Thermal stabilization of l-lactate oxidase in natural deep eutectic solvents mimicking in a living cell

Organisms maintain protein structure and function under environmental stress conditions by accumulating small organic molecules known as osmolytes. Mixtures of osmolytes can form natural deep eutectic solvents (NADESs), which have attracted attention as environmentally friendly media for biocatalysis; however, the effect of such NADESs on large, industrial enzymes and the mechanisms underlying their thermal stabilization remain unclear. In this study, we examined the effect of a NADES that mimics the osmolyte composition of the inner medulla of the rabbit kidney (RK-NADES) on the thermal stability of tetrameric l-lactate oxidase (LOX). LOX was completely inactivated at 60 °C in buffer, whereas in RK-NADES, it retained activity up to 70 °C and was inactivated above 80 °C. The apparent half-life of LOX at elevated temperatures was extended up to 15-fold compared with its individual aqueous components. Moreover, RK-NADES suppressed thermally induced changes in the tertiary structure of LOX, which indicates a structural basis for its enhanced functional stability. Molecular dynamics simulations further supported this observation, suggesting that RK-NADES components partially replace water molecules and form stabilizing hydrogen bonds with LOX. These findings provide a simple and effective approach to stabilizing enzymes under unstable conditions using NADESs designed to mimic the osmolyte composition of living cells.