Effect of ionic liquid on activity, stability, and structure ...

International Journal of Biological Macromolecules 51 (2012) 555–560

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Review

Effect of ionic liquid on activity, stability, and structure of enzymes: A review Mu. Naushad a , Zied Abdullah ALOthman a , Abbul Bashar Khan b , Maroof Ali c,∗ a

Advanced Materials Research Chair, Department of Chemistry, College of Science, King Saud University, Riyadh, Saudi Arabia Al-Falah School of Engineering and Technology, Faridabad, Haryana, India c Department of Chemistry, Indian Institute of Technology, New Delhi, India b

a r t i c l e

i n f o

Article history: Received 23 March 2012 Received in revised form 12 June 2012 Accepted 14 June 2012 Available online 23 June 2012 Keywords: Ionic liquids Enzyme activity Enzymes Proteins Lipase Biocatalysis

a b s t r a c t 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. © 2012 Published by Elsevier B.V.

Contents 1. 2.

3. 4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effect of physicochemical properties of ILs on activity and stability of enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Effect of IL polarity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Effect of alkyl chain length in the cation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Effect of anion of IL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Effect of hydrophobicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Ion specific effects: Hofmeister series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Effect of viscosity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Other factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structural and conformational dynamics of enzymes in ILs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure, stability, and activity of enzymes in protic ILs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Throughout the recent decade or so, room temperature ionic liquids (ILs) as potential environmentally friendly solvents have garnered widespread attention and curiosity from the academic and industrial research communities due to their unusual and

∗ Corresponding author at: Department of Chemistry, Indian Institute of Technology Delhi, New Delhi, India. Tel.: +91 11 26591395; fax: +91 11 26581102. E-mail address: maroof [email protected] (M. Ali). 0141-8130/$ – see front matter © 2012 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ijbiomac.2012.06.020

useful properties [1]. Combined with the fact that ILs are composed entirely of cations and anions but still exist in the liquid state at ambient conditions, so these are more desirable as solvent for reaction and processes [2]. Almost every named reaction and many additional organic/inorganic/organometallic reactions have been reported in ILs [1,2]. In contrast to conventional organic solvent, ILs have many favorable properties such as low vapor pressure, high ionic conductivity, wide liquid range, high thermal stability, and ability to dissolve a variety of the solutes [2]. Novel analytical applications of ILs are emerging daily; effective, and in some cases

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truly unique, deployment of ILs has been demonstrated within a variety of analysis modes encompassing electroanalysis, separation, extraction, mass spectrometry, and sensing [1,4–10]. Recently, several groups demonstrate the role of ILs in altering the photophysical behaviors of solutes and dyes in interesting and unique manner [2,9,11–14]. ILs have also emerged as exceptionally interesting non aqueous media for enzymatic reactions, and research interest in this area has increase widely during recent years [15]. Their interest as green chemicals resides in their high thermal stability and very low pressure, which can be helpful to overcome the problem of volatile organic emission. The additional advantage with ILs is that their physicochemical properties (density, viscosity, melting point, polarity, etc.) can be finely tuned by appropriate combination of cations and/or anions [2,3]. For example, ILs can be synthesized to be water miscible or immiscible with different polarities, and viscosities. All these tunable properties are very important for enzymatic reactions, because the stability and activity of enzymes can be enhanced by tuning the physicochemical properties [15]. Researchers have already shown that biocatalyst activity, enantioselectivity, thermal stability, and reusability can all be improved in ILs [16–21]. Many enzymatic reactions such as synthesis of Z-aspartame using thrmolysin, alcoholysis, ammoniolysis, and perhydrolysis using lipase [22–25], transesterification using ␣chymotrypsin [26–28], and ketone reduction using whole cells of Baker’s yeast have been reported in ILs [29]. Numerous other enzymatic reactions also have been reported in ILs. A careful examination of these enzymatic reactions revealed that physicochemical properties of ILs play important role in altering the structure, stability, and activity of enzymes. Many ILs (based on BF4 − , PF6 − and Tf2 N− ) show less denaturing property than organic solvents and high enantioselectivity and high catalytic activity [15,30]. While, many enzymes shows same magnitude of the activities in ILs as in conventional organic solvents. Thus, it is necessary to have proper understanding of the effects of IL’s chemistry on stability, activity, and structural/conformational dynamics of enzymes/protein, before choosing or designing an IL to serve as a solvent media for enzymatic reactions. In this context, in the present review we try to shed light on the effects of ILs’ chemistry on stability, activity, and conformational/structural dynamics of enzymes, in a systematic manner, in the view of recent findings in the field, which could be useful for the researchers in designing an IL-based solvent media for enzymatic reactions and protein preservation. Further, in the present review, we also try to explore the effect of ILs on the structure and conformational dynamics of proteins, which definitely help the researchers to understand the mechanism, how ILs stabilize the enzymes. 2. Effect of physicochemical properties of ILs on activity and stability of enzymes ILs offer diverse range of physicochemical properties depending on cations and anions. These physicochemical properties (such as, polarity, hydrophobicity, viscosity, and kosmotropicity etc.) of the ILs play important role in affecting the enzyme’s activity and stability. 2.1. Effect of IL polarity ILs are usually considered highly polar on account of their ionic nature [2,3,31]. Several polarity indicator based parameters have been used to describe the solvent polarity of ILs. Based on solvatochromic probes studies, it was observed that ILs have polarity close to low chain alcohols or formamide [32]. There are

