Effect of ionic liquid on activity, stability, and structure of enzymes: A review

Ionic liquids have shown their potential as a solvent media for many enzymatic reactions as well as protein preservation, because of their unusual characteristics. It is also observed that change in cation or anion alters the physiochemical properties of the ionic liquids, which in turn influence the enzymatic reactions by altering the structure, activity, enatioselectivity, and stability of the enzymes. Thus, it is utmost need of the researchers to have full understanding of these influences created by ionic liquids before choosing or developing an ionic liquid to serve as solvent media for enzymatic reaction or protein preservation. So, in the present review, we try to shed light on effects of ionic liquids chemistry on structure, stability, and activity of enzymes, which will be helpful for the researchers in various biocatalytic applications.     

The bioreduction of a β-tetralone to its corresponding alcohol by the yeast Trichosporon capitatum MY1890 and bacterium Rhodococcus erythropolis MA7213 in a range of ionic liquids

The stereospecific reduction of 6-Br-β-tetralone to its corresponding alcohol (S)-6-Br-β-tetralol was carried out by the yeast Trichosporon capitatum MY1890 and by the bacterium Rhodococcus erythropolis MA7213, using a range of ionic liquids chosen for the diversity of their composition. The decrease in cell viability of both types of cell upon exposure to ionic liquids was found to be between that determined for cells residing purely in fermentation media, and cells residing in a two-phase mixture of media and organic solvent (toluene). For T. capitatum MY1890 bioconversions, the water miscible hydrophilic ionic liquid [Emim][TOS] gave a reaction profile comparable to that observed in the previously studied water-ethanol (10% v/v) system, in terms of overall rate of reaction (0.2 g (prod) L^-1 h^-1) and conversion (100%). Of the hydrophobic ionic liquids evaluated, [Oc₃MeN][BTA] gave the best conversion of 60%, but at a much reduced rate, suggesting solute mass transfer from the ionic liquid phase was rate limiting. For bioconversions carried out with R. erythropolis MA7213 employing 20% v/v [Emim][TOS] as a co-solvent, the conversion yield doubled, and a four-fold increase in initial rate was found compared to the standard ethanol co-solvent. This was attributed to improved cell viability and reduced aggregation of the R. erythropolis MA7213 compared to T. capitatum MY1890. Overall, this study demonstrates the feasibility of using ionic liquids for whole cell biocatalysis, however, no obvious link is apparent between the physico-chemical properties of ionic liquids, their influence on cell viability, and their efficacy as media for bioconversions.     

Enzyme catalysis in ionic liquids

Ionic liquids offer new possibilities for the application of solvent engineering to biocatalytic reactions. Although in many cases ionic liquids have simply been used to replace organic solvents, they have often led to improved process performance. Unlike conventional organic solvents, ionic liquids possess no vapor pressure, are able to dissolve many compounds, and can be used to form two-phase systems with many solvents. To date, reactions involving lipases have benefited most from the use of ionic liquids, but the use of ionic liquids with other enzymes and in whole-cell processes has also been described. In some cases, remarkable results with respect to yield, (enantio)selectivity or enzyme stability were observed.     

Toward advanced ionic liquids. Polar,enzyme-friendly solvents for biocatalysis

Ionic liquids, also called molten salts, are mixtures of cations and anions that melt below 100°C. Typical ionic liquids are dialkylimidazolium cations with weakly coordinating anions such as (MeOSO₃) or (PF₆). Advanced ionic liquids such as choline citrate have biodegradable, less expensive, and less toxic anions and cations. Deep eutectic solvents are also included in the advanced ionic liquids. Deep eutectic solvents are mixtures of salts such as choline chloride and uncharged hydrogen bond donors such as urea, oxalic acid, or glycerol. For example, a mixture of choline chloride and urea in 1:2 molar ratio liquefies to form a deep eutectic solvent. Their properties are similar to those of ionic liquids. Water-miscible ionic liquids as cosolvents with water enhance the solubility of substrates or products. Although traditional water-miscible organic solvents also enhance solubility, they often inactivate enzymes, while ionic liquids do not. The enhanced solubility of substrates can increase the rate of reaction and often increases the regioor enantioselectivity. Ionic liquids can also be solvents for non-aqueous reactions. In these cases, they are especially suited to dissolve polar substrates. Polar organic solvent alternatives inactivate enzymes, but ionic liquids do not even when they have similar polarities. Besides their solubility properties, ionic liquids and deep eutectic solvents may be greener than organic solvents because ionic liquids are nonvolatile, and can be made from nontoxic components. This review covers selected examples of enzyme catalyzed reaction in ionic liquids that demonstrate their advantages and unique properties, and point out opportunities for new applications. Most examples involve hydrolases, but oxidoreductases and even whole cell reactions have been reported in ionic liquids.     

