Purification and characterization of an acidic protease from the viscera of bolti fish ( Tilapia nilotica)

Abstract

An acidic protease was extracted from acetone powder of the viscera of bolti fish (Tilapia nilotica) in acidified distilled water and precipitated from the resulting extract by ammonium sulfate followed by dialysis and its kinetics studied. The crude enzyme was purified using gel filtration; its homogeneity and molecular weight were studied. The enzyme showed highest activity and purification-fold when precipitated at 40–60% ammonium sulfate. Purification fold and activity were increased after purification by dialysis and gel filtration on Sephadex G-100. Homogeneity studies, by polyacrylamide gel electrophoresis, illustrated that the enzyme was homogeneous only after purification by the gel filtration step. Sodium dodecyl sulphate polyacrylamide gel electrophoresis showed a molecular weight of 31.0 kDa. The optimal pH and optimal temperature were 2.5 and 35°C, respectively. The enzyme showed pH stability between 2 and 6. It retained more than 50% of its activity after heating between 50 and 60°C for 30 min, and 40.2 and 74.9% after heating between the same temperatures for 120 min. The K m and V max values of enzyme were 0.77 mM and 2.22 mM/min, respectively, while the catalysis efficiency (V max/K m) was 2.88. A high inhibition percentage of total enzyme activity was obtained when the enzyme was incubated with 50 mM of both soybean trypsin inhibitor and ethylenediaminetetraacetic acid. The presence of either NaCl or CaCl 2 at 10 mM concentration increased the enzyme activity by 20.5% and 31.2%, respectively.