FIG 4.

Thermal resistance (upper panels) and low-temperature activity (lower panels) of stability-enhancing (A) and activity-enhancing (B) variants generated by combinations of VRs. The enzymes (1.5 μg/ml) in buffer A were incubated at 85°C for 1 h and then subjected to an azocaseinolytic activity assay at 60°C (upper panels). The residual activity is expressed as a percentage of the original activity, and the values are means ± standard deviations (bars) of the results of three independent experiments. The azocaseinolytic activities of the enzymes (0.75 μg/ml) were determined at 25°C in buffer A (lower panels). The values are means ± standard deviations (bars) of three independent experiments, and the mean value of the WT was defined as 1. The specific activities of the variants relative to the WT were calculated.