Enzymatic method for preparing Rebaudioside C

Patent metadata

Field	Value
Publication number	US-11312985-B2
Application number	US-201616343339-A
Country	US
Kind code	B2
Filing date	Oct 21, 2016
Priority date	Oct 21, 2016
Publication date	Apr 26, 2022
Grant date	Apr 26, 2022

Abstract

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

First claim

The invention claimed is:

1. A method for preparing Rebaudioside C, the method comprising: reacting rubusoside with a glycosyl donor in a reaction system in the presence of recombinant cells comprising UDP-glycosyltransferase and/or UDP-glycosyltransferase prepared from the recombinant cells, wherein:
the reaction system comprises toluene at a concentration by volume of 1-3%;
the UDP-glycosyltransferase has the amino acid sequence of SEQ ID NO: 2; and
wherein
the reaction system further comprises a UDP-glycosyltransferase having the amino acid sequence of SEQ ID NO: 4.

2. The method according to claim 1 , wherein the glycosyl donor comprises a glucosyl donor and a rhamnosyl donor, wherein the glucosyl donor is UDP-glucose or a UDP-glucose regeneration system comprising sucrose, sucrose synthase, and UDP, and the rhamnosyl donor is UDP-rhamnose.

3. The method according to claim 1 , wherein the UDP-glucosyltransferase comprises one or both of UGT-A from Stevia rebaudiana and UGT-B from Oryza sativa.

4. The method according to claim 1 , wherein the UDP-glucosyltransferase comprises UGT-A from Stevia rebaudiana and UGT-B from Oryza sativa , and wherein the UGT-B is added in a first step and the UGT-A is added in a second step.

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

6. The method according to claim 5 , wherein the reaction system comprises a phosphoric acid buffer solution.

7. The method according to claim 1 , wherein the recombinant cells are microorganism cells.

8. The method according to claim 7 , wherein the microorganism is Escherichia coli, Saccharomyces cerevisiae or Pichia pastoris.

Assignees

• Pepsico Inc

Inventors

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

Classifications

• C12P19/18

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

• 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

• C12N9/10

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

• C12Y204/01

  Hexosyltransferases (2.4.1) · CPC title

• A23L27/30 Primary

  Artificial sweetening agents · CPC title