several reports demonstrating the effect of the polarity of ILs on enzymes stability and activity. Park and Kazlauska observed that activity of lipase (from Psuedomonas capacia) increases with IL polarity in acetylation of racemic 1-phenyl ethanol with vinyl acetate [25]. Lozano and coworkers reported that ␣-chymotrypsin shows low synthetic activity within less polar ILs [26]. However, in another study by several groups, no clear trend of enzyme activity with polarity of ILs is observed [33–35]. So it is early to consider any general correlation between these two parameters because of multifaceted molecular interactions of ILs with enzyme. 2.2. Effect of alkyl chain length in the cation ILs having long chain alkyl chains in their cation behave like surfactant in aqueous solution, and have strong impact on the stability and activity of the enzyme [36,37]. There are several reports demonstrating effect of alkyl chain of ILs on stability and activity of enzymes. Yamaguchi et al. [38] also observed that increase in the alkyl chain length of N-alkyl pyridinium chloride and Nalkyl-N-methylpyrrolidinium chlorides destabilize the lysozyme. Venkatesu et al. [39] found that more hydrophobic imidazolium and phosphonium cation carrying longer alkyl chains ([Bzmim][Cl], [Bzmim][BF4 ] and tetrabutyl phosphonium bromide) were weak stablizers for ˛-chymotrypsin, while small chain ILs tetraethyl ammonium acetate and tetraethyl ammonium phosphate act as strong stabilizer. Activity of ˛-chymotrypsin also follow the same trend. Guanguan and coworkers [40] demonstrated that CALB possessed high activity and stability in ILs having hydroxyl ethyl chain in their cations. In other study, Robert et al. [41] study the effect of a series of N-ethyl-N -methylimidazolium cation-based ILs on recombinant Plasminogen activator (rPA), and reported that strongly destabilizing anion with longer alkyl substitution like e.g. hexyl sulfate were unable to promote refolding, However, anion with short alkyl chain like di-Me, and di-Et, were able to promote refolding. Hua and coworkers [42] also reported that long decyl chain on imidazolium cation destabilize the BSA more than short chain ILs. Lange et al. [43] investigate the influence of a series of N -alkyl, and N -(ω-hydroxyalkyl)-N-methylimidazolium chlorides on the behavior of two model proteins, hen egg white lysozyme (HEWL) and an antibody fragment ScFvOx, and observed that more hydrophobic imidazolium cations carrying long alkyl chain more increasingly destabilizing, while terminal hydroxylation of alkyl chain made the salt more compatible with protein stability. Bahareh et al. observed that [bmim][Cl] and [hmim][Cl] reduce the both enzymatic stabilities and activities of ␣-amylase from Bacillus amyloliquefaciens and Bacillus licheniformis. However, some contradictions were also observed, Lau and Zhang [44] reported that enantioselectivity and stability of enzymes decreased with decreasing alkyl chain length on imidazolium cations from [omim]+ to [bmim]+ . This effect may, however, also be confounded with the viscosity effect since a longer alkyl chain length in substituents on the imidazolium cation results in higher viscosity in the range mentioned above. 2.3. Effect of anion of IL The anion of IL has an impact on the enzyme stability and activity through its hydrogen bond forming capability and nucleophilicity properties, as demonstrated by several groups [45–47]. Generally, it was observed that anion having high hydrogen bond forming capability strongly interact with enzyme, causes the conformational change in the structure and affects the activity of the enzyme [45]. Baker et al. [48] observed that the addition of

