Dynamics of Enzymes at Interfaces : Lipase adsorption and mobility on solid surfaces

This thesis aimed to give more insight in the dynamics of enzymes at interfaces. The adsorption and mobility of adsorbed proteins can e.g. give a better understanding of structure-function properties of interfacially active enzymes. Studied enzyme was the lipase from Thermomyces lanuginosus (TLL). Adsorption of TLL to surfaces of different hydrophobicity was studied by Dual Polarization Interferometry (DPI), Surface Plasmon Resonance (SPR) and ellipsometry. It was found that TLL had highest affinity and adsorbed to largest adsorbed amount on a hydrophobic, C18 terminated surface. Moreover, activity studies of adsorbed TLL suggested that a larger fraction of the lipases were orientated with the active site facing the surface on hydrophobic surfaces. Mobility of adsorbed enzymes was studied by means of Fluorescence Recovery After Photobleaching (FRAP) with Confocal Laser Scanning Microscopy (CLSM). CLSM was also used as a tool to image the role of TLL in the detergency of lipids from single cotton fibers. The TLL surface mobility was measured on model surfaces of different hydrophobicity. The rate of TLL surface diffusion was strongly dependent on the surface density of lipase, which was explained by sterical hindrance and intermolecular repulsion. The diffusion was both lowest and decreased as a function of time after adsorption on the most hydrophobic surface. This was thought to be due to a larger fraction of adsorbed TLL oriented with the active site towards the hydrophobic surface and that this fraction increased as a function of time. The presence of surfactants affected the TLL mobility on hydrophobic surfaces. The diffusion increased more than tenfold when TLL was coadsorbed with C12E6/LAS above the critical micellar concentration (cmc) of the surfactant. This was thought to be due to a surfactant induced desorption-rebinding mechanism of TLL. Total Internal Reflection Fluorescence Correlation Spectroscopy (TIR-FCS) supported this theory and was implemented as a technique to quantify kinetic processes of protein-surfactant interactions at surfaces. The surface mobility of TLL was higher on a trimyristin substrate surface compared to the model hydrophobic surface. Single particle tracing of lipases could be performed by conjugation of TLL to Quantum Dots (QDs). The microscopic behavior of QD-lipases on trimyristin suggested that the enzyme operated in two different modes on the surface, which gave the trajectories of single lipase molecules a “bead on a string” appearance.QC 2010081