Stoichiometrically Engineered Hydrated Ionic Liquids Enabling Reinforcement of Enzyme Cascade with Improved Thermodynamic Stability.

While biocatalysis in ionic liquids (ILs) using a single enzyme is well known, the successful performance of enzyme cascade reactions (ECRs) using multiple enzymes in ILs is limited by the incompatible stabilization of more than one enzyme in a single IL. Here, we introduce an innovative approach where stoichiometric precision of ILs creates pH-switchable media that dynamically modulate multienzyme microenvironments and maintain the functional integrity of ECR without requiring any proximity-engineered scaffolds. Cholinium-based ILs, with phosphate and carboxylate anions, were synthesized with varying molar ratios of cholinium to realize pH-switchable aqueous platforms for ECR. Using glucose oxidase (GOx)-horseradish peroxidase as (HRP) an enzymatic cascade, we demonstrate that under optimized conditions aqueous solutions of ILs significantly enhance both the individual enzyme (GOx and HRP) activities and ECR (GOx-HRP) efficiencies compared to the control, phosphate-buffered saline (PBS) (pH 7.4). Molecular docking, molecular dynamics simulations, UV-vis, and circular dichroism spectroscopy studies reveal that ILs are involved in soft interactions with enzymes, stabilizing catalytically favorable conformations, and protecting enzymes against thermal-stress. Remarkably, a 25-fold increase in the ECR efficiency was achieved in 10 wt % of [Ch]