Ionic liquids as protein stabilizers for biological and biomedical applications: A review.

Biotechnology has revolutionized science and health care by providing new biomolecules with biological and medical applications. However, the low stability of several life-saving bioproducts still hinders their transport, storage, and application. Hence, protein-based bioproducts instability and high costs are the main bottlenecks limiting access to biopharmaceuticals in low-income countries and communities. Aiming to improve the stability of protein-based products, researchers have studied ionic liquids (ILs) as protein stabilizers due to their unique properties and ability to enhance the solubility and stability of a wide range of biomolecules. Although different classes of ILs have the potential to improve protein stability, their effects are dependent on several variables, such as the complex and intrinsic properties of proteins, the nature and concentration of ILs, and environmental conditions (e.g., temperature, pH). For medical applications, the biocompatibility of ILs can also limit their biological use. Therefore, the current state-of-the-art on ILs applications for non-enzymatic protein stabilization was carefully analyzed and discussed, considering protein properties, ILs classes, and IL solutions concentrations. Lastly, a critical perspective regarding ILs applications as protein stabilizers was presented, highlighting the current lacunas in the field while guiding future studies to answer the existing paradigms.

Ambient-Pressured Acid-Catalysed Ethylene Glycol Organosolv Process: Liquefaction Structure-Activity Relationships from Model Cellulose-Lignin Mixtures to Lignocellulosic Wood Biomass.

Raising the awareness of carbon dioxide emissions, climate global warming and fossil fuel depletion has renewed the transition towards a circular economy approach, starting by addressing active bio-economic precepts that all portion amounts of wood are valorised as products. This is accomplished by minimizing residues formed (preferably no waste materials), maximizing reaction productivity yields, and optimising catalysed chemical by-products. Within framework structure determination, the present work aims at drawing a parallel between the characterisation of cellulose-lignin mixture (derived system model) liquefaction and real conversion process in the acidified ethylene glycol at moderate process conditions, i.e., 150 °C, ambient atmospheric pressure and potential bio-based solvent, for 4 h. Extended-processing liquid phase is characterized considering catalyst-transformed reactant species being produced, mainly recovered lignin-based polymer, by quantitative 31P, 13C and 1H nuclear magnetic resonance (NMR) spectroscopy, as well as the size exclusion- (SEC) or high performance liquid chromatography (HPLC) separation for higher or lower molecular weight compound compositions, respectively. Such mechanistic pathway analytics help to understand the steps in mild organosolv biopolymer fractionation, which is one of the key industrial barriers preventing a more widespread manufacturing of the biomass-derived (hydroxyl, carbonyl or carboxyl) aromatic monomers or oligomers for polycarbonates, polyesters, polyamides, polyurethanes and (epoxy) resins.

Chitin Deacetylation Using Deep Eutectic Solvents: Ab Initio-Supported Process Optimization.

Chitin is the most abundant marine biopolymer, being recovered during the shell biorefining of crustacean shell waste. In its native form, chitin displays a poor reactivity and solubility in most solvents due to its extensive hydrogen bonding. This can be overcome by deacetylation. However, this process requires a high concentration of acids or bases at high temperatures, forming large amounts of toxic waste. Herein, we report on the first deacetylation with deep eutectic solvents (DESs) as an environmentally friendly alternative, requiring only mild reaction conditions. Biocompatible DESs are efficient in disturbing the native hydrogen-bonding network of chitin, readily dissolving it. First, quantum chemical calculations have been performed to evaluate the feasibility of different DESs to perform chitin deacetylation by studying their mechanism. Comparing these with the calculated barriers for garden-variety alkaline/acidic hydrolysis, which are known to proceed, prospective DESs were identified with barriers around 25 kcal·mol-1 or lower. Based on density functional theory results, an experimental screening of 10 distinct DESs for chitin deacetylation followed. The most promising DESs were identified as K2CO3:glycerol (K2CO3:G), choline chloride:acetic acid ([Ch]Cl:AA), and choline chloride:malic acid ([Ch]Cl:MA) and were subjected to further optimization with respect to the water content, process duration, and temperature. Ultimately, [Ch]Cl:MA showed the best results, yielding a degree of deacetylation (DDA) of 40% after 24 h of reaction at 120 °C, which falls slightly behind the threshold value (50%) for chitin to be considered chitosan. Further quantum chemical calculations were performed to elucidate the mechanism. Upon the removal of 40% N-acetyl groups from the chitin structure, its reactivity was considerably improved.

