Critical Role of\nWater beyond the Media to Maintain\nProtein Stability and Activity in Hydrated Deep Eutectic Solvent

Hydrated deep eutectic solvents (DESs)\nare recognized for their\npotential in biocatalysis due to their tunability, biocompatibility,\ngreenness, and ability to keep protein stable and active. However,\nthe mechanisms governing enzyme stability and activity in DES remain\npoorly understood. Herein, using bromelain as the model enzyme and\nacetamide (0.5)/urea(0.3)/sorbitol(0.2) as the model DES, we provide\nexperimental evidence that modulation of associated water plays a\nkey role in dictating protein stability and activity in hydrated DES.\nSpecifically, rigid associated water at higher DES concentrations\n(beyond 40% v/v) stabilizes bromelain through entropy but destabilizes\nit through enthalpy. On the other hand, flexible associated water\ndynamics at lower DES concentrations result in an opposite thermodynamic\noutcome. Importantly, the bulk water dynamics cannot explain the stability\ntrend, which emphasizes the critical role of water near the protein\nsurface. Strikingly, associated water dynamics also correlates strongly\nwith bromelain’s proteolytic activity. An increasing flexibility\nof the associated water dynamics leads to the enhancement of the activity.\nThis is the first study to experimentally link associated water dynamics\nto enzyme behavior in hydrated DES, offering insights that could guide\nfuture developments in solvent engineering for enzyme catalysis.