Critical Role of Water beyond the Media to Maintain Protein Stability and Activity in Hydrated Deep Eutectic Solvent

Hydrated deep eutectic solvents (DESs) are recognized for their potential in biocatalysis due to their tunability, biocompatibility, greenness, and ability to keep protein stable and active. However, the mechanisms governing enzyme stability and activity in DES remain poorly understood. Herein, using bromelain as the model enzyme and acetamide (0.5)/urea(0.3)/sorbitol(0.2) as the model DES, we provide experimental evidence that modulation of associated water plays a key role in dictating protein stability and activity in hydrated DES. Specifically, rigid associated water at higher DES concentrations (beyond 40% v/v) stabilizes bromelain through entropy but destabilizes it through enthalpy. On the other hand, flexible associated water dynamics at lower DES concentrations result in an opposite thermodynamic outcome. Importantly, the bulk water dynamics cannot explain the stability trend, which emphasizes the critical role of water near the protein surface. Strikingly, associated water dynamics also correlates strongly with bromelain's proteolytic activity. An increasing flexibility of the associated water dynamics leads to the enhancement of the activity. This is the first study to experimentally link associated water dynamics to enzyme behavior in hydrated DES, offering insights that could guide future developments in solvent engineering for enzyme catalysis.