Purification and Characterization of Acid Phosphatase from Lecanicillium lecanii

• Main Authors: Shu-Yi He, 何書易
• Other Authors: Ping-Lin Ong
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/74180246090990467918

碩士 === 國立嘉義大學 === 微生物免疫與生物藥學系研究所 === 96 === The strong secretion of extracellular acid phosphatase of Lecanicillium lecanii 35549 was detected by modified API ZYM method during 7 days liquid culture. The purpose of this study was to purify and biochemical characterization of the acid phosphatase secreted from L. lecanii. Five isolates of L. lecanii (33570, 33571, 35548, 35549, and 35556) were cultured in solid and liquid medium for 14 days, and the enzymatic production of acid phosphatase and chitinase were measured daily. The data have shown that acid phosphatase has better secreting efficiency in the liquid medium. According to gel activity staining of five isolates acid phosphatase by SDS-PAGE, isolate 35549 was assigned to produce acid phosphatase for the following experiment. Isolate 35549 was shaking cultured in liquid medium at 25℃ and 125 rpm for 9 days, then the medium was collected for enzyme purification. The acid phosphatase was purified to homogeneity with the process of filtrating the culture medium of L. lecanii liquid culture, concentrating medium with Amicon ultra-4 ultrafiltrator (MW cut off: 50 kDa and 10 kDa), and loading protein extracts with MW between 50 kDa and 10 kDa into DEAE-Sepharose ion exchanger. The enzyme was purified 11.2-fold, with a yield of 26%, to a maximum specific activity of 9.028 U/mg. The molecular weight of enzyme was determined to be about 30 kDa by SDS-PAGE with activity staining and silver staining. Assaying the acid phosphatase activity with p-nitrophenyl phosphate as substrate, the activity for this enzyme shown the optimal pH was 5.0. The optimal thermostability of acid phosphatase was around 20~30℃. Acid phosphatase activity was complete inhibited by NaF (5mM) and tartrate (20mM), and the residual activity was between 28.4 and 93.2% among β-mercaptoethanol, EDTA, Na2MO4, KH2PO4, NaN3, NaCl, and CaCl2. However, other metal ions will rarely inhibit the acid phosphatase activity. Kinetic parameters of acid phosphatase toward p-nitrophenyl phosphate were Vmax = 0.0004 μmol/min, Km = 0.032 mM, Kcat = 58.18 sec-1, respectively, and the Ki of NaF inhibitor was 0.87 mM. The purified protein spot of the Native-SDS two-dimensional PAGE gel was analyzed trice by LC/MS/MS for homologous sequencing, but all fail.