Sustainable combination of ionic liquid and deep eutectic solvent for protecting and preserving of the protein structure: The synergistic interaction of enzymes and eco-friendly hybrid ionic fluids.

Hybrid ionic fluids (HIFs) are one of the emerging and fascinating sustainable solvent media, a novel environment-friendly solvent for biomolecules. The HIFs have been synthesized by combining a deep eutectic solvent (DES), an ionic liquid (IL) having a common ion. The stability and activity of hen's egg white lysozyme (Lyz) in the presence of a recently designed new class of biocompatible solvents, HIFs have been explored by UV-visible, steady-state fluorescence, circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR) along with dynamic light scattering (DLS) measurements. This work emphasizes the effect of DES synthesized by using 1:2 choline chloride and glycerol [Glyn], ILs (1-butly-3-methylimidazolium chloride [BMIM]Cl and choline acetate [Chn][Ac]) and their corresponding HIFs on the structure and functionality of Lyz. Moving forward, we also studied the secondary structure, thermal stability and enzymatic activity and thermodynamic profile of Lyz at pH = 7 in the presence of varying concentrations (0.1 to 0.5) M of [BMIM]Cl, [Chn][Ac] ILs, [Glyn] DES and [Glyn][BMIM]Cl (hybrid ionic fluid1) as well as [Glyn][Chn][Ac] (hybrid ionic fluid2). Spectroscopic results elucidate that ILs affect the activity and structural stability of Lyz, whereas the stability and activity are increased by DES and are maintained by HIFs at all the studied concentrations. Overall, the experimental results studied elucidate expressly that the properties of Lyz are maintained in the presence of hybrid ionic fluid1 while these properties are intensified in hybrid ionic fluid2. This work has elucidated expressly biocompatible green solvents in protein stability and functionality due to the alluring properties of DES, which can counteract the negative effect of ILs in HIFs.

The attenuating ability of deep eutectic solvents towards the carboxylated multiwalled carbon nanotubes induced denatured ß-lactoglobulin structure.

The stabilization of proteins has been a major challenge for their practical utilization in industrial applications. Proteins can easily lose their native conformation in the presence of denaturants, which unfolds the protein structure. Since the introduction of deep eutectic solvents (DESs), there are numerous studies in which DESs act as promising co-solvents that are biocompatible with biomolecules. DESs have emerged as sustainable biocatalytic media and an alternative to conventional organic solvents and ionic liquids (ILs). However, the superiority of DESs over the deleterious influence of denaturants on proteins is often neglected. To address this, we present the counteracting ability of biocompatible DESs, namely, choline chloride-glycerol (DES-1) and choline chloride-urea (DES-2), against the structural changes induced in ß-lactoglobulin (Blg) by carboxylated multiwalled carbon nanotubes (CA-MWCNTs). The work is substantiated with various spectroscopic and thermal studies. The spectroscopic results revealed that the fluorescence emission intensity enhances for the protein in DESs. Contrary to this, the emission intensity extremely quenches in the presence of CA-MWCNTs. However, in the mixture of DESs and CA-MWCNTs, there was a slight increase in the fluorescence intensity. Circular dichroism spectral studies reflect the reappearance of the native band that was lost in the presence of CA-MWCNTs, which is a good indicator of the counteraction ability of DESs. Further, thermal fluorescence studies showed that the protein exhibited extremely great thermal stability in both DESs as well as in the mixture of DES-CA-MWCNTs compared to the protein in buffer. This study is also supported by dynamic light scattering and zeta potential measurements; the results reveal that DESs were successfully able to maintain the protein structure. The addition of CA-MWCNTs results in complex formation with the protein, which is indicated by the increased hydrodynamic size of the protein. The presence of DESs in the mixture of CA-MWCNTs and DESs was quite successful in eliminating the negative impact of CA-MWCNTs on protein structural alteration. DES-1 proved to be superior to DES-2 over counteraction against CA-MWCNTs and maintained the native conformation of the protein. Overall, both DESs act as recoiling media for both native and unfolded (denatured by CA-MWCNTs) Blg structures. Both the DESs can be described as potential co-solvents for Blg with increased structural and thermal stability of the protein. To the best of our knowledge, this study for the first time has demonstrated the role of choline-based DESs in the mixture with CA-MWCNTs in the structural transition of Blg. The DESs in the mixture successfully enhance the stability of the protein by reducing the perturbation caused by CA-MWCNTs and then amplifying the advantages of the DESs present in the mixture. Overall, these results might find implications for understanding the role of DES-CA-MWCNT mixtures in protein folding/unfolding and pave a new direction for the development of eco-friendly protein-protective solvents.

