Enzymatic method for preparing Rebaudioside J

Patent metadata

Field	Value
Publication number	US-11952604-B2
Application number	US-202217805626-A
Country	US
Kind code	B2
Filing date	Jun 6, 2022
Priority date	Oct 21, 2016
Publication date	Apr 9, 2024
Grant date	Apr 9, 2024

Abstract

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

First claim

The invention claimed is:

1. An enzymatic method for preparing Rebaudioside J, the method comprising reacting Rebaudioside A with a rhamnosyl donor in a reaction system comprising: recombinant cells comprising a UDP-glycosyltransferase, a UDP-glycosyltransferase prepared therefrom, or both the recombinant cells comprising the UDP-glycosyltransferase and the UDP-glycosyltransferase prepared therefrom, wherein the UDP-glycosyltransferase comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2, wherein Rebaudioside A is converted to Rebaudioside J at a conversion rate greater than 90%.

2. The method of claim 1 , wherein the UDP-glycosyltransferase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2.

3. The method of claim 2 , wherein the rhamnosyl donor is a UDP-rhamnose.

4. The method of claim 2 , wherein the UDP-glycosyltransferase is a UGT-B from Oryza sativa.

5. The method of claim 2 , wherein the reaction system is aqueous and has a temperature of 35-45° C. and a pH of 7.5 to 8.5.

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

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

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

9. The method of claim 2 , wherein the recombinant cell is a cell of a microorganism.

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

11. The method of claim 2 , further comprising purifying the Rebaudioside J via resin isolation.

12. The method of claim 11 , wherein the Rebaudioside J 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

• C12N9/1051

  Hexosyltransferases (2.4.1) · CPC title

• C12Y204/01017

  Glucuronosyltransferase (2.4.1.17) · CPC title

• A23L27/30 Primary

  Artificial sweetening agents · CPC title

• C12P19/18

  produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins · CPC title