Methods For Stabilizing and Activating Enzymes

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 enzymes 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 enzymes
activity and stability in ILs, major methods being explored include the enzyme immobilization (on solid support, solgel, 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: 891907
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

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.
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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
BF4-
CN
+
N
R2
N
F3C
FeCl4-
O
CF3 S
O
O _ O
F3C S N S CF3
R1
+
N
R1
PF6-
R3
R1
NC
R3
+
N
O
Cl
R2
O
RO S O
O
O
R1O P
OR2
Figure 1. Structures of some typical cations and anions in ILs.
Recently, some ILs have been shown capable of dissolving many

substrates that are normally insoluble in organic solvents, such as
up to 1020% (wt) cellulose, >100 g L1 other carbohydrates
(including -D-glucose, sucrose, lactose, and -cyclodextrin),
ascorbic acid, amino acids, fatty acids and triglycerides.24 27
The dissolution of carbohydrates in ILs has enabled the chemical modifications of these natural products in homogeneous
systems.24,28 34 Since the anions (typically, chloride (Cl ), dicyanamide (dca ), formate (HCOO ) and acetate (OAc )) of these
ILs tend to form strong hydrogen bonds with carbohydrates
for dissolving them,35 37 these ILs are also likely to denature
enzymes,38 40 making it impossible to transform these dissolved
substrates via enzymatic reactions. Therefore, there is a strong
and urgent need to increase the activity and stability of enzymes
in ILs, particularly in those enzyme-inactivating ILs. For these
reasons, many enzyme-stabilizing methods used in conventional
non-aqueous enzymology have been adopted for ionic media,
and some new approaches have been developed for stabilizing
and activating enzymes in ILs. It is the purpose of this review to
survey these methods, and to guide scientists in advancing these
methods to further flourish the biocatalytic applications in ionic
solvents.
MAJOR FACTORS THAT AFFECT ENZYME

ACTIVITY AND STABILITY IN IONIC LIQUIDS
Before the discussion of enzyme-stabilizing/activating methods, it
is important to know what physical properties are most important
to the catalytic behaviors of enzymes in ILs. It is well known that an
enzymes performance in ILs is affected by common factors such
as the water activity, pH, excipients and impurities.41 In addition,
the solvent properties of ILs are often related to the enzymes
activity, specificity and stability as being discussed below.
892
IL polarity15
Based on the solvatochromic studies, ILs were found to be moderately polar being close to lower alcohols42,43 or formamide.44 Park
and Kazlauskas45 observed the trend of lipase (from Pseudomonas
www.interscience.wiley.com/jctb
H Zhao
cepacia) activity increasing with the IL polarity during the acetylation of racemic 1-phenylethanol with vinyl acetate (for example,
the initial reaction rate in less polar [BMIM][PF6 ] is three times
slower than that in more polar [EMIM][BF4 ]). In another study,
lower synthetic activities of -chymotrypsin were also obtained
in less polar ILs.46 During Novozym 435-catalyzed esterification
of methyl--D-glucopyranoside with fatty acids, Mutschler et al.47
observed that the ester conversion increased with IL polarity
when ILs were used as liquid film-coating (under solvent-free
conditions), but decreased with IL polarity when ILs were used as
solvents. However, the IL polarityenzyme activity correlation has
not been established for other enzymatic reactions performed in
ILs.41,48 50
Hydrogen-bond (H-bond) basicity and nucleophilicity
of anions
Hydrogen-bond basicity and nucleophilicity are two different
concepts, but are often closely related. For molecules containing
the same nucleophilic atoms of the same charge, the stronger
base is usually the stronger nucleophile in aprotic solvents.
[BMIM]Cl (1-butyl-3-methylimidazolium chloride) could effectively dissolve cellulose51,52 because chloride ions (as H-acceptors)
interact with the cellulose -OH group and break the H-bonding
network of cellulose.35 As a result, this IL induced the inactivation
of cellulase (from Trichoderma reesei).38 Similarly, Lee et al.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. Based upon
multiple solvation interactions, [BMIM]Cl showed the largest
H-bond basicity among all ILs considered in the study by Anderson et al.,54 and thus could dissolve complex polar molecules
such as cyclodextrins and antibiotics.55 Lou et al.56 reported that
Novozym 435 showed no ammonolysis activity towards (R,S)-phydroxyphenylglycine methyl ester in [BMIM]Br and [BMIM][NO3 ],
implying the denaturing nature of these two ILs. Lau et al.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, which was further triggered by the
H-bonding interaction between lactate anions and peptide chains.
Dicyanamide (dca) based ILs such as [BMIM][dca] are able to
dissolve carbohydrates,24,58 however, [BMIM][dca] is an enzymedenaturing IL25,39,59 possibly due to the H-bond basicity of the
anion. The authors group26 also suggested both free and immobilized CALB in [EMIM][OTf] were about as active as in [BMIM][dca],
which were less active than in other ILs.
Kaar et al.48 observed that the free Candida rugosa lipase
was only active in hydrophobic [BMIM][PF6 ], but inactive in all
hydrophilic ILs based on NO3 , CH3 COO and CF3 COO during the
transesterification of methylmethacrylate with 2-ethyl-1-hexanol.
They indicated that the latter three anions are more nucleophilic
than PF6 , and thus could interact with the enzyme causing the
protein conformational changes. Hernandez-Fernandez et al.60
reported the stability of CALB in ILs to be in the following order:
[HMIM][PF6 ] > [HMIM][Tf2 N] > [HMIM][BF4 ], and [BMIM][PF6 ] >
[BMIM][dca], and the stability of Penicillin G acylase was in a similar
order [BMIM][Tf2 N] > [BMIM][PF6 ] > [BMIM][BF4 ]. They explained
Basicity refers to the ability of a base to accept a proton, and is a matter

