Designing a biocatalytic process involving deep eutectic solvents

During the last decade, deep eutectic solvents (DESs) have emerged as a promising alternative to traditional organic solvents, from both environmental and technological perspectives. The number of structural combinations encompassed by DESs is tremendous; thus, it is possible to design an optimal DES for each specific enzymatic reaction system. In (bio)catalytic processes, a DES can serve as solvent/co‐solvent, as an extractive reagent for an enzymatic product, and as a pretreatment solvent of enzymatic biomass. To date, hydrolases are the most studied enzymes in DESs, which is not surprising given that lipases are the most important industrial enzymes. At the same time, there are a limited number of papers dealing with synthetic reactions in DESs involving other hydrolytic enzymes (epoxide hydrolases, phospholipase, proteases and haloalkane dehalogenases), lyases, and dehydrogenases (as a part of the whole Saccharomyces cerevisae and Escherichia coli cell biocatalysis). When designing efficient biocatalytic processes involving DESs, independent of the reaction type and enzyme used, the following steps should be included: (i) preparation and characterisation of the DES, (ii) screening of the DES for optimal enzyme performance, (iii) selection and optimisation of the biocatalytic protocol, and (iv) recovery of the product/DES and DES recycling with possible scale‐up. In this paper, we will present some practical aspects that we experienced while working with these solvents, together with some major observations that are available in the literature. © 2020 Society of Chemical Industry     

Insight into enzymatic synthesis of phosphatidylserine in deep eutectic solvents

Deep eutectic solvents (DESs) have recently attracted a significant interest in numerous fields including biocatalysis because they are inexpensive, environmentally friendly, non-toxic, biodegradable, and enzyme compatible. Herein, these solvents were successfully used as the reaction media for enzyme-mediated transphosphatidylation of phosphatidylcholine with l-serine for the synthesis of phosphatidylserine for the first time. Enzymatic phosphatidylserine synthesis in various DESs was comprehensively investigated, employing phospholipase D. Our results indicated that > 90% yield of phosphatidylserine could be achieved using choline chloride/ethylene glycol as DES. Furthermore, 81% original activity of the enzyme was maintained after being used for 10 batches. This study indicates that DESs act as potential candidates for the eco-friendly solvents in biocatalysis applications.     

Investigation of CO2 solubility in monoethanolamine hydrochloride based deep eutectic solvents and physical properties measurements

Deep eutectic solvents (DESs) have drawn a growing research interest for applications in a wide range of scientific and industrial arenas. However, a limited effort has been reported in the area of gas separation processes and particularly the carbon dioxide capture. This study introduces a novel set of DESs that were prepared by complexing ethylenediamine (EDA), monoethanolamine (MEA), tetraethylenepentamine (TEPA), triethylenetetramine (TETA) and diethylenetriamine (DETA) as hydrogen bond donors to monoethanolamide hydrochloride (EAHC) salt as a hydrogen bond acceptor. The absorption capacity of CO₂ was evaluated by exploiting a method based on measuring the pressure drop during the absorption process. The solubility of different DESs was studied at a temperature of 313.15 K and initial pressure of 0.8 MPa. The DES systems 1EAHC:9DETA, 1EAHC:9TETA and 1EAHC:9TEPA achieved the highest CO₂ solubility of 0.6611, 0.6572 and 0.7017 mol CO₂·(mole DES)⁻¹ respectively. The results showed that CO₂ solubility in the DESs increased with increasing the molar ratio of hydrogen bond donor. In addition, the CO₂ solubility increased as the number of amine groups in the solvent increases, therefore, increasing the alkyl chain length in the DESs, resulted in increasing the CO₂ solubility. FTIR analysis confirms the DES synthesis since no new functional group was identified. The FTIR spectra also revealed the carbamate formation in DES-CO₂ mixtures. In addition, the densities and viscosities of the synthesized DESs were also measured. The CO₂ initial investigation of reported DESs shows that these can be potential alternative for conventional solvents in CO₂ capture processes.     

