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.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
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)
