(667c) Enzyme Deactivation Probed By Non-Isothermal Continuous Activity Assay

Enzymes have narrow temperature operating windows and complex deactivation mechanisms compared to chemical catalysts. Knowledge of the mechanism of deactivation is important for designing continuous processes incorporating biocatalysts. Improved enzyme performance can be achieved without protein engineering if the process is designed with the enzyme’s deactivation mechanism and kinetics in mind.

A novel approach for determination of a kinetic model of enzyme deactivation is presented incorporating time and temperature effects into a single, continuous assay. Unique temperature profiles enhance model descrimination with fewer experiments and man-hours compared to linear temperature scans typical of techniques such as differential scanning calorimetry. Using the model enzyme penicillin G acylase (PGA), three models are examined in detail, however, the technique can be applied to any deterministic model of enzyme deactivation. Using the Akaike information criterion, the Lumry-Eyring mechanism was found to best capture PGA deactivation behavior and the corresponding kinetic parameters are presented for the first time. Simulated experiments are also used to better inform real experiments and add more confidence to the model discrimination. The results were consistent with conventional, but tedious, isothermal batch experiments. [1]

The development of this assay provided insight into continuous enzymatic catalysis. PGA demonstrated tolerance to small perturbations in temperature, which enables the operation of unique reactor designs. One design selected for further study is a reactive crystallizer [2] with a fines dissolution loop. A fines dissolution loop is a bypass at elevated temperature that destroys small crystals, increasing the average crystal size for easier downstream handling of solid products. The process tolerance to perturbations in temperature also depends heavily on enzyme stability. Knowledge of how an enzyme will react to fluctuations in temperature is necessary for design tolerances.

References:

[1] McDonald, M. A., et al., 2018. Kinetic Model Discrimination of Penicillin G Acylase Thermal Deactivation by Non-Isothermal Continuous Activity Assay. Chem. Eng. Sci., IN REVISION

[2] McDonald, M. A., et al., Enzymatic reactive crystallization for improving ampicillin synthesis Chem. Eng. Sci., 2017. 165, 81-88