Development of a Microfluidic Platform for R-Phycoerythrin Purification Using an Aqueous Micellar Two-Phase System.

Temperature-dependent aqueous micellar two-phase systems (AMTPSs) have recently been gaining attention in the isolation of high-added-value biomolecules from their natural sources. Despite their sustainability, aqueous two-phase systems, and particularly AMTPSs, have not been extensively applied in the industry, which might be changed by applying process integration and continuous manufacturing. Here, we report for the first time on an integrated microfluidic platform for fast and low-material-consuming development of continuous protein purification using an AMTPS. A system comprised of a microchannel incubated at high temperature, enabling instantaneous triggering of a two-phase system formation, and a microsettler, allowing complete phase separation at the outlets, is reported here. The separation of phycobiliproteins and particularly the purification of R-phycoerythrin from the contaminant proteins present in the aqueous crude extract obtained from fresh cells of Gracilaria gracilis were thereby achieved. The results from the developed microfluidic system revealed that the fractionation performance was maintained while reducing the processing time more than 20-fold when compared with the conventional lab-scale batch process. Furthermore, the integration of a miniaturized ultrafiltration module resulted in the complete removal of the surfactant from the bottom phase containing R-phycoerythrin, as well as in nearly twofold target protein concentration. The process setup successfully exploits the benefits of process intensification along with the integration of various downstream processes. Further transfer to a meso-scale integrated system would make such a system appropriate for the separation and purification of biomolecules with high commercial interest.

Chitin isolation from crustacean waste using a hybrid demineralization/DBD plasma process.

Conventional isolation of chitin from crustacean waste demands the use of high amounts of hazardous chemicals, hence not leading to a sustainable process. Atmospheric-pressure dielectric barrier discharge (DBD) plasma has demonstrated an enhanced ability to remove proteins directly from the biomass without the formation of any waste. Simultaneously, organic acids have proven very efficient in the removal of inorganic minerals from crustacean waste. Therefore, a hybrid process composed of DBD plasma and demineralization using organic acids has been successfully applied for the isolation of chitin. Results showed that the integration of nitrogen-based plasma and lactic acid demineralization allowed the elimination of 90 % of the proteins and ensures the complete removal of minerals from shrimp shells waste. The isolated chitin was further characterized using distinct techniques, namely XPS, ATR-FTIR, XRD and SEM. Chitin degree of deacetylation and molecular weight were also assessed. Hence, this work presents a sustainable and feasible platform for the extraction and purification of chitin from crustacean waste with almost zero waste formed.

Unravelling the Interactions between Surface-Active Ionic Liquids and Triblock Copolymers for the Design of Thermal Responsive Systems.

