Identification, Characterization and Partial Purification of Acid Phosphatase from Cotyledons of Psoralea corylifolia L.

In the present study, characterization, isolation and purification of Acid phosphatase from the cotyledons of Psoarlea corylifolia was carried out. The characterization of acid phosphatase includes the optimization of kinetic parameters over the range of substrate concentrations, influence of different divalent metal ions, modulators, various inorganic and organic phosphates on the acid phosphatase mediated hydrolysis of p-Nitrophenyl phosphates. The purification processes included the ammonium sulphate precipitation, DEAE cellulose separation and gel filtration of enzyme through sepharose column. The crude acid phosphatase enzyme showed optimum pH of 5.5 at 37C in 0.1 M sodium acetate buffer and optimum temperature of 50C. Km for the substrate p-nitrophenyl Phosphate (p-NPP) was 0.775 mM at optimum pH. Addition of other phosphate substrates such as KH2PO4 and ATP reduced the p-NPP hydrolysis, however, NaH2PO4 and G-6-P did not influence the p-NPP hydrolysis. CuSO4, Sodium flouride (NaF) and Sodium molybdate (NaMoO4) strongly inhibited the acid phosphatase activity. Partially purified acid phosphatase protein (226.08 fold) obtained by ammonium sulphate precipitation and gel filtration exhibited very high specific activity (264211.157 mole min-1 mg-1). Acid phosphatase showed spots at ~28 kDa and ~30 kDa in two dimensional gel electrophoresis followed by western blot analysis.