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

Patent metadata

Field	Value
Publication number	US-2019264247-A1
Application number	US-201916281225-A
Country	US
Kind code	A1
Filing date	Feb 21, 2019
Priority date	Feb 23, 2018
Publication date	Aug 29, 2019
Grant date	—

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.

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 90% identical to SEQ ID NO:2 or SEQ ID NO:5.

12 . A method for producing alpha-1,3-glucan, said method comprising:
(a) contacting at least water, beta-glucose-1-phosphate (beta-G1P), an acceptor molecule, and a phosphorylase enzyme that synthesizes alpha-1,3-glucan, wherein alpha-1,3-glucan is produced; and (b) optionally, isolating the alpha-1,3-glucan produced in step (a).

13 . The method of claim 12 , wherein the alpha-1,3-glucan phosphorylase enzyme comprises an amino acid sequence that is at least 90% 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.

Assignees

• Danisco Us Inc

Inventors

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

Classifications

• C12P19/08 Primary

  Dextran · CPC title

• C12N9/1051

  Hexosyltransferases (2.4.1) · CPC title

• C12P19/04 Primary

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

• C12P19/18

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