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Review

Received: 6 October 2009

Revised: 20 January 2010

Accepted: 26 January 2010

Published online in Wiley Interscience: 22 March 2010

(www.interscience.wiley.com) DOI 10.1002/jctb.2375

Methods for stabilizing and activating enzymes
in ionic liquids – a review
Hua Zhao∗
Abstract
Ionic liquids (ILs) have evolved as a new type of non-aqueous solvents for biocatalysis, mainly due to their unique and tunable
physical properties. A number of recent review papers have described a variety of enzymatic reactions conducted in IL solutions;
on the other hand, it is important to systematically analyze methods that have been developed for stabilizing and activating
enzymes in ILs. This review discusses the biocatalysis in ILs from two unique aspects (1) factors that impact the enzyme’s activity
and stability, (2) methods that have been adopted or developed to activate and/or stabilize enzymes in ionic media. Factors
that may influence the catalytic performance of enzymes include IL polarity, hydrogen-bond basicity/anion nucleophilicity, IL
network, ion kosmotropicity, viscosity, hydrophobicity, the enzyme dissolution, and surfactant effect. To improve the enzyme’s
activity and stability in ILs, major methods being explored include the enzyme immobilization (on solid support, sol–gel, or
CLEA), physical or covalent attachment to PEG, rinsing with n-propanol methods (PREP and EPRP), water-in-IL microemulsions,
IL coating, and the design of enzyme-compatible ionic solvents. It is exciting to notice that new ILs are being synthesized to be
more compatible with enzymes. To utilize the full potential of ILs, it is necessary to further improve these methods for better
enzyme compatibility. This is what has been accomplished in the field of biocatalysis in conventional organic solvents.
c 2010 Society of Chemical Industry 

Keywords: ionic liquid; enzyme activity; enzyme stabilization; immobilization; biocatalysis

NOTATION
Cations
BMIM+ = 1-butyl-3-methylimidazolium
C2 OC1 MIM+ = 1-(2-methoxy)ethyl-3-methylimidazolium
C2 OHMIM+ = 1-(2-hydroxy)ethyl-3-methylimidazolium
EMIM+ = 1-ethyl-3-methylimidazolium
HMIM+ = 1-hexyl-3-methylimidazolium
MMEP+ = 1-methyl-1-(2-methoxyethyl)pyrrolidinium
MTOA+ = methyl trioctylammonium
OMIM+ = 1-methyl-3-octylimidazolium
PPMIM+ = 1-(31 -phenylpropyl)-3-methylimidazolium
Anions
BF4 −
= tetrafluoroborate
= dicyanamide
dca−
EtSO4 − = ethyl sulfate
MeSO4 − = methyl sulfate
= acetate
OAc−
= triflate (or trifluoromethanesulfonate)
OTf−
= hexafluorophosphate
PF6 −
= bis(trifluoromethane)sulfonimide
Tf2 N−

INTRODUCTION

J Chem Technol Biotechnol 2010; 85: 891–907

Correspondence to: Hua Zhao, Chemistry Program, Savannah State
University, Savannah, GA 31404, USA. E-mail: huazhao98@gmail.com or
zhaoh@savannahstate.edu
Chemistry Program, Savannah State University, Savannah, GA 31404, USA

www.soci.org

c 2010 Society of Chemical Industry 

891

Ionic liquids (ILs) consist of ions and are liquid at temperatures
lower than 100 ◦ C. Those salts with very low melting points, so
called room-temperature ionic liquids (RTILs), are most desirable
as solvents for reactions and processes. In contrast to conventional
organic solvents, ILs have many favorable properties including extremely low vapor pressure, a wide liquid range, low flammability,
high ionic conductivity, high thermal conductivity, good dissolution power towards many substrates, high thermal and chemical
stability, and a wide electrochemical potential window. Because

of these unique properties, ILs have been widely recognized
as solvents or co-catalysts in a variety of applications including organic catalysis,1 – 9 inorganic synthesis,10 biocatalysis,7,11 – 16
polymerization,17,18 and engineering fluids.19 – 21 Typical IL
cations are nitrogen-containing (such as alkylammonium, N,
N -dialkylimidazolium, N-alkylpyridinium and pyrrolidinium), or
phosphorous containing (such as alkylphosphonium). The common choices of anions include halides, BF4 − , PF6 − , CH3 CO2 − ,
CF3 CO2 − , NO3 − , Tf2 N− [(CF3 SO2 )2 N− ], [RSO4 ]− , and [R2 PO4 ]− .
Some representative cations and anions are shown in Fig 1.
In addition, the physical properties of ILs (such as polarity,
hydrophobicity and hydrogen-bond basicity) can be finely
designed through the proper selection of cations and anions.
For example, ILs can be synthesized to be water-miscible,
partially miscible or immiscible with different polarities, and can
also be synthesized with various viscosities targeting individual
applications.9 All these tunable properties are very important for
enzyme stabilization and activation; therefore, many enzymatic
reactions have been investigated in different types of ILs.12,13,16,22
Although some ILs (such as those based on BF4 − , PF6 − , and Tf2 N− )
are less denaturing than organic solvents and high catalytic activity
and enantioselectivity have been reported for many reactions,16,23
many enzymes show the same magnitude of activities in ILs as in
conventional organic solvents, which are considerably inferior to
those in aqueous solutions.

