Enzymatic cleavage of protein substrates at solid surfaces is important in a large number of applications in the food and detergent industries, and in biomedical applications. However, investigations of the hydrolysis of proteins immobilized at solid interfaces are sparse. We desire a mechanistic understanding of enzyme action at an immobilized protein surface, and how this interaction is affected by interactions with detergent surfactants. We use a novel model protein stain created by spin-coating protein onto an amine-functionalized silicon wafer and cross-linking the stain by reacting the lysine residues with glutaraldehyde in the vapor phase. The reproducible, multilayer, immobilized protein film provides a substrate to investigate proteolysis kinetics and enzyme adsorption of the serine protease subtilisin Carlsberg. We study two globular proteins: ovalbumin, a hen egg-white protein, and bovine serum albumin (BSA).

Proteolysis kinetics are followed by measuring the immobilized protein film thickness in time using ellipsometry. Because the multilayer protein film is homogeneous, thickness decreases linearly with time, with the slope gauging the proteolysis rate. Protease adsorption is obtained by highly cross-linking the immobilized protein film to eliminate proteolysis, and then measuring adsorption amount with a specially designed ellipsometry flow cell.

Subtilisin adsorption at the immobilized protein/water interface is reversible and obeys Langmuir kinetics. Protein cleavage kinetics by subtilisin Carlsberg follow a new Langmuir-Michaelis-Menten model. As opposed to bulk proteolysis kinetics, surfactants enhance the proteolytic degradation of the immobilized protein layers. Fit parameters obtained from combining theory and experiment lend insight into the mechanism of the enzyme's action on the protein surface, and into the effect of surfactant on the adsorption and cleavage steps of catalysis. We find that surfactants aid the enzyme in removal of the immobilized protein by increasing the inter-protein “mesh size”, i.e. loosening the surface protein layer.