Water-miscible Ionic Liquids Research Articles

Determinants for 1-alkyl-3-methylimidazolium chloride-induced modulation of thermodynamic stability and CO-association dynamics of horse ferrocytochrome c

This manuscript determines the factors by which the water-miscible ionic liquid (IL), 1-alkyl-3-methylimidazolium chloride [Rmim]Cl affects the thermodynamic stability and CO association dynamics of horse ferrocytochrome c (Cyt cII) at pH 7. Investigation of rate constant and activation thermodynamic parameters for CO-association reaction of Cyt cII measured at different concentrations of [Rmim]Cl (1-Ethyl-3-methylimidazolium chloride [Emim]Cl, 1-butyl-3-methylimidazolium chloride [Bmim]Cl, 1-hexyl-3-methylimidazolium chloride [Hmim]Cl) revealed that the hydrophobicity of alkyl group of [Rmim]Cl modulates the CO-association dynamics of Cyt cII. The CO-association association kinetic results were further supported by intermolecular docking studies between the Cyt cII and [Rmim]+ ([Hmim]Cl, [Bmim]Cl, [Emim]Cl). Analysis of thermodynamic parameters calculated from thermal denaturation transitions of Cyt cII at different concentrations of [Rmim]Cl ([Hmim]Cl, [Bmim]Cl, [Emim]Cl) revealed that the [Rmim]Cl decreases the denaturation free energy in the order: [Hmim]Cl > [Bmim]Cl > [Emim]Cl, which suggests that the hydrophobicity of alkyl group of [Rmim]Cl modulates the [Rmim]Cl-induced decrease in thermodynamic stability of Cyt cII. Analysis of entropy − enthalpy plots showed that in addition to the hydrophobic effect, the enthalpic effect of [Rmim]Cl also decreases the thermodynamic stability of the protein. The dependence of unfolding free energy on [Rmim]Clconcentration was also translated into preferential interaction coefficients, Γ23. Experimental and theoretical estimations of Γ23 for Cyt cII provided the positive values of Γ23 at all across the [Rmim]Cl concentrations, indicating a robust and favorable interaction between the [Rmim]Cl and Cyt cII. Furthermore, the [Rmim]Cl-mediated increase in Γ23 typically follows the order: [Hmim]Cl > [Bmim]Cl > [Emim]Cl. Molecular dynamics (MD) simulation studies of Cyt cII performed at different concentrations of [Rmim]Cl ([Emim]Cl, [Bmim]Cl, and [Hmim]Cl) further showed that the hydrophobicity of the alkyl group modulates the structural stability and conformational-fluctuations of the Cyt cII.

A Multistep, Multicomponent Extraction and Separation Microfluidic Route to Recycle Water-Miscible Ionic Liquid Solvents.

Recycling ionic liquid (IL) solvents can reduce the lifecycle cost of these expensive solvents. Liquid-liquid extraction is the most straightforward approach to purify IL solvents and is typically performed with an immiscible washing agent (e.g., water). Herein, we describe a recycling route for water-miscible ILs in which direct recycling is usually challenging. We use hydrophobic ILs as accommodating agents to draw the water-miscible IL from the aqueous washing stream. A biphasic slug flow of the mixed ILs and water is then separated by using a membrane. The water-miscible IL can then be drawn out from the mixed IL phase with acidified water and dried under vacuum. Both the water-miscible IL and the accommodating agent are then recycled. Here, we demonstrated a proof-of-concept of this process by recycling 1-butyl-3-methylimidazolium trifluoromethanesulfonate (BMIM-OTf) in the presence of the accommodating agent 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (BMIM-NTf2) and acidified water. We then demonstrated the capacity to recycle 1-butyl-1-methylpyrrolidinium triflate (BMPYRR-OTf) from a realistic synthetic application: Pt nanoparticle synthesis in the water-miscible IL.

