Activity of lipase in water-in-oil microemulsions

Abstract

The lipase-catalysed hydrolysis rates of several nitrophenyl alkanoate esters of varying alkyl chain length (C₄–C₁₆) have been measured both in aqueous solution and in water-in-oil (w/o) microemulsions (which are known to contain discrete droplets). Lipase retains its activity in w/o microemulsions of water, heptane and sodium bis-2-ethylhexyl sulphosuccinate (AOT); the observed rates are consistent with the intrinsic activity of the enzyme (i.e. k_cat/K_m) being the same as in water. However, the observed conversion rates for C₄ and C₆ substrates are slower in the microemulsion system because of substrate partitioning to the oil-continuous phase, which results in a reduced concentration in the aqueous pseudophase. This conclusion is reached by comparing lipase and non-enzymic-(i.e. buffer) catalysed rates in both solution media. Again for the C₄ and C₆ substrate, partition coefficients for the substrates in the limit of high molar ratio of H₂O:AOT, as determined from the kinetic results, show good agreement with measured values in heptane + water mixtures. This suggests that lipase functions effectively in the water pseudophase of the microemulsion. Lipase in the microemulsion can also catalyse the hydrolysis of longer chain alkanoates (up to C₁₆). It can be inferred from the kinetics that such substrates partition to the interface where the lipase must also be active. In the case of AOT microemulsions, the pH profile of enzyme activity is not significantly altered compared with bulk water. The lipase retains > 60% activity in the microemulsion after incubation at 35 °C for 6 days. In w/o microemulsions of water, heptane, chloroform and cetyltrimethylammonium bromide (CTAB), the observed hydrolysis rates are significantly reduced and the intrinsic activity is reduced by a factor of twenty as compared with the AOT system. This is thought to be caused by inhibitory binding of CTAB to the protein.