Biocatalytic Nanoscale Coatings Through Biomimetic Layer-by-Layer Mineralization

Abstract

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 (∼5–7 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 20–100% 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.

Live Diatom Silica Immobilization of Multimeric and Redox-Active Enzymes

“…3C). The stabilization of biosilica-immobilized GOx toward heat treatment and proteolytic attack has previously been observed also for enzymes that were immobilized in synthetic silica matrices in vitro (7,14,21). It has been hypothesized that the immobilized enzyme molecules reside within nanocavities inside the silica that provide a barrier against unfolding of the enzyme during heating and make the enzyme less accessible to proteases.…”

“…GOx-PA has the same specific activity as GOx (14). GOx-PA-saturated T. pseudonana biosilica exhibited an enzyme activity level of 1.38 ϫ 10 4 Ϯ 6.74 ϫ 10 2 U per mol silica (see Fig.…”

“…To obtain a reference point for high enzyme loading in diatom silica, GOx-PA, a silica binding derivative of A. niger GOx (14), was attached to biosilica isolated from T. pseudonana wild-type cells. GOx-PA has the same specific activity as GOx (14).…”

“…T. pseudonana biosilica was isolated by extraction with a hot solution of SDS as described previously (28). A fixed amount of biosilica (10 mol SiO 2 ) was incubated with various amounts of glucose oxidase cross-linked to protamine (GOx-PA) (14) for 20 h at room temperature. Subsequently, the biosilica was pelleted by centrifugation (5 min, 16,000 ϫ g), and the pellet and supernatant were collected (fraction Sup1).…”

Highly efficient, long life, reusable and robust photosynthetic hybrid core–shell beads for the sustainable production of high value compounds

“…These amines also act as flocculating agents encasing the silica colloid. [37][38][39][40][41][42] Furthermore, swelling of alginate capsules and hybrid alginate-silica beads could be avoided by the addition of a polycation outer layer (i.e. poly-L-lysine), which may lead to an additional mechanical resistance.…”