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.

Investigation on pollination approaches, reproductive biology and essential oil variation during floral development in German chamomile (Matricaria chamomilla L.).

German chamomile is an important medicinal and aromatic herb known for its blue essential oil. It lacks studies on anthesis, breeding systems and floral development with their impact on the essential oil. Therefore, the study investigated floral development and divided it into six reproductive stages (RS-1 to RS-6). The first four stages (5-6 days long) were identified as the floral enlargement and differentiation, followed by the fifth stage (10 days long) of three anthesis flushes, i.e., anther dehiscence, ray and disc florets' style branches flush. Anther dehiscence started 1-2 days before style branches flushes showed protandry and overlapped later with style branches flushes. Pollen production started from RS-3 and showed maximum viability (89%) at anther dehiscence (RS-5.1). Pollen showed dispersal through the air up to 0.7 m distance. Seed setting in controlled pollination experiments showed that removing disc florets could be successfully used as the emasculation alternate in German chamomile. The maximum essential oil content (0.40%) at the full blossomed floral stage (RS-4 &-5) suggested the right time for capitula harvesting. The findings on reproductive biology and breeding systems would offer several tools and techniques to support future breeding programs for genetic improvement of German chamomile.

Current understanding and insights towards protein stabilization and activation in deep eutectic solvents as sustainable solvent media.

Deep eutectic solvents (DESs) have emerged as a new class of green, designer and biocompatible solvents, an alternative to conventional organic solvents and ionic liquids (ILs) which are comparatively toxic and non-biodegradable. DESs are eutectic mixtures that are formed when a hydrogen bond acceptor (HBA) is mixed with a hydrogen bond donor (HBD) at particular molar ratios by mechanical grinding or under mild heating conditions. Very recently, these solvents have been the center of attention for researchers in biotechnology, biomedicine and various scientific applications. These environmentally benign solvents have a close analogy with ILs; however, they offer certain unique merits over traditional ILs. DESs display remarkable properties such as easy preparation, tunable composition, biodegradability, recyclability, inherently low toxicity, sustainability and biocompatibility; these special features validate DESs as new potential solvents/co-solvents for biomolecules. Mechanistically, the biocompatibility and protein friendly nature of DESs depend on various factors, which include the composition of the DES, viscosity and hydration level. Therefore, it becomes an essential task to bring together all the studies related to protein behaviour in DESs to unlock their biomolecular proficiency. This review specifically highlights recent insights into the biomacromolecular functionality in DESs, including outlines of the solubilization and stabilization of proteins, long term protein packaging, different extraction methods and enzyme activation in the presence of DESs. A literature survey reveals that DESs act as green media in which the protein structure and activity are retained. In some cases, proteins refolded and enzymatic activity was enhanced several fold in the presence of DESs. Furthermore, we have reviewed the possible mechanistic behaviour behind protein stabilization, refolding and activation in DESs. Overall, the main objective of this review is to explicate the advantages of the introduction of DESs for biomolecules and to demonstrate the versatility of these eco-friendly solvents for future bio-based applications.

Evaluation of Utilizing Functionalized Graphene Oxide Nanoribbons as Compatible Biomaterial for Lysozyme.

Graphene oxide nanoribbons with superior physicochemical properties acquired from graphene and carbon nanotubes have been used in various applications including biomedical applications. For biomedical applications, it is of utmost importance to understand how these graphene oxide nanoribbons interact with proteins and the influence they have on protein conformation and function. In this regard, an attempt has been made to evaluate the utility of graphene oxide nanoribbons as a compatible biomaterial for lysozyme (Lys) protein. In this study, graphene oxide nanoribbons (GONRs) synthesized from multiwalled carbon nanotubes (MWCNTs) were first functionalized with (3-aminopropyl)triethoxysilane (APTES) and further modified with vanillin (Val) to obtain Val-APTES-GONRs. On characterization, it was found that the Val-APTES-GONRs material had a ribbonlike morphology with abundant functionalities for interaction with protein. On evaluation of Val-APTES-GONRs as a compatible biomaterial for Lys, studies revealed that a lower concentration of the as-synthesized material has less influence on the conformation and the structure of Lys with better activity, whereas higher concentrations of the as-synthesized material had a greater influence on conformation and the structure of Lys with decreased activity. Overall, the thermal stability of Lys was maintained after introducing the Val-APTES-GONRs material. In addition, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and Fourier transform infrared (FTIR) and Raman spectroscopies were performed for Lys composites with Val-APTES-GONRs for further understanding biomolecular interactions. This study is beneficial for designing advanced graphene-based materials for numerous bioinspired applications and better biomaterials for biotechnological use.

