毕赤酵母表达重组红林蚁AChE高密度发酵工艺研究  

Study on High-density Fermentation Process of Expression of Recombinant AChE in Formica sinae by Pichia pastoris

作　　者：孙召伟 赵小中[2] 李倩 胡波[2] 翁海波 SUN Zhaowei;ZHAO Xiaozhong;LI Qian;HU Bo;WENG Haibo(School of Life Sciences,Zhengzhou University,Zhengzhou,Henan 450001;Zhengzhou Agricultural Products Quality Inspection and Circulation Center,Zhengzhou,Henan 450003,China)

机构地区：[1]郑州大学生命科学学院,河南郑州450001 [2]郑州市农产品质量检测流通中心,河南郑州450003

出　　处：《贵州农业科学》2024年第8期121-126,共6页Guizhou Agricultural Sciences

基　　金：国家自然科学基金项目(81970173)。

摘　　要：【目的】优化毕赤酵母高密度生产重组红林蚁乙酰胆碱酯酶(AChE)的发酵条件,为AChE在农药残留检测中应用提供酶源。【方法】采用毕赤酵母表达系统,选用高表达AChE的重组子X33/pPICZαAAChE,对毕赤酵母高密度生产重组红林蚁AChE的发酵条件进行优化,确定最佳发酵pH值、温度、溶解氧、补料流加速率等因素。【结果】毕赤酵母表达重组红林蚁AChE高密度发酵工艺的最佳参数:pH5.0、溶解氧30%、温度29℃、转速500 r/min、罐压0.06 MPa,甲醇诱导72~96 h时发酵液上清AChE活性增长最快。从发酵结果看,发酵结束菌体湿重可达280 mg/mL,发酵罐内甲醇诱导96 h酶活性最高,达6 000 U/mL,是摇瓶诱导表达的8倍。【结论】毕赤酵母表达系统最佳发酵工艺能实现红林蚁AChE的高密度发酵生产,降低AChE提取成本。【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 highdensity 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 ℃,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 to96 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 96hours 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.

关 键 词：乙酰胆碱酯酶 毕赤酵母 发酵工艺 酶活性 有机磷 氨基甲酸酯 农药残留 

分 类 号：S481.4[农业科学—农药学] Q946.3[农业科学—植物保护]