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Methods for synthesizing natural and non-natural protopanaxatriol ginsenosides

A technology of protopanaxatriol and ginsenoside, which is applied in the fields of biotechnology and botany, can solve the problems of poor stereoselectivity, many side reactions, and large pollution in chemical methods, and achieves the effect of enriching varieties

Active Publication Date: 2019-05-24
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Glycosylation can be achieved by chemical catalysis and biocatalysis, but the chemical method has limitations such as poor stereoselectivity, many side reactions, large pollution, and low product yield; while glycosyltransferase (UDP-glycosyltransferase, UGT, The glycosylation reaction catalyzed by EC 2.4.1.17) has the characteristics of high catalytic efficiency, strong regio- and stereoselectivity, and simple product purification, and has great application potential in the biosynthesis of new ginsenosides.

Method used

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  • Methods for synthesizing natural and non-natural protopanaxatriol ginsenosides
  • Methods for synthesizing natural and non-natural protopanaxatriol ginsenosides
  • Methods for synthesizing natural and non-natural protopanaxatriol ginsenosides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1. Cloning, expression and purification of glycosyltransferase Bs-YjiC and sucrose synthase AtSuSy

[0057] The gene accession numbers of glycosyltransferase Bs-YjiC and sucrose synthase AtSuSy in NCBI (https: / / www.ncbi.nlm.nih.gov / ) are NP_389104 and NM_001036838, respectively, and the genomic DNA of Bacillus subtilis 168 As template, use Bs-YjiC-F and Bs-YjiC-R primer pair to amplify glycosyltransferase Bs-YjiC gene; use Arabidopsis cDNA as template, use At-SuSy-F and AtSuSy-R primer pair to amplify Increased sucrose synthase AtSuSy. The nucleotide sequences of the primers used are as follows (the underlined part is the restriction enzyme site):

[0058] Bs-YjiC-F: 5′-CGCGGATCCATGAAAAAGTACCATATTTCGAT-3′ (BamHI restriction site)

[0059] Bs-YjiC-R: 5′-CGCGTCGACTTACTGCGGGACAGCGGATTTT-3′ (SalI restriction site)

[0060]AtSuSy-F: 5′-GCGTCGACAAATGGCAAACGCTGAACGTATGATAA-3′ (SalI restriction site)

[0061] AtSuSy-R: 5′-TTGCGGCCGCTTATCATACGTTCAGCGTTTGCCAT-3′(NotI ...

Embodiment 2

[0063] Example 2. Glycosyltransferase Bs-YjiC catalyzes glycosylation of protopanaxatriol

[0064] Separation and purification of glycosyltransferase Bs-YjiC according to the steps described in Example 1. The reaction system for glycosylation of protopanaxatriol catalyzed by glycosyltransferase Bs-YjiC includes: 2mM protopanaxatriol, 10mM uridine diphosphate glucose, 50mM Tris-HCl (pH 7.5), 10mM MgCl 2 , and 5mU / mL glycosyltransferase Bs-YjiC, reacted at 35°C for 0.5h.

[0065] After the reaction is completed, add an equal volume of methanol to terminate the reaction, then centrifuge at 12,000 rpm for 10 minutes, take the supernatant and filter it through a 0.22 μm filter membrane, add it to a liquid phase bottle, and pass through high performance liquid chromatography and high performance liquid chromatography-mass spectrometry Analysis and identification of glycosylation products. The analysis conditions of high-performance liquid chromatography are: mobile phase A is dist...

Embodiment 3

[0068] Example 3. Coupling reaction between glycosyltransferase Bs-YjiC and sucrose synthase AtSuSy catalyzes glycosylation of protopanaxatriol

[0069] The coupling reaction of glycosyltransferase Bs-YjiC and sucrose synthase AtSuSy to catalyze the glycosylation of protopanaxatriol is as follows: 3mM protopanaxatriol, 0.5mM uridine diphosphate, 50mM Tris-HCl (pH7.5) , 10 mM MgCl 2 , 10% dimethyl sulfoxide (v / v), 160mU / mL glycosyltransferase Bs-YjiC, 200mU / mL sucrose synthase AtSuSy, react at 35°C, and react for 1h at 200rpm. After the reaction, the reaction product was identified according to the reaction conditions in Example 2 and the detection method of high performance liquid chromatography. Such as Figure 5 It shows that, like the reaction system using expensive uridine diphosphate glucose as the glycosyl donor, the double-enzyme coupling reaction system composed of glycosyltransferase Bs-YjiC and sucrose synthase AtSuSy can use cheap sucrose as the glycosyl donor. D...

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Abstract

The invention discloses a method for synthesizing natural and non-natural protopanaxatriol ginsenosides through biological catalysis. With protopanaxatriol (PPT) being a substrate and glycosyl transferase being a catalyst, glycosylation of C3 potential, C6 potential and C12 potential hydroxyls of PPT can be efficiently catalyzed, and the multiple natural and non-natural PPT ginsenosides are generated. The invention further discloses a method for synthesizing natural and non-natural PPT ginsenosides by utilizing a coupled reaction of sucrose synthetase and glycosyl transferase to catalyze PPT.According to the method, with cheap saccharose and a little uridine diphosphate being reaction cofactors, cyclic regeneration of expensive uridine diphosphate glucose can be realized, and accordinglyby efficiently catalyzing PPT with lower costs, the natural and non-natural PPT ginsenosides are synthesized.

Description

technical field [0001] The invention belongs to the technical fields of biotechnology and botany, and relates to a group of natural and non-natural protopanaxatriol type ginsenosides and a preparation method thereof, in particular to a method of catalyzing protopanaxatriol-type ginsenosides by coupling reaction of glycosyltransferase and sucrose synthase. Alcohol (protopanaxatriol, PPT) to generate a variety of natural and non-natural ginsenoside methods and applications. Background technique [0002] Ginseng (Panax ginseng C.A.Mayer) is a perennial root plant of Araliaceae. It is a traditional precious Chinese herbal medicine in my country. It has various physiological effects such as anti-cancer, anti-tumor, anti-aging, and improving immunity. It is crowned as the "King of Herbs" . [0003] Ginsenoside is the main active substance of ginseng, which is a glycoside compound composed of aglycone and sugar group. According to different aglycones, it is mainly divided into pro...

Claims

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Application Information

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IPC IPC(8): C07J17/00C12N9/10C12P33/20
Inventor 孙媛霞戴隆海张学礼戴住波李娇
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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