Enzymatic method for preparing rebaudioside N

Patent metadata

Field	Value
Publication number	US-11976313-B2
Application number	US-202217663631-A
Country	US
Kind code	B2
Filing date	May 16, 2022
Priority date	Oct 21, 2016
Publication date	May 7, 2024
Grant date	May 7, 2024

Abstract

Provided is a method for preparing rebaudioside N using an enzymatic method, comprising using rebaudioside A or rebaudioside J as a substrate, and making the substrate, in the presence of a glycosyl donor, react under the catalysis of a UDP-glycosyl-transferase and/or a UDP-glycosyltransferase-containing recombinant cell to generate rebaudioside N.

First claim

The invention claimed is:

1. An enzymatic method for preparing rebaudioside N, the method comprising:
reacting rebaudioside A in a reaction system with a glycosyl donor and a rhamnosyl donor, in the presence of (i) recombinant cells comprising a first and a second UDP-glycosyltransferase; (ii) first and second UDP glycosyltransferase prepared from the recombinant cells; (iii) or both; wherein in either (i), (ii), or (iii), the first UDP-glycosyltransferase has the amino acid sequence of SEQ ID NO: 2, and wherein the second UDP-glycosyltransferase has the amino acid sequence of SEQ ID NO: 4.

2. The method of claim 1 , wherein the glycosyl donor is a UDP-glucose or a UDP-glucose regeneration system comprising sucrose, sucrose synthetase, and UDP.

3. The method of claim 1 , wherein the rhamnosyl donor is UDP-rhamnose.

4. The method of claim 1 , wherein SEQ ID NO: 2 is UGT-A from Stevia rebaudiana and SEQ ID NO: 4 is UGT-B from Oryza sativa.

5. The method of claim 1 , wherein the reaction is carried out in an aqueous phase system at a temperature of 35 to 45° C. and at a pH of 7.5 to 8.5.

6. The method of claim 5 , wherein the aqueous system comprises a phosphate buffer solution.

7. The method of claim 5 , wherein the aqueous system further comprises a cell permeabilizing agent.

8. The method of claim 7 , wherein the cell permeabilizing agent is toluene and wherein the toluene is present in the aqueous system at a concentration by volume of 1-3%.

9. The method of claim 1 , wherein the recombinant cell is a cell of a microorganism.

10. The method of claim 9 , wherein the microorganism is Escherichia colt, Saccharomyces cerevisiae , or Pichia pastoris.

11. The method of claim 1 , further comprising purifying the rebaudioside N via resin isolation.

12. The method of claim 11 , wherein the rebaudioside N purified via resin isolation has a purity greater than 95%.

Assignees

• Pepsico Inc

Inventors

• Tao Alex
• Li Guoqing
• Wang Wenxia
• Zheng Leilei
• Zhu Chunlei
• Liang Xiaoliang
• Chan Kuikiu

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

• C12N9/1048

  Glycosyltransferases (2.4) · CPC title

• C12N9/10

  Transferases (2.) (ribonucleases C12N9/22) · CPC title

• C12P7/40

  containing a carboxyl group {including Peroxycarboxylic acids} · CPC title

• C12Y204/01017

  Glucuronosyltransferase (2.4.1.17) · CPC title