Sucrose phosphorylase capable of realizing extracellular secretory expression as well as preparation method and application of sucrose phosphorylase
A sucrose phosphorylase and extracellular secretion technology, applied in the field of enzyme engineering, can solve the problem of low activity of extracellular sucrose phosphorylase, and achieve the effect of efficiently inducing the expression of extracellular secretion
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Embodiment 1
[0046] Example 1 Recombinant bacteria expressing sucrose phosphorylase
[0047] In the present invention, the sucrose phosphorylase gene BaSP derived from Bifidobacterium animalis subsp. lactis is connected with a signal peptide N20 to construct a recombinant expression plasmid pET-N20-BaSP and transform Escherichia coli BL21 (DE3 ), achieving the efficient induction of extracellular secretion and expression of BaSP in E. coli.
[0048] Recombinant bacteria expressing sucrose phosphorylase
[0049] Using the genome of Bifidobacterium animalis subsp.lactis BB-12 as a template, the BaSP gene was amplified by polymerase chain reaction (PCR) using primers BaSP-F and BaSP-R. PCR conditions were as follows: after denaturation at 94°C for 5 min, 25 cycles consisted of 30s at 94°C, 30s at 60°C and 1 min at 72°C, followed by extension at 72°C for 5 min. Using primers N20-F and N20-R as templates and primers for each other, the N20 signal peptide sequence was amplified by PCR. PCR co...
Embodiment 2
[0056] Example 2 Induction and secretion expression of sucrose phosphorylase
[0057] The recombinant bacteria BL21-N20-BaSP was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of liquid LB medium, and was activated and cultured in a shaker at 37°C overnight to prepare a seed solution for sucrose phosphorylase fermentation.
[0058] The seed solution was inoculated into autoinduction medium (5g / L glycerol, 10g / L peptone, 5g / L yeast extract, 2.31g / L KH2PO4, 12.54g / L K2HPO4·12H20, 2g / L Lactose, 0.4g / L glucose, 50mg / L kanamycin; liquid volume 50mL / 250mL Erlenmeyer flask), shake cultured at 37°C for 24 hours. The fermentation broth without any treatment is the whole fermentation broth. The whole fermentation broth was centrifuged at 12,000 rpm for 5 min to remove the bacterial cells of recombinant E. coli, and the fermentation broth supernatant was obtained. The centrifuged cells were washed with deionized water and resuspended in PBS buffer. The resuspended cells we...
Embodiment 3
[0060] Example 3 Effects of pH and temperature on sucrose phosphorylase
[0061] Ammonium sulfate was slowly added to the supernatant of the fermentation broth pre-cooled in an ice bath until the saturation was 60%, and the precipitate was collected by centrifugation at 4°C and 12000 rpm for 10 min. The precipitate was dissolved in an appropriate amount of MES buffer (pH 6.5), which was the crude enzyme solution of sucrose phosphorylase.
[0062] In order to study the effect of different pH and temperature on the sucrose phosphorylase, its activity was measured at different temperatures (20-70°C) and pH conditions (pH 3.5-8.0). In order to study the effect of different pH on the stability of sucrose phosphorylase, the above crude enzyme solution was diluted with different buffers (pH 3.5-8.0) and then incubated at 50°C for 1 h. In order to study the effect of different temperatures on the stability of sucrose phosphorylase, the sucrose phosphorylase was incubated at different...
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