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[bmim][Cl] to the solution decreases the thermal stability of green fluorescent protein (GFP) allowing the protein to unfold at lower temperature than in aqueous solution. Recently, Noritomi et al. [49] explore the thermal stability and activity of lysozyme in presence of [emim][BF4 ], [emim][Tf2 N], and [emim][Cl], and found very low activity in case of [emim][Cl] than that of [emim][BF4 ] and [emim][Tf2 N]. Weingartner et al. also reported destabilization effect of [chol][Cl] and [bmim][Br] on the model protein RNase A [50]. Bahareh et al. observed that [bmim][Cl] and [hmim][Cl] reduce the both enzymatic stabilities and activities of ␣-amylase from B. amyloliquefaciens and B. licheniformis [51]. Marie et al. [52] study the influence of IL anion on lipase activity in the enzymatic reaction of flavinoids with long chain fatty acids, and found that IL containing Tf2 N− , PF6 − , and BF4 − anions were successful as reaction media while ILs containing anion with stronger hydrogen bond ability resulted in decreased yield. Wolski et al. [53] reported Trichoderma reesei cellulase and ˇ-glycosidase retained activity in ILs, [mmim][dmp] and [emim][lactate]. Klaehn et al. [54] study the influences of ILs on stability of lipase B from Candida antarctica (CALB), and found that stability influenced primarily by anion in order NO3 −  BF4 − < PF6 − of increasing stability. Tavares et al. [55] reported the activity and stability of enzyme Laccase in ILs, [emim][MDEGSO4 ], [emim][EtSO4 ], and [emim][MeSO3 ], and found [emim][MDEGSO4 ] show high activity among them. However, in other study higher enzyme activity was observed in case of IL having high nucleophilic anion [56], which contradicts the earlier observations. These contradictory results may be because; the multiple factors govern the enzymatic reactions in IL-based media. 2.4. Effect of hydrophobicity

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chlorides, and found that less hydrophobic ILs such as NEt, N-Bu, and N-hexylpyridinium chlorides, and N-butyl-Nmethylpyrrolidinium chloride were effective in enhancing the refolding yields. Weingartner et al. [50] reported low thermal stability of RNase A in hydrophobic IL [emim][dca]. Venkatesu et al. [39] also reported that less hydrophobic ILs such as triethyl ammonium acetate and triethyl ammonium phosphate play role of stabilizer for ␣-chymotrypsin, while more hydrophobic IL [Bzmim][Cl], [Bzmim][BF4 ] act as destabilizer for enzyme. This may be due to more factors are responsible for stability and activity of enzymes in ILs. 2.5. Ion specific effects: Hofmeister series Several researchers, proposed Hofmeister series to explain the behavior of enzymes in aqueous solution of ILs, because it was found that ILs dissociate into individual ions similar to traditional salts. Weingartner and coworkers, in their novel work, proposed the following Hofmeister series of ions of ILs, by probing melting temperature of RNase A, and mentioned that this series of ions is only meaningful at low concentration of ILs [50,65]. Cation− K+ > Na+ > [Me4 N]+  Li+ > [chol]+ > [Et4 N]+ ≈ [emim]+ ≈ [gua]+ > [bmpyr]+ > [bmim]+ ≈ [Pr4 N]+ > [hmim]+ ≈ [Bu4 N]+ Anion− SO4 2− > dhp2− > ac− > F− > Cl− || EtSO4 − > BF4 − ≈ Br− > OTf− > I− > SCN− ≈ dca− > Tf2 N−

