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Lipase gene and recombinase thereof, and application of lipase gene in preparing optically active mandelic acid

A lipase gene and lipase technology are applied in the application field of lipase gene and its recombinant enzyme and in the preparation of optically active mandelic acid, which can solve the problems of low catalytic activity and achieve high catalytic efficiency, strong stereoselectivity, fast response effect

Inactive Publication Date: 2013-02-20
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Aiming at the defect that the overall catalytic activity of Pseudomonas sp. ECU1011 as an asymmetric catalyst to selectively catalyze 2-acetoxyphenylacetic acid is relatively low, and to provide an excellent asymmetric catalytic activity, Highly reactive and environmentally friendly lipase and its gene, recombinant expression vector containing the gene, recombinant expression transformant, preparation method of the recombinant enzyme and application of the lipase or its recombinant enzyme

Method used

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  • Lipase gene and recombinase thereof, and application of lipase gene in preparing optically active mandelic acid
  • Lipase gene and recombinase thereof, and application of lipase gene in preparing optically active mandelic acid
  • Lipase gene and recombinase thereof, and application of lipase gene in preparing optically active mandelic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 RNA extraction and reverse transcription to obtain cDNA

[0043] 1. Extraction of total bacterial RNA (Trizol method):

[0044] Aspergillus fumigatus Af293CGMCC No. 2873 was inoculated into Martin medium (peptone 5g / L, yeast extract 2g / L, glucose 20g / L, K 2 HPO 4 1g / L, MgSO 4 0.5g / L, pH 6.8) at 30°C for about 48 hours.

[0045] (1) Take the Aspergillus fumigatus cultured in the shake flask (small spheres) into a centrifuge tube, centrifuge at 10000g for 10 minutes (4℃) to discard the waste liquid, and wash twice with deionized water, drain the water, and transfer the bacteria into the liquid Grind thoroughly in nitrogen, transfer it to a 1.5mL centrifuge tube containing 1mL Trizol, mix well and use the residual temperature of liquid nitrogen to freeze and thaw several times, then place it flat in an ice box for 5 minutes to completely decompose the nucleoprotein complex Isolation (the sample volume does not exceed 10% of the volume of Trizol used);

[0046] (2) Add ...

Embodiment 2

[0069] Example 2 Cloning of lipase gene

[0070] Based on the gene sequence of the lipase AFUA_3G14960 (Gene ID: 3511767) of Aspergillus fumigatus Af293 included in Genbank, the PCR primers are designed as follows:

[0071] Upstream primer: CG CATATG GCAGACTACTCGGAAT;

[0072] Downstream primer: CGC AAGCTT CTACTTTGTTTTGATC.

[0073] Among them, the underlined part of the upstream primer is the NdeI restriction site, and the underlined part of the downstream primer is the HindIII restriction site.

[0074] The cDNA of Aspergillus fumigatus Af293CGMCC No. 2873 obtained in Example 1 was used as a template for PCR amplification. The PCR system is: 2×Taq PCR MasterMix: 10μL, upstream primer and downstream primer: 1μL each, cDNA template: 1μL, ddH 2 O: 7 μL. The PCR amplification steps are: (1) 95°C, pre-denaturation for 5 minutes; (2) 94°C, denaturation for 30s; (3) 56°C annealing for 30s; (4) 72°C extension for 70s; steps (2) to (4) cycle 30 times; (5) Continue to extend at 72°C for 1...

Embodiment 3

[0075] Example 3 Preparation of recombinant expression vector (plasmid) and recombinant expression transformant

[0076] The afl67 gene obtained in Example 2 was connected with the pMD-18T vector to construct a cloning plasmid, which was transformed into E. coli DH5α competent cells. The positive clones were screened by colony PCR, the plasmids were extracted, and restriction endonucleases were used at 37°C. NdeI and HindIII were double digested for 3 hours, purified by agarose gel electrophoresis, and the target fragment was recovered using an agarose gel DNA recovery kit. Under the action of T4DNA ligase, the digested fragment was ligated with the plasmid pET28a, which was also double digested with NdeI and HindIII, at 4℃ overnight to obtain the recombinant expression plasmid pET28a-afl67( image 3 ), transformed into E.coli DH5α competent cells, screened positive recombinants on a resistant plate containing 0.5mmol / L kanamycin, picked a single clone, cultured the recombinant ba...

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Abstract

The invention discloses a lipase and a gene thereof, a recombinant expression vector, recombinant expression transformant and recombinase containing the gene, and a preparation method of the recombinase. In addition, the invention also discloses application of thallus containing the recombinant lipase as a catalyst in preparing an optically active substance from alpha-acetoxyphenylacetic acid and2-Cl-alpha-acetoxyphenylacetic acid in an asymmetric deacylation mode. The recombinant lipase gene is derived from aspergillus fumigatus Af293. The recombinase can be used as a catalyst for asymmetric deacylation to prepare optically active mandelic acid. The recombinase has the advantages of high catalytic efficiency, strong stereoselectivity, mild applicable reaction conditions and environmental friendliness. The recombinase disclosed by the invention has high catalytic activity, and has wide prospects in industrial application and development.

Description

Technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a lipase and its gene, as well as a recombinant expression vector and a recombinant expression transformant containing the gene, and also includes a preparation method of the recombinant enzyme and its recombinant lipase transformant as a catalyst Application of α-acetoxyphenylacetic acid and 2-Cl-α-acetoxyphenylacetic acid as substrates for asymmetric deacylation to prepare optically active substances. Background technique [0002] Mandelic Acid (MA), or 2-hydroxyphenylacetic acid, is an important pharmaceutical and chemical synthesis intermediate, and has a wide range of applications in biological and chemical synthesis. Optically pure mandelic acid has become a popular drug intermediate and acidic chiral resolving agent because of its good biological activity and unique physiological function. (S)-Mandelic acid is a precursor material for the synthesis of drugs f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/20C12N15/55C12N15/63C12N1/15C12N1/19C12N1/21C12P7/42C12R1/19
Inventor 赵健上官娇娇许建和范立强
Owner EAST CHINA UNIV OF SCI & TECH
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