Purification, N-Terminal Amino Acid Sequence and Characterization of pH 2. 5 Optimum Acid Phosphatase (E.C. 3.1.3.2) from Aspergillus Ficuum

Abstract

An add phosphatase from crude culture filtrate of Aspergillus ficuum was purified to homogeneity using three ion exchange chromatographic steps. SDS-PAGE of the purified enzyme gave a single stained band at approximately 68-KDa. The mobility of the native enzyme In gel filtration chromatography, however, indicated that the molecular mass to be about 130-KDa Implying the active form to be a dimer. On the basis of a molecular mass of 68-KDa, the molar extinction coefficent of the enzyme at 280 nm was estimated to be 3. 4 X10⁵ M⁻¹ cm⁻¹. The Isoelectric point of the enzyme, as judged by chromatofocusing, was about 4. 0. The purified enzyme 1s highly stable at 0°C. Thermal inactivation studies have indicated that the enzyme is unstable at 70°C. The enzyme, however, exhibited a broad temperature optima with a maximum catalytic activity at 63°C. The K_m of the enzyme for p-nitrophenylphosphate is about 270 μ;M with an estimated turnover number of 2550 per sec. The enzyme is a glycoprotein as evidenced by the positive PAS staining; the sugar composition suggests the presence of N-linked high mannose-ollgosaccharldes. A partial N-termlnal amino add sequence up to the twenty-third residue was obtained. The enzyme was Inhibited competitively by Inorganic orthophosphate (K1 = 185 μ;M) and non- competitively by phosphomydn (K1 = 600 μM).