Double bars indicates crossover from stabilizing to destabilizing behavior. Generally, log P scale has been used to quantify the hydrobZhao et al. [35] also proposed Hofmeister Series in interpreting hobicity of ILs, which describe the hydrophobicity of a species the enzyme behavior in aqueous IL solutions, and observed that in term of the logarithm of its partition coefficient P between kosmotropic (structure-maker) anions and chaotropic (structureoctanol and water [56,57]. There are several reports how breaker) cation stabilize enzyme, while chaotropic anion and this scale can be used to optimize enzyme stability. Russel kosmotropic cation destabilize it. et al. [33] reported that free lipase (Candida rugosa) shows There are some other reports which describe the Hofmeister activity only in [bmim][PF6 ], while inactive in hydrophilic ILs effect of ILs on enzyme activity and stability. Fujita et al. [66] such as [bmim][CF3 COO], [bmim][CH3 COO], and [bmim][NO3 ]. correlated the activity of cytochrome-c (Cyt-C) in IL containing (20 wt% water) with kosmotropicity of component ions. WeinIt also has been reported by Paljevac et al. [58] that cellulose activity decreases with IL hydrophobicity with trend gartner et al. [67] study the effect of different ILs on the efficiency [bmim][PF6 ] > [bmim][BF4 ] > [bmim][Cl]. of the oxidation of ethanol catalyzed by yeast alcohol dehydroShen et al. [59] demonstrated that amino lipase has high genase, and reported that both cation and anion follows the enantioselectivity in hydrophobic [omim][PF6 ] and poor enanHofmeister series of IL’s ions. Recently, Lai et al. [68] study two tioselectivity in hydrophilic IL [bmim][BF4 ] and [bmim][Cl]. model enzymes, Penicillium expansum lipase (PEL) and mushroom tyrosinase in aqueous solution of 14 different ILs. The activity It has been reported by Fernandez group [60] that CALB and Penicillin G Acylase (PGA) are more stable in hydrophoof PEL in presence of ILs follows the Homeister series very well. bic ILs than in hydrophilic. The following trend is observed In presence of ILs holding same cation, PEL activity decreased in case of CALB. [hmim][PF6 ] > [hmim][Tf2 N] > [hmim][BF4 ], in order of [mmim][MeSO4 ] > [emim][MeSO4 ] > [bmim][MeSO4 ], [bmim][PF6 ] > [bmim][dca], [omim][PF6 ] > [hmim][PF6 ] > [bmim][PF6 ], [NMe4 ][ac] > [NBu4 ][ac], [NHMe3 ][MeSO3 ] > [NBu4 ][MeSO3 ], and while, the following trend is observed in case of PGA, [NHMe3 ][dhp] > [NHEt3 ][dhp] > [NHBu3 ][dhp], while for the ILs [bmim][Tf2 N] > [bmim][PF6 ] > [bmim][BF4 ]. A research group holding same cation, [chol][ac] > [chol][MeSO3 ] > [chol][NO3 ], [61] reported higher activity of lipase in transesterification reac[NBu4 ][ac] > [NBu4 ][MeSO3 ]. The activity of mushroom tion in hydrophobic [bmim][PF6 ] than in hydrophilic [bmim][BF4 ]. tyrosinase shows some similar results. In presence of Goto et al. [62] observed that PEG-modified lipase show high the ILs holding same anion, the initial rate of tyrosiactivity in hydrophobic ILs. Another group [63] demonstrate low nase catalyzed oxidation reaction show a decreasing order [mmim][MeSO4 ] > [emim][MeSO4 ] > [bmim][MeSO4 ], stability of Penicillin acylase in hydrophilic ILs such as [bmim][BF4 ] of and [bmim][dca]. Villora et al. also reported penicillin G shows [NMe4 ][ac] > [NBu4 ][ac], [NHMe3 ][MeSO3 ] > [NBu4 ][MeSO3 ], high stability in hydrophobic ILs than hydrophilic one [64]. and [NHMe3 ][dhp] > [NHEt3 ][dhp]. The tyrosinase stability in From above mentioned reports, it is observed that enzymes presence of ILs also follows the same. show high enantioselectivity, stability, and activity in hydrophoIn other study by Gomes et al. [69] study the effect of different ILs bic ILs than hydrophilic ILs. However, there are some reports on the stability of lysozyme, and arrange anion according to their demonstrating high enzyme stability and activity in hydrophilic effect from destablizer to stabilizers in following order: ILs. Recently, Yamomoto et al. [38] studied the protein refolding Tf2 N− < hexonate < TMA− ≈ OTf− < propionate < Cl− < lactate ≈ in N-alkyl pyridinium chloride and N-alkyl-N-methylpyrrolidinium dmp− < dhp−

