Ketoreductase and application thereof in synthesis of (1S)-2-chloro-1-(3, 4-difluorophenyl)ethanol
A technology of difluorophenyl and reductase, which is applied in the direction of oxidoreductase, microorganism-based methods, microorganisms, etc., can solve the problems of difficult scale-up production, unfriendly reaction conditions, low ee value, etc., and achieve high concentration and optical purity , easy industrial amplification, and easy operation
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Embodiment 1
[0040] Acquisition of ketoreductase MT-KRED16 gene
[0041](1) Collect soil samples from Dashushan Forest Park in Hefei City, Anhui Province and extract DNA (refer to Chroma Spin TE-1000 for gene extraction method, Clontech Laboratories, Inc., USA), digest the extracted DNA samples with Sau3AI enzyme, and electrophoresis Recover 0.5-4kb DNA fragments after cutting the gel, and connect them to the pUC19 plasmid through the BamHI site to obtain a genomic plasmid library;
[0042] (2) Transform the plasmid library described in step (1) into Escherichia coli E.coli Top10 (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), and spread it on the LB plate with the corresponding ampicillin antibiotic, and select positive clones Inoculate into a 96-deep-well plate with 500 μL LB (containing the corresponding ampicillin antibiotic), incubate at 37°C for 4 hours, and then add isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.2 mmol / L Induction, continue cu...
Embodiment 2
[0045] Cloning of Ketoreductase MT-KRED Gene
[0046] Synthesize primer pair F1 (nucleotide sequence is SEQ ID NO:3) and F2 (nucleotide sequence is SEQ ID NO:4) according to SEQ ID NO:1; PCR 20ul system is as follows: 2mmol / L dNTP 2μL, 10× PCR buffer 2 μL, PCR high-fidelity enzyme 0.5 μL, DNA template obtained in Example 1 1 μL, ddH 2 O 12μL, F1 and F2 each 1μL (5mmol / L). PCR amplification steps are: ①pre-denaturation at 95°C for 5 min; ②denaturation at 98°C for 15 s; ③annealing at 56°C for 30 s; ④extension at 72°C for 45 s; steps ②~④ were repeated 30 times; The PCR product was purified by agarose electrophoresis, and the PCR amplification electrophoresis results were as follows: figure 1 As shown, the target band appears at about 900bp, and the target band is recovered with an agarose gel recovery kit to obtain a complete sequence. After DNA sequencing, the full length is 846bp, which is shown in SEQ.ID.N01 PCR product of the ketoreductase gene.
Embodiment 3
[0048] Expression of ketoreductase MT-KRED16 gene
[0049] The PCR product and the pET21a vector described in Example 2 were simultaneously digested with NdeI / XhoI, purified by agarose gel electrophoresis, and the target fragment was recovered using an agarose gel recovery kit; under the action of T4DNA ligase, The target fragment was ligated with the double-digested pET21a vector to obtain the recombinant expression plasmid pET21a-MT-KRED16.
[0050] Transform the above-mentioned recombinant expression plasmid pET21a-MT-KRED16 into E.coli BL21(DE3) (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) competent cells, spread on LB plates containing 50 μg / mL ampicillin, 37°C After culturing overnight, select positive colonies and inoculate them into 50mL liquid LB medium for cultivation. The composition of liquid LB medium is: peptone 10g / L, yeast extract 5g / L, NaCl 10g / L, pH 7.2; Transfer 10mL of seed solution into 1L of fresh LB liquid medium and cultivate to OD 600 ...
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