Acid phosphatases excreted by Humicola lutea 120-5 in casein-containing medium

Abstract

The fungus Humicola lutea 120-5 cultivated in casein-containing media, in the presence or absence of inorganic phosphate (Pi), excretes three different molecular forms of acid phosphatase (with Mr values of approximately 140, 70 and 35 kDa). The enzyme forms were isolated and purified 30–100-fold by a procedure involving two steps of ion-exchange chromatography and Sephadex G-200 gel chromatography. It was found that the fungus excretes only one of the phosphatases with the highest Mr (140 kDa) during growth on medium with inorganic nitrogen source (NaNO3). This form (designed AcPh I) was assumed to be a constitutive, since it showed resistance to high Pi-concentrations (10 mM) and its biosynthesis was not affected by the type of nitrogen source (casein or NaNO3). The other two forms (AcPh II-70 and AcPh III-35 kDa) were competitively inhibited by Pi (K i = 0.5 and 0.2 mM, respectively) and were induced by casein. The K m values of AcPh I and AcPh II were estimated as 1.3 mM, while AcPh III showed a higher affinity for p-nitrophenylphosphate (pNPP) with K m of 0.5 mM. The AcPh I–III fractions demonstrated a pH optimum in the range of 4.5–4.8 and an optimal temperature of 55 °C using pNPP as a substrate.