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These results have been typically explained in term of ion specific effects, namely the anion kosmotropicity. To explain how and why ions affect the protein stability following Hofmeister series, many theories have been proposed, based on hydrophobic interactions, water structure changes, electrostatic attractions, and others. However, there is no single theory due to complex nature of ion–protein interactions. However, it is not right to say that specific ions effect is only factor controlling enzyme performance in ILs-based solvent systems, because behaviors of enzymes in IL-containing aqueous solution may be some what different from that in pure IL or ILs with trace amount of water [45]. So, in some reports, deviation from Hofmeister series also observed. Recently, Baker et al. [70] also study the structural behavior of TMR-labeled Cyt-C in phosphate buffer containing bmim-based ILs, and demonstrate both anion and cation play critical role in controlling protein stability. Most recently, Byrne and coworkers observed a reverse Hofmeister trend when protic ILs are listed in order of fibrilization rate of A␤ 16-22 peptides [71]. 2.6. Effect of viscosity It is documented that ILs have more viscosity than conventional organic solvents [10]. There are many reports demonstrating that viscosity of ILs may have an influence on enzyme stability and activity. Lozano et al. [26] reported high activity of ␣-chymotrypsin in less viscous [emim][Tf2 N] that high viscous [MTOA][Tf2 N]. Meyer et al. [72] observed that higher viscosity of [bmim][PF6 ] compared to [OH-mmim][PF6 ] is likely to cause lower reaction rates through mass transfer limitations in furuloyl esterase from Aspergillus niger. Lau et al. [68] observed a trend between stability and activity of P. expansum lipase and viscosity coefficient, and proposed that viscosity of IL’s cation may have an impact on stability and activity. Zhao et al. [73] study the CALB catalyzed transesterification of ethyl butyrate and 1-butanol in more than 20 ILs and suggests that IL viscosity might affect the reaction rates in some cases. However, there are also some contradictory reports [74]. In one study it was reported that viscosity of ILs [bmim][PF6 ] and [bmim][BF4 ] did not affect the immobilized Penicillium G amidase despite these have more viscosity than toluene. 2.7. Other factors Besides above mentioned properties of ILs, there are numerous factors e.g. pH of medium, co-solvent, and halide impurity. Tavares et al. [55] observed the reduction in activity of laccase when pH was reduced from 9.0 to 5.0. Byrne et al. [75] observed that denaturing temperature of both HEWL and RNase A are sensitive to the proton activity of protic ILs and more stability for more basic solution is observed. It is well documented that the presence of any impurity may influence the physicochemical properties of ILs, and hence, enzymatic reactions performed in them. It was reported by several groups that the presence of halide impurity influences the enzyme’s activity and stability. Villora et al. [76] reported higher lipase activity in impurity free IL. Lee et al. [77] reported that activity of Novozyme® 435 in [omim][Tf2 N] decrease linearly with chloride content while the activity of lipase from Rhizomer miehei drastically decrease in presence of [omim][Cl]. In other study [73], activity of Novozyme® 435 in transesterification reaction between ethyl butyrate and 1-butanol was reported very low in presence of high halide (Cl− and Br− ) content. Thus it is always important to determine and remove halide contents from ILs before using them.

There are also some reports which demonstrate the cosolvent effect on enzyme stability and activity in ILs. Recently, Shaotao et al. [78] observed esterification activity of lipase of Burkholderia cepacia was higher in cosolvent mixture of ILs (n-Hexane + [bmim][PF6 ], n-Hexane + [hmim][PF6 ], and benzene + [bmim][NO3 ], etc.), followed by conventional organic solvents and ILs. Goto et al. [79] reported higher hydrolytic activity of lipase PS in water-in-IL microemulsions than in water saturated IL. Zhou et al. [80] observed high catalytic activity of lignin peroxidase and laccase in water-in[bmim][PF6 ] microemulsion than that of pure IL [bmim][PF6 ] and water saturated [bmim][PF6 ].

