毕赤酵母高密度表达重组猪胰岛素前体的研究

High-density Expression of Recombinant Porcine Insulin Precursor by Pichia pastoris

摘要:

对摇瓶和50L罐上的重组菌毕赤酵母(Pichia pastoris)表达猪胰岛素前体(PIP)的发酵过程进行了研究。摇瓶发酵中,最佳诱导周期为60 h左右,诱导期甲醇的最佳加入量为每日2．0%～2．5%。50L发酵过程分为批发酵、补料和诱导表达3个阶段。生长期(批发酵和补料阶段)细胞干重与培养时间的关系可用模型y= 0．6525e^0．1909t来描述。在批发酵阶段和补料阶段,流加的氨水和甘油几乎全部用来合成菌体和维持,没有其他副产物产生。诱导表达阶段流加的氨水和甲醇分别约有80%和70%被菌体利用。将摇瓶与发酵罐的实验结果进行了比较,发现摇瓶发酵的限制因子很可能是溶氧,而罐发酵的限制因子为碳源,因此,将摇瓶实验的结果放大到发酵罐时调整了控制策略,加大了甲醇的补料速率,最终PIP浓度达到1．72g/L。

Abstract:

The recombinaut porcine insulin precursor(PIP)produced by Pichia pastoris in shake-flask and 501.fermenter was investigated respectively.The results indicated that 60h induction time length and 2.0%～2.5% methanol addition every day was optimum in shake- flask.The process in 50L fermenter was consisted of batch,feed-batch and induction phases.The relationship between dry cell weight(y) and culture time (t) in growth phase(batch and feed-batch phase)could be described by model y=0.6525e^0.1907t.Glycerol and ammonia were almost used for cell growth and maintain,and no by-product was observed in batch and fed-batch phase.Only 80% ammonia and 70% methanol were used by cell in induction phase. By comparison the results of shake-flask and 50L fermenter,it was concluded that the limit-ing factor in the fermentation of shake-flask and 50L fermenter was dissolved oxygen (DO) and carbon source,respectively. When scaling the result of shake-flask to 50L fermenter,the control strategy was adqpted for 50L fermenter by increasing the feed rate of methanol and the maximum PIP concentration reached 1.72g/L.