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Design and preparation method of epoxide hydrolase mutant

An epoxide and hydrolase technology, applied in the fields of genetic engineering and protein expression, to achieve the effect of great application potential, high catalytic efficiency, and high enantioselectivity

Active Publication Date: 2015-04-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Based on homology modeling, molecular docking, dynamic simulation, etc., there is no report on the molecular modification of Aspergillus usamii epoxide hydrolase (AuEH2)

Method used

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  • Design and preparation method of epoxide hydrolase mutant
  • Design and preparation method of epoxide hydrolase mutant
  • Design and preparation method of epoxide hydrolase mutant

Examples

Experimental program
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Embodiment 1

[0024] The construction of embodiment 1 mutant enzyme gene and its expression plasmid

[0025] Construction of Mutant Enzyme Gene Aueh2 Using Large Primer PCR Technique G218S and Aueh2 S247Y: G218S-F and AuEH2-R, S247Y-F and AuEH2-R were used as primers, and pET-28a(+)-Aueh2 was used as a template for PCR (94°C 4min; 94°C 30s, 45°C 30s, 72°C 30s , 2 cycles; 94°C for 30s, 55°C for 30s, 72°C for 30s, 28 cycles; 72°C for 10min), the PCR product was analyzed by 1% agarose gel electrophoresis, and the target band was recovered by tapping the gel to obtain a large primer; PCR products recovered from rubber tapping in rounds of PCR and AuEH2-F as primers for large primer PCR (94°C for 4min; 94°C for 30s, 45°C for 30s, 72°C for 30s, 2 cycles; 94°C for 30s, 55°C for 30s, 72°C for 30s, 28 cycles cycle; 72°C for 10min), the PCR product was analyzed by 1% agarose gel electrophoresis, and the target band was recovered by tapping the gel and ligated with pUCm-T (pUCm-T-Aueh2 G218S , pUCm...

Embodiment 2E

[0026] Example 2E.coli BL21(DE3) / Aueh2 G218 、E.coli BL21(DE3) / Aueh2 S247Y Construction, expression and activity assay of

[0027] pET-28a(+)-Aueh2 G218S , pET-28a(+)-Aueh2 S247Y E.coli BL21(DE3) were transformed respectively, and transformants were screened on plates containing Kan resistance. Positive transformants were picked and placed in 2 mL of LB medium containing Kan resistance, cultured with shaking at 37°C overnight, transferred to 30 mL of the same medium with a 1% inoculum size, and cultured with shaking at 28°C until mid-logarithmic growth (OD 600 0.6 ~ 0.8), adding 0.2mM IPTG to induce 8h. At the same time, E.coli BL21(DE3) containing the pET-28a(+) empty plasmid was used as a negative control, and the expression of the mutant enzyme in Escherichia coli was detected by SDS-PAGE. After the recombinant expressing strain is sonicated, the mutant enzyme is used to kinetically resolve the racemic SO, and the enantiomeric excess ee values ​​of (S)-SO produced are a...

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PUM

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Abstract

The invention provides design and construction of AuEH2G218S and AuEH2S247Y mutant enzymes with high enantio-selectivity. The mutation sites are finally determined by sequence homology analysis, homologous modeling, molecular docking and dynamic simulation. Mutant genes obtained by a megaprimer RPC technology are named Aueh2G218S and Aueh2S247Y. The invention further discloses a method for construction and efficient expression of mutant engineering bacteria; and the prepared mutant enzymes have the characteristics of high enantio-selectivity and catalytic activity, and have relatively large industrial production potential.

Description

technical field [0001] The present invention relates to the field of genetic engineering and protein expression, especially AuEH2 G218S and AuEH2 S247Y The construction of the enzyme gene, as well as the preparation and properties of the enzyme. Background technique [0002] Epoxide hydrolase (EH, EC, 3.3.2.-), also known as epoxide hydratase or epoxy hydratase, is a class of catalyzed stereoselective addition of epoxide hydrolysis to Hydrolases corresponding to 1,2-diols. Such hydrolases have good regioselectivity and enantioselectivity, so they are ideal biocatalysts for kinetic resolution of racemic epoxides to prepare chiral epoxides and diols. [0003] Chiral epoxides and vicinal diols are important chiral synthons in organic synthesis, and important intermediates for the synthesis of various active substances in industries such as chiral medicine, agriculture, spices and fine chemicals. Some chiral epoxides and diols have been used in the production of β-adrenergic...

Claims

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Application Information

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IPC IPC(8): C12N9/14C12N15/55C12N15/10
CPCC12N9/14C12Y303/02003
Inventor 邬敏辰胡蝶叶慧华李剑芳朱利娟余涛
Owner JIANGNAN UNIV
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