Separation of polysaccharide and protein by ionic liquid-based extraction techniques

Biopolymers are natural macromolecules obtained from animal, plant and microbial sources, with the potential to be used in a wide range of applications. A key process step, which is still underdeveloped, is the downstream processing. In this work, water immiscible and water miscible ionic liquids (ILs) were investigated regarding their ability to fractionate a mixture of polysaccharide and proteins. Alginate and bovine serum albumin (BSA) were used as model compounds to mimic natural polymer crude extract. Phosphonium ILs composed of different anions (bromide, dicyanamide and phosphinate) were used as water immiscible ILs while imidazolium ILs, combined with phosphate salts to form biphasic system, were selected as water miscible ILs. In water immiscible IL systems, the partitioning behavior of biopolymers depended on IL's anions and there was formation of insoluble precipitate. The insolubility of precipitate in diverse aqueous and organic solvents hindered the processibility of water immiscible phosphonium IL for fractionation of biopolymers. The partitioning of biopolymers in water miscible ILs systems also depended on the IL's anion, as well the concentration of IL. Separation of alginate (yield = 90% and purity = 99%) from BSA (yield = 89% and purity = 99%) was best achieved by the [C4mim]Cl-based extraction system. After fractionation, regeneration of IL and salt used was carried out by ultrafiltration, with recovery yields up to 100%. The high extraction yields and recyclability of phase-forming compounds confirm the potential of water miscible ILs systems to fractionate polysaccharide and protein.

Impact of Two Water-Miscible Ionic Liquids on the Temperature-Dependent Self-Assembly of the (EO)6-(PO)34-(EO)6 Block Copolymer.

There are many studies on the self-assembly of triblock poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide) copolymers in aqueous solution. These polymers display a rich phase diagram as a function of block length, concentration, temperature, and additives. Here, we present a small-angle neutron scattering study of the impact of two water-miscible ionic liquids, 1-butyl-3-methylimidazolium chloride ([C4C1mim][Cl]) and 1-butyl-3-methylpyrrolidinium chloride ([C4C1pyrr][Cl]), on the temperature-dependent self-assembly of (EO)6–(PO)34–(EO)6, also known as L62 Pluronic, in aqueous solution. Both ionic liquids depress the temperatures of the various structural transitions that take place, but ([C4C1pyrr][Cl]) has a stronger effect. The structures that the triblock copolymer self-assembles into do not dramatically change nor do they significantly change the series of structures that the system transitions through as a function of temperature relative to the various transition temperatures.

Poly(vinyl alcohol) Hydrogels with Integrated Toughness, Conductivity, and Freezing Tolerance Based on Ionic Liquid/Water Binary Solvent Systems.

In recent years, ionic conductive hydrogels have shown great potential for application in flexible sensors, energy storage devices, and actuators. However, developing facile and effective methods for fabricating such hydrogels remains a great challenge, especially for hydrogels that retain their properties in extreme environmental conditions, such as at subzero temperatures or storage in open-air conditions. Herein, a water-miscible ionic liquid (IL), such as 1-ethyl-3-methylimidazolium acetate (EMImAc), was introduced to form an IL/water binary solvent system for poly(vinyl alcohol) (PVA) to create ionic conductive PVA hydrogels. The physically crosslinked PVA/EMImAc/H2O hydrogels showed better mechanical properties and transparency than the traditional PVA hydrogel prepared by the freeze-thaw method due to the formation of homogeneous and small PVA microcrystals in the EMImAc/H2O binary solvent system. More importantly, the PVA/EMImAc/H2O hydrogel exhibited significant anti-freezing and water-retaining properties because of the presence of the IL. The hydrogels remained flexible and conductive at temperatures as low as -50 °C and retained more than 90% of their weight after storage in open-air conditions for 2 weeks. In addition, the thermal stability of the hydrogel could be increased to 95 °C through the addition of Mg(II) ions. A multimodal sensor based on the PVA/EMImAc/H2O/Mg(II) hydrogel showed high sensitivity and a quick response to changes in pressure, strain, and temperature, with both long-term stability and a wide working temperature range. This study may open a new route for the fabrication of functional PVA-based hydrogel electrolytes and provide a practical pathway for their use in multifunctional electronic and sensory device applications.

