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 CO2 (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 oddase (ChOx) obeys Michaelis-Menten kinetics. The results of our experiments also show that (1) the optimum ChOx activity occurs when the molar ratio of H2O-to-PFPE (R) exceeds similar to 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,K-app) is optimal at high R values, (4) the best-case K-m,K-app is similar to 2-fold better than the value reported alkanes, (5) there is little change in the ChOx k(cat,app) and K-m,K-app as we adjust the CO2 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.