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A group of synthetic methods of natural and unnatural protopanaxatriol-type ginsenosides

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

Active Publication Date: 2022-07-22
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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  • A group of synthetic methods of natural and unnatural protopanaxatriol-type ginsenosides
  • A group of synthetic methods of natural and unnatural protopanaxatriol-type ginsenosides
  • A group of synthetic methods of natural and unnatural protopanaxatriol-type 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, with the genomic DNA of Bacillus subtilis 168 As the template, the Bs-YjiC-F and Bs-YjiC-R primer pairs were used to amplify the glycosyltransferase Bs-YjiC gene; the Arabidopsis cDNA was used as the template, and the At-SuSy-F and AtSuSy-R primer pairs were used to amplify the gene. Increase the sucrose synthase AtSuSy. The nucleotide sequences of the primers used are as follows (restriction enzyme sites are underlined):

[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-...

Embodiment 2

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

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

[0065] After the reaction was completed, an equal volume of methanol was added to terminate the reaction, and then centrifuged at 12,000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter membrane, added to a liquid bottle, and passed through high performance liquid chromatography and high performance liquid chromatography-mass spectrometry Analysis and identification of glycosylation products. The analytical conditions of high performance liquid chromatography were: mobile phase A was ...

Embodiment 3

[0068] Example 3. Coupling reaction of glycosyltransferase Bs-YjiC and sucrose synthase AtSuSy to catalyze the glycosylation of protopanaxatriol

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

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Abstract

The invention discloses a method for biocatalytically synthesizing natural and non-natural protopanaxatriol-type ginsenosides. The invention uses protopanaxatriol (PPT) as a substrate and glycosyltransferase as a catalyst, which can efficiently It catalyzes the glycosylation of the C3, C6 and C12 hydroxyl groups of protopanaxatriol to generate a variety of natural and non-natural protopanaxatriol-type ginsenosides. The invention also discloses a method for synthesizing various natural and non-natural protopanaxatriol-type ginsenosides by utilizing sucrose synthase and glycosyltransferase coupling reaction to catalyze protopanaxatriol. The method can use cheap sucrose and a small amount of ginsenosides. Using uridine diphosphate as a cofactor for the reaction can realize the recycling and regeneration of expensive uridine diphosphate glucose, thereby catalyzing protopanaxatriol to synthesize various natural and non-natural protopanaxatriol-type ginseng in a more efficient and inexpensive way. saponins.

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 for catalyzing protopanaxatriol by coupling reaction of glycosyltransferase and sucrose synthase. Methods and applications for the production of various natural and non-natural ginsenosides from protopanaxatriol (PPT). Background technique [0002] Ginseng (Panax ginseng C.A.Mayer) is a perennial perennial root plant of the Araliaceae family. It is a traditional and 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 known as the "king of all herbs". . [0003] Ginsenoside is the main active substance of ginseng, which is a glycoside compound composed of aglycone and sugar group. According to the different aglycones, it is mainly...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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