Enzyme activity

Enzymes find increasing use in aqueous-organic systems for commercial production of specialty and intermediate-scale chemicals. The kinetics of enzyme reactions in supercritical fluids, reverse micelles and microcapsules has been the subject of earlier research in our laboratory. Current research is focused on the role and kinetics of enzymes at interfaces. The adsorption of proteins at oil/water interfaces is a complex phenomenon affecting enzymatic catalysis, biomaterials compatibility, and protein stability in formulations.


This research, in collaboration with Professor Clayton Radke, examined protein adsorption at the liquid-liquid interface using tensiometry, transmission electron microscopy (TEM), and by means of a novel total internal reflection fluorescence spectrometer (TIRFS), capable of monitoring adsorption dynamics. The activity of enzymes at the oil/water interface has been examined using a model system, hydroxynitrile lyase. Hydroxynitrile lyases (Hnls) are of particular interest for the production of enantiomerically-pure cyanohydrins, due to their acceptance of a wide range of aliphatic and aromatic substrates. 

Pendant drop tensiometry has been employed to study the dynamics of adsorption of proteins (ovalbumin, β-casein, lysozyme, Hnl, and bovine serum albumin) and copolymers of L-glutamic acid at the heptane/water interface. We have characterized the dynamic adsorption behavior into three time regimes. An initial induction regime occurs where the surface pressure remains relatively constant, and proteins diffuse to the interface. Once a critical surface concentration is attained, the surface pressure increases sharply as the interface becomes saturated. Continuous loading of the surface occurs by diffusion and relaxation of proteins at the interface. In the third regime, a slow increase in surface pressure occurs as a result of protein conformational changes in the adsorbed layer. A novel TIRFS apparatus has been developed pins the liquid-liquid interface, permitting rate measurements and conformational changes to be determined over long periods. Using extrinsic fluorescent probes, adsorption dynamics have been be followed. Resonance energy transfer (RET) using double-labeled proteins is used to probe conformational changes in the adsorbed layer. A thin film surfaces forces device has been constructed to permit disjoining pressure isotherms of proteins solutions to be measured, and differences in the conformation of proteins at the interface observed. 

© Harvey Blanch 2013