Thermal stability and thermodynamic analysis of a commercial xylanase in natural deep eutectic solvents (NADES)

Enzymes' structural and functional integrity is highly sensitive to temperature and pH in numerous industrial processes, often reducing efficiency; thus, alternative solvent systems are needed to provide a stabilizing microenvironment that sustains enzymatic activity under demanding process conditions. This study investigates the thermal and thermodynamic stability of a commercial xylanase (Viscoferm®) in selected Natural Deep Eutectic Solvents (NADES) compared to conventional aqueous buffer systems. The enzyme was incubated at six temperatures (30–80 °C) in three NADES, Betaine:Glycerol (1:3, Bet:Gly), Choline chloride:Glycerol (1:2, ChCl:Gly), and L-proline:Glycerol (1:2, Pro:Gly), each containing 20% ( w /w) water as cosolvent. Parallel tests were conducted in buffers at pH values corresponding to each NADES. Kinetic parameters (k d , t 1/2 , D, z-value) and thermodynamic properties ( E α d , ΔΗ D ∗ , Δ G D ∗ , ΔS D ∗ ) were calculated to elucidate deactivation mechanisms and solvent-enzyme interactions. Results indicated enhanced thermal stability in all NADES, while thermodynamic analysis revealed consistently higher Δ G D ∗ values and lower ΔΗ D ∗ , ΔS D ∗ values in NADES, suggesting improved resistance to thermal unfolding. Fluorescence spectroscopy supported these findings, indicating better retention of tertiary structure. Among the NADES, Bet:Gly induced the greatest thermodynamic stabilization. These findings demonstrate that NADES significantly enhance enzyme thermal stability, supporting their use as green and tunable media in industrial biocatalysis.