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Cyclohexene formate hydrolase, and mutant, encoding gene, expression vector, recombinant bacteria and application thereof

A technology of cyclohexene carboxylate and coding genes, which is applied in the field of genetic engineering, can solve the problems of unsuitability for industrial production, low enzyme activity, high catalyst cost, etc., achieve good industrial application prospects, high catalyst efficiency, and easy industrial scale-up Effect

Active Publication Date: 2020-10-16
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Compared with the traditional chemical synthesis method, the method of biocatalytic preparation of chiral cyclohexenecarboxylic acid has the advantages of environmental friendliness, mild reaction conditions, and simple operation. Not high, long reaction time and other defects, not suitable for industrial production
The preparation of chiral cyclohexenecarboxylic acid by wild bacteria method has problems such as high enzyme loading, high catalyst cost, serious emulsification of reaction solution, and low yield.

Method used

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  • Cyclohexene formate hydrolase, and mutant, encoding gene, expression vector, recombinant bacteria and application thereof
  • Cyclohexene formate hydrolase, and mutant, encoding gene, expression vector, recombinant bacteria and application thereof
  • Cyclohexene formate hydrolase, and mutant, encoding gene, expression vector, recombinant bacteria and application thereof

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Experimental program
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Embodiment 1

[0027] Embodiment 1: Cloning of cyclohexene carboxylate hydrolase AcEst1 gene

[0028]The strain Acinetobacter sp.JNU9335 was cultured in LB medium, and the total genomic DNA with high purity and large fragments was extracted by CTAB (cetyltrimethylammonium bromide) method. Add appropriate amount of Acinetobacter sp.JNU9335 cells into liquid nitrogen to freeze, grind into powder, add appropriate amount of 2×CTAB extraction buffer (100mmol / L Tris-HCl, pH8.0, 20mmol / L EDTA, 1.4mol / L NaCl, 2% (w / v) CTAB, 40mmol / L mercaptoethanol), keep warm at 65°C for 10min, and shake intermittently. Then add an equal volume of chloroform / isoamyl alcohol, gently invert the centrifuge tube to mix, centrifuge at 8000 rpm for 10 min at room temperature, transfer the supernatant to another centrifuge tube, add an equal volume of chloroform / isoamyl alcohol, and centrifuge upside down The tube was mixed and centrifuged at 8000rpm for 10min at room temperature. Transfer the upper aqueous phase into a...

Embodiment 2

[0033] Embodiment 2: the preparation of the plasmid of recombinant AcEst1 and recombinant bacterium, recombinase

[0034] Using the polymerase chain reaction technique to obtain The nucleotide sequence of AcEst1 was amplified, and the obtained DNA fragment containing the AcEst1 sequence was double-digested with NdeI and XhoI, respectively, and then ligated with the plasmid pET-28a(+) that was also double-digested with NdeI and XhoI. The recombinant plasmid pET-28a(+)-AcEst1 was obtained.

[0035] The obtained recombinant plasmid pET-28a(+)-AcEst1 was transformed into Escherichia coli E.coli BL21, and the recombinant Escherichia coli containing cyclohexene carboxylate hydrolase AcEst1 was constructed and inoculated into the culture medium containing 50 μg / mL kanamycin In LB medium (peptone 10g / L, yeast extract 5g / L, NaCl 10g / L, pH 7.0), shake culture overnight at 37°C, and insert 600mL LB medium according to the inoculum size of 1% (v / v). Place in a 2L Erlenmeyer flask at 37°...

Embodiment 3

[0036] Embodiment 3: the acquisition of mutant A202K / G326A

[0037] Mutant A202K / G326A is a random mutant. The random mutant library of AcEst1 was established by error-prone PCR technology, and the color change of the indicator described in Example 1 under different pH environments was used as a means of high-throughput screening. First design primers at both ends: forward primer 5'-gtgccgcgcggcagccatatgATGGGCGTGTTGAATCAAACTT-3' (SEQ ID NO.5) reverse primer 5'-gtggtggtggtggtgctcgagTTATTTGGCATTCTTATCCCAAAA-3' (SEQ ID NO.6), PCR system (50 μL): rTaq polymerase 0.25 μL, 10×rTaq Buffer 5μL, dNTP 5μL, MgSO 4 2 μL, template plasmid about 100ng, forward primer 2 μL, reverse primer 2 μL, MnCl 2 (10mM) 0.5μL, ddH 2 O to make up to 50 μL.

[0038] PCR reaction program: (1) Denaturation at 98°C for 5min; (2) Denaturation at 98°C for 30s, (3) Annealing at 55°C for 30s, (4) Extension at 72°C for 1min, steps (2) to (4) were performed for 30 cycles in total, Finally, extend at 72°C for 1...

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Abstract

The invention discloses cyclohexene formate hydrolase, and a mutant, an encoding gene, an expression vector, recombinant bacteria and application thereof. The cyclohexene formate hydrolase AcEst1 andthe mutant thereof have the functions of efficiently splitting 3-cyclohexene-1-formate in an enantioselective mode and preparing optically active (S)-3-cyclohexene-1-formic acid. When the concentration of a substrate is up to 2000 mM (about 280 g / L), optical purity of products is higher than 99% and the substrate / catalyst is up to 3500 g / g. Compared with other preparation methods, the product prepared by the method is high in concentration, good in optical purity, high in catalyst efficiency, mild in reaction condition, environment-friendly, simple and convenient to operate and easy to industrially amplify, and thus has a good industrial application prospect.

Description

technical field [0001] The invention relates to a cyclohexene formate ester hydrolase and its mutant, coding gene, expression vector, recombinant bacteria and application, and belongs to the technical field of genetic engineering. Background technique [0002] (S)-3-cyclohexene-1-carboxylic acid [(S)-3-cyclohexene-1-carboxylic acid, (S)-CHCA], molecular formula: C 7 h 10 o 2 , molecular weight: 126.15, boiling point: 118°C / 6mmHg (lit.), CAS No. 5708-19-0. [0003] Chiral 3-cyclohexene-1-carboxylic acid is an important building block unit for the construction of various natural products, chiral drugs and agrochemicals, and can be used to synthesize many natural products and drugs such as new immunosuppressant tacrolimus, anti Tumor drug (+)-Phyllanthocin, insect control attractant (–)-pirapine-B, neuraminidase inhibitor oseltamivir phosphate, and coagulation factor Xa inhibitor edoxaban, etc., with the chiral drug market The demand for chiral cyclohexene-1-carboxylic acid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/70C12N1/21C12P41/00C12P7/62C07C27/02C07C61/22C12R1/19
CPCC12N9/18C12N15/70C12P41/005C12P7/62C07C51/09C07C2601/16C07C61/22C40B40/08
Inventor 倪晔窦哲许国超
Owner JIANGNAN UNIV
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