| Title | [Enzyme activity during reteplase production phase by recombinant Pichia pastoris] | | Author(s) | Zou M, Chu J | | Institution | School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, China. zoum2000@sina.com | | Source | Nan Fang Yi Ke Da Xue Xue Bao 2008 Aug; 28(9):1534-7. | | MeSH | Alcohol Oxidoreductases
Fermentation
Genetic Vectors
Glucosephosphate Dehydrogenase
Methanol
Pichia
Recombinant Proteins
Tissue Plasminogen Activator
| | Abstract | 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. | | Language | chi | | Pub Type(s) | English Abstract
Journal Article
Research Support, Non-U.S. Gov't
| | PubMed ID | 18819861 |
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