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Application of a recombinant esterase, gene, engineering bacteria and resolution of (r,s)-indoline-2-carboxylate ethyl ester

A technology of recombinant esterase and indoline, applied in the direction of genetic engineering, application, plant gene improvement, etc., can solve the problems of expensive, serious pollution, split reagent price, and many steps

Active Publication Date: 2021-04-06
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Both chemical synthesis and resolution methods have the disadvantages of many steps, serious pollution or expensive resolution reagents

Method used

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  • Application of a recombinant esterase, gene, engineering bacteria and resolution of (r,s)-indoline-2-carboxylate ethyl ester
  • Application of a recombinant esterase, gene, engineering bacteria and resolution of (r,s)-indoline-2-carboxylate ethyl ester
  • Application of a recombinant esterase, gene, engineering bacteria and resolution of (r,s)-indoline-2-carboxylate ethyl ester

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1: recombinant esterase BaCE

[0060] Bacillus B8W22 (Bacillus aryabhattai), preserved in the China Center for Type Culture Collection (address China, Wuhan, Wuhan University, 430072), preservation number CCTCC NO:M 2017751, preservation date November 30, 2017, has previously applied for a patent (Application No.: 2019101300148) submitted relevant strain preservation information and disclosed the source of its genetic resources.

[0061]1. Extract the genomic DNA of Bacillus CCTCC NO:M 2017751 with a nucleic acid extraction kit, use the genomic DNA as a template, and use primer pET-28a-noc1-F (5'-TAAGAAGGAGATATACCATGGATGCCGTTAGATCCGCATATTC-3'), primer pET-28a- xho1-R(5'-GTGGTGGTGGTGGTGCTCGAGAATGGAGTCAAAT

[0062] ACTTGACGCAG-3') under the effect of PCR amplification.

[0063] The amount of each component in the PCR reaction system (total volume 20 μL):

[0064] Primer pET-28a-nco1-F and primer pET-28a-xho1-R each 0.3 μL, genomic DNA 1 μL, primesTARMax Prem...

Embodiment 2

[0071] Embodiment 2: recombinant esterase BaCE bacterial cell

[0072] The recombinant Escherichia coli BL21 / pET-28a-BaCE obtained according to Example 1 was inoculated into LB liquid medium containing 100 μg / ml kanamycin, cultivated at 37°C for 12-16h, and then inoculated at 1% (v / v) Inoculate it into fresh LB liquid medium containing 100 μg / ml kanamycin, and cultivate it at 37°C until the cell concentration is OD 600 0.6, then add IPTG with a final concentration of 0.2mM to the LB liquid medium, induce culture at 22°C for 10-12h, then centrifuge at 8000rpm at 4°C for 10min, collect the wet cells, which are cells containing recombinant esterase .

Embodiment 3

[0074] 1 g of recombinant Escherichia coli BL21 / pET-28a-BaCE wet thallus obtained in Example 2 was resuspended with 20 ml, 50 mM Tris-HCl buffer solution of pH 8.0, and then ultrasonicated (ultrasonic power was 250 W, ultrasonic 2 s, Interval 6s, effective ultrasonic time 10min), the broken suspension was centrifuged at 12000rpm at 4°C for 10min, cell debris was discarded as much as possible, and the supernatant was taken to obtain protein crude enzyme solution. According to the instructions of Ni-NTA metal chelate affinity chromatography, take the protein crude enzyme solution and load it on the pre-equilibrated Ni 2+ In the column, the impurity protein and the target protein were eluted successively with an aqueous solution containing 50 mM imidazole aqueous solution, 100 mM imidazole aqueous solution, 150 mM imidazole aqueous solution, and 200 mM imidazole aqueous solution. Collect the effluent after elution with 100mM imidazole aqueous solution, ultrafiltration membrane (1...

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Abstract

The invention provides a stereoselective recombinant esterase, encoding gene, carrier, engineering bacteria and its application for splitting (R, S)-indoline-2-formic acid ethyl ester; using recombinant Escherichia coli or recombinant esterase Simultaneous catalytic resolution of ethyl (R,S)-indoline-2-carboxylate as a biocatalyst to generate (S)-indoline-2-carboxylic acid, the conversion rate is about 45%, and the optical purity of the products is 96% above.

Description

[0001] (1) Technical field [0002] The invention relates to a stereoselective esterase and its coding gene, a carrier containing the coding gene, engineering bacteria and application thereof. [0003] (2) Background technology [0004] (S)-Indoline-2-carboxylic acid and some of its compounds are the intermediates of many new drugs in the current experimental and clinical stages. For example, perindopril, a cardiovascular therapeutic drug invented by the French Servier Company, is used for the treatment of essential hypertension and heart failure. Among these active groups with efficacy in treating cardiovascular diseases, the characteristic structure of (S)-indoline-2-carboxylic acid or its hydrogenation product is generally contained. [0005] At present, the synthesis of (S)-indoline-2-carboxylic acid reported in the literature is mainly based on the synthesis of optically active starting materials, the synthesis of chiral auxiliaries, and the resolution of chemical reagent...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/16C12N15/55C12N15/70C12N1/21C12P41/00C12P17/04C12R1/19
CPCC12N9/16C12N15/70C12P17/04C12P41/00
Inventor 章银军陈昌盛郑建永吴石金
Owner ZHEJIANG UNIV OF TECH
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