Abstract

A simple method useful for the joint evaluation of substrate partitioning and kinetic parameters for reactions catalyzed by enzymes entrapped in reverse micelles is proposed. The method is applied to the hydrolysis of 2-naphthyl acetate (2-NA) catalyzed by lipase in sodium 1,4-bis(2-ethylhexyl) sulfosuccinate (AOT)/buffer/heptane reverse micellar solutions. In the presence of micelles, the relationship between the initial reaction rate and the analytical concentration of 2-NA was dependent on AOT concentration at a constant W ([water]/[AOT]) value. The dependence of the initial reaction rate profiles with [AOT] was analyzed according with the method proposed to obtain the partition constant of 2-NA between the micelles and the external solvent, Kp. A value of Kp = 2.7 L mol-1 was obtained irrespective of the water content of the micelles (W from 5 to 20). The catalytic rate constant kcat in the micellar solutions was independent of [AOT] but slightly decreased with an increase in W from 2 × 10-6 mol g-1 s-1 at W = 5 to 1.2 × 10-6 mol g-1 s-1 at W = 20. The apparent Michaelis constant determined in terms of the analytical concentration of 2-NA increased with [AOT] at a given W and moderately decreased with W at a fixed [AOT]. The increase with [AOT] is accounted for by considering the partitioning of the substrate. After correction for the partitioning of 2-NA values of (Km)corr were obtained as 3.9 × 10-3 mol L-1 (W = 5), 4.6 × 10-3 mol L-1 (W = 10), 2.3 × 10-3 mol L-1 (W = 15), and 1.7 × 10-3 mol L-1 (W = 20). The rate parameters in the aqueous phase in the absence of micelles, were obtained as (kcat)aq = 7.9 × 10-6 mol g-1 s-1 and (Km)aq = 2.5 × 10-3 mol L-1. In order to compare the efficiency of the enzyme in the micellar solution with that in aqueous phase, the values of (Km)corr were in turn corrected to take into account differences in the substrate activity, obtaining so a set of (Km)corr* values. The efficiency of the enzyme in the micellar solution, defined as the ratio, kcat/(Km)corr*, was found to be higher than in the aqueous phase, even at high water contents (W = 20). This higher efficiency is due to a significant decrease in (Km)corr* values. © 2001 Academic Press.