltose phosphorylase, or
(ii) water, inorganic phosphate, trehalose, and a trehalose phosphorylase.

18. The method of claim 12 , further comprising isolating the alpha-1,3-glucan.

19. The method of claim 12 , wherein the alpha-1,3-glucan has at least about 50% alpha-1,3 glycosidic linkages.

20. The method of claim 12 , wherein the alpha-1,3-glucan has at least about 90% alpha-1,3 glycosidic linkages.

Assignees

• Danisco Us Inc

Inventors

• Yu Zheyong
• Kralj Slavko
• Zhang Zhenghong
• Howe Laurie A

Classifications

• C12P19/18

  produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins · CPC title

• C12P19/04 Primary

  Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds · CPC title

• C12N9/1051

  Hexosyltransferases (2.4.1) · CPC title

• C12P19/08 Primary

  Dextran · CPC title