毕赤酵母流加培养诱导生产植酸酶的流加控制策略

Substrate and Inducer Feeding Strategies for Fed-batch Phytase Expression Process by Recombinant Pichia pastoris

对毕赤酵母流加培养生产植酸酶过程中的底物和诱导剂流加策略和性能进行了比较研究。在生长期采用基于DO/pH在线测量的人工神经网络状态模式识别控制法（ANNPR—Ctrl）特别有效。它可大幅度提高细胞生产强度，并使以甲醇为诱导剂的植酸酶诱导生产时刻提前。然而，在诱导期ANNPR—Ctrl和pH—Stat法等均不能有效地提高植酸酶酶活。为此，在生长阶段采用ANNPR—Ctrl法，当菌浓达到一个较高水平后，引入一个5～6h的过渡期，并在此阶段继续利用ANNPR—Ctrl法流加葡萄糖和甲醇的混合物，使细胞对甲醇逐步产生适应。最后，利用纯甲醇和离线检测甲醇的控制方法开始诱导。在此阶段，离线控制可粗略地将甲醇浓度控制在5～15g/L的范围内，酶活高达320U/mL，比3种基本控制方式提高了5倍。同时，ANNPR—Ctrl法和离线检测甲醇控制模式的有效组合还缩短了酶活达到最高水平的时间。

In this paper, the comparison and performance evaluation of different substrate and inducer feeding strategies for fed - batch phytase expression process by Pichia pastoris were conducted. The pH/DO on - line measurements based ANNPR - CTRL （Artificial Neural Network Pattern Recognition based Control） strategy was particularly effective in the cell growth phase. It could enhance the cell productivity and shorten the phytase expression starting time. However, the ANNPR - CTRL strategy, pH- Star method, and others could not effectively increase the phytase activity. As a result, we adopted the ANNPR- CTRL strategy in cell growth phase to increase cells concentration as quick as possible. After cells concentration reaching a relatively high level, a transient period of 5 - 6h was adopted by simultaneously feeding a mixture of glucose and methanol by ANNPR - CTRL strategy so that the Pichia pastoris cell could gradually adapt the environment using methanol as the inducer. Then, the induction was formally initiated by adding pure methanol with the control mode based on off- line methanol detection. With this method, the methanol concentration could be roughly controlled in a range of 5-15g/L in the induction phase. Under this condition, the phytase activity of 320U/ml, which was about 5 - fold higher than those of other control strategies, was achieved. At the same time, the combination of ANNPR- CTRL and off- line methanol detection based control could also shorten the time when the highest phytase activity appeared.