Pronucleus expression of epoxide hydrolase gene (EH-B) and preparation of chiral epichlorohydrin

A technology of epoxide and hydrolase, which is applied in the field of bioengineering, can solve unreported problems and achieve high enantioselectivity, large-scale industrial production and application potential, economic value, and high enzymatic activity.

Inactive Publication Date: 2013-03-27
JIANGNAN UNIV
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Problems solved by technology

The genetically engineered epoxide hydrolase has high purity, does not contain other proteases, and has stronger stereoselectivity; the Escherichia coli expression system has the advantages of high-efficiency intracellular expression of recombinant proteins, low cost, high productivity, and simple operation; and because the general substrate is Organic solvents, enzyme molecules a

Method used

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  • Pronucleus expression of epoxide hydrolase gene (EH-B) and preparation of chiral epichlorohydrin
  • Pronucleus expression of epoxide hydrolase gene (EH-B) and preparation of chiral epichlorohydrin
  • Pronucleus expression of epoxide hydrolase gene (EH-B) and preparation of chiral epichlorohydrin

Examples

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Example Embodiment

[0023] Example 1 Cloning of A.usamii E001EH-B mature peptide cDNA sequence

[0024] Extract the total RNA of A.usamii E001, and perform RT-PCR according to the instructions in the RNA PCR Kit (AMV) Ver.3.0 produced by TaKaRa: Use Oligo dT-Adaptor Primer as a primer for reverse transcription to synthesize the first strand of cDNA; Carry out the first round of PCR with M13Primer M4 and AuEHB-F as primers (94°C, 2min; 94°C, 30s, 51°C, 30s, 72°C, 80s, 30 cycles; 72°C, 10min); PCR product as template, AuEHB-F and AuEHB-R as primers for the second round of PCR (94°C, 2min; 94°C, 30s, 55°C, 30s, 72°C, 70s, 30 cycles; 72°C, 10min); Two rounds of PCR products were analyzed by 1% agarose gel electrophoresis, 250bp DNA LadderMarker was used as a control, the target band was tapped and recovered and connected with pUCm-T, transformed into JM109 to obtain the recombinant plasmid pUCm-T-AusEH-B, which was digested by restriction enzyme After identification, it was sent to Shanghai Shenggong f...

Example Embodiment

[0025] Example 2 Expression of Aus EH-B mature peptide gene in E. coli Rosetta (DE3)

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Abstract

The invention provides a clone and a pronucleus expression method of a novel B class epoxide hydrolase gene mature peptide cDNA sequence from Aspergillus usamii E001. The nucleotide sequence of the novel B class epoxide hydrolase gene mature peptide cDNA sequence is SEQ ID NO:1, the corresponding amino acid sequence is SEQ ID NO:2, and the corresponding gene is named Aus EH-B. According to the invention, good stereoselectivity is achieved for (R)-epichlorohydrin through chiral gas chromatography analysis rEH, and a produced (S)-epichlorohydrin antipode excess value achieves 99%. The pronucleus expression method provided by the invention lays the foundation for the industrialized production of epoxide hydrolase and provides the basis for preparing chiral epichlorohydrin by researching an EH enzyme kinetic resolution method for biological catalysis technology industrialization.

Description

technical field [0001] The invention relates to the cloning and prokaryotic expression of a novel B-type epoxide hydrolase (Aus EH-B) gene mature peptide cDNA sequence derived from Aspergillus usamii E001 strain, and the preparation of chiral The method for epoxystyrene belongs to the technical field of bioengineering. Background technique [0002] Chiral compounds are important chiral ligands, which are widely used in various asymmetric reactions, especially in the field of drug synthesis. Chiral epichlorohydrin is an important key intermediate in organic synthesis, used to prepare a variety of high optical purity pharmaceutical intermediates and natural products, such as aryloxypropanolamines, derivatives of cyclopentanone, antifungal Drugs (S) - Meikangling, atorvastatin, L-carnitine, etc. At present, racemic epichlorohydrin is cheap and easy to obtain, while chiral epichlorohydrin is expensive, and the quotation of the latter is basically more than 6 times that of the ...

Claims

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Application Information

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IPC IPC(8): C12N9/14C12N15/55C12N15/10C12N15/70C12R1/66
Inventor 李剑芳胡蝶王春娟朱天地邬敏辰
Owner JIANGNAN UNIV
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