Biocatalytic Nanoscale Coatings Through Biomimetic Layer-by-Layer Mineralization

Recent insight into the molecular mechanisms of biological mineral formation (biomineralization) has enabled biomimetic approaches for the synthesis of functional organic-inorganic hybrid materials under mild reaction conditions. Here we describe a novel method for enzyme immobilization in thin (nanoscale) conformal mineral coatings using biomimetic layer-by-layer (LbL) mineralization. The method utilizes a multifunctional molecule comprised of a naturally-occurring peptide, protamine (PA), covalently bound to the redox enzyme Glucose oxidase (GOx). PA mimics the mineralizing properties of biomolecules involved in silica biomineralization in diatoms, and its covalent attachment to GOx does not interfere with the catalytic activity. Highly efficient and stable incorporation of this modified enzyme (GOx-PA) into nanoscale layers (similar to 57 nm thickness) of Ti-O and Si-O is accomplished during protamine-enabled LbL mineralization on silica spheres. Depending on the layer location of the enzyme and the type of mineral (silica or titania) within which the enzyme is incorporated, the resulting multilayer biocatalytic hybrid materials exhibit between 20100% of the activity of the free enzyme in solution. Analyses of kinetic properties (Vmax, KM) 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 synthesized hybrid nanoscale coatings are largely determined by substrate diffusion rather than enzyme functionality. The GOx-PA immobilized in these nanoscale layers is substantially stabilized against heat-induced denaturation and largely protected from proteolytic attack. The method for enzyme immobilization described here enables, for the first time, the high yield immobilization and stabilization of enzymes within continuous, conformal, and nanoscale coatings through biomimetic LbL mineralization. This approach will likely be applicable to a wide variety of surfaces and functional biomolecules. The ability to synthesize thin (nanoscale) conformal enzyme-loaded layers is of interest for numerous applications, including enzyme-based biofuel cells and biosensors.