Therefore.60 reported the stability of CALB in ILs to be in the following order: [HMIM][PF6 ] > [HMIM][Tf2 N] > [HMIM][BF4 ].44 Park and Kazlauskas45 observed the trend of lipase (from Pseudomonas www. and thus could interact with the enzyme causing the protein conformational changes. and β-cyclodextrin). particularly in those enzyme-inactivating ILs. there is a strong and urgent need to increase the activity and stability of enzymes in ILs..org Anion Cation R1 + R4 N R2 R1 N R1 + R4 P R2 R3 R3 + + N R2 R1 R2 R1 + R2 S N R2 N + N N N BF4- CN O + N R2 N F3C FeCl4- O O CF3 S O O O _ O F3C S N S CF3 R1 O + N R1 O PF6- R R3 R1 NC N R3 + N O Cl R2 O RO S O O O R1O P O OR2 O Figure 1. the stronger base is usually the stronger nucleophile in aprotic solvents. Lau et al. Hern´andez-Fern´andez et al.39.53 also observed a dramatic decrease of the lipase activity in [OMIM][Tf2 N] (1-octyl-3-methylimidazolium bis(trifluoromethane)sulfonimide) with the increasing concentration of [OMIM]Cl.56 reported that Novozym 435 showed no ammonolysis activity towards (R. such as up to 10–20% (wt) cellulose. but decreased with IL polarity when ILs were used as solvents. Lee et al.wiley. c 2010 Society of Chemical Industry  J Chem Technol Biotechnol 2010. However. the solvent properties of ILs are often related to the enzyme’s activity. Recently.38 Similarly. excipients and impurities. They explained † Basicity refers to the ability of a base to accept a proton.57 suggested that the low CALB (lipase B from Candida antarctica) activity in [BMIM][lactate] was caused by the secondary structure changes of the protein. most commonly involving formation of a bond to carbon. which was further triggered by the H-bonding interaction between lactate anions and peptide chains.48 observed that the free Candida rugosa lipase was only active in hydrophobic [BMIM][PF6 ].35 As a result. [BMIM][dca] is an enzymedenaturing IL25. [BMIM]Cl (1-butyl-3-methylimidazolium chloride) could effectively dissolve cellulose51.54 and thus could dissolve complex polar molecules such as cyclodextrins and antibiotics. ILs were found to be moderately polar being close to lower alcohols42. [BMIM]Cl showed the largest H-bond basicity among all ILs considered in the study by Anderson et al. formate (HCOO− ) and acetate (OAc− )) of these ILs tend to form strong hydrogen bonds with carbohydrates for dissolving them. pH. which were less active than in other ILs. >100 g L−1 other carbohydrates (including β-D-glucose.58 however. and is a matter of equilibrium. Based upon multiple solvation interactions. chloride (Cl− ).interscience.24 – 27 The dissolution of carbohydrates in ILs has enabled the chemical modifications of these natural products in homogeneous systems.com/jctb H Zhao cepacia) activity increasing with the IL polarity during the acetylation of racemic 1-phenylethanol with vinyl acetate (for example. MAJOR FACTORS THAT AFFECT ENZYME ACTIVITY AND STABILITY IN IONIC LIQUIDS Before the discussion of enzyme-stabilizing/activating methods.24.47 observed that the ester conversion increased with IL polarity when ILs were used as liquid film-coating (under solvent-free conditions). lactose.www.43 or formamide. and to guide scientists in advancing these methods to further flourish the biocatalytic applications in ionic solvents. the initial reaction rate in less polar [BMIM][PF6 ] is three times slower than that in more polar [EMIM][BF4 ]).28 – 34 Since the anions (typically. ascorbic acid. Dicyanamide (dca) based ILs such as [BMIM][dca] are able to dissolve carbohydrates. It is the purpose of this review to survey these methods. nucleophilicity is a matter of kinetics (rate).48 – 50 Hydrogen-bond (H-bond) basicity and nucleophilicity of anions Hydrogen-bond basicity and nucleophilicity are two different concepts. specificity and stability as being discussed below.46 During Novozym 435-catalyzed esterification of methyl-α-D-glucopyranoside with fatty acids. this IL induced the inactivation of cellulase (from Trichoderma reesei). but inactive in all hydrophilic ILs based on NO3 − . and [BMIM][PF6 ] > [BMIM][dca]. dicyanamide (dca− ). CH3 COO− and CF3 COO− during the transesterification of methylmethacrylate with 2-ethyl-1-hexanol. It is well known that an enzyme’s performance in ILs is affected by common factors such as the water activity. 892 IL polarity15 Based on the solvatochromic studies.52 because chloride ions (as H-acceptors) interact with the cellulose -OH group and break the H-bonding network of cellulose.S)-phydroxyphenylglycine methyl ester in [BMIM]Br and [BMIM][NO3 ]. sucrose. the IL polarity–enzyme activity correlation has not been established for other enzymatic reactions performed in ILs.41 In addition. Mutschler et al.59 possibly due to the H-bond basicity of the anion. some ILs have been shown capable of dissolving many substrates that are normally insoluble in organic solvents. it is important to know what physical properties are most important to the catalytic behaviors of enzymes in ILs. and the stability of Penicillin G acylase was in a similar order [BMIM][Tf2 N] > [BMIM][PF6 ] > [BMIM][BF4 ]. They indicated that the latter three anions are more nucleophilic than PF6 − . implying the denaturing nature of these two ILs.41. and some new approaches have been developed for stabilizing and activating enzymes in ILs. fatty acids and triglycerides. Nucleophilicity of a Lewis base refers to the relative reaction rate of different nucleophilic reagents towards a common substrate.38 – 40 making it impossible to transform these dissolved substrates via enzymatic reactions.soci.55 Lou et al.35 – 37 these ILs are also likely to denature enzymes. amino acids. 85: 891–907 . The author’s group26 also suggested both free and immobilized CALB in [EMIM][OTf] were about as active as in [BMIM][dca]. For molecules containing the same nucleophilic atoms of the same charge.† but are often closely related. many enzyme-stabilizing methods used in conventional non-aqueous enzymology have been adopted for ionic media. In another study. Kaar et al. Structures of some typical cations and anions in ILs.24. For these reasons. lower synthetic activities of α-chymotrypsin were also obtained in less polar ILs.

consistent with the increasing order of nucleophilicity (PF6 − < BF4 − < Tf2 N− < dca− ). however. a concept derived from the partition coefficient of ILs between 1-octanol and water.com/jctb 893 Ion kosmotropicity In aqueous solutions. but inactive in other hydrophilic ILs including [BMIM][CH3 COO] (log P = −2. Recently. liquid and solution states.62 measured the initial transesterification rates of three lipases (Novozym 435. and observed that the anion effect on the initial rates followed a decreasing order Tf2 N− > PF6 − > OTf− > SbF6 − ∼ BF4 − . Therefore. allowing enzymes to maintain their native structures and activity.39). where the more nucleophilic anions tend to interact with the positively charged sites in enzymes and to modify the enzyme’s conformation. hydrophilic ILs dissociate into individual ions. . Therefore.72 also confirmed that the thermal stability of ribonuclease A (RNase A) in aqueous solution of ILs follows the Hofmeister series.77 in addition.50. Constantinescu et al. which could protect the essential water of proteins and the solvophobic interactions that are critical for maintaining the native structure of proteins. A recent study59 of the lipase-catalyzed transesterification of ethyl butyrate and 1-butanol in more than 20 ILs further confirmed that the IL viscosity might affect the reaction rates in some cases. in general. it is not quite clear if such an effect exists in pure ILs or ILs with trace amounts of water. They explained that OTf− and BF4 − are more nucleophilic than PF6 − . On the other hand.48 Similarly. hydrophobicity. recent studies50. Irimescu and Kato61 observed that PF6 − based ILs afforded fastest reaction rates.34 mPa s) may lead to mass transfer limitations and lower the reaction rate.90) and [BMIM][CF3 COO]. The rather confusing results may be because the enzymatic reaction is affected by multiple factors of ILs such as nucleophilicity. In a second acylation reaction of 2-phenyl-1propylamine with 4-pentenoic acid. since enzymes are not soluble in most common ILs. Lee et al.67 as well as some concentrated ILs (such as 20 wt% water71 ).79 observed that during amide synthesis through immobilized penicillin G amidase. enzyme molecules are suspended in reaction media at low water contents (for example. same cations). The second factor could be IL hydrophobicity (see more discussion in a later section) because lipases seemed more active in hydrophobic ILs than in hydrophilic ones.6 – 70 in the author’s laboratory proposed that ion kosmotropicity (Hofmeister series) is important in interpreting the enzyme’s behaviors in aqueous IL solutions: kosmotropic anions and chaotropic cations stabilize the enzyme.16 Therefore. Hydrophobicity ‘Hydrophobicity’ may be considered as a narrower concept of ‘polarity’. the viscosities of ILs (i. [BMIM][PF6 ] and [BMIM][BF4 ]) did not affect the initial reaction rates despite their much higher viscosities than toluene.15 The hydrophobicity of ILs is usually quantified by the log P scale. and Candida rugosa lipase) in different ILs under the same water activity (aw ). Basso et al.interscience.Stabilizing and activating enzymes in ionic liquids www. higher enzyme activity was observed in [EMIM][Tf2 N] than in [MTOA][Tf2 N] (MTOA = methyl trioctylammonium) because the former IL (34 mPa s) is less viscous that the latter one (574 mPa s). IL network ILs can form so-called organized ‘nano-structures’ (hydrogenbonded polymeric supramolecules. many enzymatic reactions in ILs are heterogeneous due to the low solubility of enzymes in most pure ILs. IL network theory is not appropriate for understanding these enzymatic reactions. However. the Hofmeister effect may not be suitable for explaining the enzyme’s behavior in these hydrophobic ILs or their mixtures with water. which are similar to water molecules) with polar and non-polar regions in solid.soci. However. they also realized that the enzyme stability was in agreement with the hydrophobicity of ILs: both enzymes were more stable in hydrophobic ILs than in hydrophilic ones (see more discussion in a later section). However. Irimescu and Kato61 carried out the CALB-catalyzed enantioselective acylation of 1-phenylethylamine with 4-pentenoic acid. internal and external mass-transfer limitations should be considered.63.83 achieved higher transesterification activities of lipases in [BMIM][PF6 ] than in [BMIM][BF4 ]. 85: 891–907 Viscosity ILs are more viscous fluids than conventional organic solvents. van Rantwijk and Sheldon16 explained that the high viscosity of ILs slows down the conformational changes of proteins. and how the IL hydrophobicity may influence the kosmotropicity. viscosity and impurity.org that the decreasing stability was.78 explained that the higher enantioselectivity of lipase in [BMIM][Tf2 N] at low water activities (aw < 0.70 and denatures enzymes when dissolved in aqueous solutions as Na+ or K+ salt (more denaturing than BF4 − and MeSO4 − for mushroom tyrosinase76 ). followed by OTf− and BF4 − based ILs. This suggests higher anion nucleophilicity correlating with higher enzymatic activity.wiley. it is practically important to separate ‘hydrophobicity’ from ‘polarity’ because the former is often related to the miscibility with water. For example. Nara et al. On the other hand. and thus the solubility and degree of ion dissociation of ILs in water are limited.71 correlated the activity of cytochrome c in ILs containing 20% (wt) water with the kosmotropicity of component ions.64 Dupont64 suggested that the aqueous solution of free enzymes might be embedded in the IL network. (2) the lower solubility of substrates in the IL than in MTBE may cause a lower activation energy in the IL. [BMIM][NO3 ] (log P = −2. J Chem Technol Biotechnol 2010.73. the activity of α-chymotrypsin also depended on the IL viscosity. as a result. The above preliminary studies have shown that the kosmotropic effect of ILs on enzymes may be applicable to diluted aqueous solutions of ILs. However. PF6 − is a chaotropic anion.0) of several ILs.53) than in methyl tertbutyl ether (MTBE) was for two reasons: (1) the higher viscosity of IL (52 mPa s) than that of MTBE (0. or even in the gas phase. which may be explained by the chaotropic nature of BF4 − in solutions. Russell’s group48 measured the log P values (<−2. but is not the primary factor in controlling the enzyme’s activity. Fujita et al.80 – 82 they also observed that free lipase (Candida rugosa) was only active in hydrophobic [BMIM][PF6 ] (log P = −2.e.74 An excellent review by Yang75 systematically discussed the possible mechanisms of Hofmeister effects of ILs on the enzyme activity and stability. However. in another study. The Goto group also reported higher activities of PEG-modified lipase84 and subtilisin85 in more hydrophobic ILs c 2010 Society of Chemical Industry  www. and suggested that they are very hydrophilic in nature based on Laane’s scale. Eckstein et al. Several studies11. However.77).46 indicated that besides the IL polarity. while chaotropic anions and kosmotropic cations destabilize it.66.12 Lozano et al. and found that the reaction rate relied on the type of IL anions (reaction rate in a decreasing order of OTf− > BF4 − > PF6 − . Rhizomucor miehei lipase. PF6 − based ILs (such as [BMIM][PF6 ]) are hydrophobic. a contradictory result was reported. it is also known that PF6 − based ILs are typically enzyme stabilizing. CALB is known to be active in the absence of water59.65 ).74 reported low/no activities of β-glycosidase in aqueous solutions of [BMIM][BF4 ].

