ENZYMATIC METHOD FOR PREPARING REBAUDIOSIDE J

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.

Claims

1. A method for preparing Rebaudioside J using an enzymatic method, wherein in the method, rebaudioside A is used as a substrate; and in the presence of a glycosyl donor, Rebaudioside J is produced by means of a reaction under the catalysis of recombinant cells containing UDP-glycosyltransferase and/or UDP-glycosyltransferase prepared therefrom.

2. The method according to claim 1, wherein: the glycosyl donor is a rhamnosyl donor.

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

4. The method according to claim 1, wherein: the UDP-glycosyltransferase is a UGT-B from Oryza sativa.

5. The method according to claim 4, wherein: an amino acid sequence of UGT-B from Oryza sativa is at least 60% consistent with Sequence 2 as shown in the Sequence Listing.

6. The method according to claim 5, wherein: the amino acid sequence of UGT-B from Oryza sativa is at least 70% consistent with Sequence 2 as shown in the Sequence Listing.

7. The method according to claim 6, wherein: the amino acid sequence of UGT-B from Oryza sativa is at least 80% consistent with Sequence 2 as shown in the Sequence Listing.

8. The method according to claim 7, wherein: the amino acid sequence of UGT-B from Oryza sativa is at least 90% consistent with Sequence 2 as shown in the Sequence Listing.

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

10. The method according to claim 9, wherein: the reaction is carried out in a phosphate buffer solution.

11. The method according to claim 9, wherein: the reaction system contains recombinant cells of UDP-glycosyltransferase and a cell-permeating agent

12. The method according to claim 11, wherein: the cell-permeating agent is toluene; and the volume ratio concentration of toluene in the reaction system is 1-3%.

13. The method according to claim 9, wherein: all the raw materials used in the reaction are added into a reaction kettle to be uniformly mixed and then placed at a set temperature for reaction while stirring.

14. The method according to claim 1, wherein: the recombinant cell is a microbial cell.

15. The method according to claim 14, wherein: the microorganism is Escherichia coli, Saccharomyces cerevisiae, or Pichia pastoris.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0023] For the structural formulas of Rebaudioside A and Rebaudioside J, see Formulas I and II respectively.

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[0024] The main synthesis route of Rubadioside J as provided by the present invention is as follows:

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[0025] The UGT-B adopted in the present invention may exist in the form of lyophilized enzyme powder or in the recombinant cells.

[0026] The method for obtaining the UGT-B is as follows:

a recombinant Escherichia coli (or the other microorganisms) expression strain of UGT-B is obtained by utilizing molecular cloning technique and genetic engineering technique; then the recombinant Escherichia coli is fermented to obtain recombinant cells containing UGT-B, or to prepare and obtain lyophilized powder of UGT-B from the above noted recombinant cells.

[0027] Both the molecular cloning technique and the genetic engineering technique described in the present invention are already well-known. The molecular cloning technique may be found in Molecular Cloning: A Laboratory Manual (3rd Edition) (J. Sambrook, 2005).

[0028] The expression steps of the recombinant strain herein constructed by employing genetic engineering technique are as follows:

(1) (according to Sequence 1 and Sequence 2 as shown in the Sequence Listing) the required gene fragment is genetically synthesized, ligated into a pUC57 vector, while respectively adding NdeI and BamHI enzyme cutting sites at the two ends;
(2) each gene fragment is inserted into the corresponding enzyme cutting site of the expression vector pET30a through double digestion and ligation, so that each gene is placed under the control of T7 promoter;
(3) the recombinant plasmid is transformed into Escherichia coli BL21 (DE3); the expression of the target protein is induced by utilizing IPTG; and then the expression strains of the recombinant Escherichia coli of UGT-B is obtained.

[0029] The steps for preparing the recombinant cells containing UGT-B and the lyophilized powder of UGT-B by utilizing the expression strains of the recombinant Escherichia coli containing UGT-B are as follows:

the recombinant Escherichia coli expression strains containing UGT-B were inoculated into 4 ml of liquid LB medium according to a proportion of 1%; shake cultivation was carried out at 37 C. (at 200 rpm) overnight; the substance cultivated overnight was taken and inoculated into 50 ml of liquid medium according to a proportion of 1%; shake cultivation was carried out at 37 C. (at 200 rpm) overnight until the OD.sub.600 value reached 0.6-0.8; then IPTG with a final concentration of 0.4 mM was added in at 20 C. for shake cultivation overnight. After the induction is completed, the cells were collected through centrifugation (8,000 rpm, 10 min); then the cells were resuspended with 5 ml 2 mmol/L of phosphate buffer liquid (pH7.0), to obtain the recombinant cells; then the cells were ultrasonically disrupted in ice bath; the homogenate was centrifuged (8,000 rpm, 10 min); and the supernatant was collected and lyophilized for 24 h to obtain the lyophilized powder.

