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Phaseolus vulgaris epoxide hydrolase mutant capable of improving enantioselectivity

An epoxide and mutant technology, applied in the fields of genetic engineering and protein expression, which can solve the problems of low enantioselectivity and limited application potential, etc.

Active Publication Date: 2018-04-20
JIANGNAN UNIV
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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 epoxides is not high, which limits its application potential in the chiral synthesis of high value-added prodrugs

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1: Construction of mutant enzyme gene and its expression plasmid

[0019] 1. Acquisition of plasmid pET-28a(+)-pveh1

[0020] The medium composition of recombinant E.coli BL21(DE3) / pET-28a(+)-pveh1 is: peptone 1%, yeast extract 0.5%, NaCl 1%.

[0021] The recombinant bacteria were inoculated in a test tube with a medium filling volume of 5 mL and cultured at 37° C. and 215 rpm for 12 h with shaking. After the cultivation, the cells were centrifuged at 12,000 rpm for 1 min and the cells were collected, and the plasmid pET-28a(+)-pveh1 was extracted using the plasmid extraction kit Pure PlasmidMiniKit (Kangwei Century Biotechnology Co., Ltd.); wherein the plasmid pET-28a( The amino acid sequence of the epoxide hydrolase PvEH1 in +)-pveh1 is shown in SEQ ID NO.2 (the nucleotide sequence is shown in SEQ ID NO.4).

[0022] 2. Construction of recombinant E. coli BL21(DE3) / pET-28a(+)-pveh1(W102L)

[0023] Design and synthesize specific site-directed mutagenesis p...

Embodiment 2

[0028] Example 2: Mutant enzyme PvEH1 W102L the acquisition of

[0029] E.coli BL21-pveh1 W102L Inoculate a single colony in 2 mL of LB medium containing 100 μg / mL kanamycin, and culture overnight at 37°C and 215 r / min; transfer 1 mL of the culture solution to 50 mL of the same medium, and culture to OD 600 When it is 0.6-0.8, add IPTG (final concentration 0.5mmol / L) and induce at 20°C for 10h. Collect the thalli, use 10mL sodium phosphate buffer (Na 2 HPO 4 -NaH 2 PO 4 , 100mmol / L, pH 7.0) to obtain a bacterial suspension.

Embodiment 3

[0030] Example 3: Mutant enzyme PvEH1 W102L Determination of enantioselectivity

[0031] Add 40 μL of bacterial suspension and 910 μL of phosphate buffer to a 2 mL EP tube, incubate at 25°C for 5 min, then add 50 μL of racemic o-methylphenyl glycidyl ether (rac-o-GMPE, final concentration 10 mmol / L) and immediately The reaction was timed, and 100 μL of the reaction solution was regularly added to 1 mL of ethyl acetate to terminate the reaction. After microfiltration, samples were analyzed by normal phase HPLC, OD-H column and UV detector. The mobile phase is n-hexane / isopropanol (80:20, v / v), the flow rate is 0.8mL / min, the detection wavelength is 220nm, the peak time of (R)-o-GMPE and (S)-o-GMPE They are 6.52min and 8.02min respectively. Substrate e.e. s =[(S-R) / (R+S)]×100%; E=ln[(1-c)×(1-e.e. s )] / ln[(1-c)×(1+e.e. s )]. Where: R and S represent (R)- and (S)-o-GMPE concentration, c represents rac-o-GMPE conversion rate. The wild-type enzyme can retain the (R)-o-GMPE s...

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PUM

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Abstract

The invention discloses a phaseolus vulgaris epoxide hydrolase mutant capable of improving the enantioselectivity and belongs to the field of genetic engineering and protein expression. The mutant disclosed by the invention is obtained by mutating 102th-site tryptophan into leucine on the basis of amino acid shown as SEQ ID NO. 2. According to the phaseolus vulgaris epoxide hydrolase mutant, the enantiomer ratio (E value) of a mutated enzyme PvEH1W102L is 14.95 and is 2.03 times of that of a wild type enzyme (E=7.36).

Description

technical field [0001] The invention relates to a mutant of kidney bean epoxide hydrolase with improved enantioselectivity, belonging to the fields of genetic engineering and protein expression. Background technique [0002] Epoxide hydrolases (EHs, EC 3.3.2.-) can catalyze the hydrolytic kinetic resolution or enantionormalized hydrolysis of racemic epoxides, retaining a single configuration of epoxides or converting them into Chiral vicinal diols are extremely valuable biocatalysts for the preparation of chiral intermediates as biocatalysts. The traditional chemical splitting of epoxides often requires heavy metals and toxic substances as catalysts, which not only faces great environmental challenges, but also makes it difficult to obtain chirally pure compounds in high yields. EHs have the advantages of no need for metal ions and coenzymes, wide range of sources, and high enantioselectivity. EHs is a class of hydrolase that catalyzes the stereoselective addition of epoxi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/14C12N15/55C12N15/70C12N1/21C12P41/00C12P17/02
CPCC12N9/14C12P17/02C12P41/001C12Y303/02
Inventor 邬敏辰阚婷婷宗迅成徐雄峰李剑芳
Owner JIANGNAN UNIV
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