Atomic force microscope studies on the interactions of Candida rugosa lipase and supported lipidic bilayers

Using the Langmuir-Blodgett technique we prepared substrate supported well-defined lipid/phospholipid (1-mono-palmitoyl-rac-glycerol (MPG)/l,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC)) bilayers in which the MPG lipid leaflet was exposed to the aqueous phase. Hydrolysis of MPG performed by Candida rugosa lipase (CRL) on the upper MPG layer of these supported bilayers on mica was imaged by real time atomic force microscope (AFM) using a liquid cell, so that the area increase of the initial structural defects could be followed over time. Our data strongly suggest that the edges of the initial structural defects are the preferred activation sites for CRL once the enzyme is adsorbed onto these interfaces. When a 2.5nM bulk concentration of CRL was assayed on this planar lipid substrate, we found a long lag phase before a sharp increase of catalytic activity. The lag-burst kinetic behaviour was related to the interfacial activation phenomenon although we propose that it is also dependent on the gel-phase state of this interface.