Reactivity of α-chymotrypsin in water-in-oil microemulsions

Abstract

Detailed kinetic studies have been carried out for the reaction of a range of substrates with a-chymotrypsin in water-in-oil (w/o) microemulsion systems. The main conclusions are as follows: (a) α-chymotrypsin is active when dispersed in w/o microemulsions consisting of water/ aerosol-OT (AOT)/heptane and water/tetraethyleneglycolmono-n-dodecylether (C 12E 4)/ heptane. In both systems, the turnover number ( k cat) is similar to that measured in bulk water. (b) The maximum enzyme activity (as a function of pH of the solubilised water) is found at pH ≈ 8 in bulk water and also in the microemulsion systems. (c) For three substrates in the water/AOT/heptane system, k cat values are similar to those found in bulk aqueous solution, whereas Michaelis constant ( K M) values (with units of mol dm −3, and expressed per dm 3 of dispersed water) are found to be increased by a factor of approximately 100. (d) For the substrate N-glutaryl- L-phenylalanine- p-nitroanilide (GPNA) in the AOT system, values of Δ H ‡ cat and Δ H O M were found to be similar to the bulk water values. (c) At a pH where the enzyme is active, it is found that the enzyme slowly loses its activity with time, due to autolysis. The rate of inactivation of α-chymotrypsin was increased in both the AOT and C 12E 4 systems as compared with bulk water. For the ACT system, a minimum in the enzyme inactivation rate (i.e., maximum stability with respect to autolysis) was found at R = [H 2O]/[surfactant] ≈ 12. In the C 12E 4 system, the rate of inactivation was essentially independent of R.