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Application of a kind of glycosyltransferase and its mutant in the synthesis of ginsenoside rh2

A technology of glycosyltransferase and ginsenoside, which is applied in the field of biopharmaceuticals to achieve the effects of simplified production process, low consumption and high output

Active Publication Date: 2017-11-10
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, both methods need to obtain the glycosyltransferase gene that can catalyze the synthesis of rare ginsenoside Rh2 from protopanaxadiol, and there is no report of this functional gene so far.

Method used

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  • Application of a kind of glycosyltransferase and its mutant in the synthesis of ginsenoside rh2
  • Application of a kind of glycosyltransferase and its mutant in the synthesis of ginsenoside rh2
  • Application of a kind of glycosyltransferase and its mutant in the synthesis of ginsenoside rh2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Screening of glycosyltransferases for the synthesis of ginsenoside Rh2 by transglycosylation of protopanaxadiol

[0048] With the help of Rxnfinder, a search tool based on compounds and chemical reactions, combined with PIR, NCBI and other database resources, based on the principles of substrate similarity and catalytic reaction type, some glycosyltransferase genes that may catalyze the glycosylation of protopanaxadiol were screened. They are UGT51 from Saccharomyces cerevisiae, OleD from Streptomyces resistant and SGT2 from potato. Among them, the UGT51 clone derived from Saccharomyces cerevisiae was obtained, the OleD derived from Streptomyces resistant bacteria and the SGT2 derived from potato were obtained by total gene synthesis. The full-length ORFs of OleD and SGT2 genes were cloned into pET28a(+) plasmid with NdeI and XhoI restriction sites.

[0049] The genome of Saccharomyces cerevisiae S288c was extracted using a fungal genome extraction kit, and P...

Embodiment 2

[0061] Example 2 Separation, purification and structural identification of glycosylation products

[0062]Prepare 100 mL of reaction solution according to the ratio in Example 1. Under the condition of 30°C, react for 48 hours. Add the same volume of n-butanol to terminate the reaction. Get the upper organic phase to dry by rotary evaporation, purify with silica gel column, eluent is chloroform:methanol=85:15, every 5mL aliquots are collected, the collected sample is carried out TLC analysis (condition is as described above), obtains protopanaxadiol Glycosylation product fraction, purity <90%.

[0063] Further utilize Sep-Pak tC18 column (Waters) to carry out purification to above-mentioned collection part, water (A) and acetonitrile (B) are as eluent, adopt gradient elution (20%B, 40%B, 50%B, 60% B, 65% B, 70% B, 75% B, 80% B, 85% B, 90% B, 100% B), when eluted with 70% and 75% acetonitrile, protopanaxadiol glycosylation was obtained The product part has a purity of 98%. ...

Embodiment 3

[0064] Example 3 UGT glycosyltransferase wild-type in vitro enzymatic transformation to synthesize ginsenoside Rh2

[0065] Option One

[0066] According to Example 1, the crude enzyme solution of UGT51 glycosyltransferase was obtained.

[0067] Prepare the reaction solution with dimethyl sulfoxide (DMSO), protopanaxadiol, uridine diphosphate glucose (UDPG), Tris-HCl buffer, the proportion of organic solvent DMSO in the reaction solution is 10% (v / v), the original Panaxadiol is 0.5g / L, the molar concentration of Tris-HCl buffer is 50mmol / L, the pH is 7.5, and the ratio of UDPG to protopanaxadiol is 10:1 (w / w). Add UGT51 glycosyltransferase crude enzyme solution to the reaction solution at a ratio of 70% (v / v), react at 30° C., 180 rpm, for 48 hours.

[0068] Add the same volume of n-butanol to terminate the reaction, add methanol to dissolve the extracted organic phase after vacuum drying, and carry out HPLC detection analysis and quantification, the results are as follows ...

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Abstract

The invention discloses glycosyltransferase which can catalyze protopanaxdiol to be glycosylated to synthesize rare ginsenoside Rh2. Multiple mutants with optimized catalytic efficiency are obtained through transformation of the glycosyltransferase. The yield of the rare ginsenoside Rh2 synthesized by the mutants with an in-vitro enzyme method is obviously higher than that of wild type ginsenoside Rh2. The glycosyltransferase and the mutants thereof can be applied to total synthesis of the rare ginsenoside Rh2 in microorganism bodies.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and relates to a glycosyltransferase and its mutant, in particular to the application of the glycosyltransferase and its mutant in transglycosylation to synthesize rare ginsenoside Rh2. Background technique [0002] Ginsenoside, as the main medicinal active substance of ginseng, is a triterpenoid saponin, which belongs to sterol compounds. It has vasodilation, anti-oxidation, anti-inflammatory and anti-tumor effects. The structure of ginsenosides has different properties and functions due to the different sugar side chains. Among them, Rh2 and Rg3 are the most researched and related to tumor cell apoptosis. They have high anti-tumor activity and have no toxic side effects on normal cells. The related products of these compounds have been widely used in clinic. Therefore, obtaining a large amount of high-purity ginsenosides has become a research hotspot in modern pharmacy. [0003] A...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/63C12P33/20
CPCC12N9/1048C12P33/02C12P33/20
Inventor 杨广宇冯雁庄宇
Owner SHANGHAI JIAO TONG UNIV
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