Engineering and Directed Evolution of a Ca²⁺ Binding Site A-Deficient AprE Mutant Reveal an Essential Contribution of the Loop Leu₇₅–Leu₈₂ to Enzyme Activity

Figure 3

Thermal unfolding of subtilisin E. Fluorescence intensity unfolding data for wild-type AprE(■), AprEΔL₇₅–L₈₂ (•), and AprEΔL₇₅–L_82-T₆₆M G₁₀₂D (□). The data were obtained with a heating rate of 1 K min^-1. Solid lines represent the best fit of unfolding data with Δ𝐻𝑚=233.3±18.2 kJ mol^-1 and T_m=326.9±0.4 K, Δ𝐻𝑚=237.3±21.1 kJ mol^-1 and T_m=329.8±0.5 K, and Δ𝐻𝑚=304.5±29.2 kJ mol^-1 and T_m=329.3±0.4, respectively.