101 In general. The first category includes the enzyme immobilization (on solid support. enzyme/protein-coated micro-crystals.57 c 2010 Society of Chemical Industry  J Chem Technol Biotechnol 2010.99.86 reported low penicillin acylase stabilities in [BMIM][BF4 ] and [BMIM][dca]. rinsing with n-propanol methods (PREP and EPRP).56 found that the initial rates of Novozym 435-catalyzed ammonolysis of (R. [BMIM][PF6 ] > [BMIM][dca].92 resulting in lower conversion of the substrate. resulting in enzyme inactivation. a higher log P of the solvent also means a more thermodynamic groundstate stabilization of substrates.‡ however. interact with enzymes through two major mechanisms: (1) electrostatic interactions of the surfactant head group and charged amino acid residues of the protein. and then declined with further increase in hydrophobicity.100 Ionic surfactants. but do not inactivate the lipase (more discussion in a later section). although such a correlation is lacking for enzymes in IL systems. the stability is in decreasing order [HMIM][PF6 ] > [HMIM][Tf2 N] > [HMIM][BF4 ]. and the enzyme’s activities increased with the cation’s hydrophobicity (EMIM+ < BMIM+ < HMIM+ < OMIM+ ). A very recent study40 on the alcoholysis of vinyl butyrate and 1-butanol by free CALB suggested that the lipase activities were generally much lower in water-miscible ILs (such as BF4 − .59 For example.38 Currently. The V´ıllora group89 observed a lower stability of penicillin G acylase in [BMIM][BF4 ] than in hydrophobic ILs (Tf2 N− and PF6 − ). However.org such as [EMIM][Tf2 N].2 mg mL−1 Candida antarctica lipase B (CALB) and retain its catalytic capability. the use of additives in ILs. lactate.25. Hern´andez-Fern´andez et al. particularly in the absence of substrate.26 Therefore. ‡ There are also some exceptions. choline dihydrogen phosphate (m. Shen et al. BF4 − based ILs are hydrophilic but do not dissolve the enzyme. This could explain the decreasing reaction rate in very hydrophobic ILs.7. sol–gel. on the other hand. and [OMIM][PF6 ] > [HMIM][PF6 ] > [BMIM][PF6 ]. most of them tend to strongly interact with proteins (such as via H-bonding).) than in water-immiscible ones (PF6 − and Tf2 N− ). or show some activating/stabilizing effects on enzymes. physical or covalent attachment to PEG. there are only a few pure ILs that are known to dissolve enzymes but do not inactivate them. and CH3 COO− ) may dissolve some enzymes. ionic surfactants are considered non-specific denaturants of enzymes. in the case of PGA.88 reported that cellulase activity decreased in the order of IL hydrophobicity: [BMIM][PF6 ] > [BMIM][BF4 ] > [BMIM]Cl. the hydrophobic cations showed an adverse effect on PGA stability: [EMIM][Tf2 N] > [BMIM][Tf2 N].57. Similarly.25.soci. the enzyme dissolution in ILs is not a definite indication of enzyme denaturation. while methods in the second category intend to minimize the denaturing properties of some ILs (such as reducing the anion’s nucleophilicity and H-bonding basicity). some studies reported relatively high enzyme activities in hydrophilic ILs (such as [BMIM][BF4 ].wiley. IL coating. EtSO4 − . OAc− . the stability is in a decreasing order of [BMIM][Tf2 N] > [BMIM][PF6 ] > [BMIM][BF4 ]. given the complexity of an enzyme’s catalytic properties in ILs. it was observed59 that the lipase activity increased with the log P value to a maximum. In summary. Lou and Zong87 studied the enantioselective acylation of (R.S)-1-trimethylsilylethanol with vinyl acetate catalyzed by lipases in several ILs.com/jctb H Zhao [BMIM]Cl. Zhang et al.60 concluded that both free CALB and penicillin G acylase (PGA) are more stable in hydrophobic ILs than in hydrophilic ones: in the case of CALB. The following is a tutorial discussion of some representative methods (typical examples were summarized in Table 1).93 – 96 Therefore.97 triethylmethylammonium methyl sulfate ([Et3 MeN][MeSO4 ]) was reported able to dissolve >1. Through a systematic investigation of lipasecatalyzed transesterification in over 20 ILs. 894 Enzyme dissolution Hydrophobic ILs (typically consist of PF6 − and Tf2 N− ) do not dissolve appreciable amounts of enzymes. STABILIZATION AND ACTIVATION METHODS Overall. but poor enantioselectivities (<5% eep ) in hydrophilic [HMIM][BF4 ] and [HMIM]Cl.www.interscience. Lee et al.101 – 103 The knowledge of surfactant–protein interactions can be useful for understanding the IL effect on enzyme activity and stability in some systems (both aqueous and non-aqueous). there is no universal method that can solve all enzyme inactivation issues.62 reported that Novozym 435 was more thermally stable in hydrophobic ILs than in hydrophilic ones following the order [BMIM][Tf2 N] > [BMIM][PF6 ] > [BMIM][OTf] > [BMIM][BF4 ] > [BMIM][SbF6 ]. The second category includes water-in-IL microemulsions. but many studies also reported that they either have no effect on enzymes. hydrophilic ILs (such as those based on NO3 − . [EMIM][BF4 ].90 also found Novozym 435 was less active and stable in [BMIM][BF4 ] than in other hydrophobic ILs. and then decreased with further increase in log P (a bell shape).71.57. Ha et al. For example. Amano lipase PS showed a high enantioselectivity (80% eep ) in hydrophobic [OMIM][PF6 ]. dca− . Contradictorily. a number of methods have been adopted or developed to improve the enzyme’s stability and to increase its activity and enantioselectivity. Surfactant effect ILs bearing long alkyl chains in their cations often form selfassembly in aqueous solutions and behave like surfactants. three-phase partitioning (TPP). and indicated that the activity.45. and the design of enzyme-compatible ionic media. multiple factors must be considered in explaining the enzymatic systems like these. 119 ◦ C) containing 20% (wt) water was found to solubilize and stabilize cytochrome c (cyt c). cellulase was dissolved but inactivated in concentrated solutions of www. and lyophilization with cyclodextrins. [BMIM][OTf] and [MMIM][MeSO4 ]). For example. or CLEA).38 – 40. Paljevac et al. NO3 − . These examples implied that the high hydrophobicity (large log P) of ILs could be beneficial to enzyme stabilization. and [BMIM][PF6 ] > [OMIM][PF6 ]. However. methods to stabilize and activate enzymes in ILs may be divided into two general categories: modification of enzymes. and modification of solvent environments. Therefore. enantioselectivity and thermostability of Novozym 435 increased with IL hydrophobicity in the order ([BMIM][PF6 ] > [OMIM][BF4 ] > [C7 MIM][BF4 ] > [HMIM][BF4 ] > [C5 MIM][BF4 ] > [BMIM][BF4 ]).S)-p-hydroxyphenylglycine methyl ester increased with the hydrophobicity of BF4 − based ILs to a maximum. 85: 891–907 .91 noticed that during the kinetic resolution of racemic cyanohydrins. The following sections will address some major advances in these methods with representative examples.p. and (2) hydrophobic interactions between the alkyl chains of the surfactant and hydrophobic amino acid residues. etc. such as sodium n-dodecyl sulfate (SDS) and n-dodecyltrimethylammonium bromide (DTABr). Methods in the first category make the enzyme more tolerant to those denaturing factors of ILs discussed earlier. Lou et al.98 Recently the author’s Laboratory synthesized a series of ether-functionalized ILs that are able to dissolve enzymes (>5 mg mL−1 CALB at 50 ◦ C) and a variety of substrates.