Improved biorefinery pathways of marine diatoms using a water miscible ionic liquid and its colloidal solution: efficient lipid extraction and in situ synthesis of fluorescent carbon dots for bio-imaging applications.

In this study, a water-miscible ‘classic’ ionic liquid (IL), 1-ethyl-3-methylimidazoliumacetate ([EMIM][Ac]), has been used for lipid extraction from marine diatoms Thalassiosira lundiana CSIR-CSMCRI 001 by following a non-polar solvent partition method. The composition of lipid was determined using gas chromatography-mass spectrometry (GC-MS). In total, 91.4 mg g−1 (dry wt) of lipid was produced, out of which the percentage of docosahexaenoic acids (DHA), myristic acid, palmitic acid, and arachidonic acid was 19.6%, 15.1%, 11.2%, and 10.4%, respectively. The IL-inseparable residual waste solution was directly used to generate green fluorescent carbon dots (FCDs) by constructing a colloidal solution with the help of a surface-active IL, choline dioctyl sulfosuccinate ([Cho][AOT]). The stability of colloidal FCDs was examined using FTIR, FT-NMR, and Raman spectroscopy. FCDs were extracted from the colloidal solutions via the demicellization process and characterized using HR-TEM (2 to 5 nm) and PXRD techniques. The optical properties of colloidal FCDs were measured using UV-Vis and fluorescence spectroscopy and showed a wide range of emission (λ460 nm to λ590 nm). Such FCD stabilized colloidal solutions could be effectively used in fluorescence imaging of yeast cells, thus making the biorefinery approach more sustainable.

Liquid-liquid equilibria of aqueous biphasic systems containing 1-alkyl-3-methylimidazolium amino acid ionic liquids with different anions (L-Leucine, L-Valine, L-Lysine) and inorganic salt (tripotassium phosphate, potassium carbonate)

Aqueous biphasic systems containing water-miscible ionic liquids (ILs) and inorganic salt are considered as an effective media for liquid-liquid extraction of bioactive materials. One of the important advantages of ILs is the possibility for a modification of their chemical structure and, as a result, their properties, i.e. their multifunctionality. Among the modified ILs, the amino acid ILs (AAILs) attract much attention due to their low toxicity and biodegradability. In the present work, new experimental data on phase behavior (binodal curves and tie lines) were obtained at 298.15 K for the systems containing 1-alkyl-3-methylmidazolium AAILs, [Cnmim]Х, with different hydrocarbon chain length (n = 4, 8), and with the amino acid anions: X = [Leu] (L-Leucine), [Val] (L-Valine), [Lys] (L-Lysine), and inorganic salts (K3PO4, K2CO3). AAILs under investigation were synthesized and characterized by means of thermogravimetric analysis, 1H- and 13C-NMR spectra. The ability of the studied systems to phase separation, the lengths and the slopes of the tie lines were discussed in terms of AAIL's structure. Systems containing [C8mim][Lys] show stronger tendency for phase separation. The slopes of the tie lines increase with the length of alkyl chain of IL's cation. For the aqueous systems [Сnmim]X + inorganic salt, the slopes of the tie lines increase in the sequence: [Lys] < [Val] < [Leu] < Cl < Br. Phase behavior of the investigated ternary systems favors their use for the extraction of bio-solutes.