of equilibrium. Nucleophilicity of a Lewis base refers to the relative reaction
rate of different nucleophilic reagents towards a common substrate, most
commonly involving formation of a bond to carbon; nucleophilicity is a matter
of kinetics (rate).

Stabilizing and activating enzymes in ionic liquids
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that the decreasing stability was, in general, consistent with the

increasing order of nucleophilicity (PF6 < BF4 < Tf2 N <
dca ), where the more nucleophilic anions tend to interact with the
positively charged sites in enzymes and to modify the enzymes
conformation. On the other hand, 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,
in another study, a contradictory result was reported. Irimescu and
Kato61 carried out the CALB-catalyzed enantioselective acylation
of 1-phenylethylamine with 4-pentenoic acid, and found that the
reaction rate relied on the type of IL anions (reaction rate in
a decreasing order of OTf > BF4 > PF6 , same cations).
This suggests higher anion nucleophilicity correlating with higher
enzymatic activity. In a second acylation reaction of 2-phenyl-1propylamine with 4-pentenoic acid, however, Irimescu and Kato61
observed that PF6 based ILs afforded fastest reaction rates,
followed by OTf and BF4 based ILs. The rather confusing results
may be because the enzymatic reaction is affected by multiple
factors of ILs such as nucleophilicity, hydrophobicity, viscosity
and impurity. Lee et al.62 measured the initial transesterification
rates of three lipases (Novozym 435, Rhizomucor miehei lipase,
and Candida rugosa lipase) in different ILs under the same water
activity (aw ), and observed that the anion effect on the initial rates
followed a decreasing order Tf2 N > PF6 > OTf > SbF6
BF4 . They explained that OTf and BF4 are more nucleophilic
than PF6 . 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.
IL network
ILs can form so-called organized nano-structures (hydrogenbonded polymeric supramolecules, which are similar to water
molecules) with polar and non-polar regions in solid, liquid and
solution states, or even in the gas phase.63,64 Dupont64 suggested
that the aqueous solution of free enzymes might be embedded in
the IL network, which could protect the essential water of proteins
and the solvophobic interactions that are critical for maintaining
the native structure of proteins. However, since enzymes are not
soluble in most common ILs, enzyme molecules are suspended
in reaction media at low water contents (for example, CALB is
known to be active in the absence of water59,65 ); as a result,
IL network theory is not appropriate for understanding these
enzymatic reactions.
Viscosity
ILs are more viscous fluids than conventional organic solvents;77 in
addition, many enzymatic reactions in ILs are heterogeneous due
to the low solubility of enzymes in most pure ILs. Therefore, internal
and external mass-transfer limitations should be considered.12
Lozano et al.46 indicated that besides the IL polarity, the activity
of -chymotrypsin also depended on the IL viscosity; 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). Eckstein
et al.78 explained that the higher enantioselectivity of lipase in
[BMIM][Tf2 N] at low water activities (aw < 0.53) than in methyl tertbutyl ether (MTBE) was for two reasons: (1) the higher viscosity of IL
(52 mPa s) than that of MTBE (0.34 mPa s) may lead to mass transfer
limitations and lower the reaction rate; (2) the lower solubility of
substrates in the IL than in MTBE may cause a lower activation
energy in the IL. van Rantwijk and Sheldon16 explained that the
high viscosity of ILs slows down the conformational changes of
proteins, allowing enzymes to maintain their native structures
and activity. However, Basso et al.79 observed that during amide
synthesis through immobilized penicillin G amidase, the viscosities
of ILs (i.e. [BMIM][PF6 ] and [BMIM][BF4 ]) did not affect the initial
reaction rates despite their much higher viscosities than toluene.
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, but is
not the primary factor in controlling the enzymes activity.
Hydrophobicity
Hydrophobicity may be considered as a narrower concept
of polarity. However, it is practically important to separate
hydrophobicity from polarity because the former is often
related to the miscibility with water.15 The hydrophobicity of
ILs is usually quantified by the log P scale, a concept derived
from the partition coefficient of ILs between 1-octanol and
water. Russells group48 measured the log P values (<2.0) of
several ILs, and suggested that they are very hydrophilic in nature
based on Laanes scale;80 82 they also observed that free lipase
(Candida rugosa) was only active in hydrophobic [BMIM][PF6 ]
(log P = 2.39), but inactive in other hydrophilic ILs including
[BMIM][CH3 COO] (log P = 2.77), [BMIM][NO3 ] (log P = 2.90)
and [BMIM][CF3 COO].48 Similarly, Nara et al.83 achieved higher
transesterification activities of lipases in [BMIM][PF6 ] than in
[BMIM][BF4 ]. The Goto group also reported higher activities of
PEG-modified lipase84 and subtilisin85 in more hydrophobic ILs

893
Ion kosmotropicity
In aqueous solutions, hydrophilic ILs dissociate into individual
ions. Therefore, recent studies50,6 70 in the authors laboratory
proposed that ion kosmotropicity (Hofmeister series) is important
in interpreting the enzymes behaviors in aqueous IL solutions:
kosmotropic anions and chaotropic cations stabilize the enzyme,
while chaotropic anions and kosmotropic cations destabilize it.
Recently, Fujita et al.71 correlated the activity of cytochrome
c in ILs containing 20% (wt) water with the kosmotropicity
of component ions. Constantinescu et al.72 also confirmed that
the thermal stability of ribonuclease A (RNase A) in aqueous
solution of ILs follows the Hofmeister series. Several studies11,73,74
reported low/no activities of -glycosidase in aqueous solutions of
[BMIM][BF4 ], which may be explained by the chaotropic nature of
BF4 in solutions.74 An excellent review by Yang75 systematically
discussed the possible mechanisms of Hofmeister effects of ILs on
the enzyme activity and stability. The above preliminary studies

