Enzymatic method for preparing rebaudioside M

Patent metadata

Field	Value
Publication number	US-10428364-B2
Application number	US-201916380678-A
Country	US
Kind code	B2
Filing date	Apr 10, 2019
Priority date	Aug 14, 2013
Publication date	Oct 1, 2019
Grant date	Oct 1, 2019

Abstract

Provided is a method for preparing rebaudioside M by using an enzyme method. In the method, rebaudioside A or rebaudioside D is used as a substrate; and in the existence of a glucosyl donor, rebaudioside M is generated by means of reaction of the substrate under the catalysis of UDP-glucosyl transferase and/or recombinant cells containing the UDP-glucosyl transferase.

First claim

What is claimed is:

1. A method for preparing rebaudioside M, the method comprising reacting rebaudioside D in a reaction solution with a glucosyl donor in the presence of recombinant cells comprising a UDP-glucosyl transferase having the amino acid sequence of SEQ ID NO: 2, to obtain rebaudioside M;
wherein at least 40% of the rebaudioside D is converted to rebaudioside M after reacting for two hours.

2. The method of claim 1 , wherein the glucosyl donor is UDP-glucose.

3. The method of claim 2 , wherein the UDP-glucose is generated in situ from UDP and sucrose in the presence of a sucrose synthetase.

4. The method of claim 1 , wherein the recombinant cells are microbial cells selected from the group consisting of: Escherichia coli, Saccharomyces cerevisiae , and Pichia pastoris.

5. The method of claim 1 , wherein reacting rebaudioside D with the glucosyl donor in the presence of recombinant cells comprising the UDP-glucosyl transferase having the amino acid sequence of SEQ ID NO: 2, is carried out in an aqueous phase system having a temperature ranging from 25° C. to 35° C. and a pH ranging from 6.5 to 7.5.

6. The method of claim 5 , wherein the aqueous phase system comprises pH 7 phosphate buffer.

7. The method of claim 5 , wherein the aqueous phase system comprises a cellular permeating agent.

8. The method of claim 7 , wherein the cellular permeating agent is toluene at a concentration of 1% to 3% by volume.

9. The method of claim 1 , wherein the rebaudioside D is prepared by reacting rebaudioside A with a glucosyl donor in the presence of recombinant cells comprising a UDP-glucosyl transferase having the amino acid sequence of SEQ ID NO: 4.

10. The method of claim 9 , wherein the glucosyl donor is UDP-glucose.

11. The method of claim 10 , wherein the UDP-glucose is generated in situ from UDP and sucrose in the presence of a sucrose synthetase.

12. The method of claim 9 , wherein the recombinant cells are microbial cells selected from the group consisting of: Escherichia coli, Saccharomyces cerevisiae , and Pichia pastoris.

13. The method of claim 9 , wherein reacting rebaudioside A with the glucosyl donor in the presence of recombinant cells comprising the UDP-glucosyl transferase having the amino acid sequence of SEQ ID NO: 2, is carried out in an aqueous phase system having a temperature ranging from 25° C. to 35° C. and a pH ranging from 6.5 to 7.5.

14. The method of claim 13 , wherein the aqueous phase system comprises pH 7 phosphate buffer.

15. The method of claim 13 , wherein the aqueous phase system comprises a cellular permeating agent.

16. The method of claim 15 , wherein the cellular permeating agent is toluene at a concentration of 1% to 3% by volume.

Assignees

• Pepsico Inc

Inventors

• Tao Junhua
• Li Guoqing
• Liang Xiaoliang
• Tao Andrew

Classifications

• C12P19/56 Primary

  having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin · CPC title

• C12N9/1051 Primary

  Hexosyltransferases (2.4.1) · CPC title

• A23V2002/00

  Food compositions, function of food ingredients or processes for food or foodstuffs · CPC title

• A23L27/36

  Terpene glycosides · CPC title

• C12Y204/01017

  Glucuronosyltransferase (2.4.1.17) · CPC title