Elevated Temperature Homogeneous Liquid Phase Extraction Coupled to Ionic Liquid–Based Dispersive Liquid–Liquid Microextraction Followed by High-Performance Liquid Chromatography: Application of Water-Miscible Ionic Liquids as Extraction Solvent in Determination of Carbamate Pesticides

In the present study, an elevated temperature homogeneous liquid extraction coupled with ionic liquid‑based dispersive liquid–liquid microextraction was developed to extract five carbamate pesticides (pirimicarb, carbaryl, methiocarb, aldicarb, and carbofuran) from packed fruit juice samples. The method consisted of a two-step procedure in which cyclohexylamine was used in order to extract the target analytes. In the first step, the analytes were extracted from a sample solution thermostated at elevated temperature (80 °C) into a water-immiscible organic solvent (cyclohexylamine). In the second step, the separated organic phase was mixed with 1-butyl-3-methylimidazolium chloride (as the solvent for extraction) and the mixture was spread into deionized water. In this step, cyclohexylamine controlled the solubility of 1-butyl-3-methylimidazolium chloride in the aqueous phase and acted as a dispersive solvent and a phase separation agent, simultaneously. After centrifugation, we removed all the gathered organic phases and analyzed them by using high-performance liquid chromatography diode-array detector. Figure of merits of the method including the limits of detection and quantification, enrichment factors, extraction recoveries, and relative standard deviations were obtained within ranges 0.4–3.9 and 1.5–13.2 ng mL−1, 147–217, 59–87%, and 3.3–5.5%, respectively, indicating that the method was successful in determining the analytes.

Water molecular state in 1-hexylpyridinium hexafluorophosphate: Water mean cluster size as a function of water concentration

The water sorption behavior of representative pyridinium-based ionic liquid (IL), 1-hexylpyridinium hexafluorophosphate ([C6Py][PF6]), was studied over the whole range of the water activity a using a continuous gravimetric method. The analysis of the water sorption isotherm using the combination of a two-mode sorption (i.e. Henry-clustering) allowed to better understand [C6Py][PF6]-water interactions. At low and intermediate activity (a ≤ 0.8), the water molecules revealed a very low affinity to [C6Py][PF6] and, consequently, the water uptake was rather low. On the contrary, at high water activity (a > 0.8), the water uptake increased exponentially and the water clustering easily occurred. The constant of the Henry-clustering equation as well as the water clustering mechanism in [C6Py][PF6] were discussed and compared to those of imidazolium-based ILs: 1-hexyl-3-methylimidazolium hexafluorophosphate [C6C1im][PF6] (water-immiscible IL) and 1-butyl-3-methylimidazolium tetrafluoroborate [C4C1im][BF4] (water-miscible IL). It is shown that the sorption of water molecules by pyridinium-based ILs is controlled not only by the anion's nature, but also by the cation's nature. Moreover, the Zimm-Lundberg theory was used to determine the water mean cluster size (MCS) in [C6Py][PF6], [C6C1im][PF6] and [C4C1im][BF4]. The MCS results confirmed the strong capacity of water molecules to be aggregated in [C6Py][PF6]. In order to have a deeper insight into the water molecular state, infrared spectroscopy measurements were carried out as a function of the relative humidity value and the obtained results were correlated with the results of water sorption isotherms. It is found that at high water activity (a > 0.8), sorbed water molecules are strongly linked with ILs by hydrogen bonds and, therefore, are easily aggregated.

Aqueous-phase green synthesis of formate-based ionic liquids and their thermophysical properties

In this study, the anion exchange reaction of an organic cation chloride with excess KHCO2 to synthesize formate-based ionic liquids (ILs) was performed in water, and the water-miscible IL products were easily obtained via salting-out phase separation caused by the excess KHCO2. According to the synthesis results, a set of heuristic rules could be generated as follows. First of all, the more “hydrophobic” ILs required less amount of excess KHCO2 for salting-out phase separation. Secondly, the amphiphilic ILs with a long cationic alkyl chain (e.g., octyl) are likely to form reverse micelles to encapsulate inorganic salt (i.e., KCl), resulting in relatively high residual contents of K+ and Cl−. Lastly, cooling would exert a positive effect on the salting-out phase separation and thus expand the application range of this method. For example, [C2-MIm]HCO2 was prepared at −18 °C, because it has to be phase-separated out by cooling. The basic properties of these ILs were also thoroughly characterized and analyzed, such as melting point, thermal decomposition temperature, density, refractive index, surface tension, viscosity and conductivity etc.

