[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.
Zou
Min
M
School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, China. zoum2000@sina.com
Chu
Ju
J
D000429:Alcohol Oxidoreductases / Q000378:metabolism; D005285:Fermentation; D005822:Genetic Vectors; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D000432:Methanol / Q000494:pharmacology; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; C087896:reteplase; D000429:Alcohol Oxidoreductases; D005954:Glucosephosphate Dehydrogenase; C019643:alcohol oxidase; D010959:Tissue Plasminogen Activator; D000432:Methanol