| Summary: | 碩士 === 國立陽明大學 === 生物化學研究所 === 87 === The wild type Pichia pastoris strain GS115 was found to contain extracellular phytase activity. Cells grown in rich medium depleted of inorganic phosphate expressed higher extracellular phytase activity. After enzyme activity staining, the phytase activity was found to be originally from the secretory acid phosphatase Pho1p. It is a glycoprotein with a molecular weight of approximately 80-100kDa, and about 52kDa as a deglycosylated form. Most Pho1p was released from the cell wall with lyticase and it was precipitated from the cell-wall extract with ethanol. It was purified to homogeneity by chromatography on Q-Sepharose fast flow. The general biochemical properties of Pho1p were characterized using phytate as the substrate. The pH and temperature optima of Pho1p were 2.5 and 55℃, respectively. Thermal inactivation studies have shown that the enzyme retained 92.26% and 81.45% of its activity after being subjected to 75℃ and 77℃ for 10 minutes. The Km of the enzyme for phytate is about 292μM, with an estimated Kcat of 869 s-1. Compared with Aspergillus niger phytase, Pho1p possesses a higher specific activity but lower affinity for phytate. Thus, the PHO1 gene encoding Pho1p was cloned from the yeast, and mutants V78K and T332N were obtained by site-directed mutagenesis on the basis of sequences alignment with A. niger phytase gene to improve the substrate binding ability. Using methanol-inductive protein expression system, the two mutants produced glycosylated proteins of 67-80kDa, and deglycosylated proteins of 52kDa. Characterization of the two mutants are currently underway.
|
|---|
