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

Patent metadata

Field	Value
Publication number	US-2019322990-A1
Application number	US-201916383820-A
Country	US
Kind code	A1
Filing date	Apr 15, 2019
Priority date	Apr 23, 2018
Publication date	Oct 24, 2019
Grant date	—

Abstract

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

First claim

What is claimed is:

1 . A reaction composition comprising at least water, alpha-glucose-1-phosphate (alpha-G1P), an acceptor molecule, and a beta-1,3-glucan phosphorylase enzyme comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12, wherein the enzyme synthesizes beta-1,3-glucan.

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

3 . The reaction composition of claim 2 , wherein the beta-1,3-glucan has at least about 99% beta-1,3 glycosidic linkages.

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

5 . The reaction composition of claim 4 , wherein the DP of the beta-1,3-glucan is at least about 15.

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

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

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 beta-glucan.

10 . The reaction composition of claim 9 , wherein the beta-glucan comprises laminarin.

11 . A method for producing beta-1,3-glucan, said method comprising:
(a) contacting at least water, alpha-glucose-1-phosphate (alpha-G1P), an acceptor molecule, and a beta-1,3-glucan phosphorylase enzyme comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12, wherein beta-1,3-glucan is produced; and (b) optionally, isolating the beta-1,3-glucan produced in step (a).

12 . The method of claim 11 , wherein said alpha-G1P is provided in step (a) by providing a second reaction, wherein the products of the second reaction comprise alpha-G1P.

13 . The method of claim 12 , 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).

14 . The method of claim 12 , wherein the second reaction produces alpha-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.

15 . The method of claim 11 , wherein said acceptor molecule is provided in step (a) by providing a third reaction, wherein the products of the third reaction comprise said acceptor molecule.

16 . The method of claim 15 , wherein said acceptor molecule provided by the third reaction is laminaribiose.

17 . A method of processing beta-1,3-glucan, said method comprising:
(a) providing an aqueous composition comprising up to about 40 wt % insoluble beta-1,3-glucan having a degree of polymerization (DP) of at least 17; (b) heating the aqueous composition to at least about 75° C., thereby dissolving the beta-1,3-glucan in the aqueous composition to provide a solution; (c) subjecting the solution to at least one process that reduces the content of one or more solutes other than the dissolved beta-1,3-glucan, thereby increasing the content of the dissolved beta-1,3-glucan in the solution on a dry weight basis; and (d) optionally cooling the solution to a temperature at which the beta-1,3-glucan precipitates back to an insoluble state, and optionally isolating the precipitated beta-1,3-glucan.

18 . The method of claim 17 , wherein the process of (c) comprises nanofiltration and/or ultrafiltration.

Assignees

• Danisco Us Inc

Inventors

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

Classifications

• C07K2319/21

  containing a His-tag · CPC title

• C12N9/1051 Primary

  Hexosyltransferases (2.4.1) · CPC title

• C08B37/0003

  General processes for their isolation or fractionation, e.g. purification or extraction from biomass · CPC title

• C12Y204/01097

  1,3-Beta-D-glucan phosphorylase (2.4.1.97) · CPC title

• C12P19/04 Primary

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