PENICILLIUM-CHRYSOGENUM EXTRACELLULAR ACID-PHOSPHATASE - PURIFICATION AND BIOCHEMICAL-CHARACTERIZATION

An extracellular acid phosphatase (EC 3.1.3.2) from crude culture filtrate of Penicillium chrysogenum was purified to homogeneity using high-performance ion-exchange chromatography and size-exclusion chromatography. SDS-PAGE of the purified enzyme exhibited a single stained band at an M(r) of approx. 57 000. The mobility of the native enzyme indicated the M(r) to be 50 000, implying that the active form is a monomer. The isoelectric point of the enzyme was estimated to be 6.2 by isoelectric focusing. Like acid phosphatases from several yeasts and fungi the Penicillium enzyme was a glycoprotein. Removal of carbohydrate resulted in a protein band with an M(r) of 50 000 as estimated by SDS-PAGE, suggesting that 12% of the mass of the enzyme was carbohydrate. The enzyme was catalytically active at temperatures ranging from 20-degrees-C to 65-degrees-C with a maximum activity at 60-degrees-C and the pH optimum was at 5.5. The Michaelis constant of the enzyme for p-nitrophenyl phosphate was 0.11 mM and it was inhibited competitively by inorganic phosphate (k(i) = 0.42 mM).