The present work demonstrates a pioneering approach for the packaging of β-LG with improved stability in the presence of aqueous solutions containing cholinium-based ionic liquid mixtures.

In the past two decades, ionic liquids (ILs) have been acknowledged as potentially attractive “green” and “designer” solvents since their broader chemical space and the tunable nature of the diverse ions can finely modulate their physiochemical properties while enabling task-specific optimization. They have found numerous applications in fields ranging from enzymatic reactions to protein preservation, which focused on the development of various types of ILs and their application for providing protein stability in vitro. Among various families of ILs, some of them have serious limitations similar to organic solvents; they cause environmental toxicity as they are non-biodegradable. In other words, some of the ILs impart stress to biomolecules and ultimately denature the protein. In this regard, the need for biocompatible ILs has come to the light and cholinium-based ILs (Chn ILs) have proved themselves as the most promising ILs to support the structure of biomolecules. The family of Chn ILs has only taken growth in recent years. Despite the numerous studies, more exhaustive research in the field of Chn ILs and biomolecules still needs acceleration. Herein, we review the strategies and current progress on Chn ILs for protein and enzyme-based applications keeping in mind all crucial past and present research outcomes. Furthermore, we elucidate an overview of the various ways to enhance enzymatic activity, structural stability, and long-term storage of proteins in the presence of Chn ILs. We believe these insights will be fruitful for designing various processes based on ILs in the diverse field of biotechnology and ILs will serve as novel solvents for protein stability and enzymatic reactions, maintaining their utility in industrial and biomedical based applications. The huge varieties of biobased Chn ILs hold the promise of recent advances and developments for the correct selection of long-term storage of enzymes.

No abstract

The synthesis of nanoparticles using ionic liquids (ILs) has attracted intensive research; however, synthesis and surface tailoring of gold nanoparticles (AuNPs) using ILs for enzyme immobilization have not yet been reported. Herein, we synthesized the various IL-modified AuNPs using different ILs, which are having common cation 1-ethyl-3-methyl-imidazolium (EMIM) and variable anions [BF 4−1 (AuNP-IL1), (CH 3 OSO 3 ) −1 (AuNP-IL2), (CH 3 CH 2 OSO 3 ) −1 (AuNP-IL3), and Cl −1 (AuNP-IL4)] by reduction of gold salt. The formation of IL modified AuNPs has been confirmed using UV−vis, zeta-potential, FTIR, and transmission electron microscopy (TEM). Thereafter, the centrifuged IL-modified AuNPs are being immobilized with a lysozyme (Lyz) enzyme to evaluate the effect of different AuNP covering groups (capping agent and IL's anions) for Lyz microbial activity, thermal and structural stabilities through interaction studies, spectroscopic techniques, and morphology investigation by TEM. AuNP-IL1 has increased the microbial activity of Lyz up to 2.6 fold at the concentration of 4 nM, and AuNP-IL2 is highly efficient to dextrously preserve enzyme activity against packaging for 4 weeks. The higher Michaelis−Menten constant (K M ) has been observed for Lyz immobilized in the AuNP-IL2 due to higher binding with the AuNP-IL2. Apparently, the higher specific constant (K cat /K M ) of immobilized Lyz has been observed in the case of AuNP-IL3 and shows more specific binding of Lyz with this particular IL-mediated AuNPs. The significant thermal stability enhancement about 8.11 °C is observed for transition temperature (T m ) of Lyz in the presence of sulfur group-containing IL-modified AuNPs like AuNP-IL2 and AuNP-IL3, which depends on the specific interacting ability of these AuNPs with Lyz. Therefore, the study reveals the variant character of sulfur-containing IL-modified AuNPs for higher activity and thermal and structural stability of Lyz. Surprisingly, this has created a way to monitor sulfur and hydrophobic interactions on AuNPs for enzyme immobilization through means of controlling surface modifications and interactions.

One of the major challenges in protein stability is that proteins can easily unfold in the presence of denaturants like urea, which alters the native structure of proteins. There are numerous studies in which ionic liquids (ILs) act as promising biocompatible solvents (Bio-IL) for biomolecules. In this context, we present the refolding ability of biocompatible imidazolium-based ILs, 1-ethyl 3-methyl imidazolium ethyl sulfate [Emim]­[ESO4] (IL-1) and 1-ethyl 3-methyl imidazolium chloride [Emim]­[Cl] (IL-2) against the chemically induced structural changes in bovine and human serum albumin (BSA and HSA). The work is substantiated with several spectroscopic, thermal and docking studies. In steady-state fluorescence spectroscopy, we observe that the emission intensity quenches for the protein in urea, which is reversible with the addition of ILs. Circular dichroism spectral studies reflect the reappearance of α helical content, which is a good indicator of the refolding ability of ILs. Further, thermal fluorescence studies showed that ILs have the ability to refold the urea-induced denatured protein at a higher temperature range only up to 7 M urea concentration; however, above 7 M urea concentration, IL somehow fails to refold the protein. The work is also supported by dynamic light scattering measurements, and the degree of BSA/HSA aggregation was reduced with the introduction of Bio-IL to the urea–BSA/urea–HSA system, ensuring the aggregate-free refolding. Furthermore, molecular docking studies were employed to probe the binding sites, and the results are well corroborated with the spectroscopic and thermal folding results. Therefore, through this paper, we aim to unravel the mechanistic intricacy of ILs using experimental and docking approaches. Overall, ILs act as recoiling medium for both native and unfolded (denatured by urea) BSA/HSA native structures.