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

Patent metadata

Field	Value
Publication number	US-11180740-B2
Application number	US-201916383820-A
Country	US
Kind code	B2
Filing date	Apr 15, 2019
Priority date	Apr 23, 2018
Publication date	Nov 23, 2021
Grant date	Nov 23, 2021

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 95% identical to SEQ ID NO: 6,
wherein said amino acid sequence has beta-1,3-glucan phosphorylase activity,
wherein the acceptor molecule (i) comprises one or more glucose monomeric units, (ii) does not consist of glucose, and (iii) has one or more glycosidic linkages,
wherein the beta-1,3-glucan phosphorylase enzyme synthesizes a beta-1,3-glucan in the reaction composition and the reaction composition is cell-free.

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:
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 95% identical to SEQ ID NO: 6,
wherein said amino acid sequence has beta-1,3-glucan phosphorylase activity,
wherein the acceptor molecule (i) comprises one or more glucose monomeric units, (ii) does not consist of glucose, and (iii) has one or more glycosidic linkages,
wherein a beta-1,3-glucan is produced, and
wherein the contacting step is performed under cell-free conditions.

12. The method of claim 11 , wherein said alpha-G1P is provided in the contacting step 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 the contacting step is performed, and wherein the second reaction is performed before and/or continuously with the contacting step.

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 the contacting step 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. The method of claim 11 , further comprising isolating the beta-1,3-glucan produced in the contacting step.

18. The method of claim 11 , wherein the amino acid sequence is at least 97% identical to SEQ ID NO:6.

19. The method of claim 11 , wherein the amino acid sequence is at least 98% identical to SEQ ID NO:6.

20. The reaction composition of claim 6 , wherein the acceptor molecule comprises cellobiose or p-nitrophenyl beta-D-glucopyranoside.

21. The reaction composition of claim 1 , wherein the amino acid sequence is at least 97% identical to SEQ ID NO:6.

22. The reaction composition of claim 1 , wherein the amino acid sequence is at least 98% identical to SEQ ID NO: 6.

23. The reaction composition of claim 1 , wherein amino acid sequence is at least 99% identical to SEQ ID NO: 6.

24. The reaction composition of claim 1 , wherein the amino acid sequence is at least 99.5% identical to SEQ ID NO: 6.

Assignees

• Danisco Us Inc

Inventors

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

Classifications

• C12Y204/01097

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

• C07K2319/21

  containing a His-tag · CPC title

• C12N9/1051 Primary

  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

• C08B37/0003

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