Recombinant Pichia pastoris for producing chymosin, and application thereof
A technology of Pichia pastoris and recombinant yeast, applied in the field of enzyme engineering, to achieve the effect of improving enzyme activity, reducing loss, and simple purification steps
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Embodiment 1
[0033] (1) Cloning of Bacillus amyloliquefaciens chymosin cMCE
[0034] Grow Bacillus amyloliquefaciens JNU002 to OD in 500mL shake flasks 600 At about 3.0, take 1.5mL fermentation broth, centrifuge at 12000rpm for 30s, and wash twice with sterile water. Add 600 μL of sterile water to resuspend, then add 15 μL of lysozyme (10 mg / mL), and bathe in water at 37°C for 30 minutes. After adding 60 μL of 10% SDS and mixing, immediately add 10 μL of proteinase K (20 mg / mL), and bathe in water at 65° C. for 2 to 6 hours. Take 600 μL of the treatment solution in a new tube, add an equal volume of phenol chloroform, centrifuge at 12,000 rpm for 5 minutes, take the supernatant, add an equal volume of phenol chloroform, and extract 2 to 4 times until the white precipitate is less, take the supernatant, add an equal volume Chloroform, extracted 2 times. Mix 1 / 10 of the volume of potassium acetate (10mol / L), add 2.5 times the volume of absolute ethanol to precipitate the chromosomes, dry ...
Embodiment 2
[0052] Carry out the rennet fermentation under the same condition by the method of embodiment 1, difference is, the peptone of 2,4,6,8,10,12,14,16,18g / L is respectively fed every 12h, compares different peptone concentrations Effect on chymosin activity. Such as Figure 5 As shown, when the concentration of fed peptone was increased to 12-14g / L, the chymosin activity reached a maximum value of 122SU / L, which was 6.8 times higher than that of the control (fermented with BMMY medium). When the concentration of peptone is too high, the viscosity of the fermentation broth will increase, and the dissolved oxygen per unit volume will decrease, which will affect the growth and metabolism of bacteria, resulting in a decrease in enzyme activity.
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