Enzymatic reaction in water-in-oil microemulsions. Part 2.—Rate of hydrolysis of a hydrophobic substrate, 2-naphthyl acetate

Abstract

The catalytic hydrolysis rates of a hydrophobic substrate, 2-naphthyl acetate (NA), have been measured both in aqueous solution and in water-in-oil microemulsions (ME) formed by di(2-ethylhexyl)sodium sulfosuccinate (AOT) in heptane. The catalysts used were lipase, α-chymotrypsin and imidazole.

The dependence of W_o=[H₂O]_ov/[AOT]_ov at a constant [AOT]_ov and of [AOT]_ov at a constant W_o on the overall rate constant were discussed in terms of a reaction model. The reaction model includes three parameters, the distribution constants of NA and the catalysts and the rate constant of the local reaction field. The distribution constant of NA was evaluated by measurements of the distribution of NA between the ME in the Winsor II region and the aqueous phase, but that of catalysts was treated as a fitting parameter.

It is suggested that the reactions with these catalysts proceeds at the interfacial region of ME. For the imidazole-catalysed reaction, if the imidazole exists preferentially at the interface, the rate constant is independent of W_o, but the value was 0.2 times that in the aqueous phase.

Superactivity of lipase was observed, that is the turnover number in ME was greater than that in the aqueous phase. However, as the michaelis constant was also large, the rate constant (k_CAT/k_m) in ME was smaller than that in the aqueous phase. The rate constant increased as W_o increased. The dependence was caused by the conformation change of lipase due to the interaction of AOT molecules. The rate constants for both imidazole and lipase decreased with increase in [AOT] at constant W_o, which might be caused by the change in the structure of ME.

The turnover number for α-chymotrypsin at the interface was lower than that in the aqueous phase and approached the values in the aqueous phase as W_o and [AOT] increased. However, the Michaelis constant decreased with increase in W_o and approached a constant value, which was 25 times that in the aqueous phase.