离子液体的性能及应用

离子液体不仅可用作环境友好的“绿色溶剂”,而且在生物合成和有机反应中能表现出特殊的催化、促进效应。在介绍离子液体种类、性质、合成方法的基础上,重点综述离子液体功能化方法、离子液体／超临界CO2体系和其在生物催化反应中应用的最新研究进展。     

Retention factors and resolutions of amino benzoic acid isomers with some lonic liquids

Ionic liquids in the form of organic salts are being widely used as new solvent media. In this paper three positional isomers,o-amino benzoic acid,m-amino benzoic acid, andp-amino benzoic acids were separated with four different ionic liquids as mobile phase additives using high performance liquid chromatography (HPLC). The following ionic liquids were used: 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIm][BF₄]), 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIm][BF₄]), 1-ethyl-3-methylimidazolium methylsulfate ([EMIm][MS]), and 1-octyl-3-methylimidazolium methylsulfate ([OMIm][MS]). The effects of the alkyl group length on the imidazolium ring and its counterion, and the concentrations of the ionic liquids on the retention factors and resolutions of amino benzoic acid isomers were tested. The results of the separations with ionic liquids as the eluents were better than those without ionic liquids. Excellent separations of the three isomers were achieved using 2.0≈8.0 mM/L [OMIm][MS] and 1.0≈8.0 mM/L [EMIm][MS] as the eluent modifiers.     

Investigation of ionic liquids as reaction media for enzymatic enantioselective acylation of amines

The use of ionic liquids as reaction media for lipase-catalyzed enantioselective acylation of 1-phenylethylamine (1) and 2-phenyl-1-propylamine (2) with 4-pentenoic acid was investigated. The best performing ionic liquid for each of these amines as well as its solvent properties were very different. Preparative scale kinetic resolution of 1 was performed efficiently in 1-butyl-2,3-dimethylimidazolium trifluoromethanesulphonate.     

Design of phosphonium ionic liquids for lipase-catalyzed transesterification

Ionic liquids are now recognized as solvents for use in lipase-catalyzed reactions; however, there still remains a serious drawback in that the rate of reaction in an ionic liquid is slower than that in a conventional organic solvent. To overcome this problem, attempts have been made to evolve phosphonium ionic liquids appropriate for lipase-catalyzed reaction; several types of phosphonium salts have been prepared and their capability evaluated for use as solvent for the lipase-catalyzed reaction. Very rapid lipase PS-catalyzed transesterification of secondary alcohols was obtained when 2-methoxyethyl(tri-n-butyl)phosphonium bis(trifluoromethanesulfonyl)imide ([MEBu₃P][NTf₂]) was used as solvent, affording the first example of a reaction rate superior to that in diisopropyl ether.     

Enzyme catalysis with small ionic liquid quantities

Enzyme catalysis with minimal ionic liquid quantities improves reaction rates, stereoselectivity and enables solvent-free processing. In particular the widely used lipases combine well with many ionic liquids. Demonstrated applications are racemate separation, esterification and glycerolysis. Minimal solvent processing is also an alternative to sluggish solvent-free catalysis. The method allows simplified down-stream processing, as only traces of ionic liquids have to be removed.     

Oxidative refolding of rPA in l‐ArgHCl and in ionic liquids: A correlation between hydrophobicity,salt effects,and refolding yield

The ionic liquid 1‐ethyl‐3‐methyl imidazolium chloride (EMIM Cl) and the amino acid l‐ arginine hydrochloride (l ‐ArgHCl) have been successfully used to improve the yield of oxidative refolding for various proteins. However, the molecular mechanisms behind the actions of such solvent additives—especially of ionic liquids—are still not well understood. To analyze these mechanisms, we have determined the transfer free energies from water into ionic liquid solutions of proteinogenic amino acids and of diketopiperazine as peptide bond analogue. For EMIM Cl and 1‐ethyl‐3‐methyl imidazolium diethyl phosphate, which had a suppressive effect on protein refolding, as well as for l ‐ArgHCl favorable interactions with amino acid side chains, but no favorable interactions with the peptide backbone could be observed. A quantitative analysis of other ionic liquids together with their already published effects on protein refolding showed that only solvent additives within a certain range of hydrophobicity, chaotropicity and kosmotropicity were effective for the refolding of recombinant plasminogen activator. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1129–1140, 2014.     

