Mycosystema. 2011, 30(5): 744-752.
To investigate the characteristics of acid phosphatase (ACPase, E.C.3.1.3.2) of Hypsizygus marmoreus, the crude acid phosphatase was extracted from the pileus and stipe of H. marmoreus, and purified by ammonium sulfate fraction precipitation and chromatography on Sephadex G-200. Three enzyme components and four enzyme components were obtained from pileus and stipe of this fungus respectively. Among the all obtained enzyme components of ACPase from pileus or stipe, both enzymeⅠand enzymeⅠ′ showed single electrophoretic bands in PAGE, which indicated the same molecular weight with 65kDa determined by SephadexG-75gelatin filtration. Both enzymeⅠand enzymeⅠ′ showed the maximum UV absorbance at 220nm and 280nm, while their isoelectric point was at 5.4 and 5.0, respectively. The enzymeⅠand enzymeⅠ′ showed its optimum pH of 5.0 and 4.6 with p-nitrophenyl-phosphate (PNPP) as substrate, respectively. Both enzymes showed the same optimal catalytic reaction temperature of 50℃, and could be activated by the dispose of 60℃. The thermoduric experiments indicated that 60.2% of the highest activity of the enzymeⅠand 57.3% of the enzymeⅠ′ was remained respectively, after being heated at the optimum temperature for 45mins, and moreover, enzymeⅠshowed its highest activity when incubated for 20mins while enzymeⅠ′ for 25mins. When incubated at 60℃ for 25mins, enzymeⅠand enzymeⅠ′ just remained 19.33% and 15.25% of its highest enzyme activity, respectively, and incubated for much less time, they remained much higher activity. The initial reaction velocity of enzymeⅠand enzymeⅠ′ was 17.1×10?3μmol/min and 11.3×10?3μmol/min, respectively, and their maximum reaction velocity was 27.6×10?3μmol/min and 85.0×10?3μmol/min, respectively. The Michaelis constant (Km) of enzymeⅠand enzymeⅠ′ was 4.095×10?3mol/L and 36.3×10?3mol/L, respectively. The specificity constant of enzymeⅠand enzymeⅠ′ was Kcat/Km=3.846×105(mol/L)?1S?1 and Kcat/Km=1.11×105(mol/L)?1S?1, respectively, and the activities of the both enzymes were inhibited by heavy metal ions including Hg2+, Pb2+, Ag+ and Cd2+.
