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 (scCO₂). By using a perfluoropolyether ammonium carboxylate (PFPE) surfactant, we form reverse micelles in scCO₂ 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 H₂O-to-PFPE (R) exceeds approx. 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 approx. 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 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.