Kinetic behaviour and reaction mechanism of the hydrolysis of p-nitrophenyl palmitate in mixed micelles with Triton X-100 catalyzed by lipase from Candida rugosa

The physicochemical properties of lipase from Candida rugosa in the hydrolysis of micellized p-nitrophenyl palmitate, such as thermal stability, enzyme concentration and the effect of ionic strength on the rate of catalysis, have been characterized. As regards the specificity for a series of p-nitrophenyl esters (p-NPCn), n = 2, 4, 8, 12 and 16 being the number of carbon atoms of the hydrophobic tail, the lipase from Candida rugosa proved to be non-specific, although it did hydrolyze them at different rates, depending on n and the physicochemical nature of the substrate (mixed micelles with surfactant or simple solution). At Triton X-100 levels above the critical micelle concentration (c.m.c.), the kinetic behaviour of the hydrolysis of p-nitrophenyl palmitate in Triton X-100 mixed micelles catalyzed by Candida rugosa lipase was consistent with the Michaelis—Menten rate equation under three different experimental conditions: (i) the molar fraction of substrate held constant and the Triton X-100 concentration varied; (ii) the bulk substrate concentration held constant and the Triton X-100 concentration varied, and (iii) the Triton X-100 concentration held constant and the bulk substrate concentration varied. Kinetic analysis performed in the above conditions revealed that the simple model described by Verger et al. [J. Biol. Chem., 248 (1973), 4023] correctly interprets the kinetic behaviour of the commercial lipase from Candida rugosa used in the study and highlights the advantage that this classic mechanism may have in current lipase modelling in biocatalysis.