3. Structural and conformational dynamics of enzymes in ILs It is also subject of the interest for several researchers to know the mechanism how ILs stabilize and activate the enzymes. In this regards several groups put their efforts, and performed several study to explore the structural and conformational dynamics of protein in ILs. Hoagland et al. [81] study the dissolution and dissolved molecular states of Cyt-C in [emim][EtSO4 ] by various techniques and found that secondary structure of Cyt-C remains largely intact, but tertiary structure changes significantly. Yu et al. [82] reported the binding of 1-tetradecyl-3-methylimidazolium bromide ([C14 mim][Br]) to bovine serum albumin (BSA) and observed that IL reduces the burial of tryptophal residue in hydrophobic environment at low concentration, and lead to BSA denaturation at high concentration. Hua et al. [42] study the interaction of [Cn mim][Br] (n = 4, 6, 8, 10) with BSA and observed that hydrophobic interaction plays major role in interaction of [C10 mim][Br] with BSA, while hydrogen bond and van der walls forces play major role in interaction of [Cn mim][Br] (n = 4, 6, 8) with BSA. They also observed that [C10 mim][Br] could markedly change the secondary structure. Weaver et al. [83] study the structure and function of lysozyme and interleukin-2 in IL [chol][dhp]. No significant alteration of overall tertiary structure of lysozyme was observed at lower temperature, while at higher temperature and high concentration of [chol][dhp], small change in protein structure were seen. Bekhouche et al. [84] study the structural change of formate dehydrogenase from Candida boidini (FDH) in three imidazolium-based ILs ([mmim][dmp], [bmim][ac], and [mmim][CH3 H2 PO2 OCH3 ]) and observed ILs play role of strong denaturing agents but each one acting with different mechanism. Sasmal et al. [85] study the effect of [pmim][Br] on conformational dynamics of human serum albumin (HSA) protein with fluorescence correlation spectroscopy and observed that IL act as denaturant when protein is in native state. However, addition of [pmim][Br] to a denatured protein by guanidinium chloride (GdnHCl), causes protein to refold. Shu et al. [86] found that the interaction of [bmim][Cl], [bbim][Cl], and [bmim][NO3 ] with BSA is entropically driven, and predominantly hydrophobic and electrostatic interactions lead to unfolding of polypeptide chain. Recently, Huang et al. [87] reported the unusual refolding behavior of several designed peptide in IL. Akdogan et al. [88] observed that addition of imidazolium IL causes unfolding of protein tertiary structure in HSA-fatty acid conjugate (HSA/FA). In contrast, HSA maintains its tertiary structure when [chol][dhp] is added. Bright et al. [89] study the conformational dynamics of acrylodan labeled HSA (Ac-HSA), in [bmim][BF4 ], [bmim][PF6 ], and [bmim][Tf2 N] and found that protein behave much differently in ILs in comparison to aqueous buffer. Iborra et al. [90] study the stabilization behavior of ␣-chymotrypsin in [emim][Tf2 N], and observed that [emim][Tf2 N] show the ability to compact the native conformation of protein. A

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great enhancement of ˇ-strand in presence of IL is also observed which reflects its stabilization power. In other study, Iborra et al. [91] observed by spectroscopic study that the stabilization of CALB in [emim][Tf2 N] and [btma][Tf2 N] was associated with the maintenance of ˛-helix content and enhancement of ˇ-strands. Baker et al. [92] reported that improved thermal stability of sweet protein monellin in [bmPyr][Tf2N] may be due to change in protein hydration level and structural compactation. Bright et al. [93] study the rotational reorientation dynamics of acrylodan labeled HSA (Ac-HSA) with binary mixture of [bmim][BF4 ], [bmim][PF6 ], and [bmim][Tf2 N], and water as a function of temperature and water loading. They observed that Ac-HSA behave much differently when dissolved in IL/H2 O mixture in comparison to buffer. This behavior of Ac-HSA shows dependency on protein structure, temperature and solvent composition among other factor.

