Fig. 4.

Caseinolytic progress curve of the wild-type and combined variants of the alkaline serine protease DHAP. Data are shown for reactions performed at 15 °C (a) and 50 °C (b). The activity assay was performed in a 400 μL reaction mixture (borate/NaOH buffer, pH 9.6, 2 mM CaCl₂) containing 1% casein as substrate and 0.2 μg of enzyme. At the indicated time-points, 400 μL of reaction mixture was withdrawn and the catalytic reaction was immediately stopped by the addition of 10% trichloride acetate. The data are expressed as the average value from three independent experiments with the experimental deviation shown