New Carvone-Based Deep Eutectic Solvents for Siloxanes Capture from Biogas

During biogas combustion, siloxanes form deposits of SiO₂ on engine components, thus shortening the lifespan of the installation. Therefore, the development of new methods for the purification of biogas is receiving increasing attention. One of the most effective methods is physical absorption with the use of appropriate solvents. According to the principles of green engineering, solvents should be biodegradable, non-toxic, and have a high absorption capacity. Deep eutectic solvents (DES) possess such characteristics. In the literature, due to the very large number of DES combinations, conductor-like screening models for real solvents (COSMO-RS), based on the comparison of siloxane activity coefficient of 90 DESs of various types, were studied. DESs, which have the highest affinity to siloxanes, were synthesized. The most important physicochemical properties of DESs were carefully studied. In order to explain of the mechanism of DES formation, and the interaction between DES and siloxanes, the theoretical studies based on σ-profiles, and experimental studies including the ¹H NMR, ¹³C NMR, and FT-IR spectra, were applied. The obtained results indicated that the new DESs, which were composed of carvone and carboxylic acids, were characterized by the highest affinity to siloxanes. It was shown that the hydrogen bonds between the active ketone group (=O) and the carboxyl group (-COOH) determined the formation of stable DESs with a melting point much lower than those of the individual components. On the other hand, non-bonded interactions mainly determined the effective capture of siloxanes with DES.     

Effective Release of Intracellular Enzymes by Permeating the Cell Membrane with Hydrophobic Deep Eutectic Solvents

An efficient and green method is crucial for the recovery of intracellular biological products. The major drawbacks of the conventional cell disruption method are nonselectivity and enzyme denaturation. The permeability of hydrophobic deep eutectic solvents (DESs) to the cell membrane was studied, for the first time, and then hydrophobic DESs were innovatively applied to release intracellular enzymes from recombinant Escherichia coli. After optimization, a DES suspension of l -menthol/oleic acid (0.5 %, v/v) showed the highest release yield of intracellular enzyme. Compared with that released by sonication, a release yield of phospholipase D (PLD) of up to 114.58 % was achieved, and the specific activity was increased by 1.96 times. The microstructure of the cell membrane under different treatments was observed by using an electron microscope to understand the permeation of DESs to the cell membrane. The feasibility and applicability of the proposed release method in industrial applications were also demonstrated. The effective and green release method of intracellular enzymes developed herein has bright prospects for industrial application to replace traditional cell disruption methods. A preliminary study on the permeability of hydrophobic DESs to the cell membrane showed that there would be a potential application prospect of hydrophobic DESs not only in releasing intracellular contents, but also in seeking new green penetrating agents.     

Molecular Mechanism of the Hydration of Candida antarctica Lipase B in the Gas Phase: Water Adsorption Isotherms and Molecular Dynamics Simulations

Hydration is a major determinant of activity and selectivity of enzymes in organic solvents or in gas phase. The molecular mechanism of the hydration of Candida antarctica lipase B (CALB) and its dependence on the thermodynamic activity of water (a_w) was studied by molecular dynamics simulations and compared to experimentally determined water sorption isotherms. Hydration occurred in two phases. At low water activity, single water molecules bound to specific water binding sites at the protein surface. As the water activity increased, water networks gradually developed. The number of protein‐bound water molecules increased linearly with a_w, until at a_w=0.5 a spanning water network was formed consisting of 311 water molecules, which covered the hydrophilic surface of CALB, with the exception of the hydrophobic substrate‐binding site. At higher water activity, the thickness of the hydration shell increased up to 10 Å close to a_w=1. Above a limit of 1600 protein‐bound water molecules the hydration shell becomes unstable and the formation of pure water droplets occurs in these oversaturated simulation conditions. While the structure and the overall flexibility of CALB was independent of the hydration state, the flexibility of individual loops was sensitive to hydration: some loops, such as those part of the substrate‐binding site, became more flexible, while other parts of the protein became more rigid upon hydration. However, the molecular mechanism of how flexibility is related to activity and selectivity is still elusive.     