The tunable properties of surface-active ionic liquids (SAILs) and Pluronics are dramatically magnified by combining them in aqueous solutions. The thermo-controlled character of both, essential in the extraction of valuable compounds, can be fine-tuned by properly selecting the Pluronic and SAIL nature. However, further understanding of the nanoscale interactions directing the aggregation in these complex mixtures is needed to effectively design and control these systems. In this work, a simple and transferable coarse-grained model for molecular dynamics simulations, based on the MARTINI force field, is presented to study the impact of SAILs in Pluronics aggregation in aqueous solutions. The diverse amphiphilic characteristics and micelle morphologies were exemplified by selecting four archetypical nonionic Pluronics-two normal, L-31 and L-35, and two reverse, 10R5 and 31R1. The impact of the alkyl chain length and the headgroup nature were evaluated with the imidazolium-based [C10mim]Cl and [C14mim]Cl and phosphonium-based [P4,4,4,14]Cl SAILs. Cloud point temperature (CPT) measurements at different Pluronic concentrations with 0.3 wt % of SAIL in aqueous solution emphasized the distinct impact of SAIL nature on the thermo-response behavior. The main effect of SAIL addition to nonionic Pluronics aqueous solutions is the formation of Pluronic/SAIL hybrid micelles, where the presence of SAIL molecules introduces a charged character to the micelle surface. Thus, additional energy is necessary to induce micelle aggregation, leading to the observed increase in the experimental CPT curves. The SAIL showed a relatively weak impact in Pluronic micelles with relatively high PPG hydrophobic content, whereas this effect was more evident when the Pluronic hydrophobic/hydrophilic strength is balanced. A detailed analysis of the Pluronic/SAIL micelle density profiles showed that the phosphonium head groups were positioned inside the micelle core, whereas smaller imidazolium head groups were placed much closer to the hydrophilic PEG corona, leading to a distinct effect on the cloud point temperature for those two classes of SAILs. Herein, the phosphonium-based SAIL induces a lower repulsion between neighboring micelles than the imidazolium-based SAILs, resulting in a less pronounced increase of the CPT. The model presented here offers, for the first time, an intuitive and powerful tool to unravel the complex thermo-response behavior of Pluronic and SAIL mixtures and support the design of tailor-made thermal controlled solvents.

α-Chitin dissolution, N-deacetylation and valorization in deep eutectic solvents.

Chitin displays a highly rigid structure due to the vast intra- and intermolecular hydrogen bonding, thus hindering its dissolution and deacetylation using most solvents. Deep eutectic solvents (DESs) are special and environmentally friendly solvents composed of a hydrogen bond acceptor and a hydrogen bond donor. This allows them to dissolve chitin by disturbing its natural hydrogen bonding while establishing new bonds, hence turning the polymer more susceptible to solvents. Therefore, four distinct DESs (choline chloride-lactic acid ([Ch]Cl:LA), choline chloride:oxalic acid ([Ch]Cl:OA), choline chloride:urea ([Ch]Cl:U) and betaine-glycerol (Bet:G)) were applied in chitin dissolution, being the most performant ones further applied in its homogenous N-deacetylation with NaOH. In this work, a milder and more biocompatible approach was carried out by using 30 wt% NaOH at 80°C, instead of the typical ≥40 wt% NaOH at temperatures ≥100°C. Herein, the reaction process took up to 18 hours, being the results analyzed through ATR-FTIR. Chitin was converted into chitosan with a 70-80% degree of deacetylation (DDA) in a short period while using homogenous conditions. These promising results provide the first proof of concept of the ability of Bet:G and [Ch]Cl:LA-based DESs to be used as a greener approach for the chitin homogeneous N-deacetylation.

Impact of Surface Active Ionic Liquids on the Cloud Points of Nonionic Surfactants and the Formation of Aqueous Micellar Two-Phase Systems.

Aqueous micellar two-phase systems (AMTPS) hold a large potential for cloud point extraction of biomolecules but are yet poorly studied and characterized, with few phase diagrams reported for these systems, hence limiting their use in extraction processes. This work reports a systematic investigation of the effect of different surface-active ionic liquids (SAILs)-covering a wide range of molecular properties-upon the clouding behavior of three nonionic Tergitol surfactants. Two different effects of the SAILs on the cloud points and mixed micelle size have been observed: ILs with a more hydrophilic character and lower critical packing parameter (CPP < 1/2) lead to the formation of smaller micelles and concomitantly increase the cloud points; in contrast, ILs with a more hydrophobic character and higher CPP (CPP ≥ 1) induce significant micellar growth and a decrease in the cloud points. The latter effect is particularly interesting and unusual for it was accepted that cloud point reduction is only induced by inorganic salts. The effects of nonionic surfactant concentration, SAIL concentration, pH, and micelle ζ potential are also studied and rationalized.