Ion-Specific and Concentration-Dependent Modulation of Bromelain Kinetics by Zn²⁺, Mg²⁺, and Ca²⁺

This study aimed to evaluate the concentration-dependent effects of Ca²⁺, Mg²⁺, and Zn²⁺ on the proteolytic activity and kinetic behavior of bromelain. Bromelain activity was determined spectrophotometrically using casein at concentrations of 2, 5, 10, 15, 20, and 25 mg/mL. Ca²⁺, Mg²⁺, and Zn²⁺ were tested separately at 0.1, 0.5, 1, 2, and 5 mM. Apparent Vmax and Km values were estimated by nonlinear Michaelis–Menten regression. Protein concentration was determined by the Bradford assay, and specific activity was calculated by normalizing enzyme activity to protein concentration. Experiments were performed in triplicate. At 25 mg/mL casein, all tested concentrations of Ca²⁺, Mg²⁺, and Zn²⁺ significantly reduced bromelain reaction rates compared with the metal-free control (adjusted p < 0.0001). Ca²⁺ produced a concentration-dependent decrease in apparent Vmax, with predominantly noncompetitive-like behavior at 0.5–2 mM. Mg²⁺ reduced both apparent Vmax and Km at 0.1–1 mM, whereas these differences were not maintained at higher concentrations. Zn²⁺ also reduced both parameters at 0.1–1 mM, while its response at higher concentrations was non-monotonic. Specific activity was lower than the control under all metal-ion conditions. Ca²⁺, Mg²⁺, and Zn²⁺ reduced bromelain activity under the tested conditions and produced distinct ion- and concentration-dependent kinetic responses. These findings indicate that ionic composition should be considered when optimizing bromelain-based processes.