have shown that the kosmotropic effect of ILs on enzymes may
be applicable to diluted aqueous solutions of ILs,50,66,67 as well as
some concentrated ILs (such as 20 wt% water71 ). However, it is
not quite clear if such an effect exists in pure ILs or ILs with trace
amounts of water, and how the IL hydrophobicity may influence
the kosmotropicity. For example, PF6 is a chaotropic anion,70 and
denatures enzymes when dissolved in aqueous solutions as Na+
or K+ salt (more denaturing than BF4 and MeSO4 for mushroom
tyrosinase76 ). However, PF6 based ILs (such as [BMIM][PF6 ]) are
hydrophobic, and thus the solubility and degree of ion dissociation
of ILs in water are limited. On the other hand, it is also known that
PF6 based ILs are typically enzyme stabilizing.16 Therefore, the
Hofmeister effect may not be suitable for explaining the enzymes
behavior in these hydrophobic ILs or their mixtures with water.
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such as [EMIM][Tf2 N]. Zhang et al.86 reported low penicillin acylase
stabilities in [BMIM][BF4 ] and [BMIM][dca]. Lou and Zong87 studied
the enantioselective acylation of (R,S)-1-trimethylsilylethanol with
vinyl acetate catalyzed by lipases in several ILs, and indicated that
the activity, 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 ]). Paljevac et al.88 reported that cellulase activity
decreased in the order of IL hydrophobicity: [BMIM][PF6 ] >
[BMIM][BF4 ] > [BMIM]Cl. The Vllora group89 observed a lower
stability of penicillin G acylase in [BMIM][BF4 ] than in hydrophobic
ILs (Tf2 N and PF6 ), particularly in the absence of substrate. 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 , dca , NO3 , OAc , etc.)
than in water-immiscible ones (PF6 and Tf2 N ), and the enzymes
activities increased with the cations hydrophobicity (EMIM+ <
BMIM+ < HMIM+ < OMIM+ ). Ha et al.90 also found Novozym 435
was less active and stable in [BMIM][BF4 ] than in other hydrophobic
ILs. Lee et al.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 ]. Shen et al.91 noticed that during the kinetic
resolution of racemic cyanohydrins, Amano lipase PS showed
a high enantioselectivity (80% eep ) in hydrophobic [OMIM][PF6 ],
but poor enantioselectivities (<5% eep ) in hydrophilic [HMIM][BF4 ]
and [HMIM]Cl. Hernandez-Fernandez 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 stability is in decreasing order [HMIM][PF6 ] > [HMIM][Tf2 N]
> [HMIM][BF4 ], [BMIM][PF6 ] > [BMIM][dca], and [OMIM][PF6 ] >
[HMIM][PF6 ] > [BMIM][PF6 ]; in the case of PGA, the stability is in
a decreasing order of [BMIM][Tf2 N] > [BMIM][PF6 ] > [BMIM][BF4 ].
However, the hydrophobic cations showed an adverse effect
on PGA stability: [EMIM][Tf2 N] > [BMIM][Tf2 N], and [BMIM][PF6 ]
> [OMIM][PF6 ]. Through a systematic investigation of lipasecatalyzed transesterification in over 20 ILs, it was observed59 that
the lipase activity increased with the log P value to a maximum, and
then decreased with further increase in log P (a bell shape). These
examples implied that the high hydrophobicity (large log P) of
ILs could be beneficial to enzyme stabilization. However, a higher
log P of the solvent also means a more thermodynamic groundstate stabilization of substrates,92 resulting in lower conversion
