A kinetic study of menthol esterification reaction catalyzed by surfactant-coated lipase (from Candida rugosa) has been conducted in dry isooctane. The kinetic characteristics observed in the enantioselective esterification of menthol with lauric acid (C12) was found to conform to a ping-pong bi-bi mechanism with dead-end inhibition by excess menthol. According to the mechanism we determined the maximum reaction velocity (Vmax), Michaelis constants (Km(fatty acid) and Km(menthol)), and the inhibition constant (Ki). Results have shown that the constant values Km and Ki of (+)-menthol are comparable with the corresponding values of the (-)-isomer. However, there was significant difference in the Vmax values between the two enantiomers; the Vmax involving the (-)-isomer was a 100-fold faster than that of the (+)-isomer. The results suggest that the lipase should recognize the chirality of menthol molecule not in the binding process to the hydrophobic pocket of the lipase but in the nucleophilic attack of the OH group in (-)-menthol. To assess the substrate specificity of C. rugosa lipase for carboxylic acids, the effect of fatty acids of varying chain lengths (C14, C16) on esterification kinetics have been studied using the (-)-menthol. We found that fatty acids exhibit different Km values, whereas their Vmax values are invariably similar. The results have indicated that the catalytic activity of the surfactant-coated lipase for different fatty acid largely depends on the binding behavior of a fatty acid to the active site of the coated enzyme and this action is independent of the acylation step of the (-)-isomer by an acyl-enzyme intermediate.
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