Investigation of enzymatic hydrolysis of lipid-like substrates in monolayers

The parameters of enzymatic hydrolysis of novel lipid-like substrates assembled in monolayers at water–air interface were estimated by a simple method. The method is based on measurement of the initial velocity of the reaction registered by the decrease of the monolayer area (caused by the enzymatic hydrolysis) at surface pressure of 10 mN/m in a single–compartment trough. Hydrolysis of trilaurin and three 1,3-dilaurylpseudoglycerides acylated by phenylalanine, leucine and valine was characterized by catalytic constants k_cat and apparent Michael's constants K_m(app) (using lipase from Pseudomonas fluorescens as catalyst). It was found that k_cat of the synthetic pseudoglycerides (7–13 per s) are higher than k_cat of trilaurin (4 per s) that can be explained by the presence of positively charged primary aminogroups in the substrates. K_m(app) values were found to be similar for all the substrates studied (ca. 2×10^?6 M). The proposed method allows estimation of the kinetic constants in traditional dimensions.