[0030] The present invention is further described in details in combination with specific examples.

Example 1: Preparation of the Recombinant Escherichia Coli Cells Containing UGT-B

[0031] According to Sequence 3 and Sequence 4, the UGT-B gene fragment was genetically synthesized, while respectively adding NdeI and BamHI enzyme cutting sites at the two ends, and ligated to the pUC57 vector (produced by Suzhou Jin Wei Zhi Biotech. Co., Ltd.). The UGT gene segment was enzyme cut with restriction endonucleases NdeI and BamHI; and then the segments were recovered and purified; a T4 ligase was added to ligate the segments into the corresponding enzyme cutting sites of pET30a, in order to transform the BL21 (DE3) strains.

[0032] The UGT strains were inoculated into 4 ml of liquid LB medium according to a proportion of 1%; shake cultivation was carried out at 37 C. (at 200 rpm) overnight; the substance cultivated overnight was taken and inoculated into 50 ml of liquid LB medium according to a proportion of 1%; shake cultivation was carried out at 37 C. (at 200 rpm) overnight until the OD.sub.600 value reached 0.6-0.8; then IPTG with a final concentration of 0.4 mM was added in at 20 C. for shake cultivation overnight. After the induction is completed, the cells were collected through centrifugation (8,000 rpm, 10 min); and the collected cells were resuspended with 5 ml 2 mol/L of phosphate buffer (pH 7.0) to obtain the recombinant cells containing UGT-B for catalysis.

Example 2 Preparation of the Lyophilized Powder of UGT-B

[0033] The recombinant cells containing UGT-B prepared in example 3 were ultrasonically disrupted in ice bath; the homogenate was centrifuged (8,000 rpm, 10 min); and the supernatant was collected and lyophilized for 24 h to obtain the lyophilized powder of UGT-B.

Example 3: Synthesis of Rebaudioside J Under the Catalysis of UDP-Glycosyltransferase with Rebaudioside A as the Substrate

[0034] In this example, lyophilized powder of UGT-B prepared according to the method of Example 2 was used for the catalysis and synthesis of Rebaudioside J.

[0035] 1 L 0.05 mol/L of phosphate buffer solution (pH8.0), 2 g of UDP Rhamnose, 1 g of Rebaudioside A, 10 g of lyophilized powder of UGT-B were sequentially added into the reaction system, and placed into a water bath at 40 C. after evenly mixing, for reaction for 24 h while stirring at 300 rpm. After the reaction is completed, 500 l of the reactant solution was added into anhydrous methanol of the equal volume for uniformly mixing; then it was centrifuged at 8,000 rpm for 10 min; and a high performance liquid chromatography was used to detect the supernatant filtration membrane (chromatographic conditions: column: Agilent eclipse sb-C18 4.6150 mm; detection wavelength: 210 nm; mobile phase: acetonitrile:deionized water=24%:76%; flow rate: 1.0 mL/min; column temperature: 30 C.). The conversion rate of Rebaudioside A was greater than 90%. After the supernatant was purified by post-processing such as isolating by silica resin and crystallizing, 0.52 g of Rebaudioside J was obtained, and the purity of which was greater than 95%.

Example 4: Synthesis of Rebaudioside J Under the Catalysis of Recombinant Cells of UDP-Glycosyltransferase with Rebaudioside A as the Substrate

[0036] In this example, recombinant cells containing UGT-B prepared according to the method of Example 1 was used for the catalysis and synthesis of Rebaudioside J.

[0037] 1 L 0.05 mol/L of phosphate buffer solution (pH8.0), 2 g of UDP Rhamnose, 1 g of Rebaudioside A, 20 ml of toluene, 40% of UGT-B whole cells were sequentially added into the reaction system, and placed into a water bath at 40 C. after uniformly mixing, to react for 24 h while stirring at 300 rpm. After the reaction is completed, 500 l of the reactant solution was taken, and the supernatant was added with anhydrous methanol of the equal volume for uniformly mixing; then it was centrifuged at 8,000 rpm for 10 min; and a high performance liquid chromatography was used to detect the supernatant filtration membrane (chromatographic conditions: column: Agilent eclipse sb-C18 4.6150 mm; detection wavelength: 210 nm; mobile phase: acetonitrile:deionized water=24%:76%; flow rate: 1.0 mL/min; column temperature: 30 C.). The conversion rate of Rebaudioside A was greater than 90%. After the supernatant was purified by post-processing such as isolating by silica resin and crystallizing, 0.49 g of Rebaudioside C was obtained, and the purity of which was greater than 95%.

[0038] The above-described examples are merely for the illustration of the technical concept and features of the present invention. The object of providing examples is only to allow those skilled in the art to understand the present invention and implement it accordingly; the scope of the present invention is not limited thereto. Any equivalent variations or modifications derived from the essence of the present invention shall fall within the protection scope of the present invention.