of the substrate. This could explain the decreasing reaction
rate in very hydrophobic ILs. Similarly, Lou et al.56 found that
the initial rates of Novozym 435-catalyzed ammonolysis of
(R,S)-p-hydroxyphenylglycine methyl ester increased with the
hydrophobicity of BF4 based ILs to a maximum, and then
declined with further increase in hydrophobicity. Contradictorily,
some studies reported relatively high enzyme activities in
hydrophilic ILs (such as [BMIM][BF4 ], [EMIM][BF4 ], [BMIM][OTf]
and [MMIM][MeSO4 ]).45,7,93 96 Therefore, multiple factors must be
considered in explaining the enzymatic systems like these.
894
Enzyme dissolution
Hydrophobic ILs (typically consist of PF6 and Tf2 N ) do not
dissolve appreciable amounts of enzymes; on the other hand,
hydrophilic ILs (such as those based on NO3 , lactate, EtSO4 , and
CH3 COO ) may dissolve some enzymes, however, most of them
tend to strongly interact with proteins (such as via H-bonding),
resulting in enzyme inactivation.25,38 40,57,59 For example, cellulase
was dissolved but inactivated in concentrated solutions of
H Zhao
[BMIM]Cl.38 Currently, there are only a few pure ILs that are known
to dissolve enzymes but do not inactivate them. For example,
choline dihydrogen phosphate (m.p. 119 C) containing 20% (wt)
water was found to solubilize and stabilize cytochrome c (cyt c);71,97
triethylmethylammonium methyl sulfate ([Et3 MeN][MeSO4 ]) was
reported able to dissolve >1.2 mg mL1 Candida antarctica lipase
B (CALB) and retain its catalytic capability.57,98 Recently the authors
Laboratory synthesized a series of ether-functionalized ILs that are
able to dissolve enzymes (>5 mg mL1 CALB at 50 C) and a variety
of substrates, but do not inactivate the lipase (more discussion in
a later section).25,26 Therefore, the enzyme dissolution in ILs is not
a definite indication of enzyme denaturation.
Surfactant effect
ILs bearing long alkyl chains in their cations often form selfassembly in aqueous solutions and behave like surfactants.99,100
Ionic surfactants, such as sodium n-dodecyl sulfate (SDS) and
n-dodecyltrimethylammonium bromide (DTABr), interact with
enzymes through two major mechanisms: (1) electrostatic interactions of the surfactant head group and charged amino acid residues
of the protein, and (2) hydrophobic interactions between the alkyl
chains of the surfactant and hydrophobic amino acid residues.101
In general, ionic surfactants are considered non-specific denaturants of enzymes, but many studies also reported that they either
have no effect on enzymes, or show some activating/stabilizing
effects on enzymes.101 103 The knowledge of surfactantprotein
interactions can be useful for understanding the IL effect on enzyme activity and stability in some systems (both aqueous and
non-aqueous), although such a correlation is lacking for enzymes
in IL systems.
In summary, given the complexity of an enzymes catalytic
properties in ILs, there is no universal method that can solve all
enzyme inactivation issues. Therefore, a number of methods have
been adopted or developed to improve the enzymes stability
and to increase its activity and enantioselectivity. The following
is a tutorial discussion of some representative methods (typical
examples were summarized in Table 1).
STABILIZATION AND ACTIVATION METHODS

