基因重组毕赤酵母表达瑞替普酶过程中的酶活性分析

目的 研究毕赤酵母诱导表达瑞替普酶过程中的关键酶活性.方法 以摇瓶培养为研究对象,在用甲醇诱导后,连续取样,破碎菌体制成无细胞悬液,检测乙醇氧化酶、甲醛脱氢酶、PDC、G-6-PD、ID、α-KGD和SD的活性.结果 乙醇氧化酶比活在0～6 h逐渐增加,在第6h达到最大44.5 U/mg蛋白.随后迅速下降.在第24～48 h有所回升,然后又逐渐降低.FAD比活在第0～48h逐渐增加.在第48h达到最大值6.72U/mg蛋白,随后逐渐降低.直至放瓶.G-6-PD比活在第2h～6h逐渐增加,在第6～24h逐渐降低,在第24～48h逐渐升高,48h后又逐渐降低直至放瓶.PDC比活在第0～6h逐渐降低,随后略有升高的趋势.ID、α-KGD、SD的活性变化有相似趋势.在前6 h酶活均迅速下降,在第6～24 h缓慢下降,随后ID活性继续缓慢下降,而α-KGD和SD活性在第2～48 h逐渐升高,在第48 h均达到最高值,然后又逐渐降低,直至放瓶.结论 根据酶活变化规律,可将整个诱导期分为4个阶段:第1阶段为诱导0～6 h,是甲醇适应期;第Ⅱ阶段为诱导6～24h,是快速生长期;第Ⅲ阶段为诱导24～48h,是产物积累期;第Ⅳ阶段为诱导48～72h,是代谢缓慢期.在甲醇适应期,甲醇完全氧化代谢流占主导地位.在快速生长期和产物积累期,代谢流逐渐向糖酵解途径和TCA循环途径迁移.

Enzyme activity during reteplase production phase by recombinant Pichia pastoris

OBJECTIVE: To study the activities of the key enzymes in reteplase production by Pichia pastoris. METHODS: In shaking flasks, a series of samples were maintained after methanol induction. The cells were sonicated to prepare cell-free suspensions, in which the activities of AOX, FAD, PDC, G-6-PD, ID, alpha-KGD and SD were measured. RESULTS: The specific activity of AOX increased during the initial 6 h, reaching the maximum of 44.5 U/mg protein. The activity decreased quickly between 6 and 24 h, followed by increment in the following 24 h and decreased afterwards. The specific activity of FAD increased gradually in the initial 48 h and then decreased, with the peak level of 6.72 U/mg protein occurred at 48 h. The specific activity of G-6-PD increased at in 2-6 h and 24-48 h, but decreased in 6-24 h and after 48 h. The specific activity of PDC decreased during the initial 6 h and increased slowed afterwards. The specific activities of ID, alpha-KGD and SD all showed a rapid decrease in the initial 6 h and a slow decrease in 6-24 h. After 24 h, the activity of ID continued to decrease, but the other two increased in the following 24 h and then decreased, reaching the maximum at 48 h. CONCLUSIONS: According to the changes of these enzyme activities, the whole induction phase can be divided into 4 stages: the methanol-adaptive period in the initial 6 h, the fast growth period between 6 and 24 h, the product accumulation period in 24-48 h and the metabolism lag period in 48-72 h. In the methanol-adaptive period, complete oxidation of methanol is the dominant pathway. But in the following two stages, the metabolic pathway shifts towards glycolysis and TCA cycle.