Fig. 2.

Functional studies in the EcCI inhibitor. (A) EcCI inhibition curve on papain. Papain (1 nM) was pre-incubated at 40 °C for 10 min with various EcCI concentrations in 0.1 M NaH₂PO₄ pH 6.3 buffer; 0.01 M EDTA; 0.4 M NaCl; and 5 mM DTT. (B) EcCI thermal stability. EcCI was subjected to thermal treatment at different temperatures for 30, 60, and 120 min. (C) EcCI inhibitory activity after treatment at different pH values. EcCI was incubated for 180 min at different pH values, and activity was evaluated on papain hydrolysis of the Z-Phe-Arg-pNan substrate (0.4 mM) for 30 min at 40 °C. (D) EcCI inhibition curve in the gut extract of C. maculatus larvae. Increasing concentrations of EcCI were incubated for 10 min at 37 °C with 20 μL larvae gut extract in 0.1 M NaH₂PO₄ buffer pH 6.3; 0.01 M EDTA; 0.4 M NaCl; and 5 mM DTT. The Z-Phe-Arg-pNAn substrate (5 mM) was added, and hydrolysis followed for 1 h *Statistical significance compared to control (p-value ˂ 0.05 by One-way ANOVA).