(632f) Deactivation of TEM-1 Beta-Lactamase Investigated by Isothermal Batch and Non-Isothermal Continuous Enzyme Membrane Reactor Methods

Rogers, T. A., Georgia Institute of Technology
Bommarius, A. S., Georgia Institute of Technology
Daniel, R. M., University of Waikato

The thermal deactivation of TEM-1 beta-lactamase was examined using two experimental techniques: a series of isothermal batch assays and a single, continuous, non-isothermal assay in an enzyme membrane reactor (EMR). The isothermal batch-mode technique was coupled with the three-state ?equilibrium model? of enzyme deactivation, while the results of the EMR experiment were fitted to a four-state ?molten globule model?. The two methods both led to the conclusions that the thermal deactivation of TEM-1 beta-lactamase does not follow the Lumry-Eyring model and that the Teq of the enzyme (the point at which active and inactive states are in thermodynamic equilibrium) is at least 10°C from the Tm (melting temperature), contrary to the idea that the true temperature optimum of a biocatalyst is necessarily close to the melting temperature. After comparison of the two experimental techniques, the continuous, non-isothermal EMR method was applied in the prediction of the enzyme total turnover number (ttn) at any given process temperature. The predictions of the non-isothermal model regarding the long-term yield of TEM-1 at elevated temperatures matched closely to the results of isothermally-obtained batch data, and revealed that total turnover number may be estimated, for costing purposes of a biocatalyst, by evaluating the quotient of the enzyme catalytic constant, kcat, and the observed first-order deactivation rate constant, kD, obs. This method, which may be extended to a large number of enzyme-substrate systems, allows reliable estimation of total turnover number from quantities which are easily measured experimentally.