(2) transesterification between vinyl acetate and 2-phenyl-1-propanol (1) transesterification of ethyl butyrate and 1-butanol. PF6 − and Tf2 N− 1-butanol sol–gel immobilization of lipase PS from Burkholderia cepacia [EMIM][BF4 ]. 000–35. (2) hydrolysis of N-succinyl-L-ala-L-alaL-pro-L-phe-pnitroanilide (1) transesterification of ethyl butyrate and 1-butanol. 157. 85: 891–907 c 2010 Society of Chemical Industry  www. (2) alcoholysis of (±) -1-phenylethanol and vinyl acetate esterification of oleic acid and 1-butanol aqueous buffer oxidation of guaiacol by H2 O2 hydrolysis reaction in phosphate buffer.interscience. 130 131.com/jctb . (3) resolution 1-phenylethylamine using methyl methoxyacetate physical adsorption of lipases on PEG (average molecular weight Mn = 4. The immobilized lipase was more active than the free form. as well as a comparable or higher enantioselectivity for the enzyme complex than the free lipase. Methods for stabilizing and activating enzymes in ILs with selected examples Method Immobilization PEG modification Enzyme preparation Reaction solvent Reaction lipase B from Candida antarctica immobilized on acrylic resin. 108 The immobilized lipase showed a higher activity and enantioselectivity in IL than its free enzyme. [BMIM][PF6 ] cross-linked enzyme aggregates (CLEAs ) Candida antarctica lipase B (CALB-CL) and CLEA on a polypropylene carrier (CLEA-PP) [BMIM][dca] [BMIM][NO3 ] [BMIM][OAc] [BMIM][lactate] (1) transesterification between ethyl butyrate and 1-butanol.000) [BMIM][PF6 ] [HMIM][PF6 ] [OMIM][PF6 ] [BMIM][Tf2 N] (1) alcoholysis between vinyl cinnamate and benzyl alcohol. CALB-PP also showed high enantioselectivities in two resolution reactions. [EMIM][Tf2 N]. (2) transesterification of ethyl octanoate and alcohols.132 Lower initial activity but higher enzyme stability than the CLEA preparation CALB-PP exhibited considerably higher catalytic activities in denaturing ILs than CALB-CL and free CALB. (3) ammoniolysis of ethyl octanoate and ammonia (4) epoxidation of cyclohexene (1) transesterification of N-acetyl-Lphenylalanine ethyl ester and 1-propanol.org Table 1. 93 Enzyme-SWNT conjugates exhibited higher catalytic turnover and higher thermal stability than free form.Stabilizing and activating enzymes in ionic liquids www. 112 Enhanced activity and thermal stability was observed for IL-sol–gel enzyme. known as Novozym 435 [BMIM][BF4 ] [BMIM][PF6 ] proteinase K immobilized onto single-walled carbon nanotubes (SWNTs) [BMIM][BF4 ] [BMIM][OTf] [BMIM][PF6 ] [OMIM][PF6 ] [HMIM][PF6 ] Candida rugosa lipase adsorbed on multi-walled carbon nanotubes (MWNTs) [BMIM][PF6 ] (and hexane) Candida rugosa lipase immobilized on magnetic nanoparticles supported ILs sol–gel encapsulation of horseradish peroxidase using [BMIM][BF4 ] sol–gel encapsulation of Candida rugosa lipase using various ILs based on Cl− . 134 They observed a higher enzyme activity (as high as 14-fold) for PEG-lipase complex comparing with the free form. BF4 − . and synthetic reaction in n-hexane (1) hydrolysis of p-nitrophenyl butyrate in phosphate buffer. (2) resolution of 1-phenylethanol using vinyl acetate.158 39 895 J Chem Technol Biotechnol 2010. Greater hydrolytic and synthetic activities of IL-sol–gel encapsulated lipase were observed than that without ILs. 109 The immobilized exhibited a higher catalytic activity and thermal stability. (2) transesterification of vinyl acetate and benzyl alcohol in n-hexane acylations of secondary alcohols with vinyl acetate Comments Ref Reaction rates were comparable with or higher than those in organic solvents.wiley.soci.

85.163 Enzyme precipitated and rinsed with n-propanol(EPRP) EPRP of subtilisin and α-chymotrypsin [BMIM][PF6 ] [BMIM][BF4 ] (and n-octane) (1) esterification of N-acetylphenylalanine with ethanol. (2) horseradish peroxidase-catalyzed oxidation of pyrogallol 171.interscience. (2) transesterification of 2-phenyl-1-propanol with vinyl acetate. (Continued) Method Enzyme preparation Reaction solvent Reaction Comments − Ref covalently conjugating PM13 with subtilisin Carlsberg85. Higher operational stability of lipases was observed in water-in-IL microemulsions.com/jctb c 2010 Society of Chemical Industry  J Chem Technol Biotechnol 2010. PM13 -subtilisin and PM13 -lipase are soluble and exhibited much higher activity and stability than free enzymes. 175 178.179 47 182–185 896 www. FT-IR and CD spectroscopy confirmed that the lipase in water-in-IL microemulsions usually maintain its native conformation or adapt a more rigid structure.172 lipases from Candida rugosa. (3) transesterification of N-acetyl-Lphenylalanine ethyl ester with 1-butanol In Tf2 N based ILs.soci.170 Water-in-IL microemulsions lipase PS or horseradish peroxidase in water-in-[OMIM][Tf2 N] microemulsions waterin-[OMIM][Tf2 N] microemulsions using the anionic surfactant AOT (1) lipase-catalyzed hydrolysis of p-nitrophenyl butyrate. 84. (2) transesterification of N-acetyl-Lphenylalanine ethyl ester with n-propanol The EPRP of subtilisin and α-chymotrypsin exhibited very high catalytic activity comparing with pH-tuned lyophilized powder. (2) transesterification of (±) -3hydroxypentanenitrile and vinyl acetate. 85: 891–907 . The IL-coated lipases showed high enantioselectivity and extremely high reaction rates for several resolution reactions (up to 500to 1000-fold acceleration for some substrates). (3) transesterification of other secondary alcohols and vinyl acetate.www. (2) hydrolysis of p-nitrophenyl butyrate The lipase PS and horseradish peroxidase showed higher activities in water-in-IL microemulsions than in water-saturated IL and in water-in-isooctane microemulsions. 177 lipases coated with an imidazolium IL carrying anions of cetyl-PEG10-sulfate Novozym 435 beads coated by various ILs solvent free Novozym 435 Lipozyme RM IM Lipozyme TL IM lipase PS-D lipase AK20 tetraammoniumbased ILs such as Ammoeng 100 and 102 transesterification of various alcohols with vinyl acetate at room temperature (1) transesterification of 1-phenylethanol and vinyl acetate. High enzyme activity and triglyceride conversions were observed.org H Zhao Table 1.163 and Candida rugosa lipase. 168. (4) esterification of 2-(4-ethylphenoxy) propionic acid and 1-butanol esterification of methylα-D-glucopyranoside with fatty acids glycerolysis of triglycerides and glycerol Coating enzymes with ILs Higher conversions and enzymatic activities were detected.84 [EMIM][Tf2 N] [C2 OC1 MIM][Tf2 N] [C2 OHMIM][Tf2 N] (1) hydrolysis of p-nitrophenyl butyrate in tris-HCl buffer. Chromobacterium viscosum and Thermomyces lanuginosa water in [BMIM][PF6 ] microemulsions using non-ionic surfactants (Tween 20 or Triton X-100) (1) esterification of natural fatty acids with various aliphatic alcohols.wiley. Pseudomonas cepacia lipase coated by [PPMIM][PF6 ] toluene diisopropyl ether toluene hexane High enantioselectivity and activity were reported for this enzyme preparation.

