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

Abstract

Deep eutectic solvents (DESs) are considered as designer solvents that serve as alternatives to traditional solvents. Numerous favourable properties and advantageous characteristics promote their utility in bio-catalysis. Therefore, they have emerged as attractive sustainable media for different biomacromolecules. In the present work, we have synthesized cholinium-based DESs having a hydrogen bond acceptor (HBA) : hydrogen bond donor (HBD) molar ratio of 1 : 2 by varying the cationic ratio in the HBA, which led to the formation of the DESs such as monocholinium citrate ([Chn][Cit]), dicholinium citrate ([Chn]₂[Cit]) and tricholinium citrate ([Chn]₃[Cit]), keeping the HBD ethylene glycol (EG) constant 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 the structural and thermal stability, thermodynamic profile, colloidal stability and enzymatic activity of α-CT using different spectroscopic techniques. The spectroscopic results explicitly show enhanced structural stability and activity of the enzyme as the cationic ratio in the HBA increases. Fascinatingly, temperature-dependent studies through both fluorescence and activity measurements showed that DES-2 and DES-3 have highly beneficial effects on α-CT stability. The transition temperature (T_m) of α-CT was augmented by 12.0 °C in DES-2, 10.0 °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 (d_H) measurements and average decay time analysis, which confirmed the observed morphological similarities and perturbations. The long-term preservation ability and kinetics of DES-3 were eventually confirmed by Michaelis–Menten kinetics. Ultimately, these outcomes demonstrate that increasing the molar ratio of the cholinium cation in the HBA can enhance the ability of DESs to stabilize the α-CT structure. Our results also suggest that the effect imparted by DESs was due to DESs themselves rather than their constituent elements. Altogether, the present investigation provides a new insight into the dependence of protein's stability and conformational alterations on DES composition. Also, the biocompatibility of DESs towards enzymes can be varied by changing the molar ratios of the constituent components of DESs to facilitate the expansion of applicability of DESs in biocatalysis.