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A Mutant of Phaseolin Epoxide Hydrolase with Improved Catalytic Activity and Enantionormality

A technology of epoxide and hydrolase, which is applied in the field of genetic engineering and protein expression, can solve the problems of high randomness, heavy screening workload, unsatisfactory ee value, etc., and achieve high enantionormality, high catalytic activity, The effect of large application potential

Active Publication Date: 2019-07-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method has high randomness, heavy screening workload, and the ee value is not ideal

Method used

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  • A Mutant of Phaseolin Epoxide Hydrolase with Improved Catalytic Activity and Enantionormality
  • A Mutant of Phaseolin Epoxide Hydrolase with Improved Catalytic Activity and Enantionormality
  • A Mutant of Phaseolin Epoxide Hydrolase with Improved Catalytic Activity and Enantionormality

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Experimental program
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Embodiment 1

[0019] The construction of embodiment 1 mutant enzyme gene and its expression plasmid

[0020] The amplified nucleotide sequence was the gene pveh1 shown in SEQ ID NO.4, the amplified product was ligated with pUCm-T, transformed into E.coli JM109, and screened by blue and white spots, identified by SacI enzyme digestion and DNA sequencing. The recombinant plasmid with correct sequencing was named pUCm-T-pveh1. Digest pUCm-T-pveh1 with NdeI and XhoI, recover pveh1, and connect it with pET-28a(+) which has been digested with the same double enzymes, to obtain recombinant plasmid pET-28a(+)-pveh1.

[0021] Using the known crystal structure of potato EH (PDB: 2CJP) as a template, the three-dimensional structure of PvEH1 was obtained by homology modeling. The model substrate molecule (R)-SO was molecularly docked with PvEH1 using AutoDock4.2 software, and the catalytic (R)-SO molecule was counted The homology analysis excludes the amino acids at the conserved sites; the types an...

Embodiment 2

[0030] Example 2 Mutant enzyme PvEH1 L105I / M160A / M175I Determination of catalytic activity

[0031] Add 450 μL of bacterial suspension to a 2 mL EP tube, incubate at 25°C for 5 min, then add 50 μL of racemic styrene oxide (rac-SO, final concentration 20 mmol / L) and react immediately for 10 min, then take 200 μL of the reaction solution and add 1 mL of methanol was used to stop the reaction. After microfiltration, samples were analyzed by reverse phase HPLC (Waters, Milford, MA), C18 column and UV detector. The mobile phase was methanol / water (70:30, 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-SO per minute is defined as the activity unit (IU) of 1 epoxide hydrolase. Mutant enzyme PvEH1 L105I / M160A / M175I The catalytic activity was 10.66U / g, which was 5.6 times higher than that of the wild-type enzyme (1.61U / g).

Embodiment 3

[0032] The mensuration of embodiment 3 enantiomeric purity and enantioselectivity

[0033] Add 450 μL of bacterial suspension and 500 μL of sodium phosphate buffer (pH 7.5) to a 1.5 mL EP tube, incubate at 25 °C for 5 min, then add 50 μL of rac-SO (final concentration 10 mmol / L) for reaction. 50 μL of samples were regularly extracted to 1 mL of ethyl acetate (containing 1 mmol / L n-hexanol as an internal standard) for extraction, and the samples were analyzed using a gas chromatograph GC-2010 (Shimadzu, Japan), a chiral gas chromatography column, and a hydrogen flame ionization detector. The analysis conditions were: inlet and detector temperature 250°C; initial column temperature 100°C, rising to 195°C at 5°C / min; carrier gas nitrogen, flow rate 3.0mL / min, split ratio 1:50. n-Hexanol, (R)-SO((R)-Ethylene Oxide), (S)-SO((S)-Ethylene Oxide), (S)-PED((S)-Phenylethylene Glycol) and (R)-PED ((R)-phenylethylene glycol) retention times were 3.477, 5.959, 6.065, 16.752 and 16.866min,...

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Abstract

The invention discloses a kidney bean epoxide hydrolase mutant with improved catalytic activity and enantioconvergent property, belonging to the fields of genetic engineering and protein expression. A strain of epoxide hydrolase mutant PvEH1 <L105I / M160A / M175I> with improved catalytic activity and enantio-convergent property can be obtained by reforming the molecular structure of epoxide hydrolase (EH) by means of a site-directed mutation biotechnology on the basis of EH with certain enantio-convergent catalysis characteristic. The mutant has the advantages of high enantio-selectivity and high catalytic activity, thus having greater application potential and also laying a theoretical foundation for the study of the EH.

Description

technical field [0001] The invention relates to a mutant of kidney bean epoxide hydrolase with improved catalytic activity and enantionormality, and belongs to the fields of genetic engineering and protein expression. Background technique [0002] Chiral vicinal diols are a class of high value-added multifunctional synthons that are used in the synthesis of drugs, fine chemicals, pesticides, and functional materials. Epoxide hydrolases (EHs) can catalyze the enantionormal hydrolysis of racemic epoxides, and completely convert the substrate into the corresponding single configuration, which has the advantages of being green and economical. Enantionormal hydrolysis of racemic epoxides can be achieved by an epoxide hydrolase with complementary enantioselectivity and regioselectivity. [0003] So far, three epoxide hydrolases with single enantiounity catalytic properties have been reported, which are derived from potato, mung bean and C. crescentus respectively. With the devel...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/14C12N15/55C12N15/70C12N1/21C12P41/00C12P7/22
CPCC12N9/14C12P7/22C12P41/00C12Y303/02003
Inventor 邬敏辰叶慧华胡蝶石小玲吴芹李剑芳
Owner JIANGNAN UNIV
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