KINETIC-STUDIES OF CHROMOBACTERIUM-VISCOSUM LIPASE IN AOT WATER-IN-OIL MICROEMULSIONS AND GELATIN MICROEMULSION-BASED ORGANOGELS

Kinetic studies have shown that octyl decanoate synthesis by Chromobac terium viscosum (CV) lipase in sodium bis-2-(ethylhexyl) sulfosuccinat e (AOT) water in oil (w/o) microemulsions occurs via the nonsequential (ping-pong) bi bi mechanism. There was evidence of single substrate i nhibition by decanoic acid at high concentrations. Initial rate data y ielded estimates for acid and alcohol Michaelis constants of ca. 10(-1 ) mol dm(-3) and a maximum rate under saturation conditions of ca. 10( -3) mol dm(-3) s(-1) for a lipase concentration of 0.36 mg cm(-3). CV lipase immobilized in AOT microemulsion-based organogels (MBGs) was al so found to catalyze the synthesis of octyl decanoate according to the ping-pong bi bi mechanism. Reaction rates were similar in the free an d immobilized systems under comparable conditions. Initial rates at sa turating (but noninhibiting) substrate concentrations were first order with respect to CV lipase concentration in both w/o microemulsions an d the MBG/oil systems. Gradients yielded an apparent k(cat) = 4.4 x 10 (-4) mol g(-1) s(-1) in the case of w/o microemulsions, and 6.1 x 10(- 4) mot g(-1) s(-1) for CV lipase immobilized in the MBGs. A third syst em comprising w/o microemulsions containing substrates and gelatin at concentrations comparable to those employed in the MBG formulations, p rovided a useful link between the conventional liquid microemulsion me dium and the solid organogels. The nongelation of these intermediate s ystems stems from the early inclusion of substrate during a modified p reparative protocol. The presence of substrate appears to prevent the development of a percolated microstructure that is thought to be a pre requisite for MBG formation. FT-NMR was employed as a semicontinuous i n situ assay procedure. The apparent activity expressed by CV lipase i n compositionally equivalent liquid and solid phase gelatin-containing systems was similar. An apparent activation energy of 24 +/- 2 kJ mol (-1) was determined by H-1-NMR for esterification in gelatin-containin g w/o microemulsions. This value agrees with previous determinations f or CV lipase-catalyzed synthesis of octyl decanoate in ''conventional' ' w/o microemulsions and MEG/oil systems. The similarities in lipase b ehavior are consistent with the claim, based largely on structural mea surements, that the physicochemical properties of the lipase-containin g w/o microemulsion are to a large extent preserved on transformation to the daughter organogel. The close agreement of apparent activation energies suggests that substrate mass transfer is not rate determining in the three studied systems. (C) 1997 John Wiley & Sons, Inc.