ESTERIFICATION REACTIONS CATALYZED BY CHROMOBACTERIUM-VISCOSUM LIPASEIN CTAB-BASED MICROEMULSION SYSTEMS

Chromobacterium viscosum (CV) lipase solubilized in water-in-oil (w/o) microemulsions based on the cationic surfactant hexadecyltrimethylamm onium bromide (CTAB) have been used for multigram-scale ester synthesi s, including the kinetic resolution of a secondary alcohol. The stabil ity of CV lipase in all the CTAB microemulsions studied was excellent and was superior to that observed in aqueous buffer at the same pH and temperature. Kinetic studies were performed using the synthesis of et hylhexadecanoate as a model reaction. Under pseudo-first-order conditi ons, the synthesis rates were linearly dependent on the enzyme and fat ty acid concentrations and the R dependence shows the characteristic b ell-shaped curve (where R = [H2O]/[surfactant]). The dependence of enz yme activity toward octyldecanoate synthesis on the pH of the disperse d buffer phase is in marked contrast to that observed for the pH depen dence of CV lipase toward p-nitrophenylbutyrate hydrolysis. In the for mer case, the pH-activity profile is approximately sigmoidal, which ma y reflect the ionization state of the fatty acid substrate. In the lat ter case, the pH dependence is minimal at both R = 10 and R = 50, sugg esting the enzyme does not experience a changed pH environment. Inclus ion of a pH-sensitive probe molecule into those incubations containing fatty acid clearly demonstrates that the probe molecule experiences a changed environment consistent with that expected for the selected bu ffer. An in situ Fourier transform nuclear magnetic resonance (FT-NMR) assay has been developed which allows continuous monitoring of the es terification reactions, thereby providing an additional means of deter mining initial rates. The method may be of general value for lipase as says in microemulsions since it may provide, at the same time, informa tion regarding enzyme regioselectivity. (C) 1995 John Wiley and Sons, Inc.