Unravelling the stabilization mechanism of mono-, di and tri-cholinium citrate-ethylene glycol DESs towards α-chymotrypsin for preservation and activation of the enzyme

Deep eutectic solvents (DESs) are considered as designer solvents which serve as an alternative to traditional solvents. Numerous favourable properties and advantageous characteristics promote their utility in bio-catalysis. Therefore, these have emerged as attractive sustainable media for different biomacromolecules. In the present work, we have synthesized cholinium- based DESs having molar ratio of hydrogen bond acceptor (HBA): hydrogen bond donor (HBD) in 1:2 by varying the cationic ratio in HBA forming the DESs as monocholinium citrate ([Chn][Cit]), dicholinium citrate ([Chn]2[Cit]) and tricholinium citrate ([Chn]3[Cit]), keeping HBD constant as ethylene glycol (EG) to study their suitability for α-chymotrypsin (α-CT). Herein, we have systematically evaluated the influence of DES-1 ([Chn][Cit])-[EG]), DES-2 ([Chn]2[Cit])-[EG]) and DES-3 ([Chn]3[Cit])-[EG]) on structural and thermal stability, thermodynamic profile, colloidal stability and enzymatic activity of α-CT using different spectroscopic techniques. The spectroscopic results explicitly elucidate enhanced structural stability and activity of enzyme as the cationic ratio on HBA increases. Fascinatingly, temperature-dependent studies through both fluorescence and activity measurements supported that DES-2 and DES-3 have highly beneficial effects on α-CT stability. The transition temperature (Tm) of α-CT was augmented by 12 °C in DES-2, 10 °C in DES-3 and 9.1 °C in DES-1 when compared to the enzyme in buffer. Furthermore, transmission electron microscopy (TEM) analysis revealed that the morphology of α-CT in DES-2 and DES-3 closely mirrored the structure of α-CT, while DES-1 exhibited only minor structural deviations. These findings were corroborated by hydrodynamic size (dH) measurements and average decay time analysis, which confirmed the observed morphological similarities and perturbations. The long-term preservation ability and kinetics of DES-3 was eventually confirmed by Michaelis-Menten kinetics. Ultimately, these outcomes demonstrate that by increasing the molar ratio of cholinium cation in HBA can lead to intensify the proficiency of DESs to stabilize the α-CT structure. Our results also suggest that the effect imparted by DESs was due to DES itself rather than its composing elements. Also, how the biocompatibility of DESs towards enzymes can be varied by changing molar ratios of the constituent components of DESs to facilitate the expansion of applicability of DESs in biocatalysis.