低共熔溶剂用于萃取分离的研究进展

作为一种新型的绿色溶剂，低共熔溶剂（DES）拥有与离子液体媲美的优良特性，如挥发性小、可设计性等，且具有成本低廉和制备简单的优势，使得DES正逐步替代传统有机溶剂，在萃取分离应用方面得到广泛的关注。本文综述了近年来国内外有关DES在萃取分离方面的研究报道，阐述了DES直接用于液液萃取、在线生成DES的缔合萃取和通过DES分解完成萃取的应用，并分析比较了各过程的特点和存在的问题；介绍了DES在不同萃取体系中的稳定性和DES的回收方法；总结了DES萃取分离体系的理论发展和萃取机理的研究进展；展望了DES用于萃取分离的工业化前景，指出了目前面临的DES理论、萃取机理、循环稳定性等方面的挑战，分析了进一步的研究趋势。     

Thermodynamic and molecular insights into the absorption of H2S,CO2, and CH4 in choline chloride plus urea mixtures

Deep eutectic solvents (DESs) are a class of promising media for gas separation. In order to examine the potential application of DESs for natural gas upgrading, the solubilities of H₂S, CO₂, and CH₄ in choline chloride (ChCl) plus urea mixtures were measured in this work. The solubility data were correlated with Henry's law equation to calculate the thermodynamic properties of gas absorption processes, such as Henry's constants and enthalpy changes. Grand-canonical Monte Carlo simulations and quantum chemistry calculations were also performed to examine the mechanism of gas absorption processes. It is found that the absorption of H₂S in ChCl + urea mixtures is governed by the hydrogen-bond interaction between Cl of ChCl and H of H₂S, whereas the absorption of CO₂ and CH₄ in ChCl+urea mixtures is governed by the free volume of solvents. Based on the different behavior of gas absorption, high H₂S/CO₂, H₂S/CH₄, and CO₂/CH₄ selectivities can be achieved by adjusting the ratio of ChCl/urea in mixtures.     

Protic ionic liquid-based deep eutectic solvents with multiple hydrogen bonding sites for efficient absorption of NH3

The emerging of ionic liquids (ILs) provides an efficient and sustainable way to separate and recover NH₃ due to their unique properties. However, the solid or highly viscous ILs are not suitable for traditional scrubbing. Therefore, an effective strategy was proposed by combining the protic ILs (PILs) with acidic H and low viscous ethylene glycol (EG) to form IL-based deep eutectic solvents (DESs) for NH₃ absorption. The results indicated that these PIL-based DESs not only have fast absorption rate, but also exhibit exceptional NH₃ capacity and excellent recyclability. The highest mass capacity of 211 mg NH₃/g DES was achieved by [Im][NO₃]/EG with molar ratio of 1:3, and was higher than all the reported ILs and IL-based DESs, which was originated from multiple hydrogen bonding between acidic H and hydroxyl groups of the DESs and NH₃. This work will provide useful idea for designing IL-based solvents for NH₃ separation applications.     

Activity of horseradish peroxidase in aqueous and reverse micelles and back‐extraction from reverse‐micellar phases

Studies on the activity of the enzyme horseradish peroxidase (HRP) have been carried out in micellar as well as reverse‐micellar media. The activity of the enzyme was studied in the presence of different classes of surfactants – ionic as well as non‐ionic. In aqueous media, the activity of the enzyme varied depending on whether the concentration of the surfactant used was above or below the critical micellar concentration (CMC). The enzyme was also studied in reverse‐micellar systems. HRP was introduced into the reverse micellar phase by the injection method and its activity within the reverse micelles was determined. The effect of water to surfactant ratio (W_o) on activity within reverse micelles was studied, and an almost two‐fold increase in activity was seen when the enzyme was encapsulated within reverse micelles of aqueous phase fractional hold‐up (?) of 0.0072 (v/v) consisting of sodium bis‐(2‐ethylhexyl) sulfosuccinate (AOT) in isooctane at a W_o of 20. The activity of HRP was measured over a wide range of AOT concentrations having different W_o values. Back‐extraction of HRP from these reverse micelles was carried out at varying ionic strengths of phosphate buffer. Back extraction was found to be highest at pH 7.0 in 40 mol m^?3 phosphate buffer and 100 mol m^?3 sodium chloride. © 2001 Society of Chemical Industry     