A novel amalgamation of deep eutectic solvents and crowders as biocompatible solvent media for enhanced structural and thermal stability of bovine serum albumin.

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.

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.

How does bovine serum albumin sustain in saccharomate® derived from pine tree biomass?

Nowadays, research on renewable raw materials and bioresources is a new concern towards the promotion of sustainable process and product development. The use of various plant biomasses such as starch, lignocellulosic and saccharide can be considered as an alternative for using cheaper and less polluting raw materials. In this regard, pine tree biomass, a lignocellulosic forest residue that has various value-added importance and it acts as a model of economic value to the agro-industrial fields. On the other hand, in order to meet and address the challenges of ever-increasing demands of bioresources, there has been significant research interest in deciphering the molecular interactions between proteins and biomass derived substances. No study reports the significance of saccharomate® derived from pine tree biomass on the structural and thermal stability of proteins. There is a sizable interest in the interactions between proteins and biomass derived substances, owing to their utilization and applications. Herein, we used various biophysical techniques such as absorption spectroscopy, fluorescence spectroscopy, circular dichroism (CD) and dynamic light scattering (DLS) to study the impact of pine tree biomass derived saccharomate® (PBDS) on bovine serum albumin (BSA). Further for better understanding of morphological changes of BSA in presence of biomass, Transmission electron microscopy (TEM) was also studied. The present study revealed that the increasing concentration of saccharomate® perturbs structural stability however; the thermal stability of BSA remained unchanged. The transition temperature of BSA remained approximately same in presence of different concentrations of PBDS. Furthermore, the size of BSA increases from 9.22 nm to 135.58 nm in presence of higher concentration of PBDS as revealed by DLS studies. To the best of our knowledge, the results represent first detailed proof of the unusual effect of PBDS on the model protein BSA.

Scrutinizing the effect of various nitrogen containing additives on the micellization behavior of a triblock copolymer.

HYPOTHESIS: PEG-PPG-PEG contains hydrophobic (PPG) as well as hydrophilic (PEG) blocks have gained popularity due to their different physiochemical properties that make them useful in several scientific areas and industrial applications such as detergency, stabilizers for dispersion, foaming and many more. Scientific communities reported that additives have ability to tune the micellization/demicellization tendency of PEG-PPG-PEG which we further extended by the use of several N-containing additives. Especially, chemists and biochemists are interested to extend the potential role of PEG-PPG-PEG copolymer in biomedical sensing applications, that is why triblock copolymer is chosen with various additives in the present study. EXPERIMENTS: The work reports the results obtained through different kinds of interactions induced among the poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (PEG-PPG-PEG) and additives containing different structural moieties. In order to tune micellization tendency of PEG-PPG-PEG, several additives such as trimethylamine-N-oxide (TMAO), betaine, sarcosine, guanidinium hydrochloride (GdnHCl) and urea are introduced in the current part of work and studied using UV-visible and fluorescence spectroscopy, dynamic light scattering (DLS), differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy. FINDINGS: The methylamines facilitate the micellization to higher extent in comparison to that in aqueous PEG-PPG-PEG system, thereby decreasing the critical micellization temperature (CMT) values of PEG-PPG-PEG. Among studied methylamines, sarcosine has the highest efficacy in inducing the micellization followed by TMAO and betaine to the least extent. Direct interactions among polymeric segments and sarcosine is thought to be the main driving force for micellization of PEG-PPG-PEG. This is not possible for the case of betaine and TMAO due to the presence of the sterically hindered N atom. In contrast to these methylamines, GdnHCl and urea provided favorable binding sites for bridging interactions among polymer segments and thus lead to higher temperature values for CMT of PEG-PPG-PEG.