Performance of Cholesterol Oxidase Sequestered within Reverse Micelles Formed in Supercritical Carbon Dioxide

We report the first results on an enzyme-induced reaction within the water core of reverse micelles that have been formed in supercritical CO 2 (scCO 2 ). By using a perfluoropolyether ammonium carboxylate (PFPE) surfactant, we form reverse micelles in scCO 2 with water cores and we show that the oxidation of cholesterol by cholesterol oxidase (ChOx) obeys Michaelis−Menten kinetics. The results of our experiments also show that (1) the optimum ChOx activity occurs when the molar ratio of H 2 O-to-PFPE ( R ) exceeds ∼12, (2) the rate constant describing the conversion of the ChOx−cholesterol complex to product ( k cat,app ) is similar to values reported using reverse micelle systems formed in liquid alkanes, (3) the equilibrium constant that describes the ChOx−cholesterol complex dissociation ( K m,app ) is optimal at high R values, (4) the best-case K m,app is ∼2-fold better than the value reported using reverse micelles formed in liquid alkanes, (5) there is little change in the ChOx k cat,app and K m,app as we adjust the CO 2 pressure between 100 and 260 bar, and (6) the ChOx was active within the PFPE water pool for at least 5 h; however, after 8 or more hours within the PFPE water pool, ChOx became temporarily inactive.