Sono-electrodeposition (20 and 850 kHz) of copper in aqueous and deep eutectic solvents

This paper reports the effects of ultrasound at different frequencies and powers upon the electrodeposition of copper(II) chloride in aqueous potassium chloride and in glyceline 200 (a deep eutectic solvent - DES) on Pt electrodes in the potential range for copper deposition and dissolution. It is shown that the deposition of copper in both solvents is greatly affected by ultrasound at the two frequencies of 20 and 850 kHz employed. Limiting current densities were obtained in both solvents under sonication at 20 and 850 kHz and a 10-fold and 5-fold increase in currents in aqueous potassium chloride and glyceline 200 compared to silent conditions was observed respectively. The difference in viscosity of water (KCl) and glyceline 200 was found to be a crucial parameter in the evaluation of limiting current densities.     

Aromatic volatile organic compounds absorption with phenyl-based deep eutectic solvents: A molecular thermodynamics and dynamics study

The suitability of phenyl-based deep eutectic solvents (DESs) as absorbents for toluene absorption was investigated by means of thermodynamic modeling and molecular dynamics (MD). The thermodynamic models perturbed-chain statistical associating fluid theory (PC-SAFT) and conductor-like screening model for real solvents (COSMO-RS) were used to predict the vapor–liquid equilibrium of DES–toluene systems. PC-SAFT yielded quantitative results even without using any binary fitting parameters. Among the five DESs studied in this work, [TEBAC][PhOH] consisting of triethyl benzyl ammonium chloride (TEBAC) and phenol (PhOH), was considered as the most suitable absorbent. Systems with [TEBAC][PhOH] had lowest equilibrium pressures of the considered DES–toluene mixtures, the best thermodynamic characteristics (i.e., Henry's law constant, excess enthalpy, Gibbs free energy of solvation of toluene), and the highest self-diffusion coefficient of toluene. The molecular-level mechanism was explored by MD simulations, indicating that [TEBAC][PhOH] has the strongest interaction of DES–toluene compared to the other DESs under study. This work provides guidance to rationally design novel DESs for efficient aromatic volatile organic compounds absorption.     

Enzymatic alcoholysis of triolein to produce wax ester

An alcoholysis reaction between triolein and oleyl alcohol catalyzed by Lipozyme and Novozyme was carried out to produce oleyl oleate, a wax ester. The effects of various reaction parameters such as time, reaction temperature, amount of enzyme, molar ratio of substrates (oleyl alcohol/triolein), various organic solvents used and the initial water activity, a_w of the reaction system were studied. The best conditions tested to produce wax ester were respectively, incubation time, 5 h; temperature, 50 °C for Lipozyme and 60 °C for Novozyme; weight of enzyme, 0.30 g and molar ratio of oleyl alcohol to triolein, 6:1. The use of organic solvents greatly influenced the activity of lipase. Generally, the activity of lipase was high in nonpolar solvents with log P values greater than 2.50. Heptane and hexane were the best solvents tested. The enzymatic synthesis of oleyl oleate was best carried out at a_w 0.32. Analysis of the yield of the products of the reaction at optimized reaction condition using Lipozyme showed that 75.66% oleyl oleate was produced. © 2001 Society of Chemical Industry     

Removal of Dibenzothiophene from Diesels by Extraction and Catalytic Oxidation with Acetamide‐Based Deep Eutectic Solvents

A series of acetamide‐based deep eutectic solvents (DESs) with different proportions were prepared. Extraction and catalytic oxidation desulfurization (ECODS) of the acetamide‐based DESs were investigated and the process was optimized. Such DESs with a molar ratio of acetamide and p‐TsOH of 1/3 (C₂H₅NO/3p‐TsOH) exhibits such a remarkable catalytic activity that the dibenzothiophene (DBT) removal could reach 100 % under optimized conditions. C₂H₅NO/3p‐TsOH was used for the oxidative desulfurization of actual commercial diesel. The sulfur removal of diesel achieved up to 98 %. C₂H₅NO/3p‐TsOH could be recycled six times and the desulfurization activity was slightly decreased. Evaluation of the mechanism indicated that oxidative desulfurization (ODS) was realized via dual activation of acetamide‐based DESs. A novel and effective way for deep desulfurization of diesel is provided.     

