Meena Bisht

Biocompatibility of ionic liquids towards protein stability: A comprehensive overview on the current understanding and their implications

Over the past years since the discovery of ionic liquids (ILs), there is an increased demand to consider ILs as novel biocompatible co-solvents for proteins. Due to their tunable physical properties ILs can adjust themselves in any required experimental conditions starting from protein extraction to enzyme catalysis at elevated temperature. In recent years, large numbers of ILs have been synthesized and their effect on protein stability has been illustrated. With the rapid growth in various kinds of ILs, our understanding of protein stability in ILs has substantially increased. It is not necessary that a particular IL that is biocompatible to a protein will behave same for the other. Therefore, it is extremely essential to collect the literature dealing with the direct involvement of ILs in protein folding/unfolding studies under the same roof. This review focuses the tremendous accomplishments achieved in recent years in the field of protein stability in ILs. We hope that this would also help to set a stage where we can identify, explore and compare the mechanistic behavior of protein folding/unfolding in ILs. This review will surely bring a new boost in protein folding studies from the chemical biology perspective.

Long-term protein packaging in cholinium-based ionic liquids: improved catalytic activity and enhanced stability of cytochrome c against multiple stresses

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 sites 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.

Analysis of the driving force that rule the stability of lysozyme in alkylammonium-based ionic liquids.

Ionic liquids (ILs) have found various applications in the field of biotechnology that involves protein extraction from the aqueous phase. However, the stability of biomolecules in ILs is still unpredictable. Therefore, this work aims to understand the effect of ammonium-based ILs with a fixed (trifluoromethylsulfonyl)imide [NTf2](-) anion and variable ammonium cations such as butyltrimethylammonium (IL-1), ethyldimethylpropylammonium (IL-2), diethylmethyl(2-methoxyethyl)ammonium (IL-3) and methyl-trioctylammonium (IL-4) on the stability of lysozyme. The spectroscopic analysis (UV, fluorescence and circular dichroism (CD)) revealed the existence of native structure of lysozyme in the presence of ILs at 25°C. Evidently, the presence of α-helix structure in lysozyme was confirmed using CD spectroscopy. In contrary, the thermal stability of the protein gradually decreased with increase in the concentration of the ILs. This was due to the strong favorable interactions of the ILs with the amino acid residues of the protein. Further, Nile red fluorescence revealed existence of the hydrophobic interactions between ILs and the lysozyme. Hence, due to its immense hydrophobic character, IL-4 thereby, decreased the catalytic activity and stability of the lysozyme to a greater extent.

Influence of cholinium-based ionic liquids on the structural stability and activity of α-chymotrypsin

In recent years, the potential of α-chymotrypsin (CT) as biocatalysts has expanded new areas of its application ranging from pharmaceutical to chemical industries. However, attaining high thermal stability is one of the major challenges to the use of this enzyme in biocatalysis. In this regard, ionic liquids (ILs) have been used as promising media for the stabilization and preservation of proteins, enzymes, DNA and other biomolecules. In the present study, it was found that a series of cholinium-based ILs such as choline acetate ([Ch][Ac]), choline chloride ([Ch][Cl]), and choline dihydrogen phosphate ([Ch][Dhp]) stabilized the CT structure against thermal denaturation. The transition temperature (Tm) of CT was increased from ∼48.9 °C (in the buffer) to 58 °C (in the ILs media). The enzymatic activity of CT in the presence of ILs was also monitored by using casein as the substrate. It was found that choline dihydrogen citrate ([Ch][Dhc]) and choline hydroxide ([Ch][OH]) dramatically decreased the enzyme activity. Both structural stability and enzymatic activity were retained in [Ch][Ac], [Ch][Cl] and [Ch][Dhp], indicating the suitability of these ILs as a high-temperature bio-catalytic reactor systems. Our results revealed that [Ch][Ac] is the best stabilizer among all studied ILs for the native structure of CT, whereas [Ch][OH] is the strongest destabilizer for the CT structure. The outcome of our results can be helpful to overcome some of the major limitations found in the development of biocatalytic processes.

Exploring the structure and stability of amino acids and glycine peptides in biocompatible ionic liquids

Amino acids (AAs) are vital components for a variety of biological systems and can be linked through covalent bonds (or peptide bonds) to form a protein structure. Essentially, the interactions of these AAs with solvents as well as co-solvents/solutes determine the thermodynamic stability of the protein. In this context, this review represents an overview of the current status of the thermodynamic effect of ionic liquids (ILs) on AAs and glycine peptides (GPs). Moreover, ILs are considered as green solvents for many chemical and biological processes due to their tunable physical properties. Interestingly, these ILs can adjust themselves in any required experimental conditions such as protein extraction to enzyme catalysis. In this review, we attempt to assess the status of our current understanding on the biocompatible nature of the ions of ILs on AAs and protein model compounds and their functional groups with some precisely available experimental data in the literature. Examples of current applications of the ILs on protein model compounds are also covered in this review.

Does 1-Allyl-3-methylimidazolium chloride Act as a Biocompatible Solvent for Stem Bromelain?

