There is a considerable interest in the use of structurally stable and catalytically active enzymes, such as cytochrome C (Cyt C), in the pharmaceutical and fine chemical industries. However, harsh process conditions, such as temperature, pH, and presence of organic solvents, are the major barriers to the effective use of enzymes in biocatalysis. Herein, we demonstrate the suitability of bio-based ionic liquids (ILs) formed by the cholinium cation and dicarboxylate-based anions as potential media for enzymes, in which remarkable enhanced activity and improved stability of Cyt C against multiple stresses were obtained. Among the several bio-ILs studied, an exceptionally high catalytic activity (> 50-fold) of Cyt C was observed in aqueous solutions of cholinium glutarate ([Ch][Glu]; 1g/mL) as compared to the commonly used phosphate buffer solutions (pH 7.2), and > 25-fold as compared to aqueous solutions of cholinium dihydrogen phosphate ([Ch][Dhp]; 0.5g/mL) —the best known IL for long term stability of Cyt C. The catalytic activity of the enzyme in presence of bio-ILs was retained against several external stimulus, such as chemical denaturants (H2O2 and GuHCl), and temperatures up to 120 °C. The observed enzyme activity is in agreement with its structural stability, as confirmed by UV–Vis, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopies. Taking advantage of the multi-ionization states of di/tri-carboxylic acids, the pH was switched from acidic to basic by the addition of the corresponding carboxylic acid and choline hydroxide, respectively. The activity was found to be maximum at a 1:1 ratio of [Ch][carboxylate], with a pH in the range from 3 to 5.5. Moreover, it was found that the bio-ILs studied herein protect the enzyme against protease digestion and allow long-term storage (at least for 21 weeks) at room temperature. An attempt by molecular docking was also made to better understand the efficacy of the investigated bio-ILs towards the enhanced activity and long term stability of Cyt C. The results showed that dicarboxylates anions interact with the active site’s amino acids of the enzyme through H-bonding and electrostatic interactions, which are responsible for the observed enhancement of the catalytic activity. Finally, it is demonstrated that Cyt C can be successfully recovered from the aqueous solution of bio-ILs and reused without compromising its yield, structural integrity and catalytic activity, thereby overcoming the major limitations in the use of IL-protein systems in biocatalysis.

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Since the introduction of deep eutectic solvents (DESs), numerous reports have surfaced describing its tunable properties and environmentally benign nature. Due to such favorable properties of DESs, they have found a wide variety of applications. Moreover, in order to harness the potential of proteins in numerous industries, there is an emergent need to find a suitable cosolvent that is biocompatible with protein and is also environmentally safe. In this context, this work presents a systematic evaluation of effect of two deep eutectic solvents (DESs), namely, choline chloride-urea (ChCl-urea) and choline chloride-glycerol (ChCl-gly) on the structural and thermal stability along with activity of enzyme α-chymotrypsin (CT) using circular dichroism (CD), UV–visible, steady state, and thermal fluorescence spectroscopy. It was observed that the presence of DESs does lead to enhancement in the thermal stability of CT along with the preservation of activity. The enzymatic activity was well maintained in both the DESs, and the deleterious effect of urea was overcome by ChCl-urea on the enzyme. Also, desirable results were observed for ChCl-urea, despite having urea as one of its major components. Thus, the negative outcome of urea was overpowered by the combination of ChCl and urea. Furthermore, all the biomolecular studies were also performed with the individual constituents of DESs. It was found that the effect imparted by both the ChCl-based DESs on CT is by the virtue of DES itself rather than its individual constituent. Overall, both the DESs can be described as potential biocompatible, sustainable, and promising cosolvents for CT with enhanced structural and thermal stability along with preservation of its activity.