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Epoxide hydrolase as well as encoding gene and application thereof

A technology of epoxides and coding genes, which is applied in the field of enzyme catalysis and can solve problems such as inability to prepare

Active Publication Date: 2015-03-25
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problem in the above-mentioned prior art that optically pure R-configuration phenyl glycidyl ether cannot be prepared as a substrate by highly selective epoxide hydrolase enzyme racemic phenyl glycidyl ether, the present invention aims at Provide a kind of epoxide hydrolase and its coding gene and application

Method used

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  • Epoxide hydrolase as well as encoding gene and application thereof
  • Epoxide hydrolase as well as encoding gene and application thereof
  • Epoxide hydrolase as well as encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Cloning containing epoxy hydrolase gene

[0024] Purchase a standard strain freeze-dried tube numbered DSM20162 from DSMZ, Germany.

[0025] 1LTsukamurella paurometabola medium is as follows: Trypticase Soy Broth (BBL11768, Oxoid CM129 or Merck 5459) 30.0g; distilled water 1000ml; PH7.3. Sterilize at 121°C for 15min. Incubate at 28°C in a constant temperature shaker at 200 rpm for 1 to 3 days, collect the bacteria by centrifugation, and extract the Tsukamurella paurometabola genome according to the instructions of the Gerui Bacterial Genome Extraction Kit, and amplify it with the following two primers:

[0026] F: 5’-CGC GGATCC ATGACGATCACCCCGCACACCGTG-3' (SEQ ID NO: 3);

[0027] R: 5’-CCC AAGCTT TCAGCCCGCGGGGTGGTTCTCC-3' (SEQ ID NO: 4);

[0028] The full length of the gene of the epoxy hydrolase is obtained.

[0029] Toyobo KOD enzyme PCR reaction system is: H 2 O 32.5ul, buffer 5ul, 2mm dNTP5ul, MgCl 2 2.5ul, template 2ul, 20mM F / R primer 1ul, KOD enzyme 1ul. Aft...

Embodiment 2

[0030] Example 2: Obtaining whole cells containing epoxy hydrolase

[0031] The obtained transformants were inoculated into LB liquid medium containing 50μg / ml kanamycin resistance, cultured at 37°C for 12h, and then inoculated with 1% of the inoculum amount (v / v) to freshly containing 50ug / ml kanamycin In the LB liquid medium resistant to mycin, culture at 37°C until the cell concentration OD600 is about 0.6, and then add IPTG with a final concentration of 0.1mM to the LB liquid medium. After inducing and culturing at 20°C for 20 hours, the culture solution is incubated at 4 Centrifuge at 8000 rpm for 10 min, discard the supernatant, and collect the precipitate, which is the recombinant E. coli BL21 / pET28a-EH wet bacteria containing the intracellular expression recombinant plasmid. Freeze-dried cells were obtained after the wet cells were freeze-dried for 4 hours. The formula of 1000ml LB medium is: peptone 10g, sodium chloride 10g, yeast extract 5g. The wet bacteria are broke...

Embodiment 3

[0032] Example 3: Application of the epoxy hydrolase in the preparation of R-phenyl glycidyl ether

[0033] The freeze-dried bacterial cells obtained in Example 2 were used as a catalyst. Dissolve 50 mg of lyophilized bacteria in 45 mL of 100 mM Trish-Hcl, mix well at 200 rpm for 5 min, and add 5 ml of racemic phenyl glycidyl ether dissolved in 4M DMSO at 200 rpm at 30° C. and terminate the reaction after 1 hour. Use 15ml n-hexane to extract three times to get ee> 99% R-phenyl glycidyl ether, then 15ml ethyl acetate extraction three times to get ee> 83% S-phenoxy glycol.

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Abstract

The invention relates to an epoxide hydrolase having amino acid sequences as shown in SEQ ID NO: 2. The invention relates to an encoding gene for the epoxide hydrolase which has amino acid sequences as shown in SEQ ID NO: 1. The invention also relates to an application of the epoxide hydrolase, by the epoxide hydrolase, chiral aromatic epoxide is prepared by adopting racemic aromatic epoxide as a substrate. The epoxide hydrolase provided by the invention can be in a form of wet thallus or lyophilized thallus of the whole cell and can also be in a form of crude enzyme liquid or pure enzyme, by adopting racemic aromatic epoxide as the substrate, the enantiomerically pure chiral aromatic epoxide is prepared (ee is greater than 99%).

Description

Technical field [0001] The present invention relates to the field of enzyme catalysis, and more specifically to an epoxide hydrolase and its coding gene and application. Background technique [0002] Epoxide hydrolase (EC 3.3.2.3) is a group of enzymes with similar functions, which can stereoselectively catalyze epoxy compounds to produce optically pure epoxides and corresponding vicinal diols. As a kind of hydrolase, it has the characteristics of general hydrolase such as: the reaction does not require the assistance of coenzyme; it has a wide range of sources; it still has catalytic activity in non-aqueous solvents; and these reactions often show chemical, regio and stereo enantioselectivity It can act on a wide range of substrates of the same type. [0003] Epoxide hydrolases are widely found in nature, and are found in mammals, insects, microorganisms and plants. Due to the diversity of microorganisms, fast growth, easy cultivation and high yield, screening and obtaining epox...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/14C12N15/55C12P41/00C12P17/02
CPCC12N9/14C12P17/02C12P41/001C12Y303/02003
Inventor 王华磊吴锴魏东芝
Owner EAST CHINA UNIV OF SCI & TECH
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