Quantification of amino acids and peptides in an ionic liquid based aqueous two-phase system by LC\u2013MS analysis

Aqueous two-phase systems (ATPS) occur by the mixture of two polymers or a polymer and an inorganic salt in water. It was shown that not only polymers but also ionic liquids in combination with inorganic cosmotrophic salts are able to build ATPS. Suitable for the formation of ionic liquid-based ATPS systems are hydrophilic water miscible ionic liquids. To understand the driving force for amino acid and peptide distribution in IL-ATPS at different pH values, the ionic liquid Ammoeng 110™ and K2HPO4 have been chosen as a test system. To quantify the concentration of amino acids and peptides in the different phases, liquid chromatography and mass spectrometry (LC–MS) technologies were used. Therefore the peptides and amino acids have been processed with EZ:faast™-Kit from Phenomenex for an easy and reliable quantification method even in complex sample matrices. Partitioning is a surface-dependent phenomenon, investigations were focused on surface-related amino acid respectively peptide properties such as charge and hydrophobicity. Only a very low dependence between the amino acids or peptides hydrophobicity and the partition coefficient was found. Nevertheless, the presented results show that electrostatic respectively ionic interactions between the ionic liquid and the amino acids or peptides have a strong impact on their partitioning behavior.

Evaluation of Biocompatible Ionic Liquids for Their Application in Phytosterols Bioconversion by Mycobacterium sp. Resting Cells

The application of ionic liquids (ILs) as novel solvents in whole-cell bioconversion has a practical significance in innovation of green process engineering for sustainable environment. Therefore, a systematical evaluation of ILs based on their cationic and anionic composition is urgently demanded in the whole-cell system. In this work, production of androst-4-ene-3,17-dione (AD) via side-chain cleavage of phytosterols catalyzed by Mycobacterium sp. resting cells was employed to investigate the relationships of ILs structures with their biocompatibility and bioconversion performances. For 13 of biocompatible ILs, the viscosity, hydrophobicity, polarity and hydrogen-bond basicity, as well as IL/water composition at phase equilibrium were measured. Based upon cell membrane integrity and metabolic activity retention, some ILs with anions of [PF6] or [NTf2] showed proper Mycobacterium sp. biocompatibility. Among bioconversion systems containing water-immiscible or water-miscible ILs, [PrMIM][PF6] generated th...

Isolation of ionizable red Monascus pigments after extractive fermentation in nonionic surfactant micelle aqueous solution

Production of red Monascus pigments can be enhanced by extractive fermentation in a nonionic surfactant micelle aqueous solution. Using excess MSG (monosodium glutamate) as the sole nitrogen source, several kinds of red Monascus pigments, including red Monascus pigments with MSG residue as major components, were produced during the fermentation process. However, the corresponding downstream processing, i.e., isolation of red Monascus pigments from the nonionic surfactant aqueous solution, remains unsolved. In the present work, a novel ionic liquid-nonionic surfactant aqueous two-phase system, such as coacervate phase: ionic liquid ([Bmim]Cl) = 1:3 (W/W) and temperature = 60 °C, was developed for transferring of the anionic red Monascus pigments with MSG residue from nonionic surfactant micelle aqueous solution into ionic liquid aqueous solution. Thus, the challenging isolation of red Monascus pigments from nonionic surfactant aqueous solution was converted to the relatively easy separation between red Monascus pigments and water-miscible ionic liquid. By screening various organic solvents, water-chloroform two-phase system was selected for recovery of red Monascus pigments from the water-miscible ionic liquid. Finally, the isolated red Monascus pigment with MSG residue was confirmed by UPLC–MS analysis.