Overall, methods to stabilize and activate enzymes in ILs may
be divided into two general categories: modification of enzymes,
and modification of solvent environments. The first category
includes the enzyme immobilization (on solid support, solgel,
or CLEA), physical or covalent attachment to PEG, rinsing with
n-propanol methods (PREP and EPRP), three-phase partitioning
(TPP), enzyme/protein-coated micro-crystals, and lyophilization
with cyclodextrins. The second category includes water-in-IL
microemulsions, IL coating, the use of additives in ILs, and the
design of enzyme-compatible ionic media. Methods in the first
category make the enzyme more tolerant to those denaturing
factors of ILs discussed earlier, while methods in the second
category intend to minimize the denaturing properties of some
ILs (such as reducing the anions nucleophilicity and H-bonding
basicity). The following sections will address some major advances
in these methods with representative examples.
There are also some exceptions. For example, BF4 based ILs are hydrophilic
but do not dissolve the enzyme.57

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Table 1. 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, 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)

immobilized on
magnetic
nanoparticles
supported ILs
solgel encapsulation of
horseradish
peroxidase using
[BMIM][BF4 ]
solgel encapsulation of
using various ILs
based on Cl , BF4 ,
PF6 and Tf2 N
1-butanol
solgel immobilization
of lipase PS from
Burkholderia cepacia
[EMIM][BF4 ],
[EMIM][Tf2 N],
[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; (2)
resolution of
1-phenylethanol using
vinyl acetate; (3)
resolution
1-phenylethylamine
using methyl
methoxyacetate
physical adsorption of
lipases on PEG
(average molecular
weight
Mn = 4, 00035,000)
[BMIM][PF6 ]
[HMIM][PF6 ]
[OMIM][PF6 ]
[BMIM][Tf2 N]
(1) alcoholysis between

vinyl cinnamate and
benzyl alcohol; (2)
transesterification
between vinyl acetate
and
2-phenyl-1-propanol
(1) transesterification of
ethyl butyrate and
1-butanol; (2)
transesterification of
ethyl octanoate and
alcohols; (3)
ammoniolysis of ethyl
octanoate and
ammonia (4)
epoxidation of
cyclohexene
N-acetyl-Lphenylalanine ethyl
ester and 1-propanol;
(2) hydrolysis of
N-succinyl-L-ala-L-alaL-pro-L-phe-pnitroanilide
ethyl butyrate and
1-butanol; (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,
and synthetic
reaction in
n-hexane
(1) hydrolysis of
p-nitrophenyl butyrate
in phosphate buffer;
(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. The
immobilized lipase
was more active than
the free form.
93
Enzyme-SWNT
conjugates exhibited
higher catalytic
turnover and higher
thermal stability than
free form.
108
The immobilized lipase