85: 891–907 25–27 heme-containing proteins/enzymes (myoglobin. the immobilized enzyme exhibited greater catalytic activity towards the oxidation of guaiacol with hydrogen peroxide in 1 : 1 (v/v) mixture of water and 3-methylpyrazolium tetrafluoroborate than in water. this report will not replicate the work. and horseradish peroxidase) onto SWNTs coated with ILs (such as [BMIM][BF4 ] or [BMIM][PF6 ]). (3) transesterification of D-glucose. one of which is carbon nanotubes. One main reason is that the immobilization techniques (both physical adsorption and covalent attachment) are straightforward and easy to accomplish via regular laboratory procedures. A recent comprehensive review16 has summarized many of these examples. Modifying the enzymes Enzyme immobilization The most common method for enzyme stabilization and activation in ILs is the use of immobilized entities instead of free forms. which can be synthesized through the polymerization of 1-vinyl-3-ethylimidazolium bromide using the crosslinker N. (4) regioselective transesterification of cellulose.196 . myoglobin. Very recently. The binding to a solid support is a routine method for improving the enzyme stability in conventional organic solvents as well as in ILs. (6) lipase-catalyzed synthesis of glucose fatty acid esters.108 Similarly.N -methylenebis(acrylamide) in a concentrated water-in-oil emulsion. therefore. (Continued) Method Manipulation/design of IL-structures Enzyme preparation Reaction solvent Reaction Comments Ref alcohol dehydrogenases (ADHs) aqueous solutions of Ammoeng 110 reduction acetophenone in 10% (wt/wt) IL solution 191 lipase PS [MEBu3 P][Tf2 N] Novozym 435 free CALB ether-functionalized ILs based on acetate transesterification of (E) 4-phenylbut-3-en-2-ol and vinyl acetate (1) transesterification of sorbic acid and sorbate ester with n-propanol 25. In addition.112 New nanoparticles formed from cross-linked fluoroalkyl end-capped acrylic acid cooligomer containing poly(oxyethylene) units were shown to be effective in encapsulating cytochrome c (cyt-c). fatty acid.110 immobilized 23 . The reaction rate in IL was superior to that in diisopropyl ether.com/jctb 897 a) Immobilization on a solid carrier. Another new carrier developed by the Goto group111 is polymerized ionic liquid microparticles (pIL-MP).113 As an interesting application in electrocatalysis.107 The high surface area and unique nanoscopic dimensions of carbon nanotubes enable high protein loading and low mass-transfer resistance. However. sol–gel encapsulation.109 Du et al.org Table 1.soci. the enzyme-SWNT conjugates were well dispersed in ILs. ascorbic acid. it is interesting to highlight some new carriers for enzyme immobilization. or catalase) in agarose hydrogel films. cytochrome c. glucose. (7) transesterification of triglycerides and methanol The use of IL as a co-solvent enhanced the solubility of hydrophobic substrates. and protein cross-linking (a carrier-free technique). (5) enzymatic synthesis of methyl-phthalate of betulinic acid. another new enzyme support named ‘magnetic nanoparticles supported ILs’ was constructed by covalent bonding of ILs-silane on magnetic silica nanoparticles. These immobilized methods generally fall into three categories: binding to a solid carrier. and stabilized the ADH. the Pang group114 – 116 designed new glassy carbon electrodes by entrapping heme proteins (hemoglobin. amino acids. and many immobilized enzymes are commercially available (such as lipase B from Candida antarctica immobilized on acrylic resin. horseradish peroxidase (HRP. (2) transesterification of ethyl butyrate and 1-butanol. known as Novozym 435). chemically modified with comb-shaped polyethylene glycol) encapsulated in this carrier was more than twice as active in aqueous solutions than that encapsulated in polyacrylamide microparticles. The noncovalent binding of proteinase K onto single-walled carbon nanotubes (SWNTs) resulted in higher enzyme activity and higher thermal stability than its native form in ILs. and triglycerides).wiley. Candida rugosa lipase immobilized on this support showed higher catalytic activity and thermal stability.Stabilizing and activating enzymes in ionic liquids www. and observed that the encapsulated enzymes retained their bioelectrocatalytic activities toward the reduction of oxygen and hydrogen peroxide. Candida rugosa lipase adsorbed on multi-walled carbon nanotubes (MWNTs) displayed a higher transesterification activity and enantioselectivity than the pHtuned free form in [BMIM][PF6 ].interscience.104 – 106 J Chem Technol Biotechnol 2010. betulinic acid. and observed high electrocatalytic activities of the heme protein–agarose films in both [BMIM][PF6 ] and c 2010 Society of Chemical Industry  www. The ether-functionalized ILs could dissolve a variety of substrates (such as sugars. CALB (particularly the immobilized form) exhibited high catalytic properties in these ILs.

