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]2[PAA] through [Ch]2[PAA] assisted improved substrate channeling and reduced transition-state energy barriers. Moreover, an ∼16% increase in the half-life temperature (T50) of GOx–HRP cascade in the presence of 10 wt % [Ch]2[PAA] with an enhanced melting temperature (Tm) of the enzymes suggested improved thermal stability relative to PBS. The results of improved enzyme stability in hydrated ILs were further investigated by the thermodynamic stability curves (ΔG vs T). Overall, this work provides a basis for multienzyme biocatalysis in aqueous solution of ILs with an accelerated ECR rate and improved thermodynamic stability, envisaging sustainable biocatalysis and metabolic engineering.