Extraction of gingerols and shogaols from ginger (Zingiber officinale Roscoe) through microwave technique using ionic liquids

An ionic liquid-based microwave-assisted extraction (ILMAE) method was developed to extract 6-, 8-, 10-gingerols and 6-, 8-, and 10-shogaols from ginger (Zingiber officinale Roscoe). A series of water-miscible ionic liquids (ILs) with different kinds of anions and cations were investigated. Results indicated that among the investigated ILs, the carbon chains of alkyl cations remarkably affected the extraction efficiency of the six compounds and 1-decyl-3-methylimidazolium bromide ([C10MIM]Br) was the optimal IL. The selected conditions for ILMAE were as follows: [C10MIM]Br concentration, 0.80M; extraction temperature, 75°C; irradiation time, 30min; and irradiation power, 400W, in the case of solid–liquid ratio of 0.1:10g/mL. This study showed that compared with the total extraction yield of 0.595±0.031% by methanol marinated extraction and 0.673±0.043% by methanol-based microwave-assisted extraction, ILMAE attained higher yield of 0.716±0.051%; and ILMAE obtained a shorter time of 30min for extraction of gingerols and shogaols from ginger. This study provides a basis for separating and purifying bioactive components from ginger. Moreover, the use of small amounts of [C10MIM]Br as extraction solution in the method was considered environmentally friendly.

Redox Reaction: A New Route for the Synthesis of Water-Miscible Imidazolium Ionic Liquids

A novel chemical redox route was developed for the preparation of water-miscible imidazolium ionic liquids (ILs). In this method, the reaction between 1-alkyl-3-methylimidazolium bromides or 3-butyl-1-phenylimidazolium bromide and the appropriate acid reactant was promoted by the redox reaction between the bromide ion and aqueous hydrogen peroxide, with hex-1-ene as both solvent and bromine scavenger. The residual bromide ion and water contents of the prepared ILs were determined by ion chromatography and the Karl–Fischer test, respectively. This method not only produces water-miscible ILs in high purity and high yield, but also simplifies the reaction conditions in comparison with previous routes.

Impact of imidazolium-based ionic liquids on the structure and stability of lysozyme

ABSTRACTVarious types of water-miscible aprotic ionic liquids (ILs) with different cations (1-ethyl-3-methylimidazolium, 1-butyl-3-methylimidazolium, 1-octyl-3-methylimidazolium) and anions (ethylsulfate and chloride) were used as co-solvents to investigate the stability of lysozyme. Different techniques such as fluorescence, thermal absorption, and circular dichroism (CD) spectroscopy have been used for the study. Fluorescence results reveal that the addition of ILs (1-ethyl-3-methylimidazolium ethyl sulfate and 1-ethyl-3-methylimidazolium) increases the hydrophobicity around the tryptophan environment in lysozyme. CD analysis and temperature-dependent studies were done to investigate the stability of the protein. From the CD analysis, it was observed that the ILs keep the native structure of protein intact. Thermal denaturation studies depicted that the melting temperature of the protein increased in the presence of ILs (1-ethyl-3-methylimidazolium ethyl sulfate and 1-ethyl-3-methylimidazolium), which indicates the stabilization of the protein.

Asymmetric reduction of ethyl acetoacetate catalyzed by immobilized Acetobacter sp. CCTCC M209061 cells in hydrophilic ionic liquid hybrid system

The asymmetric reduction of ethyl acetoacetate (EAA) to ethyl (R)-3-hydroxybutyrate [(R)-EHB] using immobilized Acetobacter sp. CCTCC M209061 cells was successfully conducted in a hydrophilic ionic liquid (IL)-containing system. The best one of all the tested watermiscible ILs was 1-(hydroxyethyl)-3-methylimidazolium hydrochloride (C2OHMIM·Cl). In C2OHMIM·Cl-aqueous buffer hybrid system, it was found that the optimal IL concentration, substrate and co-substrate concentration, reaction temperature, buffer pH and shaking rate were 0.5mol/L, 45 mmol/L, 80 mmol/L, 35°C, pH 5.5 and 200 rpm, respectively. Under the optimized reaction conditions, the initial reaction rate, the yield and the product e.e. reached 4.90 µmol/min, 95.3 and > 99.0%, respectively, which were much higher than the corresponding values reported previously. The efficient biocatalytic process mediated by the immobilized cells was feasible on 500 mL preparative scale, and the biocatalysts showed good operational stability and could be recycled for at least 10 batches.

