Purification and characterization of secreted acid phosphatase under phosphate-deficient condition in Pholiota nameko

Activity of acid phosphatase secreted by mycelia of Pholiota nameko on cultivation for 30 d in Pi-depleted medium was 88-fold higher than the corresponding activity in the Pi-supplied medium. One isozyme of the secreted acid phosphatases was purified from the culture filtrate of Pi-depleted medium by ammonium sulfate fractionation and cation exchange chromatography. The purified enzyme was homogeneous on electrophoresis. Gel filtration analysis showed that the native molecule had a molecular weight of 117,000. The molecular weight on gel electrophoresis with SDS was 52,000, indicating that the native form of the enzyme was a homodimer. The optimum pH and temperature of the enzyme were 5.5 and 45°C respectively, and the isoelectric point of the enzyme was pH 6.9. Adsorption on Con ASepharose and periodic-Schiff stain suggested that the enzyme is a glycoprotein. The enzyme hydrolyzed a wide variety of phosphate esters, nucleoside phosphates, sugar phosphates, and phosphorylated amino acids. Cu2+, Fe2+ Hg2+, iodoacetate, molybdate, tartaric acid, and SDS inhibited the enzyme activity. Fe3+ (1 mM), Triton X-100, methanol, and ethanol activated it. Fifteen residues of the N-terminal amino acid sequence were determined.