(618f) Application of a Continuously-Operated Enzyme Membrane Reactor In the Determination of the Operational Stability of An Enzyme Undergoing Reversible Denaturation

Rogers, T. A., Georgia Institute of Technology
Bommarius, A., Georgia Institute of Technology

While many enzyme-catalyzed reactions offer clear advantages, such as high enantioselectivity and relatively mild process conditions, most enzymes suffer from denaturation when subjected to the elevated temperatures which are often used in commercially-viable processes. It is useful to model the combined effects of temperature on the activation (increased intrinsic kinetic rate) and deactivation (thermally induced degradation) of a biocatalyst and optimize the total turnover number in order to assess whether a biocatalyst will be cost-effective under a given set of process conditions. The correlation of short-term biochemical measurements to the accurate prediction of the productivity of an enzyme over its useful life has only recently been mentioned in the literature and has not yet been proven. Here, wild-type TEM-1 beta-lactamase was subjected to a temperature-time gradient in an enzyme membrane reactor (EMR), and the total turnover number was predicted by fitting conversion data to the Lumry-Eyring model. This has been attempted previously in the literature, but for an enzyme which deactivates irreversibly at high temperature. [1] The EMR results were correlated to kinetic data and thermal deactivation data collected in isothermal experiments. At temperatures well below the melting temperature of the enzyme, for which the population of unfolded protein in the reactor is very small compared to the population of native protein, it was demonstrated that the total turnover number is simply a function of the catalytic constant (kcat) and the observed deactivation rate (kD,obs), two quantities which are readily measured by experiment.

[1] P. R. Gibbs, et al. Biotechnology Progress (2005), 21, 762-774.