PURIFICATION AND CHARACTERIZATION OF AN ALKALINE PROTEASE FROM THE VISCERA OF BOLTI FISH (TILAPIA NILOTICA)

Abstract

An alkaline protease was extracted from acetone powder of the viscera of bolti fish (Tilapia nilotica) with distilled water, precipitated from the extract by 40–60% (NH4)2SO4and then dialyzed. Enzyme homogeneity studies by polyacrylamide gel electrophoresis (PAGE) illustrated that the enzyme was homogenous. Sodium dodecyl sulfate–PAGE showed a molecular weight of 23.0 kDa. The optimal pH and optimal temperature were 8.0 and 45C, respectively, with casein as a substrate. A remarkable stability was observed under alkaline conditions (pH 6.0–10.0), where more than 90% of the enzyme activity was maintained. In the acidic region (pH 2.0–6.0), the enzyme retained more than 50% of its activity. Furthermore, the enzyme retained 85.4 and 41.8% of its activity after heating at 50 and 60C for 120 min, respectively, while the relative activity after heating at 70C for 120 min was 5.25%. The alkaline enzyme lost most of its activity when incubated at 80C for 30 min. A high percentage of total enzyme activity was inhibited when the enzyme was incubated with 50 mM of either soybean trypsin inhibitor or ethylenediaminetetraacetic acid. The presence of NaCl and CaCl2at 10 mM concentration increased the enzyme activity by 6.9 and 10.6%, respectively.