Extracellular acid protease from Aspergillus niger I1: purification and characterization

Abstract

A new strain of Aspergillus niger producing acid protease was isolated and identified by universal primers NL1 and NL4. The acid protease from A. niger I1 was purified to homogeneity by ultrafiltration using a 10-KDa cut-off membrane, gel filtration on Sephadex G-75 and ion exchange chromatography on CM-Sephadex C-50, with a 3.55-fold increase in specific activity and 56% recovery. The molecular weight of the protease was estimated to be 50 kDa on SDS-PAGE and gel filtration, which is higher than those from other A. niger strains. Carbohydrate content of the purified protease, determined by the chemical anthrone method, was calculated to be 16%. The Km and Vmax for caseinolytic activity of the purified enzyme were found to be 1.02 mM and 2.2 µmol/min, respectively. The enzyme was optimally active at 60°C and pH 3.0. The most metal ions tested had no significant effect on protease activity. The enzyme activity was inhibited by pepstatin A, suggesting that the purified enzyme is an aspartic protease.