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High-enantiomer selectivity epoxide hydrolase and gene coded thereby

A technology of epoxides and hydrolytic enzymes, which is applied in the fields of hydrolytic enzymes, plant genetic improvement, genetic engineering, etc., and can solve problems such as inability to meet large-scale industrial applications, high prices, and limited scope of substrate action

Inactive Publication Date: 2009-12-02
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are epoxide hydrolase enzyme products derived from Aspergillus niger, Agrobacterium radiobacter, Rhodococcus rhodochrous and people on the market, but on the one hand, these epoxides The scope of substrate action of hydrolytic enzymes is limited. On the other hand, the production of these enzyme preparations is small and the price is very expensive, which cannot meet the requirements of large-scale industrial applications.

Method used

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  • High-enantiomer selectivity epoxide hydrolase and gene coded thereby
  • High-enantiomer selectivity epoxide hydrolase and gene coded thereby
  • High-enantiomer selectivity epoxide hydrolase and gene coded thereby

Examples

Experimental program
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Effect test

Embodiment 1

[0014] Example 1: Cloning of the sequence of the epoxide hydrolase PchEHA coding region from P.chrysosporium

[0015]Cultivation and RNA extraction of P.chrysosporium BKM-F1767: Inoculate P.chrysosporium BKM-F1767 on a PDA plate and culture at 38°C for 5 days. After a large number of conidia are formed on the plate, use 0.1% sterile Tween- 20 solution to wash the spores from the plate, vortex to disperse and filter with gauze. Centrifuge the filtrate at 4000rpm for 5min, wash twice with sterile water, resuspend the spores, and adjust the concentration to A 600 ≈1.0. Inoculate 3ml of the prepared spore suspension into 50mL of Kirk low-nitrogen medium, respectively, and inoculate it with oxygen at 39°C, set up 3 parallel samples, and collect the mycelium by filtration at 48h and 72h after cultivation For the extraction of total cellular RNA. The total RNA of P. chrysosporium was extracted using TRIZOL kit (Invitrogen Company), and the operation steps were carried out accordin...

Embodiment 2

[0020] Embodiment 2: Construction of epoxide hydrolase PchEHA expression vector

[0021] Plasmid pTEHA was double digested with BamH I and Hind III, and the coding region fragment of the epoxide PchEHA was recovered after agarose electrophoresis detection, and pET-28a(+) (Novagen) was digested with BamH I and Hind III Gel recovery was carried out to prepare the carrier. Ligate the coding region fragment of recovered epoxide PchEHA with the prepared pET-28a(+) vector, transform the ligation product into E.coliJM109, select positive clones and extract the plasmid, and perform agarose gel electrophoresis detection on the plasmid, and after electrophoresis The detection confirmed that the plasmid containing the inserted fragment was identified by restriction digestion with BamH I and Hind III. Thus, the expression plasmid containing PchEHA was obtained, and it was named pET28EHA.

Embodiment 3

[0022] Example 3: Expression and purification of epoxide hydrolase PchEHA in Escherichia coli

[0023] The recombinant expression plasmid pET28EHA was transformed into E.coli BL21(DE3) competent cells to obtain the genetically engineered strain E.coli BL21(DE3) / pET28EHA expressing the recombinase, and after overnight culture, a single colony was picked to induce expression. The induced expression operation refers to the PET System operation manual. The specific steps are: inoculate a single colony of E.coli BL21(DE3) containing pE28EHA in 3ml LB liquid medium (containing 50μg / ml kanamycin), and cultivate overnight at 37°C with shaking at 220r / min; take 2.5ml The culture solution (1%) was inserted into 250ml of new LB liquid medium (8 bottles were inoculated, 2L in total), at 37°C; 220r / min shaking culture to OD 600 About 0.5; add 0.5mM IPTG, induce expression at 18°C; after culturing for 16-20h, centrifuge for 10 minutes (4000r / min) to collect the bacteria, and resuspend the ...

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Abstract

The invention belongs to the field biotechnology and discloses use of an RT-PCR method in cloning an epoxide hydrolase gene (PchEHA) from a BKM-F1767 strain (Phanerochaete chrysosporium BKM-F1767) of fungus Phanerochaete chrysosporium and a nucleotide sequence and an amino acid sequence of a coding region of the PchEHA. Cloned plasmid pTEHA containing the gene, a recombinant expression plasmid pET28EHA containing the gene and a genetic engineering strain Escherichia coli E.coli BL21(DE3) / PeT28EHA containing the PchEHA gene are provided to prepared the recombinant epoxide hydrolase. The epoxide hydrolase of the invention has catalytic activity for various epoxides and shows enantiomer selectivity of different degrees, particularly high enantiomer selectivity for epoxyethylbenzene. The recombinant epoxide hydrolase of the invention can be used in the fields of the kinetic resolution of a chiral epoxide racemic mixture, the asymmetric catalysis of epoxides, and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an enantioselective epoxide hydrolase derived from the white-rot fungus Phanerochaete chrysosporium (BKM-F1767), including the nucleus of the epoxide hydrolase Nucleotide sequences and recombinant epoxidases, and the application of recombinant enzymes in the resolution and asymmetric catalysis of chiral epoxide racemic mixtures. Background technique [0002] Epoxide hydrolase (EC 3.3.2.3) is a general term for a class of enzymes that catalyze the hydrolysis of epoxides to generate corresponding vicinal diols. In enzymatic classification, epoxide hydrolase and esterase, protease , lipase, and dehalogenase belong to the α / β-fold hydrolase class (Fretland and Omiecinski, 2000). Epoxide hydrolases are widely found in nature and found in a variety of animals, plants, bacteria and fungi (Barth et al.2004a, b), and play different physiological functions in different organisms, mainly includi...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N9/14C12N1/21C12P41/00C12P7/18C12R1/19
Inventor 冯红李念张琳丰张义正
Owner SICHUAN UNIV
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