Abstract

Specifically designed co-solvent mixtures are an efficient way to enhance the kinetics of enzyme-catalyzed reactions without compromising enzyme stability; among them, several deep eutectic solvents have emerged as exciting co-solvent mixtures for biocatalytic reactions. DESs nature allows one to tailor the enzyme-co-solvent interactions by using DESs constituents of diverse functional groups. In this work, the influence of co-solvents (betaine, glycerol, and sorbitol) and two DESs (betaine:glycerol and betaine:sorbitol) on the kinetics of candida boidinii Formate dehydrogenase was evaluated. The results showed a 30% increase in catalytic efficiency by adding 15 wt.-% of betaine to the buffered aqueous reaction media. Further, cbFDH folded-state stability was evaluated using differential scanning fluorimetry to finally obtain the binding affinity, unfolding curves, and thermodynamic unfolding parameters. The addition of glycerol, sorbitol, and DESs increased cbFDH protection against thermal stress, and this effect could be improved by increasing co-solvent concentrations. Moreover, DESs showed the ability to reduce the irreversibility of the unfolding process. Betaine was the only co-solvent that had a negative stability effect, which was offset by using betaine-based DESs. The latter was a result of the additivity of certain individual co-solvent effects on thermal stability. Non-monotonous stability effects were obtained by adding sorbitol to the buffer solutions, probably because hydrogen bond dynamics between cbFDH/co-solvent/water change dramatically with the amount of water present. Finally, DESs improved NAD+ binding affinity with cbFDH interestingly without direct correlation with the results obtained for kinetics.