基因重组毕赤酵母表达瑞替普酶过程中的酶活性分析  

Enzyme activity during reteplase production phase by recombinant Pichia pastoris

作　　者：邹敏[1] 储炬[2] 

机构地区：[1]南方医科大学药学院,广东广州510515 [2]华东理工大学生物反应器工程国家重点实验室,上海200237

出　　处：《南方医科大学学报》2008年第9期1534-1537,共4页Journal of Southern Medical University

基　　金：国家高技术研究发展计划"863计划"(2002AA217021;2002AA2Z3451)

摘　　要：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6 h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0～48 h逐渐增加,在第48 h达到最大值6.72 U/mg蛋白,随后逐渐降低,直至放瓶。G-6-PD比活在第2 h～6 h逐渐增加,在第6～24 h逐渐降低,在第24～48 h逐渐升高,48 h后又逐渐降低直至放瓶。PDC比活在第0～6 h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24～48 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Ⅰ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。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-bee suspensions, in which the activities of AOX, FAD, PDC, G-6-PD, ID, ct-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, α-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.

关 键 词：毕赤酵母 瑞替普酶 酶活性 

分 类 号：Q789[生物学—分子生物学] R977.6[医药卫生—药品]