High-throughput screening for ionic liquids dissolving (ligno-)cellulose

The recalcitrance of lignocellulosic biomass poses a major challenge for its sustainable and cost-effective utilization. Therefore, an efficient pretreatment is decisive for processes based on lignocellulose. A green and energy-efficient pretreatment could be the dissolution of lignocellulose in ionic liquids. Several ionic liquids were identified earlier which are capable to dissolve (ligno-)cellulose. However, due to their multitude and high costs, a high-throughput screening on small scale is essential for the determination of the most efficient ionic liquid. In this contribution two high-throughput systems are presented based on extinction or scattered light measurements. Quasi-continuous dissolution profiles allow a direct comparison of up to 96 ionic liquids per experiment in terms of their dissolution kinetics. The screening results indicate that among the ionic liquids tested EMIM Ac is the most efficient for dissolving cellulose. Moreover, it was observed that AMIM Cl is the most effective ionic liquid for dissolving wood chips.     

Application of ionic liquids as mobile phase modifier in HPLC

Ionic liquids are receiving an upsurge of interest as ‘green’ solvents; primarily as replacements for conventional media in chemical processes. Although ionic liquids are rather “young” modifier, their great potential in high-performance liquids chromatography (HPLC) has already been demonstrated. This review presents an overview of the applications of ionic liquids as mobile phase modifiers in HPLC.     

Development of Ionic Liquid‐Water‐Based Thermomorphic Solvent (TMS)‐Systems for Biocatalytic Reactions

The applicability of ionic liquid‐water‐based thermomorphic solvent (TMS)‐systems with an upper critical solution temperature for homogeneous biocatalysis is investigated. Cholinium‐ and imidazolium‐based ionic liquids are used to facilitate a temperature‐dependent phase change, which can be easily fine‐tuned by adding salts or polar organic solvents. Within the TMS‐system, a high enzymatic activity and subsequent full conversion is achieved in the intermittent monophasic reaction system of the TMS‐system. Therefore, the biocatalyst can be easily recycled after separating the phases at lower temperatures.     

Buffer-mediated activation of Candida antarctica lipase B dissolved in hydroxyl-functionalized ionic liquids

Ionic-liquid buffer having phosphate anion was synthesized for the development of buffered enzymatic ionic liquid systems. Both the conformation and transesterification activity of Candida antarctica lipase B (CALB) dissolved in the hydroxyl-functionalized ionic liquids were buffer dependent. Intrinsic fluorescence studies indicated that the CALB possessed a more compact conformation in the medium consisted of ionic liquid buffer having phosphate anion and hydroxyl-functionalized ionic liquids like 1-(1-hydroxyethyl)-3-methyl-imidazolium tetrafluoroborate and 1-(1-hydroxyethyl)-3-methyl-imidazolium nitrate. High activity and outstanding stability could be obtained with the CALB enzyme in the buffered ionic liquids for the transesterification.     

Recent advances of enzymatic reactions in ionic liquids

The tremendous potential of room temperature ionic liquids as an alternative to environmentally harmful ordinary organic solvents is well recognized. Ionic liquids, having no measurable vapor pressure, are an interesting class of tunable and designer solvents, and they have been used extensively in a wide range of applications including enzymatic biotransformation. In fact, ionic liquids can be designed with different cation and anion combinations, which allow the possibility of tailoring reaction solvents with specific desired properties, and these unconventional solvent properties of ionic liquids provide the opportunity to carry out many important biocatalytic reactions that are impossible in traditional solvents. As compared to those observed in conventional organic solvents, the use of enzymes in ionic liquids has presented many advantages such as high conversion rates, high enantioselectivity, better enzyme stability, as well as better recoverability and recyclability. To date, a wide range of pronounced approaches have been taken to further improve the performance of enzymes in ionic liquids. This review presents the recent technological developments in which the advantages of ionic liquids as a medium for enzymes have been gradually realized.     

