重组Pichia酵母(Muts)发酵过渡阶段关键酶活分析

重组Pichia酵母表达系统的发酵存在从利用甘油为碳源生长到利用甲醇为碳源表达外源蛋白的发酵表达过渡阶段。Pichia酵母过渡阶段碳源代谢途径关键酶酶活分析表明：甲醇诱导3h AOX2酶活为0，4h时突然增加到0．05U，继续诱导，酶活缓慢增加；在过渡阶段甲醛脱氢酶和6-P-葡萄糖脱氢酶分别增加了6．1倍、2．5倍，而丙酮酸脱氢酶和异柠檬酸脱氢酶分别下降为原酶活的29．4％及16．4％，表明甲醇诱导后甲醇完全氧化代谢途径得到强化，而糖酵解途径和三羧酸循环途径代谢作用减弱。

Analysis of Key Enzymes in Metabolic Pathways of Recombinant Pichia pastoris(Muts) at Transition Phase

There exists a transition phase from growth phase with glycerol as carbon source to expression phase with methanol as carbon source when Pichia pastoris was used as heterogous protein expression system. The key enzymes of main metabolism pathways at the transition phase were analyzed in this (paper.) It was shown that alcohol oxidase(AOX2) activity was not detected at 0 h and 3 h when induced with methanol, whereas its activity sharply increased to 0.05 U at 4 h after induction, and AOX2 activity (increased) slowly afterwards. During transition phase formaldehyde dehydrogenase(EC 1.2.1.1 FAD) and glucose-6-phosphate dehydrogenase(EC 1.1.1.49, G-6-P) increased 6.1 times and 2.5 times, while pyruvate dehydrogenase complex(PDC) and isocitrate dehydrogenase(EC 1.1.1.42,IDE) decreased by 70.6% and 83.6%, respectively compared with the activities at the end of growth phase. Therefore, we inferred that the main methanol metabolic pathway was that methanol was completely oxidized to exhaust CO_2 and that metabolic activities of glycolysis pathway and tricarboxylic acids cycle were attenuated when carbon source was changed from glycerol to methanol.