An engineering bacterium producing human gastric alcohol dehydrogenase and its preparation method
A technology for alcohol dehydrogenase and ethanol, applied in the biological field, can solve the problems of complicated procedures, high cost, long process time and the like, and achieve the effects of simplifying operation steps, improving efficiency and reducing ethanol content
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Embodiment 1
[0037] Embodiment 1: Construction of recombinant expression vector
[0038] In order to facilitate the subcloning of the target gene adh7, first connect it to the pMD19T-simple vector, transform E.coliJM109, select the positive clone for enzyme digestion verification, name the positive clone pMD19-T-adh7 and perform sequencing verification, and sequence The correct plasmid was digested with double enzymes to obtain the target gene (the amino acid sequence of the target gene is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 2), and it was combined with pET-32a ( +) After ligation, transform E.coli BL21(DE3) and smear the bacterial solution on LB solid medium containing 100 μg / L ampicillin, culture at 37°C overnight, and select transformants for colony PCR verification. PCR reaction conditions: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 1 min and 20 s, 29 cycles; final extension ...
Embodiment 2
[0039] Example 2: Induced expression in Escherichia coli
[0040]Pick the recombinant expression plasmid (pET-32a-adh7) and inoculate it in 3 mL of LB liquid medium containing 100 μg / L ampicillin, culture overnight at 37°C with shaking at 200 r / min, and then expand the culture at an inoculum size of 1:100 , when the bacterial solution concentration reaches OD 600 When the value is 0.6, add IPTG to a final concentration of 0.1 mmol / L, and continue culturing at 30°C for 4h.
Embodiment 3
[0041] Example 3: Preparation of inclusion bodies
[0042] The bacterial solution was centrifuged to remove the supernatant, and the bacterial cells were resuspended with 50mmol / L Tris-HCl, 150mmol / LNaCl, 10mmol / L EDTA, pH8.0 disruption buffer and ultrasonically disrupted in an ice bath for 10min (ultrasound 3s, interval 6s, power 350W), centrifuge at 8000r / min for 15min at 4°C, and harvest the crude inclusion body precipitate. The precipitate was washed once with 50mmol / L Tris-HCl, 150mmol / LNaCl, 0.5% Triton X-100, 1mmol / L EDTA, pH 8.0 washing buffer, then washed once with water, centrifuged at 8000r / min for 15min at 4°C, The harvested precipitate is the purified δδ-ADH inclusion body.
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