The development, characterization, and application of biomimetic nanoscale enzyme immobilization

The utilization of enzymes is of interest for applications such as biosensors and biofuel cells. Immobilizing enzymes provides a means to develop these applications. Previous immobilization efforts have been accomplished by exposing surfaces on which silica-forming molecules are present to solutions containing an enzyme and a silica precursor. This approach leads to the enzyme being entrapped in a matrix three orders of magnitude larger than the enzyme itself, resulting in low retention of enzyme activity.;The research herein introduces a method for the immobilization of enzymes during the layer-by-layer buildup of Si-O and Ti-O coatings which are nanoscale in thickness. This approach is an application of a peptide-induced mineral deposition method developed in the Sandhage and Kröger groups, and it involves the alternating exposure of a surface to solutions containing the peptide protamine and then an aqueous precursor solution of silicon- or titanium-oxide at near-neutral pH.;A method has been developed that enables in situ immobilization of enzymes in the protamine/mineral oxide coatings. Depending on the layer and mineral (silica or titania) within which the enzyme is incorporated, the resulting multilayer biocatalytic hybrid materials retain 20 – 100% of the enzyme activity. Analyses of kinetic properties of the immobilized enzyme, coupled with characterization of physical properties of the mineral-bearing layers (thickness, porosity, pore size distribution), indicates that the catalytic activities of the enzymes immobilized in the different layers are largely determined by substrate diffusion. The enzyme was also found to be substantially stabilized against heat-induced denaturation and largely protected from proteolytic attack.;These functional coatings are then developed for use as antimicrobial materials. Glucose oxidase, which catalyzes production of the cytotoxic agent hydrogen peroxide, was immobilized with silver nanoparticles, can release antimicrobial silver ions. It is demonstrated that these two antimicrobial agents work in a synergistic manner for enhanced antimicrobial efficacy. Evidence of the proposed mechanism of synergy, namely enhanced release of silver ions by reaction of H2O2 with silver nanoparticles, is provided. Finally, the deployment of these materials in silk fibroins for development as wound dressings is also presented.;Protamine cross-linking was then extended to the oxygen-reducing enzyme laccase to explore the use of this modified enzyme in an enzymatic biocathode. In this application laccase accepts electrons from the electrode and uses them to reduce oxygen to water molecules. The protamine-cross-linked enzyme exhibits a higher degree of immobilization, better retention of activity once immobilized, and superior electrochemical activity versus the native enzyme.;Finally, preliminary research on the structure-function relationships of 16-mer peptides which adsorb to surfaces and deposit titanium oxide is presented. Specifically, the effect of content and distribution of arginine residues on the ability of peptides to adsorb to surfaces and subsequently deposit mineral oxides was investigated. The data demonstrate that surface adsorption of the peptides relies on both a critical number of arginine residues and their position within the peptide. Furthermore, the exchange of serine against arginine residues in surface-adsorbed peptides is detrimental to Ti-O deposition.