| Proteolysis at solid/liquid interfaces is a phenomenon important in a wide variety of commercial applications, including food processing, contact lens cleaning, and automatic dishwashing and laundry detergents. The detergent industry in particular dominates the market as a consumer of industrial enzymes, accounting for approximately 40% of the over ;The focus of this work is to obtain a mechanistic understanding of protease adsorption on and hydrolysis of immobilized substrates relevant to detergency applications, as well as the effect of surfactant on proteolysis. In particular, we study a novel "model stain" consisting of an immobilized, multilayer ovalbumin protein film. Subtilisin Carlsberg, a serine protease isolated from Bacillus licheniformis, is the model protease used to catalyze hydrolysis of the immobilized ovalbumin substrate. The novel multilayer substrate permits study of interfacial proteolysis unhindered by the solubilization and surface heterogeneity of other monolayer and sub-monolayer protein films presented in the literature for surface proteolysis studies to date.;Reduction of the protein-film thickness by subtilisin Carlsberg was measured over time using ellipsometry. Resulting kinetic and in situ adsorption data as a function of aqueous enzyme concentration and temperature were well-fit by a proposed Langmuir-Michaelis-Menten model for surface proteolysis, yielding parameters describing the activation energy, kinetics, enzyme surface coverage, and equilibrium adsorption constant for surface proteolysis. As temperature increased, the adsorbed enzyme surface concentration decreased at a given aqueous enzyme concentration. However, the enzyme cleaved the substrate more rapidly leading to a net increase in the ovalbumin film degradation rate.;The effect of a detergent-relevant anionic surfactant, sodium dodecylbenzene sulfonate (SDBS), on proteolysis was also assessed. Surfactant increased the measured film thickness, absorbing into the protein film and causing swelling. Surfactant-induced film-swelling was found to be reversible upon rinsing with buffer. Nevertheless, even after rinsing, subsequent exposure to enzyme caused a three-fold increase in the film degradation rate, most likely due to conformational changes induced in the substrate film by SDBS. To assess the effect of surfactant on substrate conformation and proteolysis, aqueous studies were conducted with the protein bovine serum albumin (BSA) and various nonionic and ionic surfactants. Interestingly, in aqueous proteolysis, all surfactants reduced the proteolysis rate. Circular dichroism (CD), tryptophan fluorescence spectra, and tryptophan fluorescence thermograms indicate that BSA undergoes conformational changes in the presence of surfactants, more notably so for ionic surfactants and for surfactant concentrations near and above the critical micelle concentration (CMC). Enzyme-surfactant interactions also reduced the activity of subtilisin Carlsberg. While the surfactant may decrease enzyme activity and form micelles along the partially unfolded protein (hence, blocking the enzyme from binding to those sites), the surfactant-induced loosening of the protein substrate makes the protein more susceptible to enzyme digestion. This latter effect apparently dominates at the protein film/aqueous interface, where substrate accessibility is a limiting factor in proteolysis. |
