Molecular insights into the interaction between graphene oxide and β-galactosidase: From multispectral experiment, enzyme kinetics experiment to computational simulations

• Graphene oxide was synthesized by an improved Hummer method. • A combination of computational simulation and multispectral experiments was used. • Graphene oxide and β-galactosidase interacted through van der Waals force , hydrophobic force , and electrostatic interaction . • Graphene oxide can improve the stability and activity of β-galactosidase (about 1.5 times). β-galactosidase has potential applications in various scientific disciplines, including the food industry, genetic engineering, and enzyme engineering. In this study, multi-spectral experiments combined with enzyme kinetics experiments and computational simulation were used to explore the interaction mechanism, structural and functional changes, and effects on the microenvironment of graphene oxide and β-galactosidase. Graphene oxide was synthesized using the improved Hummer method, and the characterization experiments demonstrated that the structure was flat and free of cracks, and the functional groups satisfied the required amount. Zeta potential experiments and molecular docking results jointly confirmed that graphene oxide and β-galactosidase were combined by van der Waals forces, electrostatic forces, and hydrophobic forces. Fluorescence quenching experiments showed that the quenching constant decreased from 2.90 × 10 12 mLμg -1 s −1 to 1.76 × 10 12 mLμg −1 s −1 with the increase of temperature, indicating that the quenching mode was static quenching, accompanied by increased polarity and decreased hydrophilicity of β-galactosidase. Circular Dichroism Spectrum, Fourier Transform infrared spectroscopy, the ONPG experiment, and molecular dynamics simulation proved that the stability and activity of β-galactosidase were improved after binding with graphene oxide. These findings provide support for the potential applications of β-galactosidase in addressing lactose intolerance, optimizing gene expression efficiency, and developing innovative biosensors or bioprocessing systems.