SYNTHESIS OF GLUCAN COMPRISING BETA-1,3 GLYCOSIDIC LINKAGES WITH PHOSPHORYLASE ENZYMES

• US 20190322990A1
• Filed: 04/15/2019
• Published: 10/24/2019
• Est. Priority Date: 04/23/2018
• Status: Active Grant

• Current Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.), DuPont Nutrition Biosciences ApS (International Flavors & Fragrances Incorporated)
• Original Assignee: Danisco US, Inc. (International Flavors & Fragrances Incorporated)
• Inventors: YU, ZHEYONG, KRALJ, SLAVKO, BEHABTU, NATNAEL, ZHANG, ZHENGHONG, HOWE, LAURIE A
• Granted Patent: US 11,180,740 B2
• Time in Patent Office: Days
• CPC Class Codes: C07K 2319/21 containing a His-tag
• CPC Class Codes: C08B 37/0003 General processes for their isolation or fractionation, e.g. purification or extraction from biomass
• CPC Class Codes: C08B 37/0024 beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12P 19/04 Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
• CPC Class Codes: C12Y 204/01097 1,3-Beta-D-glucan phosphorylase (2.4.1.97)

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.

Claims

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.