Medium and method for improving the yield of phospholipase c produced by fermentation of Pichia pastoris
A fermentation medium, the technology of Pichia pastoris, which is applied in the field of medium for improving the production of phospholipase C produced by fermentation of Pichia pastoris, and can solve the problem of high total osmotic pressure of the medium
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Embodiment 1
[0160] Embodiment 1, bacterial strain preparation
[0161] In this experiment, a new eukaryotic expression system of Pichia pastoris was used. According to the partial tropism of codons in the expression system of Pichia pastoris, the artificially synthesized Bacillus cereus (Bacillus cereus) phospholipase C was matured by PLC. The nucleotide (sequence shown in SEQ ID No: 1) is used as a template, and the PLC gene is amplified by polymerase chain reaction (PCR). The target fragment was connected with the expression vector pAO815 plasmid to obtain the recombinant expression plasmid pA0815-PLC, and the recombinant expression plasmid pAO815-PLC containing a single copy of PLC was constructed. ) The auxotrophic type screened out the recombinant yeast GS115-pAO815-PLC. Refer to the specific process: Pichia pastoris expression handbook released by Invitrogen.
Embodiment 2
[0163] 2.1. Preparation of seed solution:
[0164] The recombinant yeast GS115-pAO815-PLC stored in a -80°C refrigerator was streaked on a YPD plate, and cultured at a constant temperature of 30°C for 2 days. Pick a ring of colonies and inoculate them into a 250ml Erlenmeyer flask with baffles containing 30ml of YPD-glycerol liquid medium, and cultivate at 30°C and 200rpm for 20-24h to obtain a seed solution for inoculation in a 6.7L fermenter.
[0165] 2.2. Fermentation culture
[0166] Recombinant Pichia pastoris was cultured in a 6.7L fermenter (RALF-duett6.7L, Biou, Switzerland) and phospholipase C was induced to express with methanol. The expression strategy adopted a three-step fermentation method: glycerol batch culture stage, glycerol fed-batch culture phase and methanol fed-batch induced expression phase.
[0167] (1): Glycerin batch culture stage to amplify the bacteria
[0168] With a 2% inoculum size, the above seed solution was pumped into a sterilized fermenta...
Embodiment 3
[0177] Using fermentation medium 1-1, in which MgSO 4 . 77H 2 The O content was 35g / L, and the rest were the same as in Example 2. The results showed that the fermentation end point was reached after 83 hours of induced expression, and the induced expression period was 83 hours.
[0178] The wet weight test results of the bacteria during the fermentation process are as follows: Figure 1-2 As shown, the detection results of phospholipase activity are as follows Picture 1-1 As shown, the phospholipase activity at the end of the fermentation, the protease activity, and the detection results of the total protein content are shown in Table 1, and the SDS electrophoresis results of the fermentation broth (10-fold dilution) of cultivating 78h, 96h and 114h are shown in Table 1. Figure 2-2 shown.
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