Enzymatic reaction in water-in-oil microemulsions. Part 1.—Rate of hydrolysis of a hydrophilic substrate: acetylsalicylic acid

Abstract

The rates of catalytic hydrolysis of a hydrophilic substrate, acetylsalicylic acid (AA), have been measured both in aqueous solution and in water-in-oil (w/o) microemulsions formed by AOT in n-heptane. Catalysts used were lipase from Rhizopus delemar, α-chymotrypsin from Bovine pancreas and imidazole as an acid–base catalyst.

Since the hydrolysis of AA proceeds in aqueous solution without catalyst (intramolecular catalysis), the rate of hydrolysis was measured in microemulsions. The rate constant for the hydrolysis of AA increased with increasing concentration ratio of water to AOT, W₀=[H₂O]_ov/[AOT]_ov, at constant [AOT]_ov, and was asymptotic to the value obtained in aqueous solution. For constant W₀ the rate constant was almost independent of [AOT]_ov over the range 0.15–0.65 mol dm^–3.

The overall rate constant for the catalytic hydrolysis of AA in microemulsions was obtained by subtracting the rate constant for the intramolecular catalysis of AA from the rate constant observed. The intrinsic rate constant in the water pools of the microemulsion was then evaluated by considering the volume fraction of water determined by Karl Fischer titration.

For W₀ > 10 at [AOT]_ov= 0.5 mol dm^–3, the intrinsic rate constant for imidazole-catalysed hydrolysis of AA was equal to that obtained in the aqueous phase. As W₀ decreased below 10, the intrinsic rate constant decreased and the value at W₀= 6.5 was 0.6 times that in the aqueous phase. The intrinsic rate constant was independent of [AOT]_ov.

The addition of lipase to the aqueous phase did not enhance the rate of hydrolysis of AA. However, the reaction in microemulsions was catalysed by the lipase and was linearly dependent on lipase concentration. Lipase in microemulsions exhibits activity for the hydrolysis of AA owing to a change in the conformation of lipase. The intrinsic rate constant in the water pool is a maximum at W₀= 7 and is independent of [AOT]_ov.

The rate of hydrolysis of AA was little affected by the presence of α-chymotrypsin either in the aqueous phase or in microemulsions.