Mechanistic insights into nitrile hydratase immobilization on nanodiamonds: Protein corona thermodynamics and...

Nitrile hydratases (NHases) are key biocatalysts for the industrial production of amides, yet their practical application is often limited by insufficient stability and difficulties in enzyme recovery. Here, we report the immobilization of nitrile hydratase on carbon nanodiamonds and investigate the physicochemical mechanisms governing enzyme adsorption and catalytic performance. Adsorption measurements reveal bimodal behavior consistent with the formation of distinct hard and soft protein coronas, whose thermodynamic characteristics were quantified using a Langmuir-Freundlich model. Infrared spectroscopy shows that NHase adsorption is primarily mediated by interactions between surface carboxylate groups of nanodiamonds and polar regions of the protein without significant perturbation of its secondary structure. The immobilized enzyme exhibits high catalytic activity and remarkable operational stability during repeated nitrile hydration cycles. In situ spectroscopic analysis further reveals the formation of proton-shared water complexes at the enzyme-nanodiamond interface, consistent with Zundel-type structures that may facilitate proton transfer during catalysis. These findings demonstrate that NHases immobilized on nanodiamond surfaces as hard coronas provide a unique interfacial environment capable of stabilizing catalytically active water species and offer new insights into the role of interfacial hydration in enzymatic reactions.