Fig 5.

Properties of mature enzymes. (A) Thermal stability of mature enzymes. The enzymes (0.5 μg/ml) were incubated at 95°C for the indicated time periods and then subjected to an azocaseinolytic activity assay. The residual activity is expressed as a percentage of the original activity. The values are expressed as means ± SD (bars) of the results of three independent experiments. (B) Temperature dependence of enzyme activity. Using azocasein as the substrate, activity assays were performed for 10 min at the indicated temperatures. The values are expressed as means ± SD (bars) of the results of three independent experiments. (C) Acid resistance of the mature enzymes. The purified mature enzyme samples were precipitated with 20% TCA or treated with 5 M formic acid (FA) and then subjected to SDS-PAGE (s) and gelatin overlay assay (g) at 95°C. (D) Digestion patterns of β-casein cleaved by mature enzymes. The reaction was carried out at 85°C in 50 mM sodium phosphate buffer (pH 7.5) containing 0.5 mg/ml of β-casein (Sigma) and 2 nM enzyme for different time periods, and then the samples were subjected to Tricine–SDS-PAGE analysis.