The power of NADES: Boosting enzyme stability towards thermal degradation

In recent years, natural deep eutectic solvents (NADESs) have gained increasing attention as promising nontoxic solvents for biotechnological applications, due to their attractive properties including low flammability, low volatility, facile preparation, high solvability, compatibility with enzymes and the ability to enhance their activity. In this regard, one of the challenges in the implementation of enzymes in industrial processes is their ability to remain active for a longer time or survive harsh operative conditions, resulting in the need of reengineer enzymes to fine-tune their properties toward the end application. Betaine-based NADESs at a concentration of 25 wt % in a buffered aqueous solution were used as media to inhibit thermal inactivation of POXA1b laccase and its five variants when incubated at 70 and 90 °C. All the tested laccases showed higher residual activity when incubated in NADES solutions, with a further enhancement achieved also for the most thermostable variant. Furthermore, the residual activity of laccases in the presence of NADESs showed a clear advantage over the use of NADESs’ individual components. A computer-aided approach based on molecular docking simulation was performed to understand the role of NADESs in the stabilization of laccases toward thermal inactivation, evaluating the interaction between each enzyme and NADESs’ individual components gaining more insights into this structure− function relationship. A correlation within the binding energies between laccases and NADES components and the stabilization of the enzymes was also demonstrated. These findings establish the possibility of preincubating enzymes in NADESs as a facile and cost-effective solution to inhibit thermal inactivation of enzymes when exposed to high temperatures. The precise combination of interactions and molecule orientation determined a different stabilizing effect for each enzyme, suggesting the possibility to tailor the NADES composition for every enzyme of interest, taking advantage of computer-aided approaches for a preliminary screening of different combinations of NADES’ components