Purification and properties of a 4-nitrophenylphosphatase from Aspergillus niger.
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 phosphate-limiting 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 pI of 4.6. The activity was optimal at pH 8.0 and was stimulated by Mg2+ and to a lesser extent by Mn2+ but was inhibited by Zn2+ and Ca2+. The enzyme was highly specific for 4-nitrophenyl phosphate as substrate, having a Km of 0.77 mM 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.
Versaw
W K
WK
Department of Biochemistry, University of Nebraska-Lincoln 68583-0718.
Bevins
M A
MA
Markwell
J
J
D009597:4-Nitrophenylphosphatase / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001234:Aspergillus niger / Q000201:enzymology* / Q000254:growth & development; D002417:Cattle; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007422:Intestines / Q000201:enzymology; D013379:Substrate Specificity
D003470:Culture Media; D009597:4-Nitrophenylphosphatase