85: 891–907 . the subsequent cross-linking condensation forms a SiO2 matrix to encapsulate the biomolecules. gel shrinkage and pore collapse have been major drawbacks of this method. improved enzyme activity. and N-methylglycine) have been added to reduce the gel shrinkage and adjust the protein hydration. As an early version of carrier-free immobilization. and obtained higher enzyme stability in [EMIM][BF4 ]. [BMIM][NO3 ].39 immobilized CALB through two methods: a CLEA method without a solid support (resulting in CALB-CL). The use of CLEAs has such advantages as ease of preparation and recycling. amino acids.com/jctb H Zhao c) Cross-linked enzyme aggregates (CLEAs ). However. [EMIM][Tf2 N] and [BMIM][PF6 ] than the enzyme preparation via the cross-linked enzyme aggregates (CLEAs) method (see next section).119 This technique involves the acid.140 This technique offers many advantages such as enhanced thermal. and resolution of 1-phenylethylamine in [BMIM][NO3 ] and [BMIM][dca]).0 µmol L−1 with a detection limit of 0. there is an increasing interest in utilizing ILs as additives124 for protein/enzyme sol–gel immobilization. Toral et al. high stability and selectivity in organic solvents.128 An independent study by the Deng group129 further confirmed that anions of ILs had a strong impact on the pore structures of silica-gel materials. silica xerogels were prepared with various morphologies via the sol–gel method in the presence of ether-functionalized ILs (such as 1-triethylene glycol monomethyl ether-3-methylimidazolium methanesulfonate). d) Enzyme immobilization with ILs in biosensors/electrodes. CALB-PP exhibited considerably higher catalytic activities in denaturing ILs than CALB-CL and free CALB.0 to 6.121 The structural rigidity of the sol–gel matrix protects the integrity of encapsulated enzyme molecules and prevents their leaching. Several groups have clearly demonstrated the advantages of enzyme encapsulation in IL sol–gel hybrid carrier. This method involves the addition of salts. sodium alginate.or base-catalyzed hydrolysis of tetraalkoxysilanes. the use of ILs as solvents was not demonstrated. This is because room-temperature ILs are non-volatile. the cross-linked enzymes (CLEs) can be created by reacting glutaraldehyde with NH2 groups on the protein surface.5 µmol L−1 at a signal/noise ratio of 3. and graphite.132 examined a variety of ILs and their mixtures as additives during the sol–gel immobilization of Candida rugosa lipase.151 A disposable biosensor was constructed from the composite material based on N-butylpyridinium hexafluorophosphate. www. in aqueous solutions. A more recent development in cross-linking enzymes has been pioneered by the Sheldon group. Earlier studies125.133 encapsulated subtilisin in sol–gel derived silica glasses using alkoxysilane precursors carrying different chain lengths.4 mmol L−1 ).interscience.www. the preparation of CLECs requires laborious purification and crystallization of enzymes. the novel biosensor showed good electrochemical response to glucose and high enzyme compatibility.104. [BMIM][OAc] and [BMIM][lactate]). with the potential inclusion of metal catalysts. these ILs act as both morphology controller and acid pre-catalyst. different additives (such as sugars. enzymes and antibodies. mechanical and pH stability. and high activity toward peptide synthesis in an IL ([BMIM][PF6 ]).1 µmol L−1 . It is a relatively simple method (see protocols117. and has been widely used for entrapping a variety of biological molecules such as proteins. or organic solvents (such as hexane). and ease of recycling. the enzyme aggregates are then cross-linked by glutaraldehyde. the solvents used in the above assay reactions catalyzed by IL sol–gel immobilized enzymes were either aqueous solutions. CALB-PP also showed high enantioselectivities in two resolution reactions (resolution of 1-phenylethanol in [BMIM][NO3 ]. The native structures of heme proteins were retained in agarose film as indicated by UV-visible and Fourier transform infrared (FT-IR) spectroscopy.137 At the same time.wiley.123 To overcome these hurdles.120. and adsorbed and cross-linked on a polypropylene carrier (resulting in CALB-PP). Shah and Gupta148 observed higher enzymatic activity of Burkholderia cepacia lipase by the CLEA preparation than the pH-tuned lyophilized free enzyme in [BMIM][PF6 ]. immobilizing biocatalysts containing more than one enzyme.123 Recently. More recently.139.02 to 0. this new biosensor could detect H2 O2 in a linear calibration range of 1. such as tetraethyl orthosilicate (TEOS) or tetramethyl orthosilicate (TMOS).138 eventually leading to a commercial immobilization technology called cross-linked enzyme crystals (CLECs or CLCs). The Li group149 developed an amperometric biosensor by immobilizing horseradish peroxidase on [BMIM][BF4 ]-based sol–gel. The Koo group131.137 However. after optimization.118 ). organic solvents or non-ionic polymers to precipitate the enzyme as physical aggregates from aqueous solutions. a wide linear range (0. and to further increase the activity and stability of enzymes. intermolecular cross-linking of these biomolecules often results in low enzyme activity.136 However. although the cross-linked lipase was not active in some ILs (such as [BMIM][OAc] and [BMIM][lactate]). the mesoporous structures and high pore volume of sol–gel polymer afford the free diffusion of small substrate molecules and their effective interactions with the enzyme. The actively pursued encapsulation method for the enzyme stabilization in ILs is the sol–gel technology. poor reproducibility and low mechanical stability. as well as a high sensitivity and stability. a low detection limit (3. designable particle sizes.5 µmol L−1 ).105. The Li group130 observed improved activity and thermal stability of horseradish peroxidase after immobilizing in [BMIM][BF4 ] based sol–gel.127.org [BMIM][BF4 ] solutions containing a small amount of water. They carried out several reactions using these two enzyme preparations in denaturing ILs (such as [BMIM][dca].137.141 so called crosslinked enzyme aggregates (CLEAs ).soci.26 mmol L−1 and a low detection sensitivity of 1. the cross-linking of a crystalline enzyme via glutaraldehyde was reported.01–7.152 Another peroxidase biosensor was assembled by mixing [BMIM][Tf2 N] c 2010 Society of Chemical Industry  J Chem Technol Biotechnol 2010. and achieved greater hydrolytic and esterification activities of sol–gel encapsulated lipase coated with ILs than that without ILs.122 However. there are other issues such as the alcohol release during the hydrolysis of silicon alkoxide. In addition.150 A glucose biosensor was fabricated by immobilizing glucose oxidase in thin films of polyethylenimine-functionalized IL containing carbon nanotubes and gold nanoparticles.104. etc.135.126 focused on the preparation of mesoporous silica through high dispersion of ILs (such as [BMIM][BF4 ]) in sol–gel. the resulting immobilized enzyme showed enhanced thermal stability. The Kanerva group134 immobilized lipase PS from Burkholderia cepacia in a sol–gel.104 – 106. Sangeetha et al. the biosensor exhibited high stability and sensitivity with linear calibration ranging from 0. and are tunable to be enzyme-compatible. in addition. During the transesterification between ethyl butyrate and 1-butanol. the new biosensor exhibited a short response time (2 s). Encapsulation is a technique for entrapping biomolecules in a polymer matrix via non-covalent interactions between the network and the biomolecule. 898 b) Sol–gel encapsulation. thermally stable. An electrochemical H2 O2 biosensor was constructed by entrapping horseradish peroxidase in [BMIM][BF4 ] doped DNA network casting on a gold electrode.142 – 147 Higher enzyme stability is expected for CLEAs in ILs than free enzymes.

155 designed a glucose microsensor from a composite paste of mesoporous carbon. The EPRP of α-chymotrypsin showed two orders of magnitude higher esterification activity than lyophilized powder in n-octane. As shown in Fig.com/jctb 899 PEG-modification The modification of enzymes with poly(ethylene glycol) (PEG) through either physical complexation or covalent interaction is a routine method for enzyme stabilization in denaturing environments. PEG p-nitrophenyl carbonate is commonly used to connect PEG units with amino residues of proteins to form stable carbamates. Shah and Gupta148 conducted the enzymatic resolution of 1-phenylethanol in [BMIM][PF6 ] catalyzed by various lipase preparations. By strict definition.158 applied PEG with different molecular weights (average Mn = 4000–35 000) as the enzymecoating amphiphile for the preparation of PEG–lipase complexes. the Goto group171 reported the use of water-in-IL microemulsions as a new medium for dissolving various enzymes and proteins (such as lipase c 2010 Society of Chemical Industry  www.84 respectively. The surfactant formation of micelles in ILs is being actively studied and has been reviewed.soci.166 The PREPs of subtilisin Carlsberg.. PM13 . Kaar et al. and H .166 α-chymotrypsin. The second strategy for associating enzymes with PEG is through covalent attachment.158 Turner et al.166 and papain167 have shown very high enzymatic activities in organic media (such as acetonitrile and t-butanol). The most common PEGmodification is achieved through physical adsorption because of its simple procedures. The following several methods. which minimizes protein denaturation and keeps the majority of enzyme molecules in an active conformation. PEG-modification is one type of enzyme immobilization method.164 The PM13 -lipase complex showed a higher activity and stability in benzene than its free form.170 In another study.160. and demonstrated its high sensitivity over a concentration range from 10. PEG-modification of cytochrome c (cyt.169 The EPRP technique is a combination of several methods: enzyme precipitation by alcohols. and was more enantioselective than pH-tuned lyophilized CRL and PREP preparation.48 used PEG-monoisocyanate to link PEG with lysine residues of the protein to form a PEG–lipase complex. and the use of salt tuning during co-precipitation.. mild conditions.154 Sun et al.157. J Chem Technol Biotechnol 2010. They investigated the PEG–lipases in several alcoholysis reactions in hydrophobic ILs (PF6 − and Tf2 N− ).148 Modifying the solvent environments Water-in-IL microemulsions The above several methods focus on the modification of enzymes to improve their adaptability in ionic environments. On the other hand. pH-tuned lyophilized subtilisin showed no activity in [BMIM][BF4 ]. the preparation could be cumbersome. they observed that the EPRP of Candida rugosa lipase (CRL) was more active than pH-tuned lyophilized CRL and CLEA preparation. PM13 is a copolymer of PEG derivatives with maleic anhydride (molecular weight ∼15.org with the immobilized peroxidase (tissue from pine nuts) onto chitosan crosslinked with citrate. On the other hand. which was further developed into a sensitive biosensor for glucose through the inclusion of glucose oxidase. rinsing the precipitate with 1-propanol.162 Meanwhile. which is achieved through repeatedly rinsing the silica-immobilized enzyme with dry 1-propanol. Ltd. showed a high catalytic activity and storage stability in these ionic solvents.e. and [MMEP][OAc] compared with the free form.163 and Candida rugosa lipase. Japan) for covalently conjugating PEG with subtilisin Carlsberg85. however.168 The EPRP of subtilisin exhibited >4000 times higher initial rate (for transesterification) than pH-tuned lyophilized powder in [BMIM][PF6 ].interscience.158 as well as comparable or higher enantioselectivity of the enzyme complex than that of free lipase. and different enzymes involved.38 reported higher enzyme activity of PEG–cellulase complex (PEG average Mn = 1000) in aqueous solution and [BMIM]Cl solutions than that of free cellulase. A further development of PREP is a preparation method called ‘enzyme precipitated and rinsed with n-propanol’ (EPRP). 85: 891–907 C CH2 CH2 O CH C O O(C2H4O)33CH3 CH C O 8 Figure 2.0 to 80.85. These biosensor examples suggest that various immobilized enzymes have a high compatibility with ILs.153 A new composite electrode was constructed using multiwall carbon nanotubes (MWCNT) and the IL n-octylpyridinium hexafluorophosphate. 2. only small amounts of water may be added during the reaction to activate catalysis. this complex showed no improvement in lipase activity in [BMIM][PF6 ].166 The principle of this technique is that 1propanol removes water from protein. glucose oxidase and [BMIM][PF6 ]. toluene and chlorinated hydrocarbons)156 and ILs. enzyme precipitated and rinsed with acetone (EPRA) and acetone-rinsed enzyme preparation (AREP)) resulted in high enzyme activity and enantioselectivity. the Goto group adopted an unusual comb-shaped PEG. For example. the PEG-modified α-chymotrypsin (PEG average Mn = 1000) via physical adsorption showed no or marginal increase in the reaction rate in PF6 − based ILs than its free form.0 µmol L−1 .c) allowed the protein to become soluble in [EMIM][Tf2 N] without denaturation.168.100 Recently. on the other hand. intend to change the ionic media to improve their enzyme compatibility. PEG has both hydrophilic and hydrophobic properties. [BMIM][NO3 ]. whereas the EPRP gave a moderately high initial rate in this IL. PM13 -subtilisin is soluble and exhibited much higher transesterification activity and stability than its free enzyme. For example. modified enzymes become soluble in some organic solvents (such as benzene. (SUNBRIGHT AM-1510K from NOF Co. and the unchanged protein structures. and the enzyme catalytic properties may vary in different immobilization batches. 000). the acid anhydrides react with amino groups of the enzyme.Stabilizing and activating enzymes in ionic liquids www. The Goto group157. different PEG molecular weights. but two alternative formulations (i. and observed higher enzyme activity (as high as 14-fold) of the PEG–lipase complex than that of the free form.wiley. PM13 -lipase is also soluble in Tf2 N− based ILs.84 However. Enzyme precipitated and rinsed with 1-propanol (EPRP) The propanol rinsed enzyme preparation (PREP) is a method for stabilizing the immobilized enzymes.161 Alternatively. Structure of comb-shaped poly(ethylene glycol) PM13 . therefore.163 Similarly.165 In Tf2 N− based ILs. the EPRP of Burkholderia cepacia lipase (BCL) lost substantial amounts of activity. there are also some disadvantages associated with PEG-modification.85 and show an increased stability in these solvents.159 These differences could be due to a number of factors such as different preparation conditions.

