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Epoxide hydrolase mutant, engineering bacteria and application of epoxide hydrolase mutant

An epoxide and hydrolase technology, which is applied in the field of bioengineering technology, can solve the problems of limited application potential and low enantioselectivity, and achieves good industrial application prospects, high catalytic activity, and improved enantioselectivity. Effect

Active Publication Date: 2015-07-01
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the enantioselectivity of the recombinant enzyme to the substrate is not high, which limits its application potential in the chiral synthesis of high value-added prodrugs

Method used

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  • Epoxide hydrolase mutant, engineering bacteria and application of epoxide hydrolase mutant
  • Epoxide hydrolase mutant, engineering bacteria and application of epoxide hydrolase mutant
  • Epoxide hydrolase mutant, engineering bacteria and application of epoxide hydrolase mutant

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Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1: the preparation of epoxide hydrolase mutant

[0032] Site-directed saturation mutagenesis and site-directed mutagenesis refer to the protocol described in (Current Protocols in Protein Science 2011, 26.6.1-26.6.10; Anal. Biochem. 2008, 375: 376-378). First, design the mutation primers containing the mutation points, as shown in Table 1. The epoxide hydrolase (GenBank no.JX467176) derived from Agromyces mediolanus CCTCC M 2012299 consists of 288 amino acid residues (the amino acid sequence is shown in SEQ ID NO.13), and the expression vector pET28-AmEH has been successfully constructed. The plasmid pET28a -AmEH was used as template for whole plasmid amplification. The PCR system is: 5×PS Buffer 10 μl, dNTP (2.5mM each) 4 μl, mutant primers F and R 0.5 μl each, plasmid pET28-AmEH 0.5 μl, PrimeSTAR DNA polymerase 0.5 μl, and water to 50 μl. PCR conditions were pre-denaturation at 98°C for 2 minutes, 27 cycles: 98°C for 10s, 65°C for 10s, 72°C for 7min, and ...

Embodiment 2

[0035] Example 2: Induced Expression and Purification of Epoxide Hydrolase Parents and Mutants

[0036] 1. Expression of Epoxide Hydrolase Parents and Mutants

[0037] The starting strain E.coli BL21(DE3) / pET28-AmEH of Example 1 and the mutant strain in Example 1 were respectively inoculated into LB liquid medium containing 50mg / L kanamycin, cultivated at 37°C for 12h, and then Inoculate into fresh LB liquid culture medium containing 50mg / L kanamycin with 1% inoculum size (v / v), cultivate to bacterium concentration OD 600 About 0.6, then add IPTG with a final concentration of 0.1mM to the LB liquid medium, induce culture at 28°C for 10h, then centrifuge at 8000g for 10min at 4°C, and collect the bacterial cells containing recombinant epoxide hydrolase, which can be used for enzyme production Purification and biocatalytic preparation of (S)-ECH.

[0038] 2. Purification of Epoxide Hydrolase Parents and Mutants

[0039] After the epoxide hydrolase bacterial cells were ultraso...

Embodiment 3

[0045] Activity, catalytic constant and stereoselective determination of embodiment 3 epoxide hydrolase mutants

[0046] The substrate and product in the reaction mixture are separated by gas chromatography, the decrease of substrate concentration in the reaction system before and after the reaction is measured, and the catalytic activity of the epoxide hydrolase is determined.

[0047] Gas chromatography GC-14C column type: BGB-175 capillary column; chromatographic conditions: column temperature 90°C, injection chamber temperature 220°C, FID detector 220°C, helium flow rate 1.6mL / min; split ratio 40: 1.

[0048] Enzyme activity unit (U) is defined as: the amount of enzyme required to catalyze 1 μmol epichlorohydrin within 1 min at 30°C and pH 8.0 is defined as 1U. Specific enzyme activity is defined as the number of units of activity per milligram of protein.

[0049] Epoxyhydrolase activity assay method is: total reaction system is 0.4ml, wherein contains 50mM substrate, p...

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Abstract

The invention discloses an epoxide hydrolase mutant, an engineering bacteria and application of the epoxide hydrolase mutant. The epoxide hydrolase mutant is obtained by carrying out single-point mutation or multi-point mutation on amino acid in the 182rd position, the 207th position and / or the 240th position of an amino acid sequence represented by SEQ ID NO.13. Compared with the wild type epoxide hydrolase, the epoxide hydrolase mutant provided by the invention has higher catalytic activity and stereoselectivity; and the epoxide hydrolase mutant is particularly applied to preparing (S)-epoxy chloropropane by splitting racemic epichlorohydrin.

Description

(1) Technical field [0001] The present invention relates to the technical field of bioengineering, in particular to an epoxide hydrolase mutant and its coding gene, a recombinant expression vector containing the epoxide hydrolase mutant gene and a recombinant engineering bacterium, its recombinase and the recombinant The preparation method of the enzyme, and the application of the epoxide hydrolase in enantioselective hydrolysis and resolution of racemic epichlorohydrin to prepare highly optically active (S)-epoxychlorohydrin. (2) Background technology [0002] Epoxide hydrolases (EC 3.3.2.3) are a group of functionally similar enzymes that stereoselectively catalyze the formation of optically active epoxides and corresponding vicinal diols from epoxides. As a hydrolytic enzyme, it has the characteristics of general hydrolytic enzymes such as: 1. The reaction does not need to add cofactors. 2. Has a wide range of sources. 3. It can still retain certain activity in non-aque...

Claims

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Application Information

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IPC IPC(8): C12N9/14C12N15/55C12N15/70C12N1/21C12P17/02
Inventor 柳志强郑裕国薛锋朱杭芹王亚军沈寅初
Owner ZHEJIANG UNIV OF TECH
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