Genetic recombination escherichia coli and method for efficiently producing pullulanase
A technology of recombinant Escherichia coli and pullulanase, applied in the direction of microorganism-based methods, biochemical equipment and methods, genetic engineering, etc., can solve the problems of low extracellular enzyme activity and rising production cost of pullulanase
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Embodiment 1
[0053] LB medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0. When needed, add kanamycin (50 μg / mL) before use, and add 15 g / L agar powder to the solid medium.
[0054] pick recombinant Escherichia coli E. coli BL21(DE3) / pET28a(+)-PelB- pul A single colony of positive clones was inoculated in 3 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured overnight at 37 °C with shaking at 200 rpm. Take 1 mL of the culture medium and transfer it to 50 mL of LB liquid medium containing 50 μg / mL kanamycin, and culture at 37°C and 200 rpm until OD 600 About 0.6. The inducer IPTG was added to the culture to a final concentration of 0.8 mmol / L, and the induction culture was carried out at a culture temperature of 30°C for 12 hours. After the cultivation, the fermentation broth was centrifuged at 10,000 rpm for 10 min to obtain the fermentation supernatant for the determination of the extracellular enzyme activity of the strain. Using LB medium and IPTG i...
Embodiment 2
[0056]LB medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0. When needed, add kanamycin (50 μg / mL) before use, and add 15 g / L agar powder to the solid medium.
[0057] pick recombinant Escherichia coli E. coli BL21(DE3) / pET28a(+)-PelB- pul A single colony of positive clones was inoculated in 3 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured overnight at 37 °C with shaking at 200 rpm. Take 1 mL of the culture medium and transfer it to 50 mL of LB liquid medium containing 50 μg / mL kanamycin, and culture at 37°C and 200 rpm until OD 600 About 0.6. The inducer IPTG was added to the culture to a final concentration of 0.4 mmol / L, and the induction culture was carried out at a culture temperature of 30°C for 12 hours. After the cultivation, the fermentation broth was centrifuged at 10,000 rpm for 10 min to obtain the fermentation supernatant for the determination of the extracellular enzyme activity of the strain. Using LB medium and IPTG in...
Embodiment 3
[0059] Autoinduction medium (g / L): β-lactose 10, anhydrous glucose 0.5, glycerol 5, KH 2 PO 4 6.8, MgSO 4 0.24, tryptone 10, yeast extract 5, Na 2 HPO 4 7.1, Na 2 SO 4 0.71, NH 4 Cl 2.67, trace element solution 400 μL / L, pH 7.5~8.0. Trace element solution (g / L): FeCl 3 8.125, CaCl 2 2.22, MnCl 2 2.52, ZnSO 4 1.61, CoCl 2 0.26, CuCl 2 0.27, NiCl 2 0.26, Na 2 MoO 4 0.41, Na 2 SeO 3 0.346,H 3 BO 3 0.124, HCl 2.19.
[0060] Recombinant bacteria E. coli BL21(DE3) / pET28a(+)-PelB- pul The bacterial solution was inoculated in 50 mL of autoinduction medium containing 50 mg / L kanamycin, and cultured at 37°C and 200 rpm for 3 hours with shaking, then added glycine with a final concentration of 6 g / L, and the temperature was changed to 20°C for 60 hours . After the cultivation, the fermentation broth was centrifuged at 10,000 rpm for 10 minutes to obtain the fermentation supernatant for the determination of the extracellular enzyme activity of the st...
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