Graphene oxide (GO), having a large specific surface area and abundant functional groups, provides an ideal substrate for study enzyme immobilization. We demonstrated that the enzyme immobilization on the GO sheets could take place readily without using any cross-linking reagents and additional surface modification. The atomically flat surface enabled us to observe the immobilized enzyme in the native state directly using atomic force microscopy (AFM). Combining the AFM imaging results of the immobilized enzyme molecules and their catalytic activity, we illustrated that the conformation of the immobilized enzyme is mainly determined by interactions of enzyme molecules with the functional groups of GO.

Biochemical and biomedical applications of graphene oxide (GO) critically rely on the interaction of biomolecules with it. It has been previously reported that the biological activity of the GO-enzyme conjugate decreases due to electrostatic interaction between the enzymes and GO. Herein, the immobilization of horseradish peroxidase (HRP) and oxalate oxidase (OxOx) on chemically reduced graphene oxide (CRGO) are reported. The enzymes can be adsorbed onto CRGO directly with a tenfold higher enzyme loading than that on GO, and maximum enzyme loadings reach 1.3 and 12 mg mg(-1) for HRP and OxOx, respectively. Significantly, the more CRGO is reduced, the higher the enzyme loading. The CRGO-HRP conjugates also exhibit higher enzyme activity and stability than GO-HRP. Excellent properties of the CRGO-enzyme conjugates are attributed to hydrophobic interaction between the enzymes and the CRGO. The hydrophobic interaction mode of the CRGO-enzyme conjugates can be applied to other hydrophobic proteins, and thus could dramatically improve the performance of immobilized proteins. The results indicate that CRGO is a potential substrate for efficient enzyme immobilization, and is an ideal candidate as a macromolecule carrier and biosensor.

Composition, morphology, and surface characteristics of solid substrates play critical roles in regulating immobilized enzyme activity. Grapheme oxide (GO), a novel nanostructured material, has been illustrated as an ideal enzyme immobilization substrate due to its unique chemical and structural properties. Physical properties and catalytic activity of GO immobilized horseradish peroxidase (HRP) and its application in phenolic compound removal are described in the present study. HRP loading on GO was found to be much higher than that on reported substrates. The GO immobilized HRP showed improved thermal stability and a wide active pH range, attractive for practical applications. The removal of phenolic compounds from aqueous solution using the GO immobilized HRP was explored with seven phenolic compounds as model substrates. The GO immobilized HRP exhibited overall a high removal efficiency to several phenolic compounds in comparison to soluble HRP, especially for 2,4-dimetheoxyphenol and 2-chlorphenol, the latter a major component of industrial wastewater.

In this study, zinc oxide (ZnO) nanocrystals with different morphologies were synthesized and used as substrates for enzyme immobilization. The effects of morphology of ZnO nanocrystals on enzyme immobilization and their catalytic activities were investigated. The ZnO nanocrystals were prepared through a hydrothermal procedure using tetramethylammonium hydroxide as a mineralizing agent. The control on the morphology of ZnO nanocrystals was achieved by varying the ratio of CH3OH to H2O, which were used as solvents in the hydrothermal reaction system. The surface of as-prepared ZnO nanoparticles was functionalized with amino groups using 3-aminopropyltriethoxysilane and tetraethyl orthosilicate, and the amino groups on the surface were identified and calculated by FT-IR and the Kaiser assay. Horseradish peroxidase was immobilized on as-modified ZnO nanostructures with glutaraldehyde as a crosslinker. The results showed that three-dimensional nanomultipod is more appropriate for the immobilization of enzyme used further in catalytic reaction.

Oxalate oxidase (OxOx), a well known enzyme catalyzes the cleavage of oxalate to carbon dioxide with reduction of dioxygen to hydrogen peroxide, however its catalytic process is not well understood. To define the substrate binding site, interaction of Fe(3+) ions with OxOx was systemically investigated using biochemical method, circular dichrosim spectroscopy, microscale thermophoresis, and computer modeling. We demonstrated that Fe(3+) is a non-competitive inhibitor with a milder binding affinity to OxOx, and the secondary structure of the OxOx was slightly altered upon its binding. On the basis of the structural properties of the OxOx and its interaction with Fe(3+) ions, two residue clusters of OxOx were assigned as potential Fe(3+) binding sites, the mechanism of the inhibition of Fe(3+) was delineated. Importantly, the residues that interact with Fe(3+) ions are involved in the substrate orienting based on computer docking. Consequently, the interaction of OxOx with Fe(3+) highlights insight into substrate binding site in OxOx.