Effective separation of aromatic hydrocarbons by pyridine‐based deep eutectic solvents

Many promising qualities of deep‐eutectic solvents made them suitable solvents in separation process. In this work, the pyridine‐based deep eutectic solvents were designed and synthesized with N‐ethylpyridinium bromide and two HBDs (N‐formyl morpholine and levulinic acid). Two ternary systems, benzene + cyclohexane + DES and toluene + n‐heptane + DES, were studied by the liquid‐liquid extraction. The effect of different HBDs, extraction time, volume ratio of DES to system solution, and the initial concentration of aromatic were studied. The DES with N‐formyl morpholine showed better separation performance than that with levulinic acid. The liquid‐liquid extraction equilibrium could be obtained in 10 minutes. The volume ratio of DES to system solution was set as 1:1. Both DESs showed their best separation performance at low temperatures (20°C) and low aromatic concentration system. For the benzene + cyclohexane system, the distribution coefficient of benzene was 1.733 and the selectivity was 23.8 at 20°C. For the toluene + n‐heptane system, the distribution coefficient of toluene was 0.853 and the selectivity was 40.7. Tie‐lines for two ternary systems were obtained, and the Othmer‐Tobias correlation was used to check the reliability of the obtained liquid‐liquid extraction experimental data. The experimental LLE data were correlated using the NRTL model and the calculated data correlated significantly with the experimental data.     

Investigation of the effects of ternary deep eutectic solvent composition on pretreatment of sorghum stover

Biomass-derived deep eutectic solvents (DESs) have been introduced as promising pretreatment and fractionation solvents because of their mild processing conditions, easy synthesis, and green solvent components from biomass. In recent DES studies, solvent-based third constituents like water, ethanol, and others improve the processibility of typical binary DESs. However, the impacts of these components are not well understood. Here, two solvent-based constituents, including water and ethylene glycol, were applied to 3,4-dihydroxybenzoic acid (DHBA)-based DES system for improving the conversion efficiency of cellulose-rich fraction and the properties of lignin fraction. Chemical composition, enzymatic digestibility, degree of polymerization of cellulose and physicochemical properties of lignin were used to evaluate the impact of each third constituent on biomass processing. Ternary ChCl-DHBA DESs exhibited better performances in delignification, fermentable sugar production, and preservation of β-O-4 ether linkage in lignin compared with binary ChCl-DHBA DES.     

Facile Synthesis of 7-Aryl-benzo[h]tetrazolo[5,1-b]quinazoline-5,6-dione Fused Polycyclic Compounds by Using a Novel Magnetic Polyurethane Catalyst

Novel Brönsted acid functionalized magnetic polymeric nanocomposite, Ba_0.5Sr_0.5Fe₁₂O₁₉@PU-SO₃H, has been successfully synthesized and characterized by field-emission scanning electron microscopy (FE-SEM), X-ray diffraction (XRD), vibrating sample magnetometer (VSM), Fourier transform infrared spectroscopy (FT-IR), energy-dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM) and thermogravimetric analysis (TGA) techniques. The catalytic activity of the nanocomposite was tested in the regioselective synthesis of 7-aryl-benzo[h]tetrazolo [5,1-b] quinazoline-5,6-diones in an eco-friendly and recyclable media, deep eutectic solvent (DES), based on choline chloride and urea. This novel method offers several advantages, such as high yields, short reaction time, environmentally friendly reaction media, easy isolation of the product, and also offers an easy method for the synthesis of nanocatalysts. The catalyst was readily recycled by the use of an external magnetic field and could be reused 6 times without significant loss of activity or mass. DES as one of the most promising environmentally benign and cost-effective alternatives to conventional ionic liquids and volatile organic solvents was recovered from the aqueous filtrate by evaporating the water under vacuum. The recycled DES was used up to 4 runs without any loss in activity.     

