Enhancing protein stability under stress: osmolyte-based deep eutectic solvents as a biocompatible and robust stabilizing medium for lysozyme under heat and cold shock.

In biomedical and biotechnological domains, liquid protein formulations are vital tools, offering versatility across various fields. However, maintaining protein stability in a liquid form presents challenges due to environmental factors, driving research to refine formulations for broader applications. In our recent study, we investigated the relationship between deep eutectic solvents (DESs) and the natural presence of osmolytes in specific combinations, showcasing the effectiveness of a bioinspired osmolyte-based DES in stabilizing a model protein. Recognizing the need for a more nuanced understanding of osmolyte-based DES stabilization capabilities under different storage conditions, here we broadened the scope of our osmolyte-based DES experimental screening, and delved deeper into structural changes in the enzyme under these conditions. We subjected lysozyme solutions in DESs based on various kosmotropic osmolytes (TMAO, betaine, sarcosine, DMSP, ectoine, GPC, proline, sorbitol and taurine) paired either with another kosmotropic (glycerol) or with chaotropic osmolyte urea to rigorous conditions: heat shock (at 80 °C) and repetitive freeze-thaw cycles (at -20 and -80 °C). Changes in enzyme activity, colloidal stability, and conformational alterations were then monitored using bioassays, aggregation tests, and spectroscopic techniques (FT-IR and CD). Our results demonstrate the remarkable effectiveness of osmolyte-based DES in stabilizing lysozyme under stress conditions, with sarcosine- and betaine-based DESs containing glycerol as a hydrogen bond donor showing the highest efficacy, even at high enzyme loadings up to 200 mg ml -1 . Investigation of the individual and combined effects of the DES components on enzyme stability confirmed the synergistic behavior of the kosmotrope-urea mixtures and the cumulative effects in kosmotrope-glycerol mixtures. Additionally, we have shown that the interplay between the enzyme's active and stable (but inactive) states is highly influenced by the water content in DESs. Finally, toxicity assessments of osmolyte-based DESs using cell lines (Caco-2, HaCaT, and HeLa) revealed no risks to human health.