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

## TL;DR

This paper introduces a new method using ionic liquids to stabilize and enhance the performance of enzyme cascades, leading to faster and more stable reactions.

## Contribution

The novel approach uses stoichiometrically engineered ionic liquids to create pH-switchable media for multienzyme stabilization.

## Key findings

- A 25-fold increase in ECR efficiency was achieved using [Ch]2[PAA].
- Thermal stability of the enzyme cascade improved with a 16% increase in half-life temperature.
- Ionic liquids stabilized enzymes by protecting against thermal stress and enhancing substrate channeling.

## Abstract

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 (T
50) of GOx–HRP cascade in the presence of 10 wt % [Ch]2[PAA] with an enhanced melting temperature (T
m) 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.