4. Structure, stability, and activity of enzymes in protic ILs Similar to aprotic ILs, the protic ILs (pILs) also draw the attention of scientific and engineering community due to their unique features and applications [1–3]. Hence, many enzymatic reactions have been performed in pILs, which offer enhanced activity and stability of enzymes in biocatalytic reactions. In recent work, Angell and coworkers [94] reported on the multiyear stabilization against aggregation and hydrolysis for highly concentrated (>200 mg mL−1 ) hen egg white lysozyme (HEWL) in ice avoiding solvents containing pIL ethylammonium nitrate (EAN) and triethylammonium methyl sulfonate (TEAMS). Most recently, Byrne and Angell showed that selected pIL are able to initiate the formation of HEWL amyloid fibrils which are subsequently be dissolved with up to 72% restoration of the native enzymatic activity [95]. Fumino et al. [96] observed that EAN is most biocompatible IL investigated, as tetramethyl rhodamine labeled Cyt-C (TMR-Cyt-C) appear to adopt a native like global folded even in 2.5 M EAN. Persumably, this is a reflection of the fact that EAN shares with water ability to form three-dimensional hydrogen-bonded network. Recently, Danielson and Wei [97] study the structural stability of Cyt-C in pIL, methyl ammonium formate and ethyl ammonium formate, and found that the structure of Cyt-C maintained in high IL concentration (50–70% IL/water). Most recently, Byrne and coworkers [71] found that pILs containing kosmotropic anions like phosphate and sulfate promote fibrilization Aˇ 16-22 with mature fibrils, while pIL containing mesylate anion completely suppress amyloid fibrilization. Mann et al. [98] observed an increase in stability and activity of HEWL in the presence of four pILs (ethylammonium formate (EAF), propylammonium formate (PAF), 2-methoxyethylammonium formate (MOEAF) and ethanolammonium formate (EtAF)). In other study, by Summers and Flowers [99], EAN was used to enhance the recovery of denatured-reduced HEWL, which show the ability of EAN to prevent aggregation of the denatured protein. Recently, Venkatesu et al. [39] observed high stability and activity of ␣-chymotrypsin in pILs, triethyl ammonium acetate (TEAA), and triethyl ammonium phosphate (TEAP). Francesco et al. [100] reported the activity of subtilisin in diethanol ammonium chloride against the model substrate APEE (N-acetyl-L-phenyl alanine ethyl ether). Recently, Attri et al. [101] study the behavior of ␣chymotrypsin in TEAA, and observed that IL play a role to attenuates the denaturation action of non-ionic chaotropic urea. Thus, from the study done so far, it is observed that pILs also play important role in enhancing the activity, and stability of enzymes, which shows their potential to serve as non aqueous media for enzymatic reactions.

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5. Conclusion From the studies done so far, in most of cases it is observed that ILs having hydrophobic nature, less viscosity, kosmotropic anion and chaotropic cation usually enhance the activity and stability of enzymes. However, a general correlation could not be established because of many contradictory results. Overall, the information in present review could be helpful for the researchers to choose or design a compatible IL to serve as solvent for enzymatic reaction, protein preservation, and other bioapplications, as well. Further, information regarding structural and conformational dynamics of proteins could be helpful for engineering and scientific communities to understand how ILs enhance the stability and activity of enzymes. Abbreviations IL cations [emim]+ [bmim]+ [hmim]+ [mmim]+ [Bzmim]+ [omim]+ [pmim]+ [bbim]+ [bmPyr]+ [OH-emim]+ [Me4 N]+ [Et4 N]+ [Pr4 N]+ [Bu4 N]+ [Me3 NH]+ [Et3 NH]+ [Bu3 NH]+ [MTOA]+ [btma]+ [Chol]+ [gua]+ IL anions [BF4 ]− [PF6 ]− [Tf2 N]− [dmp]− [MDEGSO4 ]− [MeSO3 ]− [EtSO4 ]− [CF3 OO]− [dca]− [dhp]2− [OTf]− [MeSO4 ]− [TMA]−

1-ethyl-3-methylimidazolium 1-butyl-3-methylimidazolium 1-hexyl-3-methylimidazolium 1,3-dimethylimidazolium 1-benzyl-3-methylimidazolium 1-octyl-3-methylimidazolium 1-propyl-3-methylimidazolium 1,3-dibutylimidazolium 1-butyl-3-methylpyrrolidinium 1-(2-hydroxyethyl)-3-methylimidazolium tetra methyl ammonium tetra ethyl ammonium tetra propyl ammonium tetra butyl ammonium tri methyl ammonium tri ethyl ammonium tri butyl ammonium methyl trioctyl ammonium butyl trimethyl ammonium choline guanidinium tetrafluoroborate hexafluorophosphate bis(trifluromethane)sulfonimide dimethyl phosphate 2-(2-methoxyethoxy) ethyl phosphate methyl sulfonate ethyl sulfate trifluoro acetate dicyanamide dihydrogen phosphate trifluoromethane sulfonate methyl sulfate trimethyl acetate

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