A serine protease and its application in loquat preservation
A serine protease and loquat technology, applied in the field of serine protease and its application in loquat preservation, can solve the problems of pathogenic bacteria that are prone to drug resistance, pollute the environment, and endanger human health
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Embodiment 1
[0026] The Bacillus amyloliquefaciens MG-3 stored in glycerin was taken out from the ultra-low temperature refrigerator, quickly thawed, spread on LB plates, and placed in a biochemical incubator at 37°C for 24 hours. The colony MG-3 on the plate was picked and inoculated into the seed medium, and cultured in a shaking incubator at 37°C and 170r / min for 12h. Take 3% of the seed solution and inoculate it into the fermentation medium, and culture it in a shaking incubator at 37°C and 170r / min for 72h to prepare the fermentation solution.
[0027] The prepared fermentation broth was centrifuged at low temperature (4°C, 10000g) for 10min, the supernatant was collected, and the bacteria and residue were discarded. Put the collected supernatant into ice water, and add solid ammonium sulfate while stirring slowly until the ammonium sulfate reaches 20% saturation. After standing still in the refrigerator at 4°C for 2h, centrifuge at low temperature and high speed (4°C, 11000g) for 10m...
Embodiment 2
[0032] (1) Sample preparation: chop up the single strip with glue obtained after separation and purification and transfer it to an EP tube, add 400 μL 100 mmol / L NH 4 HCO 3 / 30%ACN decolorization solution, wash repeatedly until the decolorization becomes transparent, discard the supernatant, add 100mM NH 4 HCO 3 , incubate at room temperature for 15 min; discard the supernatant, lyophilize, then add 5 μL of 10 ng / μL sequencing-grade Trypsin (Promega) solution (the mass ratio of enzyme to protein to be analyzed is generally 1:20-1:100), react overnight at 37°C; aspirate Transfer the enzymatic solution to a new EP tube, add 100 μL of 60% ACN / 0.1%TFA to the original tube, sonicate for 15 minutes, and freeze-dry the combined enzymatic solution; if there is a lot of salt, use Ziptip (millipore) to desalt.
[0033] (2) Mass spectrometry operation steps: take 2 μL of 20% acetonitrile to redissolve the freeze-dried enzymatic sample. Take 1 μL of dissolved sample and directly spot o...
Embodiment 3
[0037] (1) Effect of temperature on the antibacterial effect of antibacterial protein
[0038] Dissolve the purified antimicrobial protein freeze-dried into powder in phosphate buffer (20mmol / L pH7.4) with a concentration of 40ug / mL, distribute it in 1.5mL EP tubes, each tube contains 1mL, and store them at 0°C , 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, and 100°C for 30 minutes, and the same batch of untreated samples was used as a control to determine the antibacterial activity of the antibacterial protein. The result is attached by Figure 5 It can be seen that the antibacterial activity of the treatment temperature from 0°C to 30°C gradually increased, indicating that low temperature has a certain inhibitory effect on the antibacterial protein, but the antibacterial protein can also maintain more than 70% of the activity at low temperature; with the increase of the treatment temperature, the antibacterial protein The antibacterial activity of the protein has a certain failure, ...
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