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

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.