Effect of Temperature on Salt–Salt Aqueous Biphasic Systems: Manifestations of Upper Critical Solution Temperature

Water-miscible ionic liquids (ILs) may be salted out using kosmotropic salts such as potassium phosphate (K3PO4) to form salt–salt aqueous biphasic systems (ABS). The effect of temperature on these systems has been studied using phase diagrams and it is observed that the degree of binodal shift decreases (requiring lower IL and kosmotropic salt concentrations) with the increase of temperature following the trend [C4mim]Cl > [C4py]Cl > [C4mmim] Cl ~ [N4444]Cl. This trend can be correlated with the decreasing hydrogen bonding abilities of each salt. The phase behavior was also interpreted on the basis of critical solution temperature behavior of pure aqueous ionic liquid solutions. Additionally, the distribution of alcohols in these systems was studied as a function of temperature and it was found that the distribution ratios did not change with changes in temperature. The Gibbs energy of transfer of a methylene group in these systems and correlation to tie-line length was also determined. It was concluded that while the miscibility of alcohols increases in the ILs with increasing temperature, phase divergence in the aqueous biphasic system decreases, and thus these competing forces tend to cancel each other out for small polar molecules. A comparison is provided for the response to temperature in the currently studied salt–salt systems and analogous ABS formed by the addition of hydrophilic polymers to kosmotropic salts (polymer–salt) or other polymers (polymer–polymer).

Directed evolution of a formate dehydrogenase for increased tolerance to ionic liquids reveals a new site for increasing the stability.

The formate dehydrogenase (FDH) from Candida boidinii is a well-known enzyme in biocatalysis for NADH regeneration. Nevertheless, it has low activity in a water-miscible ionic liquid (1,3-dimethylimidazolium dimethyl phosphate, [MMIm][Me2 PO4 ]). In this work, this enzyme was subjected to directed evolution by using error-prone PCR, and a mutant (N187S/T321S) displaying higher activity was obtained following selection based on the formazan-based colorimetric assay. The mutation N187S is responsible for improved activity both in aqueous solution and in [MMIm][Me2 PO4 ], through an enhancement of the kcat value by a factor of 5.8. Fluorescence experiments performed in the presence of a quenching agent revealed that the mutant does not unfold in the presence of 50 % (v/v) [MMIm][Me2 PO4 ] whereas the wild-type enzyme does. Molecular modelling revealed that the mutation is located at the monomer-monomer interface and causes an increase in the pKa of residue E163 from 4.8 to 5.5. Calculation of the pKa of this residue in other microbial FDHs showed that thermostable FDHs have a highly basic glutamate at this position (pKa up to 6.2). We have identified a new site for improving FDH thermostability and tolerance to ionic liquids, and it is linked to the local charge of the enzymes in this class.

Improving the catalytic performance of porcine pancreatic lipase in the presence of [MMIm][MeSO4] with the modification of functional ionic liquids

Porcine pancreatic lipase (PPL) was chemically modified with various functional ionic liquids (ILs) to increase its catalytic performance in water-miscible IL. Catalytic activity and thermostability were tested with a p-nitrophenyl palmitate (pNPP) hydrolysis reaction. The native enzyme lost 18% of its initial activity in 0.4M [MMIm][MeSO4], whereas the activities of all the modified enzymes increased. The [HOOCBMIm][Cl] modification led to a 2-fold increase in activity in 0.3M [MMIm][MeSO4] than in aqueous. All the modified enzymes exhibited higher thermostability compared with the native enzyme at high temperature. In particular, the [HOOCBMIm][Cl] modification led to a 6-fold increase in thermostability at 60°C. Conformational changes were confirmed by fluorescence spectroscopy and circular dichroism spectroscopy to elucidate the mechanism of catalytic performance alteration.