(593aq) Enzyme Immobilization Onto Various Nanosupports: A Critical Study

Due to their high catalytic properties and selectivity, enzymes are widely used in multiple industries including food and paper processing, brewery, and biofuel generation. However, enzymes possess low storage and operational stability, and may leach into solution. Thus, there is a critical need to improve enzyme stability and retention when such applications are envisioned. Immobilization of enzymes onto nanosupports, such as nanotubes or nanoparticles, is a possible solution. These nanosupports posses high surface-area-to-volume ratios and high aspect ratios, which allow for high enzyme loadings upon immobilization and enzyme retention when the support is exposed in solution. The attachment of enzymes to the nanosupport surface has also been shown to enhance enzyme stability under storage and operational conditions in some cases. However, the process of immobilization will decrease the activity of the enzymes due to interactions at the protein-nanomaterial interface. Herein we are presenting a critical study focused on outlining what properties influence activity loss. Briefly, we have immobilized various enzymes (i.e. soybean peroxidase (SBP), chloroperoxidase (CPO), and glucose oxidase (GO_X)) onto multiple nanosupports (i.e. multi-walled carbon nanotubes (MWNTs), single-walled carbon nanotubes (SWNTs), titanium dioxide nanobelts (TiO₂), tungsten oxide nanoparticles (WO₃), platinized tungsten oxide nanoparticles (PtWO₃), and graphene oxide nanosheets (GON)) using either physical or covalent binding. In the cases of MWNTs and TiO₂, covalent binding and covalent binding with the utilization of an amino-dPEG12-COOH (PEG) linker were also tested. In order to test the interactions at the protein-nanomaterial interface, the derived conjugates were characterized in terms of enzyme loading, retained activity, and thermostability. Protein concentration after attachment, or loading, was determined using a standard colorimetric assay. The retained activity refers to the activity of the immobilized enzyme, determined via colorimetric reactions, compared to the activity of free enzyme in solution at the corresponding concentration. The Michaelis-Menten parameters of all conjugates were also studied and compared to those of free enzymes to determine any influence of the nanosupport on the enzyme kinetics. Thermal inactivation was tested at 75°C. Our data show that enzyme activity loss upon immobilization can be attributed to enzyme denaturation at the interface, reduced enzyme surface area available for substrate interaction, and unwanted protein-protein interactions due to unspecific binding. It was demonstrated that as the diameter of the nanosupport increases (i.e. rate of curvature decreases) protein inactivation is more dramatic because more enzyme surface area comes into contact with the nanosupport; such observations are also dependent on the surface properties of the enzyme and the interface reactions with the nanosupport. Our studies can be implemented to improve upon current uses of enzymes as well as new applications such as active surface decontamination and biosensors.