Bioconjugation of lipase and cholesterol oxidase with graphene or graphene oxide

The catalytic behavior of lipase and cholesterol oxidase (ChOx) in the absence and in the presence of graphene (G) or graphene oxide (GO) was investigated at 24 ± 1 °C and pH 6.5. GO flat sheets (0.5–2 μm) were ~2-nm thick, while G formed aggregates. The maximum reaction velocity (V max) values and turnover numbers (k cat) determined for reactions catalyzed by physical mixtures of lipase (at 0.01 g l−1) or ChOx (at 0.03 g l−1) and G (0.012 g l−1) increased six-fold or doubled, respectively, in comparison to neat enzymes. Circular dichroism (CD) and photoluminescence (PL) spectroscopic measurements revealed the preservation of native secondary structures of enzymes and bioconjugation driven by hydrophobic interaction and energy transfer (redshift) between lipase or ChOx and G, corroborating with the enhanced catalytic behavior. On the other hand, the interactions between GO, which has hydrophilic moieties on the basal plane, and ChOx caused enzyme deactivation, as evidenced by the absence of typical CD signal. At low GO concentration (<0.012 g l−1), bioconjugates of lipases with GO led to V max and k cat values four-fold lower than their counterparts with G, but the GO hydrophilic groups probably favored the affinity for the substrate, because the Michaelis constant (Km) values decreased in comparison to that of neat lipase. Upon increasing the GO concentration, lipases lost secondary structure and the typical lipase PL bands disappeared.