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Proteins have immense untapped potential in numerous industries as a green catalyst. Thus, there is an emergent need to find a suitable co-solvent which is biocompatible with proteins and environmentally safe. In this context, the present study investigates the effect of a novel solvent medium designed by an amalgamation of macromolecular crowders and deep eutectic solvents (DESs) on bovine serum albumin (BSA). It was discovered that the presence of crowding agents such as polyethylene glycol (PEG) 12 kDa (P12), 20 kDa (P20), and Ficoll 70 kDa (F70) marginally destabilizes the conformational stability of BSA. While the thermal stability of BSA was dramatically enhanced in the presence of choline chloride (ChCl)-based deep eutectic solvents (DESs) namely ChCl-urea (DES1) and ChCl-glycerol (DES2), the order of stability was (DES1) > (DES2). It was interesting to note that DES1 possessing urea as a hydrogen bond donor leads to the exceptional thermal stability of the BSA structure. Taking a cue from this, an innovative crowded DES medium was prepared by mixing a synthetic crowder and DES1 in optimum concentration. Fascinatingly, the combination of DES1 with PEG delivers promising results, as they elevate the thermal stability of BSA by approximately 16 °C. Thus, crowded DES medium confers structural compactness to the BSA. Altogether, this work reports for the first time the potential of DESs to attenuate the adverse effects of macromolecular crowding on protein stability.

Ionic liquids (ILs) present a vast number of solvents capable of replacing toxic organic solvents in chemical, biotechnology and biomedical applications. ILs are inexpensive and environmentally friendly as the materials can be recycled conveniently. Chemists use a variety of cation and anion combinations to produce an IL that fits the requirements of the sustainable future through the pursuit of greener chemical processes. As such, the development of various types of ILs has been recognized as the emergence of environmentally friendly solvents to attain enhanced protein stability in vitro. The literature survey reveals that there exist a large number of scholarly articles as well as elegant reviews on protein stability in ILs. Biomolecules have adapted to antagonistic environmental stresses that normally denature proteins, and the mechanism of adaptation that protects the cellular components against denaturation involves the intracellular concentration of co-solvents. In this regard, recent experimental results distinctly demonstrated that ILs are stabilizing proteins against denaturing stresses, and their presence in the cells does not alter protein functional activities. However, a review focusing particularly on the refolding and counteracting effects of the ILs against denatured proteins by multiple stresses is still missing. This perspective unveils the studies that have been conducted to improve protein stabilities with ILs as well as the refolding and counteracting abilities of these ILs against the denatured proteins under the influence of multiple stresses. We believe that ILs can provide significant environmental and economic advantages for biochemical processes in the near future. Essentially, numerous investigations are required to allow us to further explore the stabilizing properties of ILs over proteins.