Kinetic properties of α-chymotrypsin in water-in-oil microemulsions: studies with a variety of substrates and microemulsion systems

Abstract

(1) A study has been made of the catalytic activity of α-chymotrypsin towards a number of substrates in various water-in-oil microemulsion systems containing dispersed water droplets. (2) When solubilised in the dispersed aqueous phase of a microemulsion consisting of water, n-heptane and the anionic surfactant Aerosol®-OT (dioctyl sodium sulphosuccinate, DSS), α-chymotrypsin was active towards the substrate N-trans-cinnamoylimidazole and Michaelis-Menten kinetics were observed. The steady-state kinetic parameters, Km and kcat (the former referred to concentrations in the aqueous phase volume) were practically independent of the aqueous phase volume fraction for a constant composition ratio. R, where R is the molar ratio [H2O]/[DSS]. As R is increased, the size of the water droplets also increases and kcat approaches that measured in bulk water. In contrast, values of Km were typically 100-fold greater than observed in bulk water. Corresponding behaviour was previously observed for the substrate N-glutaryl-l-phenylalanyl-p-nitroanilide (GPNA). (3) α-Chymotrypsin was also active towards a range of substrates (N-trans-cinnamoylimidazole, GPNA and N-acetyltryptophan methyl ester) in a water-in-oil microemulsion consisting of the cationic surfactant cetyltrimethylammonium bromide (CTAB) in 50% (v/v) n-heptane/chloroform. (The pH optimum of the enzyme was not displaced significantly from that observed in bulk water.) The enzyme was readily inactivated in this medium, with up to 50% of activity being lost after 1 h. α-Chymotrypsin in CTAB microemulsions also displayed Michaelis-Menten kinetics towards several substrates; as for the DSS system, values of kcat were comparable to those found in bulk water, whereas values of Km were again increased. (4) α-Chymotrypsin is very unstable in a water-in-oil microemulsion system consisting of water/toluene/sodium dodecyl sulphate (SDS)/pentanol. At low water contents (where droplets are formed) and at low temperatures, the rate of inactivation is minimised. In such microemulsions, α-chymotrypsin is active towards GPNA; kcat is again comparable to that in bulk water, whereas Km is considerably increased. (5) In each microemulsion system, the increase in Km compared to water is essentially independent of substrate. The order of Km values, using the different surfactants, is bulk water /2/lt DSS /lt CTAB $SDS/pentanol. It is possible that the phenomenon arised from an inhibitory effect of surfactant on the enzyme.