Unraveling the Role of Deep Eutectic Solvents with Varying Hydrogen-Bond Acceptors on the Thermoresponsive Polymer Poly(N-isopropylacrylamide).

Deep eutectic solvents (DESs) have emerged as promising tools for crafting polymeric materials across diverse domains. This study delves into the impact of a series of DESs on the phase behavior of poly(N-isopropylacrylamide) (PNIPAM) in aqueous environments, presenting compelling insights into their performance. Specifically, we explore the conformational phase behavior of PNIPAM in the presence of four distinct lactic acid (LA)-based DESs: LA-betaine (LA-BET), LA-proline (LA-PRO), LA-choline chloride (LA-CC), and LA-urea (LA-U). By maintaining a consistent hydrogen-bond donor (HBD) while varying the hydrogen-bond acceptor (HBA), we unravel how different DES compositions modulate the phase transition behavior of PNIPAM. Our findings underscore the profound influence of DESs comprising LA as the HBD and diverse HBAs-BET, PRO, CC, and U on the thermoresponsive behavior of PNIPAM. Employing spectroscopic techniques such as ultraviolet-visible (UV-vis) spectroscopy, steady-state fluorescence, Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), ζ-potential, and transmission electron microscopy (TEM), we elucidate the preferential interactions between the HBA groups within DESs and the hydration layer of PNIPAM. Notably, temperature-dependent DLS analyses reveal a discernible decrease in the lower critical solution temperature (LCST) of PNIPAM with increasing DES concentration, ultimately disrupting the hydrogen-bond interactions and resulting in early hydrophobic collapse of the polymer, which can be clearly seen in the TEM micrographs. Furthermore, the formation of polymer composites within the mixed system leads to notable alterations in the physiochemical properties of PNIPAM, as evidenced by shifts in its LCST value in the presence of DESs. This perturbation disrupts hydrogen-bond interactions, inducing hydrophobic collapse of the polymers, a phenomenon vividly captured in TEM micrographs. In essence, our study sheds new light on the pivotal role of varying HBA groups within DESs in modulating the conformational transitions of PNIPAM. These insights not only enrich our fundamental understanding but also hold immense promise for the development of smart polymeric systems with multifaceted applications spanning bioimaging, biomedical science, polymer science, and beyond.

Suitability of Adenosine Derivatives in Improving the Activity and Stability of Cytochrome c under Stress: Insights into the Effect of Phosphate Groups.

It is well known that adenosine and its phosphate derivatives play a crucial role in biological phenomena such as apoptosis and cell signaling and act as the energy currency of the cell. Although their interactions with various proteins and enzymes have been described, the focus of this work is to demonstrate the effect of the phosphate group on the activity and stability of the native heme metalloprotein cytochrome c (Cyt c), which is important from both biological and industrial aspects. In situ and in silico characterizations are used to correlate the relationship between the binding affinity of adenosine and its phosphate groups with unfolding behavior, corresponding peroxidase activities, and stability factors. Interaction of adenosine (ADN), adenosine monophosphate (AMP), adenosine 5'-diphosphate (ADP), and adenosine 5'-triphosphate (ATP) with Cyt c increases peroxidase-like activity by up to 1.8-6.5-fold compared to native Cyt c. This activity is significantly maintained even after multiple stress conditions such as oxidative stress and the presence of a chaotropic agent such as guanidine hydrochloride (GuHCl). With binding affinities on the order of ADN < AMP < ADP < ATP, adenosine derivatives were found to stabilize Cyt c by varying the secondary structural features of the protein. Thus, in addition to being a fundamental study, the current work also proposes a way of stabilizing protein systems to be used for real-time biocatalytic applications.

Assessing the compatibility of choline-based deep eutectic solvents for the structural stability and activity of cellulase: Enzyme sustain at high temperature.

