Kinetic model discrimination of penicillin G acylase thermal deactivation by non-isothermal continuous activity assay

Abstract

A novel approach for the determination of a kinetic model of enzyme deactivation is presented incorporating time and temperature dependence into a single, continuous assay. A generic method for a non-isothermal, continuous activity assay is developed. Unique temperature profiles that enhance model discrimination with fewer experiments compared to linear temperature scans were used to differentiate kinetic models of penicillin G acylase (PGA) deactivation. Three models are examined in depth but the technique is generalizable to any kinetic deactivation model. Using the Akaike information criterion (AIC), the Lumry-Eyring model was found to best capture PGA deactivation behavior and the corresponding kinetic parameters are presented for the first time. Additionally, simulated experiments on lysozyme and TEM-1 beta-lactamase were used to develop temperature profiles that best differentiate the studied kinetic models. The results from the proposed approach were consistent with conventional, but tedious, isothermal batch experiments.