Enzyme catalytic activity in reversed micelle microreactors with bile salt co-surfactant

The first part of this dissertation focuses on the influence of bile salt cosurfactant, sodium taurocholate (NaTC) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) on the catalytic activity of yeast alcohol dehydrogenase (YADH) solubilized by Aerosol-OT (AOT) reversed micelles. NaTC and CHAPS modify the interfacial properties of the reversed micelles and increase their water capacity, resulting in a more favorable environment for hydrophilic enzyme catalysis. The reaction velocity for the oxidation of ethanol substrate catalyzed by YADH can be modeled using a two-substrate Michaelis-Menten relation and shows slight enhancement upon inclusion of either bile salt in the reversed micelles. NaTC and CHAPS also cause changes in the Michaelis constant, KM. The smaller K M values for reactions in bile salt-containing reversed micelles indicate enhanced substrate and enzyme/coenzyme affinity upon inclusion of bile salts.;The second portion of this dissertation involves the characterization of reversed micelles formed by the nonionic surfactant polyoxyethylene-4-laurylether (Brij-30). Molecular probes, including methyl orange, methylene blue, 1,8-anilinonaphthalene and tris (2,2'-bipyridine) ruthenium dichloride hexahydrate, were used to probe the water pool properties of the reversed micelles. The water inside the polar core of the reversed micelles has much lower polarity than bulk water. Lipase activity was studied in this nonionic surfactant microemulsion system. Enhanced enzymatic activity was observed compared with lipase in AOT reversed micelles. The catalytic efficiency, kcat/K M, was increased 3-fold. Although introduction of NaTC or CHAPS to Brij-30 reversed micelles showed no apparent increase in lipase activity, enzyme stability was greatly improved in the Brij-30 reversed micelles compared with bile salt containing AOT reversed micelles. The different interfacial property between the two reversed micelle systems determined lipase activity.;The final part of this dissertation describes studies of the dynamic properties of AOT/isooctane reversed micelles using temperature-induced percolation and size determinations using dynamic light scattering (DLS). The percolating temperature of reversed micelles decreased upon inclusion of bile salts, indicating increased water uptake of reversed micelles. The size measurement using DLS also showed consistent enlargement of reversed micelles with bile salts. The results show that addition of 10 mM CHAPS to AOT/isooctane reversed micelles results in a 6-fold increase in the overall size of the aggregates. In addition, DLS studies indicate that solubilizing YADH into the aqueous cores of the reversed micelles distorts the spherical structure of the micelles, while adding bile salts to the enzyme-containing reversed micelles restores the spherical structure and increases the overall size of the aggregates.