As a new generation of 'green solvents' deep eutectic solvents (DESs) represents a promising alternative to the conventional solvents. Their environmental-benign nature and designer properties promote their utility in biocatalysis. Enzymes are marginally stable when exposed to physical/chemical disturbances. One such enzyme is cellulase which is a propitious catalyst for the depolymerization of cellulose under mild conditions. Therefore, their stability is a prerequisite condition to match demands of biorefineries. To address this issue of low stability, activity and thermal denaturation of cellulase, there is a need to find a sustainable and suitable co-solvent that is biocompatible with enzymes ultimately to facilitate their application in bio-industries. In this regard, we synthesized three choline-based DESs, choline chloride (ChCl)-glycerol, ChCl-ethylene glycol and ChCl-lactic acid and employed them to analyze their suitability for cellulase. The present study systematically evaluates the influence of the mentioned DESs on stability, activity and thermal stability of cellulase with the help of various spectroscopic techniques. The spectroscopic analysis revealed that the structural stability and activity of the enzyme were improved in presence of ChCl-glycerol and ChCl-ethylene glycol. The thermal stability was also very well maintained in both the DESs. Interestingly, the relative activity of cellulase was >80 % even after incubation at 50 °C after 48 h for both the DESs. This activity preservation behaviour was more pronounced for ChCl-ethylene glycol than ChCl-glycerol. Moreover, temperature variations studies also reveal promising results by maintain conformational intactness. On the other side, ChCl-lactic acid showed a deleterious effect on the enzyme both structurally as well as thermally. The dynamic light scattering (DLS) analysis provides more specific information about the negative influence of ChCl-lactic acid towards cellulase native structure. This DES induces unavoidable alterations in the enzyme structure which leads to the unfolding of enzyme, ultimately, destabilizing it. Overall, our results present a physical insight into how the enzyme stability and activity depend on the nature of DES. Also, the findings will help to facilitate the development and application of DESs as biocatalytic process.

Designing protein nano-construct in ionic liquid: a boost in efficacy of cytochrome C under stresses.

Herein, we present a simple approach to fabricate protein nanoconstructs by complexing cytochrome C (Cyt C) with silk nanofibrils (SNF) and choline dihydrogen phosphate ionic liquid (IL). The peroxidase activity of the IL modified Cyt C nanoconstruct (Cyt C + SNF + IL) increased significantly (2.5 to 10-fold) over unmodified Cyt C and showed enhanced catalytic activity and stability under harsh conditions, proving its potential as a suitable protein packaging strategy.

Presenting B-DNA as macromolecular crowding agent to improve efficacy of cytochrome c under various stresses.

Existence of numerous biomolecules results in biological fluids to be extremely crowded. Thus, Macromolecular crowding is an essential phenomenon to sustain active conformation of proteins in biological systems. Herein, double helical deoxyribonucleic acid (B-DNA) is presented for the first time as a biomacromolecular crowding system for sustainable packaging of cytochrome c (Cyt C). The peroxidase activity of Cyt C was investigated in the presence of various concentrations of B-DNA (from salmon milt). At an optimized concentration of 0.125 mg/mL B-DNA, an 11-fold higher catalytic activity was found than in native Cyt C with improved stability. Molecular docking and spectroscopic analyses revealed that electrostatic and H-bonding are the main interactions between DNA and Cyt C that affect the structural stability and activity of the protein. Moreover, the catalytic activity and stability of the protein were further investigated in the presence of severe process conditions by UV-visible, circular dichroism, and Fourier-transform infrared spectroscopies. Molecularly crowded Cyt C showed significantly higher activity and stability under severe environments such as high temperature (110 °C), oxidative stress, high pH (pH 10) and biological (trypsin) and chemical denaturants (urea) compared to bare Cyt C. The observed results support the suitability of DNA-based macromolecular crowding media as a viable and effective stabilizer of proteins against multiple stresses.

Unveiling the potential of water as a co-solvent in microwave-assisted delignification of sugarcane bagasse using ternary deep eutectic solvents.

