<br/>Proteolytic enzymes in liquid detergents suffer from lack of stability in the sense that activity diminishes with time. Although the phenomenon could be attributed to several factors, the influence of colloidal surfaces on the enzymatic stability was investigated. Besides the types of surfaces that are present in the usual detergent systems, other surfaces have been also studied. The enzyme under investigation was wild-type Savinase <sup>TM</SUP>, which belongs to the class of microbial alkaline serine proteinases. This thesis contains a set of experiments and discussions on the interactions, viz. adsorption and inactivation, between Savinase and several types of surfaces.<p>In order to understand the mechanism of adsorption of Savinase from aqueous solution, adsorption from solution was achieved by stagnation point flow towards an oxidized silicon strip. The adsorbed amount was monitored continuously as a function of time by reflectometry. In order to prevent autodigestion during the experiments, the enzyme was irreversibly inhibited by reaction of the active site with a small inhibitor. The adsorbed amount was monitored as a function of time. The experimental variables were the amphipathicity of the solid surface, the pH, the ionic strength, the type of monovalent ions used and the protein concentration. It was concluded that electrostatic interactions dominate the adsorption of Savinase on a hydrophilic surface and that unfolding of the protein molecule does not take place. On the other hand, for a hydrophobic surface, adsorption is dictated by unfolding of the protein structure and/or hydrophobic dehydration.<p>Another type of surface that was relevant for this study, was a negatively charged model surface, carrying chemically grafted short poly(ethylene) oxide chains. To that end, a method was developed to prepare charge- stabilized polystyrene latex particles, carrying these moieties. The latices had a surface charge and possessed on the surface a PEO-350/methacrylate molecule, where the number refers to the molecular weight of the ethylene(oxide) part. The surface properties of this latex (PS-B) were compared to those of a similar latex but without the PEO-surface group (PS- A). The presence of the PEO-moiety on the PS-B particles could be established by the colloidal stability against salt, complexation with molybdatophosphoric acid and proton NMR.<p>The loss of activity of wild-type Savinase was determined both in solution and in the presence of colloidal particles, which provided a surface area for adsorption of 25% of the enzyme population. It was demonstrated that the intact protein was always converted into autolytic degradation products at the expense of biological activity. The different particles, however, deactivated the enzymes to different extents. In the presence of particles that have hydrophobic surface properties (PS-A or teflon latex) autodigestion was substantially enhanced as compared to that in solution. It was inferred that hydrophobic inactivation was due to an increased sensitivity of the adsorbed enzyme towards digestion by the enzyme remaining in solution. On the other hand, PS-B and hydrophilic silica rendered the enzyme more stable against autodigestion than in solution. It is thus concluded that the type of surface determines the mode (conformation/ orientation) in which the enzyme is adsorbed on a particle which, in turn, affects the autocatalytic rate. As the colloidal interfaces present in the usual liquid detergents, i.e. the zeolite-water and vesicle-water interfaces, are modelled by those of silica and PS-B, respectively, the surfaces in the detergents probably <em>increase</em> the enzymatic stability as well.<p>The rapid hydrophobic inactivation was rationalized by a conformational change of the adsorbed protein which increases its autolytic susceptibility. The experimental approach to verify structural changes consisted of time-resolved and steady-state fluorescence of tryptophan residues and of circular dichroism (CD) of the protein. These properties were measured for inhibited Savinase in situ at the hydrophobic teflon latex. The results are compared with those obtained from the protein at the hydrophilic silica suspension and in solution. In the case of fluorescence it is reasoned that the average excited-state lifetime and short internal rotation correlation times are parameters indicative of structural changes in the protein.<p>Fluorescence and CD both proved that Savinase altered its conformation when it adsorbed at low surface coverage on hydrophobic teflon particles. In that case, the tryptophan fluorescence lifetime was decreased which was accompanied by an increase in the amount of α-helix. The fluorescence of the protein on silica was not affected, irrespective of the surface occupation. At monolayer coverage on teflon, the protein maintained its original structure although significant changes in fluorophore dynamics occurred.<p>Hence, as hydrophobic inactivation of the wild-type enzyme occurred at full surface occupation, the altered dynamics may render the adsorbed protein more liable to proteolytic attack. This assumption should be qualified, since the conformational change that is required for autodigestion may not be the same as that involved in tryptophan dynamics.<p>Therefore, the experimental data on solution- and hydrophobic inactivation were subjected to kinetic analysis regarding the possible occurrence of a critical conformational change. In addition, the inactivation data of Savinase were extended to two other types of teflon latices which were different with respect to the plateau value of protein adsorption.<p>A mathematical model for autodigestion was developed. It involved transport and attachment to the particle surface as well as processes that describe the chemical conversion of the molecules. By analytical or numerical simulation, the effect of each step in the model was investigated. The order of the inactivation reaction with respect to time was used for the detection of a conformational change, although this was not a rigourous procedure. Following this method and considering the distinctly different deactivation behaviour of the various hydrophobic latices it was concluded that the model for surface-enhanced autodigestion, including a conformational change of the enzyme at the surface, has good validity.
|Qualification||Doctor of Philosophy|
|Award date||9 Jan 1996|
|Place of Publication||S.l.|
|Publication status||Published - 1996|