A Ketoreductase and Its Application in Asymmetric Synthesis of Chiral Hydroxyl Compounds
A carbonyl compound and reductase technology, applied in the field of bioengineering, can solve problems such as low yield, low optical activity of products, and high purity requirements of substrates
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Embodiment 1
[0049] Example 1 Environmental Ketoreductase Gene Isolation
[0050] Soil samples were collected from Zhujia Village, Situan Town, Fengxian District, Shanghai, and DNA was extracted (extraction method refers to ChromaSpin TE-1000, Clontech Laboratories, Inc., USA), partially digested with Sau3AI, and 0.5-4kb fragments were collected by electrophoresis, recovered and Ligated to the BamHI site of pUC19 to obtain a plasmid library. Transform the library into E.coli DH5α and smear it on an LB plate containing 100 μg / mL ampicillin, select positive clones and inoculate them into a 96-deep-well plate containing 500 μL LB (containing 100 μg / mL ampicillin), and culture at 37°C for 4 Add 1mM IPTG after 1 hour for induction, and continue culturing overnight at 30°C; then take 50μL of each deep-well culture to a new 96-well plate with 50mM sodium phosphate buffer (pH7.5), freeze and thaw repeatedly at -80°C to lyse the bacteria Add 1mM acetophenone as substrate, 10mM glucose, 1 unit of g...
Embodiment 2
[0051] Example 2 Ketoreductase expression
[0052] Primer pairs P1 (nucleotide sequence: SEQ ID NO:3) and P2 (nucleotide sequence: SEQ ID NO:4) were synthesized according to SEQ ID NO:1.
[0053] Use P1 and P2 to amplify the full-length ORF sequence, the PCR system is as follows: 10×KOD-Plus PCR buffer 2μL, 25mMMgSO 4 1.2 μL, 2 mM dNTP 2 μL, KOD-Plus PCR high-fidelity enzyme 0.3 μL, DNA template obtained in Example 1 0.5 μL (containing 0.1 μg DNA template), ddH 2 O 13μL, P1 and P2 each 0.5μL (10mmol / L). PCR amplification steps are: (1) Pre-denaturation at 95°C for 3 minutes; (2) Denaturation at 98°C for 15 seconds; (3) Annealing at 56°C for 30 seconds; (4) Extension at 72°C for 1 minute; steps (2) to (4) repeated 35 times; (5) Continue to extend at 72°C for 10 minutes, then cool to 4°C. PCR products were purified by agarose gel electrophoresis (see figure 1 ), using an agarose gel DNA recovery kit to recover the target band in the 800-900 bp interval, and obtain a complete...
Embodiment 3
[0055] Embodiment 3 enzyme activity assay method
[0056] Enzyme activity (U) was defined as the amount of enzyme required to consume 1 μmol NADPH per minute.
[0057] The method for measuring the enzyme activity (U) is: in 2mL reaction solution, add 2mM acetophenone as substrate, 0.1mM NADPH as cofactor, then add 20μL crude enzyme solution, and measure the decrease rate of OD340 within 1 minute as ΔA340. Specific enzyme activity per mL of enzyme solution U=ΔA 340 ×1000 / (6220×20), that is, the specific enzyme activity per mL of lysate.
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