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Application of gynostemma pentaphyllum glycosyl transferase in synthesis of rare ginsenosides

A technology of glycosyltransferase and ginsenoside, which is applied in the application field of the gynostemma glycosyltransferase in the synthesis of rare ginsenoside CK, which can solve the problems of low yield, environmental pollution, and complicated ginsenoside process, and achieve high yield High efficiency, simple process and low consumption

Active Publication Date: 2017-05-24
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the growth cycle of ginseng is long, and the process of extracting ginsenosides is complicated, the yield is low, and it is easy to cause environmental pollution.

Method used

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  • Application of gynostemma pentaphyllum glycosyl transferase in synthesis of rare ginsenosides
  • Application of gynostemma pentaphyllum glycosyl transferase in synthesis of rare ginsenosides
  • Application of gynostemma pentaphyllum glycosyl transferase in synthesis of rare ginsenosides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Cloning of Glycosyltransferase UGTGp5 from Gynostemma pentaphyllum

[0049] The glycosyltransferase genes that may catalyze the glycosylation of protopanaxadiol were screened with the help of search tools based on compounds and chemical reactions, combined with NCBI and other database resources, and plant PSPG boxes. Using primers SEQ ID NO.1-SEQ ID NO.2, and using a polymerase to amplify the gene from the Gynostemma cDNA library by PCR. The amplified fragment was gel-cut and purified, and double-digested with XhoI and BamHI. The digested fragment was ligated with the plasmid pET-28a (+) that had also been double-digested with XhoI and BamHI. The carrier: the target fragment Mix according to the molar ratio of 1:3, add T4 DNALigase, enzyme ligate at 22°C for 5 hours, and transform the ligated product E. coli DH5α, and positive clones were screened on the Kanna plate and verified by sequencing. The recombinant plasmid pET-28a(+)-comp22398 was obtained.

Embodiment 2

[0051] Establishment of Escherichia coli expression strain and purification of Gynostemma glycosyltransferase UGTGp4

[0052] Transform recombinant expression plasmids into host bacteria E. coli BL21(DE3) (NEB Company) competent cells were used to obtain recombinant expression strains of Gynostemma glycosyltransferase. When the recombinant bacteria were cultured until the OD was 0.6-0.8, 0.1 mM IPTG was added, and the expression was induced at a low temperature of 16° C. for 20 h. The cells were collected by centrifugation at 5500 rpm at 4°C, and the cells were ultrasonically disrupted. It was purified using Ni-NTA His-binding resin.

Embodiment 3

[0054] in vitro enzyme experiment

[0055] According to the preparation of the in vitro reaction system, the experiment of glycosyltransferase was carried out.

[0056]

[0057] After incubating the reaction mixture for 3 h at 30° C., the same volume of n-butanol was added to terminate the reaction.

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Abstract

The invention discloses an application of gynostemma pentaphyllum glycosyl transferase in synthesis of rare ginsenoside. The gynostemma pentaphyllum glycosyl transferase can be used for catalyzing protopanaxadiol to synthesize the rare ginsenoside CK. The name of the gynostemma pentaphyllum glycosyl transferase is UGTGp5. The invention also discloses a method for synthesizing the rare ginsenoside CK and an application. The application has the advantages that a gynostemma pentaphyllum glycosyl transferase gene is subject to heterologous expression in escherichia coli, and the function of enzymatic reaction in vitro is identified; after a product is subject to ESI (electro-spray ionization) mass spectrometry and HPLC (high performance liquid chromatography) detection, a higher signal is displayed. The prepared ginsenoside has the advantages that the yield rate is high, the number of byproducts is fewer, the industrialized production is easy, and the foundation is laid for the heterologous biosynthesis of saponin of gynostemma pentaphyllum.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and relates to a method for synthesizing rare ginsenosides in vitro by expressing gypenosyltransferase heterologously in Escherichia coli, and also relates to the application of the gypenosyltransferase in synthesizing rare ginsenoside CK. Background technique [0002] Gynostemma Gynostemma is a perennial herbaceous vine of Cucurbitaceae Gynostemma Gynostemma. Gynostemma Gynostemma contains a variety of medicinal ingredients, the most important of which is Gypenoside. The structure of Gypenoside is a tetracyclic triterpene dammarane type, which is the same as that of ginsenoside. Gypenoside is composed of two parts, which is a biomacromolecule formed by the combination of glycoside and glycoside. So far, it has been found that eight kinds of gypenosides are identical in structure to protopanaxadiol-type ginsenosides, and the total amount of these eight kinds of saponins reaches about ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N9/10C12P33/20C12P33/02C12R1/19
CPCC12N9/1048C12P33/02C12P33/20
Inventor 王磊刘斌许莹莹徐艳丽田鑫黄笛
Owner NANKAI UNIV
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