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Epoxide hydrolase sourced from Phaseolus vulgaris and application thereof

A technology of epoxide and hydrolase, which is applied in the biological field to achieve the effect of strong specificity

Inactive Publication Date: 2017-09-15
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although the epoxide hydrolases reported so far all have certain asymmetric activity of hydrolyzing epoxides, only more than 40 kinds of EHs show high enantioselectivity for one or more epoxides (i.e. Enantioselectivity E value > 30), as far as we know, except for the potato EH studied by foreign scholars, there is no plant-derived EH that can efficiently split rac-SO to prepare highly chiral pure R-SO. , mining the EH gene with high selectivity to construct its expression system by means of molecular biology, and applying it to the research work such as the preparation of optically pure epoxides has important economic and social benefits

Method used

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  • Epoxide hydrolase sourced from Phaseolus vulgaris and application thereof
  • Epoxide hydrolase sourced from Phaseolus vulgaris and application thereof
  • Epoxide hydrolase sourced from Phaseolus vulgaris and application thereof

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Experimental program
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Effect test

Embodiment 1

[0028] The cloning of embodiment 1 epoxide hydrolase gene and the construction of expression plasmid

[0029] The total RNA of kidney bean was extracted (using TRIZOL kit, Invitrogen Company), and the operation was performed according to the instructions of the kit. The reverse transcription process refers to the RT-PCR kit PCR PrimeScriptTM RT reagent Kit with gDNAEraser (Perfect Real Time) manual of Takara Company, and then the cDNA obtained by reverse transcription is used as a template, and the upstream and downstream primer sequences are respectively SEQ ID NO: 3 and SEQ ID NO.4 (Nde I and Xho I restriction endonuclease sites were introduced into the primers, and the primers were all synthesized by Shanghai Shenggong Co., Ltd.), the pveh2 full-length gene was amplified by conventional PCR method, and its nucleotide The sequence is shown as SEQ ID NO.1, and the PCR reaction conditions are: pre-denaturation at 94°C for 2 minutes, denaturation at 94°C for 30 seconds, anneali...

Embodiment 2

[0030] The preparation of embodiment 2 epoxide hydrolase PvEH2

[0031] The genetically engineered bacteria E.coli BL21(DE3) / pET28a(+)-pveh2 in Example 1 was inoculated in LB liquid medium (containing 0.1mg / ml kanamycin sulfate), and cultivated at 37°C for 10~16h ( Optimizing, be 12h), then transfer in the LB culture medium that contains same composition with the inoculum size of 1% (v / v) to continue expanding culture, when thalline concentration OD 600 When the value reaches 0.8-1.0 (optimized, 0.8), add IPTG to its final concentration of 0.2mmol / l, induce culture at 25°C for 4-9h (optimized, 7h), and then centrifuge at 8000r / min for 5min at 4°C , collect the bacterial cell precipitate, and according to the above steps, 0.5-0.8 g of wet bacterial cell can be obtained by fermentation per 100 ml of culture medium. The results of SDS-PAGE analysis of bacterial cells are as follows: figure 2 As shown, the apparent molecular weight of the target protein is about 37.9kDa. .

Embodiment 3

[0032] Example 3 Application of Epoxide Hydrolase PvEH2 in the Preparation of Chiral Styrene Oxide

[0033] The wet thalli obtained through centrifugation in embodiment 2 is according to every g corresponding to 10ml NaH 2 PO 4 -Na 2 HPO 4 (100mM, pH7.2) buffer ratio re-suspended, bacterial cell suspension directly as a biological enzyme catalyst, racemic styrene oxide as a substrate for conversion to prepare R-type styrene oxide. The specific operation is: add 2.5ml bacterial cell suspension (bacterial concentration 50mg / ml, activity is 0.04U / ml), 2.25ml buffer solution, 0.25ml racemic epoxy with a concentration of 200mmol / l in a 10ml Ep plastic tube Phenylethane (final concentration in the catalytic system is 10 mmol / l) was converted at 25° C. for 120 min while shaking, and finally the same volume of ethyl acetate was added to extract and terminate the reaction.

[0034] The reaction process of PvEH2 under the above conditions for styrene oxide is as follows image 3 sh...

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Abstract

The invention discloses an epoxide hydrolase sourced from Phaseolus vulgaris and an application thereof and belongs to the technical field of biology. The invention discloses an epoxide hydrolase gene (pveh2) sourced from a leguminous plant, Phaseolus vulgaris, as well as a corresponding amino sequence. The invention provides a recombinant plasmid pET28a(+)-pveh2 and a genetic engineering bacteria E.coli BL21(DE3) / pET28a(+)-pveh2, which comprise the gene. A recombinant epoxide hydrolase is prepared through expression by the bacterial strain. The invention provides a method of biocatalysis through fermentation with an enzyme produced by the recombinant bacterial strain constructed in the invention, thus producing enantiomerically pure R-type styrene oxide (ee > 99%); in addition, final yield can reach 49.3%, which is approximate to a theoretical value, 50%.

Description

technical field [0001] The invention relates to an epoxide hydrolase derived from kidney bean and its application, belonging to the field of biotechnology. Background technique [0002] Chirally pure epoxides and their corresponding hydrolysis products, vicinal diols, are considered to be a class of multifunctional chiral building blocks with high added value, and are widely used in the synthesis of chiral drugs and fine chemicals. However, in the preparation process of some chiral epoxides, the reaction results of chemical methods such as Sharpless asymmetric epoxidation and Jacobsen epoxidation often cannot reach the desired chiral purity, and the product (referring to the remaining single-configuration ring The enantiomeric excess (ee) value of the oxide and the resulting diol) is usually only 30% to 80%, and the catalysts such as heavy metals used also pose a huge threat to the environment. In recent years, enzyme-mediated kinetic resolution and enantiomerically normali...

Claims

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Application Information

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IPC IPC(8): C12N9/14C12N15/55C12N1/21C12P41/00C12P17/02C12R1/19
CPCC12N9/14C12P17/02C12P41/001C12Y303/02
Inventor 邬敏辰李闯宗迅成王瑞李剑芳
Owner JIANGNAN UNIV
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