and their combinations) considerably influence the IL physical properties that are crucial for ILenzyme interactions and enzyme stabilization. The operational stability of these lipases in water-in-IL microemulsions was much higher than that in other microheterogeneous media. 1.173 formed the water-in-[BMIM][PF6 ] microemulsions using nonionic surfactant Triton X-100. [BMIM][BF4 ]. each of these ILs is an ionic mixture containing multiple alkyloxy groups.186 – 188 De Diego et al. depending on the solvating environment.180 Lozano et al. Several studies have tailored the IL structures to dictate the compatibility of enzymes. and observed that both lignin peroxidase and laccase were catalytically active in the new medium while little activity was detected in pure [BMIM][PF6 ] or water-saturated [BMIM][PF6 ]. Xu and co-workers183. [EMIM][Tf2 N]. FT-IR and circular dichroism (CD) spectroscopy further confirmed that the lipase in water-in-IL microemulsions usually maintain their native conformation or adapt a more rigid structure compared with that incubated in other microheterogeneous media. and observed that synthetic activity could be improved up to www. [PPMIM][PF6 ] ([PPMIM] = 1-(31 -phenylpropyl)-3-methylimidazolium). onto lipase from Pseudomonas cepacia. the enzymatic activity rapidly decreased with the increase of IL concentration.81. it is important to customize the structures of ILs for particular biocatalytic applications based on current knowledge of IL structure and enzyme activity relationship. It is interesting to mention that oxygencontaining ILs (such as Ammoeng series.189 have further demonstrated the higher transesterification activities of both free and immobilized CALB in [CPMA][MeSO4 ] than in several PF6 − and BF4 − based ILs. the Stamatis group175 developed water-in-IL microemulsions through using non-ionic surfactants (Tween 20 or Triton X-100) in [BMIM][PF6 ]. Ammoeng 100 (also known as [CPMA][MeSO4 ]‡ ) and 102 are capable of dissolving triglycerides and have shown to be lipase compatible during the glycerolysis reaction. they also found the increase in activity followed the hydrophobicity of ILs ([OMIM][PF6 ] > [HMIM][PF6 ] > [BMIM][PF6 ]).interscience. 4. which resulted in higher conversions in lipase-mediated esterification of methyl-α-D-glucopyranoside with fatty acids than uncoated lipase. while in homogeneous solutions. and less than 1% of these ILs and 10% isopropanol/water were sufficient to improve the activity of PLE (up to four times) as well as the enantioselectivity. and achieved high enantioselectivity and activity from this enzyme preparation. followed by the addition of aqueous buffer to prepare a microemulsion. and [EMIM][BF4 ] respectively. The IL-coated lipase PS is also commercially available. the model also provides guidance in designing the structures of cations and anions. Similarly. 1. IL network. Candida antarctica lipase B. however. Structure of 1-butyl-2. The Pandey group176 developed a visual method for examining the formation of water-in-IL microemulsions: Co (II) salt (CoCl2 ·H2 O) was added into the microemulsions formed by using [BMIM][PF6 ] as the oil phase and nonionic TX-100 as the surfactant.60 for [BMIM][PF6 ].179 prepared an imidazolium IL carrying anions of cetyl-PEG10-sulfate (Fig.www. kosmotropicity.wiley. As shown in Fig.soci. The new medium was created by dissolving anionic surfactant sodium bis(2ethyl-1-hexyl)sulfosuccinate (AOT) in hydrophobic [OMIM][Tf2 N] containing 10% (v/v) 1-hexanol (as a co-surfactant). the structures of ILs (as defined by the types of cations and anions. 85: 891–907 . viscosity and hydrophobicity.to 1000-fold acceleration for some substrates). the other two lipases from Thermomyces lanuginosus (TLL) and Rhizomuncor miehei (RML) seemed less active in [CPMA][MeSO4 ] than in PF6 − and BF4 − based ILs. H-bonding and van der Waals interaction energy) to understand the multiple interactions in ILs. 3).184 trioctylmethylammonium bis(trifluoromethylsulfonyl)imide ([TOMA][Tf2 N]) and its mixture with Ammoeng 102 have also been demonstrated to be suitable solvents for enzymatic glycerolysis.66. the color change can be determined by UV-Visible absorbance spectra. in waterin-[BMIM][PF6 ] microemulsions containing Triton X-100. Mutschler et al.23 Mucor javanicus lipase coated with various ILs was found to be more active and more stable than the untreated lipase in aqueous solution. and the activation factors were 1.185 utilized the conductorlike screening model for real solvents (COSMS-RS) to derive various parameters (such as misfit. The Itoh group178.172 Zhou et al.56 and 1. and found that the reaction in IL microemulsions was much more effective than that in a conventional AOT/water/isooctane microemulsion.org PS. horseradish peroxidase and enhanced green fluorescent protein). The Xu group182 – 187 judiciously selected a group of commercial tetraammonium-based ILs as reaction media for the enzymatic glycerolysis. and the hydrolysis of p-nitrophenyl butyrate. H-bond basicity. Chromobacterium viscosum and Thermomyces lanuginosa in these novel microemulsions were investigated through the esterification of natural fatty acids with various aliphatic alcohols. two-fold by using the IL coating onto lipase. Therefore. and coated it on lipases to stabilize the enzymes in organic solvents (such as diisopropyl ether). The lipase PS showed a higher hydrolytic activity in water-in-IL microemulsions than in water-saturated IL and in water-inisooctane microemulsions. In particular. 5) – is quite effective in forming aqueous two-phase (ATP) for the purification of active enzymes (two different alcohol dehydrogenases). alcohol dehydrogenase from yeast maintained a high catalytic activity. anion nucleophilicity. Co (II) salt was chosen as the probe because of different colors of the hexa-coordinated and tetra-coordinated complexes of the cation. Manipulation/design of IL structures As mentioned previously.192 Based on the lyoprotectant effect of tris(hydroxymethyl) aminomethane (Tris) as excipient in horseradish peroxidase ‡ From the name cocosalkyl pentaethoxy methylammonium methylsulfate.171 The same group optimized the oxidation of pyrogallol in water-in-IL microemulsions catalyzed by horseradish peroxidase.183. α-chymotrypsin. c 2010 Society of Chemical Industry  J Chem Technol Biotechnol 2010. The IL-coated lipases exhibited high enantioselectivity and high reaction rates in several resolution reactions (up to 500. These properties include the polarity. The catalytic properties of lipases from Candida rugosa. and [C2 OHmim]Cl) were used as additives in the enantioselective hydrolysis of diester malonates by pig liver esterase (PLE).com/jctb H Zhao O N + N O S O O 10 n-C15H33 O Figure 3.3-dimethylimidazolium cetyl-PEG10sulfate. which have both hydrophilic and hydrophobic properties like PEG.190 The Kragl group191 found that an IL in the Ammoeng family – Ammoeng 110 (Fig. 900 Coating enzymes with ILs Lee and Kim177 coated an ionic liquid. the IL is capable of stabilizing the enzymes and enhancing the solubility of hydrophobic substrates.174 More recently.47 coated various ILs onto Novozym 435 beads.181 carried out the enzymatic synthesis of citronellyl esters mediated by Novozym 435.

