FIG 4.

Effects of mutation of the residues of Ca1 (A, C, and E) and Ca2 (B, D, and F) on pyrolysin stability. (A and B) SDS-PAGE analysis of the purified samples of the mature mutants. The major and minor species in the purified enzyme sample are indicated by black and white arrowheads, respectively. Lanes M, molecular mass markers. (C to F) Thermal stability of the mutants. The purified samples of mPls and its mutants (5.0 μg/ml) in buffer A were incubated at 95°C in the absence (C and D) or presence (E and F) of 2 mM EDTA. At the time intervals indicated, samples were withdrawn and subjected to an azocaseinolytic activity assay (C to F) and SDS-PAGE analysis (E and F). The residual activity is expressed as a percentage of the original activity of each enzyme sample. The values are expressed as means ± SDs (bars) of the results of three independent experiments.