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Epoxide hydrolase mutant and application thereof

A technology of epoxides and mutants, applied in the direction of hydrolytic enzymes, enzymes, biochemical equipment and methods, etc., can solve the problems of low substrate concentration, low activity, low yield, etc., and achieve improved E value and half-life , Stereoselectivity and stability improvement

Inactive Publication Date: 2016-07-06
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some progress has been made in the production of chiral epichlorohydrin by epoxide hydrolase catalysis, the reported epoxide hydrolase has generally low activity, low substrate concentration and low yield, which is far from practical application. there is still a distance

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

[0027] The gene synthesis of embodiment 1 parental epoxide hydrolase

[0028] In order to express the protein with His-tag after the gene of the parental epoxide hydrolase (GenBank: Y12804.1) is connected to the vector pET-28b, its stop codon was excised, and the codon preference of E.coli The sequence was optimized for reference, and NcoI was introduced at the start codon, thereby inserting glycine after methionine. The sequence of the newly designed epoxide hydrolase gene is shown in SEQ ID NO.1 (the amino acid sequence is shown in SEQ ID NO.2). The gene synthesis work was entrusted to Shanghai Xuguan Biotechnology Development Co., Ltd., and the gene synthesis was connected to the expression vector pET -28b.

Embodiment 2

[0029] Construction of embodiment 2 mutant library

[0030] Using the parental sequence of the nucleotide sequence shown in SEQ ID NO.1 obtained in Example 1 as a template, PCR amplification was performed with the following three pairs of nucleotide sequences as primers respectively, and the 108-position isoleucine was mutated into leucine respectively. Acid single mutant IIe108-Leu (nucleotide sequence is shown in SEQIDNO.3, amino acid sequence is shown in SEQIDNO.4), 247th threonine is mutated to lysine single mutant Thr247-Lys (nucleoside The acid sequence is shown in SEQ ID NO.5, the amino acid sequence is shown in SEQ ID NO.6) and the single mutant Asp131-Ser (the nucleotide sequence is shown in SEQ ID NO.7, and the amino acid sequence is shown in SEQ ID NO.8).

[0031] Seven pairs of oligonucleotide primers are as follows (5'-3'):

[0032] Forward primer (108IIe): GTAGGCCATGATNNKGCAGCGATCGTCC

[0033] Reverse primer (108IIe): GGACGATCGCTGCNNKATCATGGCCTAC

[0034] For...

Embodiment 3

[0045] Example 3: Screening of Mutant Library

[0046] (1) Primary screening of mutant library

[0047] Pick a single colony cultured in Example 2 and place it in a 96-well plate containing 1mL of LB culture medium, add Kan (final concentration 50μg / mL), and culture at 37°C until OD 600 When it reaches 0.6-0.8, add IPTG (final concentration 0.1mM) and induce at 28°C for 12h. Centrifuge at 9000rpm for 10min, remove the supernatant, and obtain the cells. Pick wet bacteria and resuspend with 1mL (pH8.5, 0.1M) phosphate buffer to prepare bacteria suspension. The reaction was carried out in Ep tube, in 1mL system: 900μl (pH8.5, 0.1M) Tris-HCl buffer solution, 100μL of the above bacterial suspension, final concentration of 100mM epichlorohydrin, temperature 35℃, rotation speed 1000rpm, reaction After 5 minutes, 10 μl of 6M HCl was used to terminate the reaction, and 200 μl of the reaction solution was extracted in 800 μl of ethyl acetate, centrifuged at 8000 rpm for 5 minutes, an...

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Abstract

The invention discloses an epoxide hydrolase mutant and its application. The mutant is the 108th isoleucine or the 131st aspartic acid or the 247th aspartic acid in the amino acid sequence shown in SEQ ID NO.2. Threonine is obtained by carrying out single mutation, double mutation or triple mutation; compared with the unmutated epoxide hydrolase mutant, the enzymatic activity, stereoselectivity and stability of the epoxide hydrolase mutant according to the present invention Significantly improved; Among them, the specific enzyme activity of the mutant was improved by 5.4 times compared with the original enzyme, reaching 315.2U / mg, the half-life was improved by 12.8 times, the enantioselective E value was improved by 2.1 times, and the epoxide hydrolase mutant was In the two-phase system, 800mM racemic epichlorohydrin can be catalytically resolved, and the yield of R-epichlorohydrin reaches more than 90% of the theoretical yield, and the enantioselectivity is greater than 99%.

Description

(1) Technical field [0001] The invention relates to an epoxide hydrolase, in particular to a mutant epoxide hydrolase and its application in the synthesis of R-epichlorohydrin. (2) Background technology [0002] Epoxide hydrolases (EC3.3.2.3) are an important class of industrial enzymes. Epoxide hydrolase can stereoselectively and asymmetrically hydrolyze racemic epoxides to prepare optically pure epoxides and vicinal diols, and can produce important pharmaceutical intermediates. The source of epoxide hydrolase is very extensive, and it is found in bacteria, filamentous fungi, yeast, plants, animals, etc. The catalytic process of epoxide hydrolase does not require the participation of prosthetic groups and coenzymes, and has the advantages of wide substrate spectrum, high catalytic efficiency, and high enantioselectivity. It is one of the most widely studied and applied biocatalysts. Most epoxide hydrolases are α / β-sheet enzymes, which contain two functional domains (core ...

Claims

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Application Information

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IPC IPC(8): C12N9/14C12P41/00C12P17/02
CPCC12N9/14C12P17/02C12P41/001C12Y303/02003
Inventor 郑裕国邹树平王志才吴群
Owner ZHEJIANG UNIV OF TECH
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