in two ILs under optimal conditions).p.7%. C18 acyl group. they reported that horseradish peroxidase in this new IL was 10 times more active than in methanol and at least 30–240-fold more active than in conventional ILs. Lourenc¸o et al. 6). = 302 ◦ C) and urea (m.soci. Structures of tetraammonium-based ILs (AmmoengR series). OH O CH3CH2 + N H3C CH2CH3 Figure 5. m + n = 4-14 Tallow O + N m Et Et + N OH OH EtSO4HO O O m OH H2PO4- Et n (d) Ammoeng 112 m = 50-60 (c) Ammoeng 102 Tallow. The possible explanation is that the denaturing anion’s molar concentration in [aliq][dca] is much lower than that in [BMIM][dca] due to much higher molar mass of the former IL.26. and achieved high conversion yields (72.com/jctb 901 lyophilization.org Et + N OH MeSO4- OH HO O O m OH CH3COO- Et n (b) Ammoeng 111 m = 50-60 (a) Ammoeng 100 Cocos. Abe et al. but high activity and enantioselectivity in [aliq][dca] (aliq = trioctylmethylammonium (Aliquat 336 )). tetrakis(2-hydroxyethyl)ammonium tri- et al.interscience.p. C18 acyl group. R'. respectively. Recently. so called ‘deep eutectic ILs’ (DEILs). m. Structure of flouromethanesulfonate. n. unavailable Figure 4.5% and 76. m + n = 14-25 O R O + N m R' O MeSO4O R' O n O (e) Ammoeng 120 R.198 – 200 A good example is the mixture of choline chloride (m. and have relatively low viscosities. Structure of Ammoeng n = 5-15 OH n HO Cl CF3SO3OH 110. HO Figure 6.Stabilizing and activating enzymes in ionic liquids O Cocos + N m www.23 synthesized an alkyloxy-containing hydrophobic IL named 2-methoxyethyl(tri-n-butyl)phosphonium bis(trifluoromethane)sulfonamide ([MEBu3 P][Tf2 N]).194 mimicked the structure of Tris and rationally designed a new IL known as tetrakis(2hydroxyethyl)ammonium triflouromethanesulfonate (Fig.wiley.196 Vafiadi J Chem Technol Biotechnol 2010. the Abbott group has demonstrated that a mixture of solid organic salt and a complexing agent can form a liquid at temperatures below 100 ◦ C.198 The major advantage of this approach is that inexpensive and non-toxic compounds can be used and the properties of the liquid can c 2010 Society of Chemical Industry  www. 85: 891–907 + N . C14 alkyl group. These two ILs are considered as amphiphilic (hydrophilic cation and hydrophobic anion). and observed a faster reaction rate (lipase PS-catalyzed transesterification of secondary alcohols) in this IL than in diisopropyl ether.25.197 employed two functionalized ILs [C2 OHmim][PF6 ] and [C5 O2 mim][PF6 ] as solvents for the feruloyl esterase-catalyzed esterification of glycerol with sinapic acid.195 measured little Novozym 435 activity in denaturing [BMIM][dca]. = 133 ◦ C) in a 1 : 2 molar ratio resulting in a room-temperature IL (Tf = 12 ◦ C).193 Das et al.

more systematic studies on these subjects are necessary to explore the full potentials of ILs. phosphonium. pp 289–318 (2003). ACKNOWLEDGEMENTS The Donors of the American Chemical Society Petroleum Research Fund (46776-GB1) are thanked for partial support of this research. 7 Jain N. The H-bond network in DEILs is suspected responsible for reducing the chemical potential of the components of DEILs and making them less reactive. and we may find new opportunities to design more enzyme-compatible solvents. 19 Brennecke JF and Maginn EJ.215 At present. Christensen MW and Kirk O. 3 Seddon KR. Chem Rev 107:2757–2785 (2007). respectively) (n=2. ed. pyrimidinium. ed by Wasserscheid P and Welton T.579. 12 Kragl U.25 – 27. It is important to realize that new ILs based on other cations (such as triazolium.25 It was found that ILs can be designed to dissolve many substrates (such as cellulose. pp 319–335 (2003). pyrazinium. oxazolium.interscience. Chauhan S and Chauhan SMS. other techniques have also been explored to improve the enzyme’s adaptability to ionic media including three-phase partitioning (TPP) of subtilisin. 1&2. Weinheim. Aldrichimica Acta 35:75–83 (2002). This could significantly improve the catalytic efficiency of these reactions as some substrates may not be soluble in conventional organic media.214 and lipase lyophilization with cyclodextrins. and to be able to dissolve a variety of compounds.www. Application of ionic liquids as solvents for polymerization processes. J Chem Technol Biotechnol 80:1089–1096 (2005). Trends Biotechnol 21:131–138 (2003). Sorgedrager MJ. Those ILs that are enzyme-compatible and can dissolve substrates that are not soluble in conventional organic solvents. Appl Catal A: Gen 222:101–117 (2001).203 In addition. these ILs can be optimized to be compatible with CALB. ascorbic acid. 3. these enzyme-compatible ILs have some common structure features: (1) they usually have relatively large molecular structures so that H-bond basicity and nucleophilicity of anions are minimized. Weinheim (2008). many of these methods have not been investigated in-depth in terms of the variety of enzymes and different types of ILs. 2 Houlton S. the author’s group synthesized a series of alkyloxyalkyl-containing ILs based on acetate (Fig. glycerol. Chem Rev 99:2071–2083 (1999). Eckstein M and Kaftzik N. .207 – 212 It will be interesting to expand our scope of media to these new ILs for biocatalysis. and pyrrolidinium salts.25 – 27 Therefore. Madeira Lau R and Sheldon RA. Xia S and Ma P. Ionic liquids – new ‘solutions’ for transition metal catalysis.201 reported that several hydrolases (CALB. and their physical properties have been characterized. Financial support by the Research Fund Grant from Royal Society of Chemistry is also acknowledged. in Ionic Liquids in Synthesis. WileyVCH. AngewChemIntEd 39:3772–3789 (2000).343 (2003). 15 van Rantwijk F. c 2010 Society of Chemical Industry  J Chem Technol Biotechnol 2010. by Wasserscheid P and Welton T.org H 3C O n H3C O N + N OAc- + N(CH2CH3)3 OAcn Figure 7. As shown by discussions above and the illustration in Table 1. Wiley-VCH Verlag. amino acids. Inorganic synthesis. Olivier-Bourbigou H and Wasserscheid P. Na2 CO3 or triethylamine). When being used as co-solvents. Gorke et al. Curr Opin Biotechnol 13:565–571 (2002). 13 Park S and Kazlauskas RJ. the types of ILs used in biocatalysis are usually limited to quaternary alkyl-substituted ammonium. Enzyme catalysed synthesis in ambient temperature ionic liquids.170 the use of additives (such as NaHCO3 . and glycerol was >600-fold less reactive than 1-butanol in CALB-catalyzed transesterifications of butyl valerate. Forestier A. urea. 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