Investigation of kinetics of immobilized liver esterase by flow calorimetry

Abstract

Flow calorimetry (FC) was shown to be a powerful tool for investigation of the kinetics of phenyl acetate hydrolysis catalyzed by pig liver carboxyl esterase. The enzyme was immobilized in alginate gel particles that were placed in a calorimetric flow column and the heat effect of enzyme reaction was followed in single flow and total recirculation conditions. It was shown that the registered temperature change was proportional to molar amount of substrate transformed in the column. A mathematical model describing the enzyme reaction, mass transfer, and heat effects in the calorimetric system was developed and used for the kinetic data evaluation. By combining data from single flow and recirculation modes true kinetic parameters were evaluated by the proposed mathematical procedure based on the model solution and successive approximations.The kinetic data for carboxyl esterase showed a slide substrate inhibition by phenyl acetate. The obtained kinetic parameters were as follows: Michaelis constant Km=2mmoldm−3 and substrate inhibition constant Ki=42mmoldm−3. The method can be applied to kinetic study of immobilized enzymes directly in the flow calorimeter without any requirement of an independent analytical technique.