Integrative simulation and spectroscopy illuminates L-asparaginase stability on nanocomposite surface for biosensing.

The design and functional immobilization of enzymes on nanomaterial supports are key aspect in advancing biosensor applications. In this work, we have presented an integrated experimental and computational study of L-asparaginase (asnB) covalently immobilized onto reduced graphene oxide-molybdenum disulfide (rGO@MoS₂) nanocomposites via glutaraldehyde crosslinking. Hydrothermal synthesis and spectroscopic (XRD, FTIR, CD, SEM-EDX) characterization confirmed successful nanocomposite formation and enzyme attachment. All-atom molecular dynamics (MD) simulations have been further performed to complement the experimental findings. Key results from computational analysis reveal that immobilization constrains global mobility and induces localized protein compaction, as evidenced by a substantial reduction in solvent accessible surface area post-immobilization of asnB. Angle deviation analyses showed the asnB adopts a stable orientation on the nanocomposite surface, supporting high orientational stability. Further secondary structure analyses demonstrated that native α-helix and β-sheet content are largely retained. The enhanced shelf-life and stability are evidenced by the retention of 78% residual activity after 45 days, effectively addressing a key limitation in industrial applications. The bio-nanocomposite also shows sensing metrics of LoD 30 ± 5 nM and LoQ 86 ± 2 nM, and reusability of 5 times, with reference to the retention of catalytic ability of asnB. This work highlights how the integrated spectroscopic and simulation approach guides engineering of robust, activity-preserving enzyme nanomaterial interfaces for industrial applications like biosensing and biocatalytic technologies.