Method for preparing high-yield leucine aminopeptidase through fermentation of recombinant bacillus subtilis
A technology of Bacillus subtilis and leucine aminopeptidase, which is applied in the fields of fermentation and enzyme preparations, can solve the problems of difficult industrial application, low yield and the like, and achieves the effect of improving capacity
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Embodiment 1
[0038] Bacterial species: Recombinant Bacillus subtilis (containing the PMA5 plasmid linked to the full sequence of the aminopeptidase gene from Bacillus subtilis Zj016, the host strain Bacillus subtilis WB600).
[0039] Seed medium: sodium chloride 10 g / L, tryptone 10 g / L, yeast extract 5 g / L.
[0040] Seed culture: Inoculate the recombinant Bacillus subtilis strains stored in glycerol tubes at -40°C into the seed medium, and cultivate them on a rotary constant temperature shaker with the speed of the shaker controlled at 220 rpm and the culture temperature at 37°C for 10 hours.
[0041] Add enzyme production promoters (osmotic pressure regulators, surfactants, organic acid salts) in a certain amount to the unadjusted fermentation medium (soluble starch 6 g / L, soybean meal 20 g / L, yeast extract 18 g / L, glycerin 2mL / L, dipotassium hydrogen phosphate 6 g / L, cobalt chloride 0.6 mmol / L, potassium chloride 0.1 mmol / L), after inoculating 5% seed solution, use a rotary Cultivate ...
Embodiment 2
[0049] Bacterial classification, seed culture condition and seed culture medium are with embodiment 1.
[0050] Fermentation medium: soluble starch 20 g / L, soybean meal 20 g / L, yeast extract 18 g / L, glycerin 2mL / L, dipotassium hydrogen phosphate 6 g / L, cobalt chloride 0.6 mmol / L, potassium chloride 0.1 mol / L, sodium glutamate 0.6%, potassium tartrate 0.7%, Tween-80 2.4 mL / L.
[0051] (1) Effect of initial sugar concentration on fermentation culture
[0052] Cultivation method: Inoculate 5% of the cultivated seed culture solution into a 7L fermenter for fermentation (the liquid volume is 4L), the fermentation temperature is 37°C, and sterile air is introduced during the fermentation process, and the ventilation rate is 1.2:1, the minimum dissolved oxygen is maintained at 30%, the dissolved oxygen is coupled with the rotation speed, the stirring speed is controlled at 250-450rpm, and the fermentation and cultivation time is 30h. During the fermentation process, samples were ta...
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