Epoxide hydrolase mutant and application thereof

A technology of epoxide and hydrolase, which is applied in the field of genetic engineering and enzyme engineering, can solve the problems of low catalytic activity, poor thermal stability, and sudden drop in enzyme activity, so as to improve the activity and thermal stability of the enzyme, improve the activity and The effect of service life

Active Publication Date: 2020-06-12
ZHEJIANG UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Abstract
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Problems solved by technology

However, the thermal stability of the reported epoxide hydrolase is poor, and when the temperature exceeds 50°C, the enzyme activity begins to drop suddenly (JMicrobiol Biotechnol 2010, 20(4):659-665)
In industrial production, the temperature of enzyme conversion will reach 55-60°C, which greatly limits the service life of biocatalysts
In addition, the catalytic activity of the enzyme is low, and the maximum reaction rate is 94.34 μM / min / mg (J Microbiol Biotechnol 2010, 20(4):659-665), while the epoxide hydrolase producing L(+)-tartrate The maximum reaction rate (2.24 mM / min / mg) is 23 times that of the enzyme (Appl MicrobiolBiotechnol, 2007, 74:99-106), which greatly limits the efficiency of biocatalysts

Method used

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  • Epoxide hydrolase mutant and application thereof
  • Epoxide hydrolase mutant and application thereof
  • Epoxide hydrolase mutant and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1: the synthesis of epoxide hydrolase gene and the construction of genetically engineered bacteria

[0031] According to the coding gene of Bacillus alcaligenes epoxide hydrolase (GenBank accession number E50984), Sangon Bioengineering (Shanghai) Co., Ltd. was commissioned to synthesize the gene sequence, its nucleotide sequence is shown in SEQ ID No.8, and Add 6 histidine tags to the N-terminus of the protein, use Nde I and BamHI restriction endonuclease sites, insert it into pET22b vector, and transform Escherichia coli BL21 (DE3). Then, a genetically engineered bacterium containing Alcaligenes epoxide hydrolase was obtained from Sangon Bioengineering (Shanghai) Co., Ltd.

Embodiment 2

[0032] Example 2: Error-prone PCR (error-prone PCR) method to construct a random mutation library of epoxide hydrolase

[0033] (1) The above-mentioned epoxide hydrolase gene was randomly mutated using the GeneMorph II Random Mutagenesis kit (purchased from Agilent, Code No. 200550). The primers used are T7 and T7t, the nucleotide sequences of which are shown in SEQ ID No.9 and SEQ ID No.10. The reaction conditions were: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, a total of 25 cycles. After PCR, run 1% agarose gel electrophoresis and use a gel recovery kit (purchased from Sangon, CodeNo.B518131) to recover gene fragments.

[0034] (2) Digest the recovered fragment with Nde I and BamH I double enzymes, and perform ligation reaction with the pET 22b(+) vector (ampicillin resistance) that has undergone the same enzyme digestion. The reaction conditions are: vector and fragment according to 1: M...

Embodiment 3

[0039] Embodiment 3: Preparation of double mutants

[0040] Extract the plasmid of the single mutant L8I strain in Example 2, transform Escherichia coli DH5α competent cells, then extract the plasmid as a template in this bacterial strain, design site-directed mutagenesis primers W26F (+) and W26F (-), its nucleotide The sequences are shown in SEQ ID No.11 and SEQ ID No.12. Fasta-II rapid site-directed mutagenesis kit (Beijing Saibaisheng Gene Technology Co., Ltd.) was used for site-directed mutagenesis to obtain double mutants (L8I / W26F), and passed Sequencing verification, its nucleotide sequence is as shown in SEQ ID No.7.

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Abstract

An epoxide hydrolase mutant and application thereof belong to the technical field of gene engineering and enzyme engineering. The epoxide hydrolase mutant is obtained by carrying out single-site mutation or multi-site mutation on the 8th site and / or the 26th site of an amino acid sequence of wild-type epoxide hydrolase shown as SEQ ID No.4, wherein the single-site mutation is to mutate leucine atthe 8th site into isoleucine or mutate tryptophan at the 26th site into phenylalanine. According to the epoxide hydrolase mutant obtained by the invention, the activity and the thermal stability of the enzyme are greatly improved. Some genetically engineered bacteria expressing the mutant epoxide hydrolase are obtained through a genetic engineering technology, and the genetically engineered bacteria are used for producing D (-)-tartaric acid, so that the activity of cells is greatly improved, the service life of the cells is greatly prolonged, and the requirements of current industrial application are met.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and enzyme engineering, and specifically relates to an epoxide hydrolase mutant and application thereof. Background technique [0002] Epoxide hydrolase (EC 3.3.2.3) is a general term for a class of enzymes that catalyze the hydrolysis of epoxides or their salts to corresponding adjacent diols or their salts without the participation of any coenzymes, prosthetic groups or metal ions. Epoxide hydrolases are widely distributed in mammals, plants and microorganisms. Microbial-derived epoxide hydrolases have good stereospecificity, fast enzymatic reaction, high optical purity and yield of products, and simple separation and purification of products. Its characteristics have attracted people's attention. [0003] D(-)-tartaric acid [(2S,3S)-2,3-dihydroxybutane-1,4-dicarboxylic acid] is an isomer of natural L(+)-tartaric acid, which rarely occurs in nature , mainly used as a chiral source...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/14C12N15/55C12N15/70C12N1/21C12P7/46C12R1/19
CPCC12N9/14C12N15/70C12P7/46C12Y303/02003
Inventor 鲍文娜刘士旺陈怡廖鸿秀陈航
Owner ZHEJIANG UNIVERSITY OF SCIENCE AND TECHNOLOGY
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