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Applications of glycosyltransferase and mutants thereof to synthesis of ginsenoside Rh2

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

Active Publication Date: 2015-02-18
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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  • Applications of glycosyltransferase and mutants thereof to synthesis of ginsenoside Rh2
  • Applications of glycosyltransferase and mutants thereof to synthesis of ginsenoside Rh2
  • Applications of glycosyltransferase and mutants thereof to 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 database resources such as PIR and NCBI, and 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 derived from Saccharomyces cerevisiae, OleD derived from Streptomyces antibiotics and SGT2 derived from potato. Among them, UGT51 derived from Saccharomyces cerevisiae was obtained by cloning, OleD derived from Streptomyces antibiotics and SGT2 derived from potato were obtained by whole 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 extr...

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, the reaction was carried out for 48 hours. The reaction was terminated by adding the same volume of n-butanol. The upper organic phase was dried by rotary evaporation, purified by silica gel column, the eluent was chloroform:methanol=85:15, and each 5mL aliquot was collected, and the collected samples were analyzed by TLC (conditions were the same as above) to obtain protopanaxadiol Glycosylation product fraction, purity <90%.

[0063] The collected fractions were further purified using a Sep-Pak tC18 column (Waters) with water (A) and acetonitrile (B) as eluents, using a gradient elution (20%B, 40%B, 50%B, 60%B) B, 65% B, 70% B, 75% B, 80% B, 85% B, 90% B, 100% B), at 70% and 75% acetonitrile elution, to obtain protopanaxadiol glycosylation Product fraction, the purit...

Embodiment 3

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

[0065] Option One

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

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

[0068] The same volume of n-butanol was added to terminate the reaction. The extracted organic phase was vacuum-dried and dissolved in methanol. The HPLC analysis and quantification were c...

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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 a mutant thereof, in particular to the application of a glycosyltransferase and a mutant thereof in the synthesis of rare ginsenoside Rh2 by transglycosylation. Background technique [0002] As the main medicinal active substance of ginseng, ginsenoside is a triterpenoid saponin, which belongs to the sterol compound. It has vasodilatory, antioxidant, anti-inflammatory and anti-tumor effects. The structures of ginsenosides differ greatly in properties and functions due to different glycosyl side chains. Among them, Rh2 and Rg3 are the most studied 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 pharm...

Claims

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Application Information

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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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