Enzymatic Method for Preparing Rebaudioside M

Patent metadata

Field	Value
Publication number	US-2016298159-A1
Application number	US-201314911876-A
Country	US
Kind code	A1
Filing date	Sep 29, 2013
Priority date	Aug 14, 2013
Publication date	Oct 13, 2016
Grant date	—

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

1 . A method for preparing rebaudioside M by using an enzyme method, characterized in that, 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.

2 . The method according to claim 1 , characterized in that, the glucosyl donor is UDP-glucose, or a UDP-glucose regeneration system composed of sucrose, sucrose synthetase and UDP.

3 . The method according to claim 1 , characterized in that, the UDP-glucosyl transferase is UGT-A from Stevia rebaudkma and/or UGT-B from Oryza sativa.

4 . The method according to claim 3 , characterized in that, the amino acid sequence of UGT-A has at least 80% identity to sequence 2.

5 . The method according to claim 4 , characterized in that, the amino acid sequence of UGT-A has at least 90% identity to sequence 2.

6 . The method according to claim 3 , characterized in that, the amino acid sequence of UGT-B has at least 80% identity to sequence 4.

7 . The method according to claim 6 , characterized in that, the amino acid sequence of UGT-B has at least 90% identity to sequence 4.

8 . The method according to claim 1 , characterized in that, the reaction is carried out in an aqueous phase system at a temperature from 25° C. to 35° C. and a pH value from 6.5 to 7.5.

9 . The method according to claim 8 , characterized in that, the reaction is carried out in a phosphate buffer at pH 7.0.

10 . The method according to claim 8 , characterized in that, the catalysis is carried out by employing recombinant cells containing the UDP-glucosyl transferase, and the reaction system further contains toluene at a concentration from 1% to 3% according to the ratio by volume.

11 . The method according to claim 8 , characterized in that, the method is implemented as follows: all the raw materials employed in the reaction are added into a reaction kettle, mixed uniformly, then placed at a set temperature, and stirred for reaction.

12 . The method according to claim 1 , characterized in that, the recombinant cells are microbial cells.

13 . The method according to claim 12 , characterized in that, the microorganism is Escherichia coli, Saccharomyces cerevisiae or Pichia pastoris.

14 . The method according to any one of claims 1 to 13 , characterized in that, the substrate is rebaudioside A, the UDP-glucosyl transferase is a mixture of UGT-A from Stevia rebaudkma and UGT-B from Oryza sativa , wherein the amino acid sequence of UGT-A from Stevia rebaudkma has at least 80% identity to sequence 2, and the amino acid sequence of UGT-B from Oryza sativa has at least 80% identity to sequence 4.

15 . The method according to claim 14 , characterized in that, in the mixture, UGT-A from Stevia rebaudkma and UGT-B from Oryza sativa have a ratio by weight of 1:0.8 to 1.2.

16 . The method according to any one of claims 1 to 13 , characterized in that, the substrate is rebaudioside D, and the UDP-glucosyl transferase is UGT-A from Stevia rebaudkma , wherein the amino acid sequence of UGT-A from Stevia rebaudkma has at least 80% identity to sequence 2.

Assignees

• Pepsico Inc

Inventors

• Tao Junhua
• Li Guoqing
• Liang Xiaoliang
• Lee Thomas
• Yep Gregory
• Tao Andrew

Classifications

• C12N9/1051 Primary

  Hexosyltransferases (2.4.1) · CPC title

• A23L27/36

  Terpene glycosides · CPC title

• C12Y204/01017

  Glucuronosyltransferase (2.4.1.17) · CPC title

• A23V2002/00

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