Enzymatic Oxidation of Cholesterol in Reverse Micelles

We explore the use of the microheterogeneous system of reverse micelles as the reaction medium for the enzymatic oxidation of cholesterol to cholestenone catalyzed by cholesterol oxidase. Toward this goal, the activity of the enzyme has been measured in three reverse micellar systems based on the anionic AOT, the cationic CTAB, and the nonionic Triton X-100 surfactants as a function of the pH, the molar ratio R of water-to-surfactant in the reverse micelles, and the surfactant concentration. The Michaelis-Menten kinetic constants k_cat and K_m have been estimated in the three reverse micellar systems and are compared to those obtained in the aqueous medium. For the AOT system, the kinetic constants have been estimated also as a function of R and the surfactant concentration. Further, the kinetic constants in the reverse micelles are related to those in the aqueous medium using a model which assumes the partitioning of the substrate between the three microdomains of the reverse micelle system (the water pool, the interface, and the organic continuous phase) as well as the physical partitioning of the enzyme and/or the noncompetitive inhibition of the enzyme by the surfactant in the interfacial microdomain.