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Application of glycosyl transferase mutant in directional synthesis of non-natural ginsenoside

A technology of glycosyltransferase and ginsenoside, which is applied in the application field of glycosyltransferase BcGT1 mutant in the enzymatic preparation of unnatural ginsenoside, which can solve the problems of low substrate concentration, poor reaction selectivity and expression of glycosyltransferase To achieve the effects of high selectivity, increased yield, and simple preparation method

Active Publication Date: 2022-08-02
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Aiming at problems such as poor expression of glycosyltransferases, low substrate concentration, and poor reaction selectivity, the present invention provides the application of glycosyltransferase mutants in the directional synthesis of unnatural ginsenosides. Good selectivity, good stability and high expression in the preparation of non-natural ginsenoside system

Method used

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  • Application of glycosyl transferase mutant in directional synthesis of non-natural ginsenoside
  • Application of glycosyl transferase mutant in directional synthesis of non-natural ginsenoside
  • Application of glycosyl transferase mutant in directional synthesis of non-natural ginsenoside

Examples

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Embodiment 1

[0030] Example 1: This example illustrates the cloning of the glycosyltransferase BcGT1

[0031] Microbial strain genome extraction: take the standard strain: Bacillus cereus ATCC14579 as the starting strain, and extract the target genome with reference to the extraction method of the microbial genome extraction kit (TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0). Store at -20°C for short-term use.

[0032] The cloning primers were designed with reference to the target glycosyltransferase gene in the genome information of Bacillus cereus NC7401 in the NCBI database as a template. Using the extracted Bacillus cereus genome as a template, the target glycosyltransferase gene fragment BcGT1 was obtained by PCR amplification with the corresponding cloning primers. After the target glycosyltransferase gene fragment BcGT1 obtained by PCR amplification was verified by agarose gel electrophoresis, the target gene fragment was purified and recovered according to the AxyPr...

Embodiment 2

[0033] Example 2: This example illustrates the construction of a glycosyltransferase gene expression vector

[0034] In order to achieve the high-efficiency expression of glycosyltransferase, in the construction of the gene expression vector, the SUMO solubilization tag and the intein NHT intein NHT gene sequence are designed to be added as SEQ ID NO: 3, and the SUMO gene sequence is SEQ ID NO: 4 .

[0035] SUMO can be used as a fusion tag and molecular chaperone for recombinant protein expression, which not only can further improve the expression of fusion protein, but also has the functions of resisting protease hydrolysis, promoting the correct folding of target protein, and improving the solubility of recombinant protein. Intein NHT exists in the same open reading frame (ORF) as the host protein gene, and is transcribed and translated synchronously with the host protein gene. After translation to form a protein precursor, the intein NHT self-splicing and fusion Protein, t...

Embodiment 3

[0045] Example 3: This example illustrates site-directed mutagenesis of the glycosyltransferase BcGT1

[0046] The three-dimensional structural model of the glycosyltransferase BcGT1 was established with the swiss-model online server, and hotspotwizard online software was used to scan and predict the hot spots. The predicted amino acid residues around the active center were mutated to alanine, and the glycosyltransferase BcGT1 was mutated to alanine. The amino acid sites are P18A, L59A, S60A, I64A, H80A, E86A, N108A, F133A, E142A, N181A, T325A, G326A, respectively. The mutants of glycosyltransferase BcGT1 were respectively induced and expressed, and the mutants were prepared. The wild-type glycosyltransferase BcGT1 or its mutants were used to catalyze the synthesis of unnatural ginsenosides, the products were detected by high performance liquid chromatography, and the conversion rate and product selectivity of the mutant catalyzed substrates were calculated. Two mutants P18A ...

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Abstract

The invention relates to an application of a glycosyl transferase mutant in directional synthesis of non-natural ginsenoside. The mutant is obtained by mutating one or more of 18th and 133rd amino acid residues in an amino acid sequence of glycosyl transferase BcGT1 into another amino acid residue; the amino acid sequence of the glycosyl transferase is as shown in SEQ ID NO: 2. According to the glycosyl transferase mutant and sucrose synthase AtSuSy double-enzyme coupled catalytic system constructed by the invention, non-natural ginsenoside 12-O-beta-Glc-PPT or 12-O-beta-Glc-PPD can be directly and directionally synthesized by a one-step method, and the preparation process of the non-natural ginsenoside is simplified; and the product is relatively single, so that the subsequent separation step is reduced, and the purification process of the product is simplified. The non-natural ginsenoside has a stronger lung cancer cell proliferation inhibition effect than natural ginsenoside Rg3, Rh2 and the like, so that the non-natural ginsenoside has a great prospect in the development of new drugs.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals of traditional Chinese medicine, and particularly relates to the application of a BcGT1 mutant of a Bacillus cereus-derived glycosyltransferase in the enzymatic preparation of unnatural ginsenosides. Background technique [0002] Ginseng (Panax ginseng C.A. Mayer) is a perennial herb of the Araliaceae family and is a traditional and precious traditional Chinese medicine in my country. Traditional Chinese medicine ginseng has a wide range of pharmacological activities such as anti-cancer, anti-fatigue, improving immunity, and protecting cardiovascular and cerebrovascular. The main medicinal ingredient contained in traditional Chinese medicine ginseng, ginsenoside, is a class of tetracyclic triterpenoids. So far, 180 ginsenosides have been identified from 17 species of ginseng, most of which are tetracyclic dammarane-type structures. Dammarane-type ginsenosides can be further divided in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12P33/20
CPCC12N9/1051C12P33/20Y02P20/55
Inventor 何冰芳徐晓丽储建林赵璐钦松高振吴斌
Owner NANJING TECH UNIV
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