Purification and properties of a 4-nitrophenylphosphatase from Aspergillus niger

Abstract

A 4-nitrophenylphosphatase (EC 3.1.3.41) was identified in extracts of Aspergillus niger. The production of this activity was decreased by growth on a phosphatelimiting medium and was greatest in a medium supplemented with corn steep liquor. The phosphatase activity was purified by hydrophobic, ion-exchange, and molecular sieve chromatography. The purified enzyme has a native size of approximately 80,000, polypeptide subunits with sizes of 37,000 upon denaturation, and a p I of 4.6. The activity was optimal at pH 8.0 and was stimulated by Mg 2+ and to a lesser extent by Mn 2+ but was inhibited by Zn 2+ and Ca 2+. The enzyme was highly specific for 4-nitrophenyl phosphate as substrate, having a K m of 0.77 m m and a turnover number of 108 s −1. The purified enzyme did not hydrolyze any of 22 sugar phosphates, mononucleotides, or other phosphocompounds tested. A small, but reproducible, amount of activity was measured using 5′-DNA phosphate as a substrate. Although some similarities exist to three previously characterized 4-nitrophenylphosphatases from Saccharomyces cerevisiae, the enzyme from A. niger is distinctly different from at least two of these activities.