Kinetic studies of Chromobacterium viscosum lipase in AOT water in oil microemulsions and gelatin microemulsion-based organogels

Abstract

Kinetic studies have shown that octyl decanoate synthesis by Chromobacterium viscosum (CV) lipase in sodium bis‐2‐(ethylhexyl) sulfosuccinate (AOT) water in oil (w/o) microemulsions occurs via the nonsequential (ping‐pong) bi bi mechanism. There was evidence of single substrate inhibition by decanoic acid at high concentrations. Initial rate data yielded 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 also found to catalyze the synthesis of octyl decanoate according to the ping‐pong bi bi mechanism. Reaction rates were similar in the free and immobilized systems under comparable conditions. Initial rates at saturating (but noninhibiting) substrate concentrations were first order with respect to CV lipase concentration in both w/o microemulsions and the MBG/oil systems. Gradients yielded an apparent kcat = 4.4 × 10−4 mol g−1 s−1 in the case of w/o microemulsions, and 6.1 × 10−4 mol g−1 s−1 for CV lipase immobilized in the MBGs. A third system comprising w/o microemulsions containing substrates and gelatin at concentrations comparable to those employed in the MBG formulations, provided a useful link between the conventional liquid microemulsion medium and the solid organogels. The nongelation of these intermediate systems stems from the early inclusion of substrate during a modified preparative protocol. The presence of substrate appears to prevent the development of a percolated microstructure that is thought to be a prerequisite for MBG formation. FT‐NMR was employed as a semicontinuous in situ assay procedure. The apparent activity expressed by CV lipase in compositionally equivalent liquid and solid phase gelatin‐containing systems was similar. An apparent activation energy of 24 ± 2 kJ mol−1 was determined by 1H‐NMR for esterification in gelatin‐containing w/o microemulsions. This value agrees with previous determinations for CV lipase‐catalyzed synthesis of octyl decanoate in “conventional” w/o microemulsions and MBG/oil systems. The similarities in lipase behavior are consistent with the claim, based largely on structural measurements, that the physico‐chemical properties of the lipase‐containing w/o microemulsion are to a large extent preserved on transformation to the daughter organogel. The close agreement of apparrent activation energies suggests that substrate mass transfer is not rate determining in the three studied systems. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54:416–427, 1997.

Biocatalysis using lipase encapsulated in microemulsion-based organogels in supercritical carbon dioxide

“…The same mechanism has been proposed for esterification or transesterification reactions catalyzed by both free lipases in microemulsions [47] as well as immobilized lipases (lipozyme) in non-polar organic solvents [48] and scCO 2 [49]. Furthermore, it has also been suggested for lipases immobilized in MBGs [13,24].…”

“…The preparation of MBGs was first reported in 1986 [6][7][8] and subsequent spectroscopic investigations [9,10] have shown that the microemulsion structure is well preserved in the network of the gels, which are believed to contain a more or less bicontinuous phase that may co-exist with conventional w/o microemulsion droplets. MBGs containing immobilized lipase were applied for the preparative scale synthesis of miscellaneous esters and both regio-and stereoselectivity have been observed [11][12][13][14][15][16][17][18][19][20][21].…”

Esterification reactions catalyzed by lipases immobilized in organogels: effect of temperature and substrate diffusion

“…In aqueous solutions E a is an intrinsic feature of the enzyme itself (Jenta et al 1997). However, immobilizing the enzyme, diffusion may become rate limiting and the reaction may switch from kinetic to diffusion controlled resulting in a change in the measured apparent E a .…”

Surfactant tail length-dependent lipase activity profile in cationic water-in-oil microemulsions

“…They found that both the activity of Chromobacterium viscosum lipase (CV-lipase) and the interfacial concentration of water, [H 2 O] i (28.1-31.8 M) [33] were essentially unchanged across W 0 = 12-44. Presumably, the scarcity of [H 2 O] i (almost half of the bulk water concentration) with remarkably high interfacial concentration (8.1-6.4 M) of n-hexanol (competitive inhibitor of lipase [37][38][39][40]) plays an important role in significantly reducing lipase efficiency. Chart 1. Keeping in mind the potentials of micellar enzymology, improvement of lipase activity is indispensable in cationic w/o microemulsions.…”