Investigation of CO2 solubility in monoethanolamine hydrochloride based deep eutectic solvents and physical properties measurements

Deep eutectic solvents (DESs) have drawn a growing research interest for applications in a wide range of scientific and industrial arenas. However, a limited effort has been reported in the area of gas separation processes and particularly the carbon dioxide capture. This study introduces a novel set of DESs that were prepared by complexing ethylenediamine (EDA), monoethanolamine (MEA), tetraethylenepentamine (TEPA), triethylenetetramine (TETA) and diethylenetriamine (DETA) as hydrogen bond donors to monoethanolamide hydrochloride (EAHC) salt as a hydrogen bond acceptor. The absorption capacity of CO₂ was evaluated by exploiting a method based on measuring the pressure drop during the absorption process. The solubility of different DESs was studied at a temperature of 313.15 K and initial pressure of 0.8 MPa. The DES systems 1EAHC:9DETA, 1EAHC:9TETA and 1EAHC:9TEPA achieved the highest CO₂ solubility of 0.6611, 0.6572 and 0.7017 mol CO₂·(mole DES)^-1 respectively. The results showed that CO₂ solubility in the DESs increased with increasing the molar ratio of hydrogen bond donor. In addition, the CO₂ solubility increased as the number of amine groups in the solvent increases, therefore, increasing the alkyl chain length in the DESs, resulted in increasing the CO₂ solubility. FTIR analysis confirms the DES synthesis since no new functional group was identified. The FTIR spectra also revealed the carbamate formation in DES-CO₂ mixtures. In addition, the densities and viscosities of the synthesized DESs were also measured. The CO₂ initial investigation of reported DESs shows that these can be potential alternative for conventional solvents in CO₂ capture processes.     

Liquid-Liquid Equilibrium Data for the System N-Octane + Toluene + DES at 293.15 and 313.15 K and Atmospheric Pressure

Deep eutectic solvents (DESs) are promising alternative for ionic liquids due to their low cost and sustainability. Considerable attention is paid on the ability of DES to replace ionic liquids in the separation of organic liquid mixtures via extraction. In this sense, it is important to determine the physicochemical parameters of liquid-liquid equilibrium for the industrially significant mixtures and deep eutectic solvents. In the present work a mixture of n-octane, toluene and DES based on choline chloride and malonic acid was studied at 293.15, 313.15 K and atmospheric pressure. Tie-lines were obtained and ability of deep eutectic solvents to separate aliphatic-aromatic mixture was analyzed. Experimental liquid-liquid equilibrium data were fit with the NRTL model, and interaction parameters of components were obtained and discussed.     

Liposome Microencapsulation for the Surface Modification and Improved Entrapment of Cytochrome c for Targeted Delivery

In this study, we established a procedure based on the microencapsulation vesicle (MCV) method for preparing surface‐modified liposomes, using polyethylene glycol (PEG) and a site‐directed ligand, with high entrapment efficiency of cytochrome c (Cyt c). For preparing a water‐in‐oil (W/O) emulsion, egg phosphatidylcholine and cholesterol were dissolved in organic solvents (O phase) and emulsified by sonication with aqueous solution of Cyt c (W₁). Although the dispersion stability of the W₁/O emulsion was low when n‐hexane was used to dissolve the lipids in the O phase, it was substantially improved by using mixed solvents consisting of n‐hexane and other organic solvents, such as ethanol and dichloromethane (DCM). The W₁/O emulsion was then added to another water phase (W₂) to prepare the W₁/O/W₂ emulsion. PEG‐ and/or ligand‐modified lipids were introduced into the W₂ phase as external emulsifiers not only for stabilizing the W₁/O/W₂ emulsion but also for modifying the surface of liposomes obtained later. After solvent evaporation and extrusion for downsizing the liposomes, approximately 50% of Cyt c was encapsulated in the liposomes when the mixed solvent consisting of n‐hexane and DCM at a volume ratio of 75/25 was used in the O phase. Finally, the fluorescence‐labeled liposomes, with a peptide ligand having affinity to the vasculature in adipose tissue, were prepared by the MCV method and intravenously injected into mice. Confocal microscopy showed the substantial accumulation of these liposomes in the adipose tissue vessels. Taken together, the MCV technique, along with solvent optimization, could be useful for generating surface‐modified liposomes with high drug entrapment efficiency for targeted delivery.