Study on High-density Fermentation Process of Expression of Recombinant AChE in Formica sinae by Pichia pastoris
[Objective]The fermentation condition of acetylcholinesterase(AChE)in Formica sinae that was produced and recombined with high-density by Pichia pastoris was optimized to provide the enzyme source when applying AChE in the detection of pesticide residue.[Method]The expression system of P.pastoris was used and the high-level expressed recombinant X33/pPICZαA-AChE from AChE was chosen to optimize the fermentation condition of high-density production and recombination of AChE in F.sinae by P.pastoris,so that the factors influencing the optimal fermentation,including pH value,temperature,dissolved oxygen and feeding flow rate,were determined.[Result]The optimal parameters of high-density fermentation process for expression of recombinant AChE in F.sinae by P.pastoris were as follows:pH at 5.0,dissolved oxygen at 30%,temperature at 29 oC,rotating speed at 500 r/min,tank pressure at 0.06 Mpa.And the AChE activity in the supernatant fermentation broth increased the fastest when induced by methanol for 72 to 96 hours.The fermentation result revealed that the wet weight of thalli reached to 280 mg/mL when the fermentation finished,and the enzyme activity was the highest(6 000 U/mL)as the methanol induced in the fermentation tank for 96 hours and was 8 times more than that induced by shaker bottle.[Conclusion]The optimal fermentation process for expression system of P.pastoris can realize the high-density fermentation and production of AChE in F.sinae and reduce the extraction costs of AChE.
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