Deep eutectic solvents (DESs) have become popular owing to their biodegradability and recyclability. In this study, the influence of water as a co-solvent is demonstrated to enhance the properties of choline based ternary DESs. A fast and energy-efficient microwave-assisted pre-treatment process was developed for delignification of sugarcane bagasse (SB). The effectiveness of SB fractionation was revealed by incorporating Lewis acids (MgCl2.6H20, NiCl2.6H20) with the DESs for pre-treatment and Choline chloride: Ethylene glycol: NiCl2.6H20 (CC:EG:NI) at a molar ratio 1:2:0.016 with 20w% water as a co-solvent provided the most promising result, with 84% delignification and 99% enzyme digestibility. Water was also employed as an anti-solvent to facilitate lignin solubility and exhibited up to 26w% lignin yield from DES liquor with maximum DES recovery of 95% (w/w). Water distinctly affects the density, viscosity, and intermolecular hydrogen bonding of the DES and its impact on the process dynamics is worth further exploration.

Protein-olive oil-in-water nanoemulsions as encapsulation materials for curcumin acting as anticancer agent towards MDA-MB-231 cells.

The sustainable cellular delivery of the pleiotropic drug curcumin encounters drawbacks related to its fast autoxidation at the physiological pH, cytotoxicity of delivery vehicles and poor cellular uptake. A biomaterial compatible with curcumin and with the appropriate structure to allow the correct curcumin encapsulation considering its poor solubility in water, while maintaining its stability for a safe release was developed. In this work, the biomaterial developed started by the preparation of an oil-in-water nanoemulsion using with a cytocompatible copolymer (Pluronic F 127) coated with a positively charged protein (gelatin), designed as G-Cur-NE, to mitigate the cytotoxicity issue of curcumin. These G-Cur-NE showed excellent capacity to stabilize curcumin, to increase its bio-accessibility, while allowing to arrest its autoxidation during its successful application as an anticancer agent proved by the disintegration of MDA-MB-231 breast cancer cells as a proof of concept.

Multifunctional solvothermal carbon derived from alginate using 'water-in-deep eutectic solvents' for enhancing enzyme activity.

Herein, the use of a "water-in-deep eutectic solvent (DES)" system has been shown as an alternative platform for the low temperature conversion of alginic acid (AA) to a multifunctional aliginate derived carbon (AAC) material with variable oxygen functionalities. Using the in-built oxygenated functionalities in AAC, cytochrome-c (Cyt-c) was immobilised on the surface of AACs and the peroxidase activity of the enzyme was studied. Remarkably, the enzymatic activity was enhanced up to 5.5-fold compared to the native protein without compromising the structural stability of Cyt-c. Altogether, the present study demonstrates a sustainable process for the preparation of AACs and shows their beneficial effect on the activity and stability of Cyt-c, and thus, their application as a protein-friendly material for biotechnological applications.

Designing biological fluid inspired molecularly crowded ionic liquid media as a sustainable packaging platform for cytochrome c.

Biological fluids are highly crowded due to the presence of various biomolecules. Therefore, it is essential to study the effects of molecular crowding agents to probe the behavior of a protein in a cell-like environment. Inspired by biofluids, herein, a molecularly crowded environment is created in the presence of an ionic liquid (IL), envisaging sustainable protein packaging. Interestingly, the molecularly crowded IL media enhanced the stability and catalytic activity (1.5-fold higher) of cytochrome c (Cyt c) as compared to the IL alone and the crowding agents without the IL. A similar trend was observed when the activity was recorded at 100 °C and when stored at room temperature for 30 days. Moreover, Cyt c dissolved in the molecularly crowded IL media was regenerated successfully without affecting the melting temperature of the protein, confirming the suitability of the molecularly crowded IL media as a potential and ecofriendly packaging system for Cyt c.

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.

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.

Long-term protein packaging in bio-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 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.

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.

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.

Refolding effects of partially immiscible ammonium-based ionic liquids on the urea-induced unfolded lysozyme structure.

The activity of lysozyme over a Micrococcus lysodeikticus cell suspension increased to 13% of the initial value in the presence of 1% v/v ammonium-based ionic liquids after deactivation with 4.0 M urea. This increase in activity reflects the refolding ability of the ionic liquids against the denaturation effects of urea on lysozyme.

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.