Recombinant escherichia coli for improving enzyme activity of bile salt hydrolase
A technology of recombinant Escherichia coli and bile salt hydrolyzing enzyme, which is applied in the field of bioengineering, can solve the problems of low expression level, unsuitable growth environment and fermentation intensity for large-scale industrial production, easy formation of inclusion bodies, etc., and achieve the effect of simple operation
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Embodiment 1
[0025] The construction of embodiment 1 recombinant escherichia coli
[0026] Using the plasmid pET-28a(+) as the carrier, the bifidobacterium ATCC 27673 bile salt hydrolase gene (NCBI accession number is CP007522.1) was synthesized to construct the recombinant plasmid pET-28a(+)-bsh, which was transformed into E.coil JM109 Competent cells, pick positive colonies. After culturing overnight on a shaker at 37°C, the plasmid was extracted, and after sequencing verification, the correctly sequenced plasmid pET-28a(+)-bsh was transformed into E. coli E.coil BL21(DE3) to obtain recombinant E.coli BL21(DE3) pET-28a(+)-bsh.
Embodiment 2
[0027] The screening of embodiment 2 bile salt hydrolase gene
[0028] (1) Bifidobacterium ATCC 27672 (purchased from ATCC) was used as the starting strain, inoculated into MRS medium containing 0.5% (mass fraction) cysteine, cultured anaerobically at 37°C until logarithmic phase, and 10 μL The bacterial suspension was dropped on the sterilized and cooled glass slide, and the plasma mutagenesis was carried out with the ARTP mutagenesis breeding instrument (Siqingyuan Biotechnology), and the mutagenesis time was 0s, 5s, 10s, 15s, 20s, 40s, 60s.
[0029] (2) Transfer the mutated bacterial suspension to a sterile tube containing 1 mL of normal saline, and then dilute the sample to 10 -1 ~10 -3 Three concentrations were taken, and the diluted solution was spread on the screening medium (LB solid plate containing 0.1g / L porcine bile salt), placed in a constant temperature incubator at 37°C for anaerobic culture for 24-48h, and each treatment was repeated three times.
[0030] (3...
Embodiment 3
[0032] The construction of embodiment 3 recombinant escherichia coli
[0033] Using the plasmid pET-28a(+) as the vector, construct the recombinant plasmid pET-28a(+)-bsh06, transform it into the competent cells of E.coilJM109, and pick the positive colonies. The plasmid was extracted after overnight culture on a shaker at 37°C. After sequencing verification, the correctly sequenced plasmid pET-28a(+)-bsh06 was transformed into E. coli E.coil BL21(DE3) to obtain recombinant E. coli E.coilBL21(DE3)pET -28a(+)-bsh06.
[0034] Induced expression and enzyme activity determination of enzyme protein in embodiment 3 recombinant bacteria
[0035] (1) Recombinant Escherichia coli E.coil BL21(DE3)pET-28a(+)-bsh and E.coil BL21(DE3)pET-28a(+)-bsh06 expressing bile salt hydrolase were inoculated into Cultivate in LB liquid medium at 37°C and 200rpm for 12h, transfer to TB medium with an inoculum size of 1%, and cultivate at 37°C and 200rpm until the cells grow to OD 600 When the temper...
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Abstract
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