Improving the activity of horseradish peroxidase in betaine-based natural deep eutectic systems

In this work, the activity of horseradish peroxidase (HRP) in betaine-based natural deep eutectic systems (NADESs) was measured and studied by molecular simulations. Focus was laid on enzymatic activity in the NADESs under thermal stress as well as under the influence of water. Furthermore, the structure of HRP under these different conditions was measured by circular dichroism (CD). As a result, HRP remains enzymatically active in all NADESs upon incubation for 24 h at 37 °C and 60 °C and after 4 h at 80 °C, especially when incubated in a NADES composed of betaine, trehalose, glycerol, and water, in a molar ratio of 2 : 1 : 3 : 5. The CD studies have shown that high activity is obtained in the systems that promoted higher α-helix contents. The molecular simulations showed that using a NADES instead of buffer solvent reduces HRP flexibility, and we found that enzymatic activity correlates with Gibbs energy of solvation of HRP. Finally, hydrophobic hydration interactions govern the stabilization mechanism of the HRP folded state as shown by a drastic enzymatic activity drop upon 5 wt% water addition using a betaine: glycerol NADES as solvent.