Thermodynamically based solvent design for enzymatic saccharide acylation with hydroxycinnamic acids in non-conventional media

Enzyme-catalyzed synthesis has been widely studied with lipases (EC 3.1.1.3), but feruloyl esterases (FAEs; EC 3.1.1.73) may provide advantages such as higher substrate affinity and regioselectivity in the synthesis of hydroxycinnamate saccharide esters. These compounds are interesting because of their amphiphilicity and antioxidative potential. Synthetic reactions using mono- or disaccharides as one of the substrates may moreover direct new routes for biomass upgrading in the biorefinery. The paper reviews the available data for enzymatic hydroxycinnamate saccharide ester synthesis in organic solvent systems as well as other enzymatic hydroxycinnamate acylations in ionic liquid systems. The choice of solvent system is highly decisive for enzyme stability, selectivity, and reaction yields in these synthesis reactions. To increase the understanding of the reaction environment and to facilitate solvent screening as a crucial part of the reaction design, the review explores the use of activity coefficient models for describing these systems and - more importantly - the use of group contribution model UNIFAC and quantum chemistry based COSMO-RS for thermodynamic predictions and preliminary solvent screening. Surfactant-free microemulsions of a hydrocarbon, a polar alcohol, and water are interesting solvent systems because they accommodate different substrate and product solubilities and maintain enzyme stability. Ionic liquids may provide advantages as solvents in terms of increased substrate and product solubility, higher reactivity and selectivity, as well as tunable physicochemical properties, but their design should be carefully considered in relation to enzyme stability. The treatise shows that thermodynamic modeling tools for solvent design provide a new toolbox to design enzyme-catalyzed synthetic reactions from biomass sources.     

Kinetic study on the enzymatic resolution of homophenylalanine ester using ionic liquids

Two ionic liquids (ILs) were investigated as novel media for the enzymatic resolution of amino acid ester to obtain enantiomeric amino acid homophenylalanine. The effects of solvent nature, polarity, and concentration on the kinetic resolution were investigated. With change in solvent concentration, a systematic study shows that an improved enzyme activity can be obtained by adjusting these solvent parameters.     

Penicillin acylase catalysis in the presence of ionic liquids

Several ionic liquids were used as reaction media for penicillin G acylase catalysis. In all the assayed ionic liquids, [bmim]PF6 proved good media for PGA-catalyzed hydrolysis. A novel [bmim]PF6/water two-phase system is provided for 6-aminopenicillanic acid (APA) production, which will be more benefical than aquous batch systems used widely in industrial production of APA.     

Evaluation of parallel milliliter-scale stirred-tank bioreactors for the study of biphasic whole-cell biocatalysis with ionic liquids

As clear structure-activity relationships are still rare for ionic liquids, preliminary experiments are necessary for the process development of biphasic whole-cell processes involving these solvents. To reduce the time investment and the material costs, the process development of such biphasic reaction systems would profit from a small-scale high-throughput platform. Exemplarily, the reduction of 2-octanone to (R)-2-octanol by a recombinant Escherichia coli in a biphasic ionic liquid/water system was studied in a miniaturized stirred-tank bioreactor system allowing the parallel operation of up to 48 reactors at the mL-scale. The results were compared to those obtained in a 20-fold larger stirred-tank reactor. The maximum local energy dissipation was evaluated at the larger scale and compared to the data available for the small-scale reactors, to verify if similar mass transfer could be obtained at both scales. Thereafter, the reaction kinetics and final conversions reached in different reactions setups were analysed. The results were in good agreement between both scales for varying ionic liquids and for ionic liquid volume fractions up to 40%. The parallel bioreactor system can thus be used for the process development of the majority of biphasic reaction systems involving ionic liquids, reducing the time and resource investment during the process development of this type of applications.     

Unexpected improvement in stability and utility of cytochrome c by solution in biocompatible ionic liquids

Proteins generally are only stable in vitro for short periods of time. This results in challenges during isolation and purification of recombinant proteins and reduces the shelf life of protein-based pharmaceuticals. Here we show that certain novel, biocompatible ionic liquids provide a stabilizing solvent for proteins, for example, cytochrome c, such that structure and activity are maintained even after 6 months of storage at room temperature. Normally, this protein would be rendered inactive after only 1 week in buffered aqueous solution. The effect of the ionic liquid solvent appears to be related to protection against hydrolysis.