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Halohydrin dehalogenase mutant and application thereof in synthesis of chiral epichlorohydrin

A technology of halohydrin dehalogenase and mutants, which is applied in the application field of preparing epichlorohydrin, can solve the problems such as low e.e. value of halohydrin dehalogenase catalysis, product racemization, etc., and achieve industrial production, Enzyme activity improvement, the effect of enzyme activity improvement

Active Publication Date: 2020-09-11
ZHEJIANG UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The problem to be solved by the present invention is to overcome the problems of low e.e. value and product racemization of halohydrin dehalogenase catalysis in the existing method, and provide a halohydrin dehalogenase mutant derived from radioactive Agrobacterium and the Application of Mutants in Preparation of High Yield and e.e. Values ​​of (S)-ECH in Biphasic Buffer Solutions

Method used

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  • Halohydrin dehalogenase mutant and application thereof in synthesis of chiral epichlorohydrin
  • Halohydrin dehalogenase mutant and application thereof in synthesis of chiral epichlorohydrin
  • Halohydrin dehalogenase mutant and application thereof in synthesis of chiral epichlorohydrin

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1: Homology Modeling and Molecular Dynamics Simulation of Halohydrin Dehalogenases

[0030] Using the three-dimensional structure of HheC (PDB ID: 1ZO8) derived from Agrobacterium radiobacterstrain AD1 in GenBank AAK92099 as a template, the three-dimensional structure of 1,3-dcp was drawn using ChemDraw 8.1, and the molecular docking program was aligned according to the AutoDock 4.6.2 program. Docking of HheC and substrate 1,3-dcp. All the above structures were visually analyzed using the PyMOL program, and it was found that the 105th, 134th, 136th, 137th, 178th, and 186th positions are the key sites that affect the enzyme activity ( figure 1 ).

Embodiment 2

[0031] Example 2: Construction of Halohydrin Dehalogenase Site-Directed Saturation Library

[0032]According to the conclusion of Example 1, design based on the gene sequence (nucleotide sequence shown in SEQ ID NO.1, amino acid sequence shown in SEQ ID NO.2) of the wild-type halohydrin dehalogenase HheC included in GenBank Primers (see Table 1). Respectively with primers L105X-F / L105X-R, A134X-F / A134X-R, P136X-F / P136X-R, F137X-F / F137X-R, Y178X-F / Y178X-R, T186X-F / T186X-R The parental HheC gene (nucleotide sequence is SEQ ID NO.1) was subjected to site-directed saturation mutation, and pET-28b(+) was used as the expression vector to obtain mutant plasmids with the target gene, and the target gene The mutant plasmid was transformed into E.coli BL21(DE3), and the mutants of recombinant bacteria containing the halohydrin dehalogenase mutant gene were respectively obtained, which were respectively E.coli BL21(DE3)-L105X (referred to as mutant L105X), E. .coli BL21(DE3)-A134X (ref...

Embodiment 3

[0047] Example 3: Screening of Halohydrin Dehalogenase Mutant Library

[0048] 1. Screening of the halohydrin dehalogenase mutant library Taking wild-type HheC before mutation as a reference, pick a single colony clone (the mutant library constructed in Example 2) and culture it in a 2mL deep 96-well plate. 600 μL of LB culture solution with a concentration of 50 μg / mL kanamycin, and at the same time pick two parental strains in the last two wells of a 96-well plate as controls. Place a 2 mL 96-well plate at 37°C for 8 hours as a seed solution, then add 200 μL of the seed solution to a new sterile 600 μL LB culture solution containing a final concentration of 50 μg / mL kanamycin and 0.1 mM IPTG , after induction of expression at 28°C containing the seed solution for 12 hours, centrifuge at 4000 rpm for 20 minutes, discard the supernatant, collect the wet cells, and carry out the next step of high-throughput screening.

[0049] 2. According to the characteristics of the catalyt...

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Abstract

The invention discloses a halohydrin dehalogenase mutant and application of the halohydrin dehalogenase mutant in synthesis of chiral epichlorohydrin. The mutant is characterized in that the mutant isobtained by carrying out single mutation or multiple mutation on the 105th site, the 134th site, the 136th site, the 137th site, the 178th site or the 186th site of an amino acid sequence shown in SEQ ID NO.2. The e.e. Value of the product (S)-epichlorohydrin of the halohydrin dehalogenase mutant exceeds 99% by utilizing a water-organic double-phase buffer solution system (the ratio of ethyl acetate to a sodium phosphate buffer solution is 6: 4), and the enzyme activity of wet cells is respectively improved to 100-600U / mL from 23.56 U / mL; the yield reaches 15.9 percent to 80.2 percent.

Description

[0001] (1) Technical field [0002] The invention relates to a halohydrin dehalogenase mutant and its application in preparing (S)-epichlorohydrin. [0003] (2) Background technology [0004] As important intermediates in pharmaceutical and chemical industries, halohydrin compounds are widely used in organic synthesis and pharmaceutical research and development. However, most halides in nature have disadvantages such as poor degradation ability, high toxicity and potential carcinogenicity. It has become one of the main environmental pollutants, so people pay more and more attention to its degradation treatment in order to reduce the pollution of this kind of organic halides to the natural environment and the potential threat to human beings. The use of traditional physical and chemical methods not only has harsh reaction conditions, but also may cause secondary pollution, while the use of biodegradation methods has environmental pollution-free, mild reaction conditions; normal ...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N1/21C12P17/02C12R1/19
CPCC12N9/88C12P17/02C12Y405/01Y02P20/584
Inventor 汤晓玲万欣雨郑仁朝郑裕国
Owner ZHEJIANG UNIV OF TECH
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