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Kidney bean epoxide hydrolase mutant with improved catalytic activity

A technology of epoxides and mutants, applied in the fields of genetic engineering and protein expression, can solve problems such as low enzyme activity and limitations

Active Publication Date: 2018-06-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although epoxide hydrolases have been widely valued in the preparation of optically pure substrates and products due to their advantages of no cofactors or metal ions, mild reaction conditions, and environmental friendliness, they are widely used in laboratories and industries. In terms of large-scale applications, the further development of epoxide hydrolase is limited by its low enzyme activity and other defects.

Method used

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  • Kidney bean epoxide hydrolase mutant with improved catalytic activity

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: Construction of mutant enzyme gene and its expression plasmid

[0021] 1. Acquisition of plasmid pET-28a(+)-pveh1

[0022] The medium composition of recombinant E.coli BL21(DE3) / pET-28a(+)-pveh1 is: peptone 1%, yeast extract 0.5%, NaCl 1%.

[0023] 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 PlasmidMini Kit (Kangwei Century Biotechnology Co., Ltd.); 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).

[0024] 2. Construction of recombinant Escherichia coli E. coli BL21(DE3) / pET-28a(+)-pveh1(V106I)

[0025] Design and synthesize specific site-directed mut...

Embodiment 2

[0029] Example 2: Mutant enzyme PvEH1 V106I the acquisition of

[0030] E.coli BL21-pveh1 V106I 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

[0031] Example 3: Mutant enzyme PvEH1 V106I Determination of catalytic activity

[0032] Add 30 μL of bacterial suspension and 920 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) immediately After timing the reaction for 10 min, take 200 μL of the reaction solution and add 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 was n-hexane / isopropanol (80:20, v / v), the flow rate was 0.8 mL / min, and the detection wavelength was 220 nm. Definition of enzyme activity unit: Under the conditions of this assay, the amount of enzyme required to decompose 1 μmol rac-o-GMPE per minute is defined as an activity unit (IU) of epoxide hydrolase. Mutant enzyme PvEH1 V106I The catalytic activity was 234.75U / g, which was 1.49 times that of the wild-type enzyme (15...

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Abstract

The invention discloses a kidney bean epoxide hydrolase mutant with improved catalytic activity and belongs to the fields of gene engineering and protein expression. The mutant mutates valine at the 106th site into isoleucine on the basis of amino acid shown in SEQ ID NO.2. The catalytic activity of mutant enzyme PvEH1V106I obtained by shaking flask induction for 10h, disclosed by the invention, is 234.75U / g, which is 1.49 times that of wild type enzyme (157.15U / g).

Description

technical field [0001] The invention relates to a mutant of kidney bean epoxide hydrolase with improved catalytic activity, belonging to the fields of genetic engineering and protein expression. Background technique [0002] Epoxide hydrolases (epoxide hydrolases, EHs, EC 3.3.2.-) widely exist in natural environments such as animals, plants, microorganisms, etc. It can add a molecule of water to the epoxy ring to make the epoxide ring open and hydrolyze The corresponding diols are formed without the participation of cofactors and metal ions. Because epoxide hydrolase has the characteristics of high stereoselectivity, mild reaction conditions and less environmental pollution when catalyzing hydrolysis, people have paid more and more attention to its application value in biocatalytic hydrolysis of racemic epoxides in recent years. [0003] The vast majority of EHs can be divided into three categories according to different catalytic mechanisms: one is the limonene-1,2-epoxide...

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 邬敏辰阚婷婷王婷婷苏永君李剑芳
Owner JIANGNAN UNIV
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