The broader scope of ILs in chemical sciences particularly in pharmaceutical, bioanalytical and many more applications is increasing day by day. Hitherto, a very less amount of research is available in the depiction of conformational stability, activity, and thermal stability of enzymes in the presence of ILs. In the present study, the perturbation in the structure, stability, and activity of stem bromelain (BM) has been observed in the presence of 1-allyl-3-methylimidazolium chloride ([Amim][Cl]) using various techniques. This is the first report in which the influence of [Amim][Cl] has been studied on the enzyme BM. Fluorescence spectroscopy has been utilized to map out the changes in the environment around tryptophan (Trp) residues of BM and also to discuss the variations in the thermal stability of BM as an outcome of its interaction with the IL at different concentrations. Further, the work delineates the denaturing effect of high concentration of IL on enzyme structure and activity. It dictates the fact that low concentrations (0.01-0.10 M) of [Amim][Cl] are only changing the structural arrangement of the protein without having harsh consequences on its activity and stability. However, high concentrations of IL proved to be totally devastating for both activity and stability of BM. The observed decrease in the stability of BM at high concentration may be due to the combined effect of cation and anion interactions with the protein residues. The present work is successful in dictating the probable mechanism of interaction between BM and [Amim][Cl]. These results can prove to be fruitful in the studies of enzymes in aqueous IL systems since the used IL is thermally stable and nonvolatile in nature thereby providing a pathway of alteration in the activity of enzymes in potentially green systems.

Effect of Imidazolium-Based Ionic Liquids on the Structure and Stability of Stem Bromelain: Concentration and Alkyl Chain Length Effect

In the present work, changes in the structure and stability of stem bromelain (BM) are observed in the presence of a set of four imidazolium-based ionic liquids (ILs) such as 1-ethyl-3-methylimidazolium chloride ([Emim][Cl]), 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]), 1-hexyl-3-methylimidazolium chloride ([Hmim][Cl]), and 1-decyl-3-methylimidazolium chloride ([Dmim][Cl]), using various biophysical techniques. Fluorescence spectroscopy is used to observe the changes taking place in the microenvironment around the tryptophan (Trp) residues of BM and its thermal stability because of its interactions with the ILs at different concentrations. Near-UV circular dichroism results showed that the native structure of BM remained preserved only at lower concentrations of ILs. In agreement with these results, dynamic light scattering revealed the formation of large aggregates of BM at higher concentrations of ILs, indicating the unfolding of BM. In addition to this, the results also show that higher alkyl chain length imidazolium-based ILs have a more denaturing effect on the BM structure as compared to the lower alkyl chain length ILs because of the increased hydrophobic interaction between the ILs and the BM structure. Interestingly, it is noted that low concentrations (0.01-0.10 M) of short alkyl chain ILs only alter the structural arrangement of the protein without any significant effect on its stability. However, high concentrations of all five ILs are found to disrupt the structural stability of BM.

Exploring the Effect of Choline-Based Ionic Liquids on the Stability and Activity of Stem Bromelain

Enzymes are very important components which are vital for the existence of every cellular life. There is significant interest in the use of structurally stable and catalytically active enzymes in pharmaceutical, food, fine chemicals industries, and in various industrial processes as catalysts. Stem bromelain (BM) is a proteolytic enzyme which is widely used in chemical, medical, and pharmaceutical fields. However, harsh process conditions are the main barriers to the effective use of this enzyme in different applications. To overcome these drawbacks, biocompatible bio-based ionic liquids (ILs), composed of the choline cation (an essential nutrient) and different anions are used. The ILs namely choline chloride [Ch]+[Cl]-, choline acetate [Ch]+[Ac]-, choline dihydrogen phosphate [Ch]+[Dhp]-, choline bitartrate [Ch]+[Bit]-, choline iodide [Ch]+[I]-, and choline hydroxide [Ch]+[OH]- are chosen for the current work. Therefore, in the present study, structural stability and activity of BM have been evaluated in the presence of choline-based ILs using various biophysical techniques at different concentrations. The present work demonstrated that [Ch]+[OH]- is the strongest destabilizer, whereas [Ch]+[Cl]- is the best stabilizer for the native structure of BM among all studied ILs. This work revealed the suitability of some choline-based ILs as potential media for sustained stability and activity of BM.

Direct Conversion of Lignocellulosic Biomass to Biomimetic Tendril-Like Functional Carbon Helices: A Protein Friendly Host for Cytochrome C

There is sizable interest in the fabrication of carbon helices owing to their uses in several applications. Methods have been developed for the preparation of such carbon helices from nonrenewable hydrocarbons by a metal-catalyzed chemical vapor deposition process. However, such methods require high temperature and toxic gases besides multi-step processes. Herein we report the preparation of tendril-like functional carbon helices (TLFCHs) directly from lignocellulosic biomass using a green solvothermal method employing a deep eutectic solvent as both soft template and catalyst. Under optimized conditions biomimetic helical carbons with fibre widths of 1.5–2.0 μm, coil diameters of 8–10 μm, coil lengths of 30–50 μm and having a high degree of oxygenated functionalities were obtained. Taking advantage of both helicity and in-built chemical functionalities, cytochrome C (Cyt C) was immobilized on the surface of TLFCHs to probe their protein friendly nature. The results demonstrated that TLFCHs showed significant potential as a host for an enzyme without compromising the catalytic activity and thus can be envisaged as a protein friendly biomaterial for facile biocatalysis.

The Role of Ionic Liquids in Protein Folding/Unfolding Studies

Ionic liquids (ILs) have emerged as novel solvent medium for several biotechnological processes in vitro. The use of ILs starts from protein extraction to catalysis to folding/ unfolding studies. ILs are becoming the most favorite non-aqueous medium for protein studies due to their unique ionic combinations (cation + anion) and tunable physical properties. In this context, several research results have been published that use of pure or aqueous IL solutions as stabilizer for proteins. Hence, herein, in this chapter, we present a collection of research work that focuses on the importance of ILs (and their mixture) in protein stabilities. In addition, we have also reviewed the unique properties of ILs as counteracting solvents for cold-induced denaturation and also their refolding properties. This report will definitely generate a new understanding for the ILs, their importance and applicability in protein folding studies.