Kinetics of Immobilized Enzyme with Nonuniform Activity Distribution

This paper presents a method to evaluate kinetics for an immobilized enzyme with nonuniform activity distribution. Kinetic studies were conducted for esterification of caproic acid with methanol by immobilized lipase (Lypozyme) at 40 °C. Effective diffusivity (De) and Michaelis constant (Km) were determined independently and then the activity distribution was determined by analysing the relationship between the reaction rate and the substrate concentration. First De was determined by step response experiments that were conducted in a packed-bed column using carbonic acid as a tracer and hexane as a solvent. In the analysis of the response curves, intraparticle mass transfer, De, and adsorption of tracer in the particles was taken into account. The adsorption constant, Ka, was evaluated by adsorption experiments. Then, Km was determined by analysing the relationship between the reaction rate and the substrate concentration under chemical reaction control. The final step was the evaluation of active enzyme distribution in the particles using De, Km and Ka. The enzyme activity was observed only in the vicinity of the surface of the support. By using these evaluated constants and considering the nonuniform activity distribution of the immobilized enzyme, kinetics of the reaction could be successfully described over a wide range of concentrations and particle sizes.