showed a higher
activity and
enantioselectivity in IL
than its free enzyme.
109
The immobilized
exhibited a higher
catalytic activity and
thermal stability.
112
Enhanced activity and

thermal stability was
observed for
IL-solgel enzyme.
Greater hydrolytic and
synthetic activities of
IL-solgel
encapsulated lipase
were observed than
that without ILs.
130
131,132
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. CALB-PP also
showed high
enantioselectivities in
two resolution
reactions.
134
They observed a higher

enzyme activity (as
high as 14-fold) for
PEG-lipase complex
comparing with the
free form, as well as a
comparable or higher
enantioselectivity for
the enzyme complex
than the free lipase.
157,158
39
895

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H Zhao
Table 1. (Continued)
Method
Enzyme preparation
Reaction solvent
Reaction
Comments
Ref
covalently conjugating
PM13 with subtilisin
Carlsberg85,163 and
Candida rugosa
lipase,84
[EMIM][Tf2 N]
[C2 OC1 MIM][Tf2 N]
[C2 OHMIM][Tf2 N]
(1) hydrolysis of
p-nitrophenyl butyrate
in tris-HCl buffer; (2)
2-phenyl-1-propanol
with vinyl acetate; (3)
ester with 1-butanol
In Tf2 N based ILs,

PM13 -subtilisin and
PM13 -lipase are
soluble and exhibited
much higher activity
and stability than free
enzymes.
84,85,163
Enzyme precipitated
and rinsed with
n-propanol(EPRP)
EPRP of subtilisin and

-chymotrypsin
[BMIM][PF6 ]
[BMIM][BF4 ] (and
n-octane)
(1) esterification of
N-acetylphenylalanine
with ethanol; (2)
ester with n-propanol
The EPRP of subtilisin

and -chymotrypsin
exhibited very high
catalytic activity
comparing with
pH-tuned lyophilized
powder.
168,170
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; (2)
horseradish
peroxidase-catalyzed
oxidation of pyrogallol
171,172
lipases from Candida

rugosa,
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; (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.
Higher operational
stability of lipases was
observed in
water-in-IL
microemulsions. 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.
Pseudomonas cepacia
lipase coated by
[PPMIM][PF6 ]
toluene
diisopropyl ether
toluene hexane
High enantioselectivity
and activity were
reported for this
enzyme preparation.
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).
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
various alcohols with
vinyl acetate at room
temperature
1-phenylethanol and
vinyl acetate; (2)
() -3hydroxypentanenitrile
and vinyl acetate; (3)
other secondary
alcohols and vinyl
acetate; (4)
esterification of
2-(4-ethylphenoxy)
propionic acid and
1-butanol
esterification of methyl-D-glucopyranoside
with fatty acids
glycerolysis of
triglycerides and
glycerol
Coating enzymes
with ILs
Higher conversions and

enzymatic activities
were detected.
High enzyme activity and
triglyceride
conversions were
observed.
175
178,179
47
182185
896

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Table 1. (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
sorbic acid and sorbate
ester with n-propanol
25,196 ; (2)
ethyl butyrate and
1-butanol; (3)
D-glucose; (4)
regioselective
cellulose; (5)
enzymatic synthesis of
methyl-phthalate of
betulinic acid; (6)
lipase-catalyzed
synthesis of glucose
fatty acid esters; (7)
triglycerides and
methanol
The use of IL as a
co-solvent enhanced
the solubility of
hydrophobic
substrates, and
stabilized the ADH.
The reaction rate in IL
was superior to that in
diisopropyl ether.
The ether-functionalized
ILs could dissolve a
variety of substrates
(such as sugars,
glucose, ascorbic acid,
amino acids, betulinic
acid, fatty acid, and
triglycerides). In
addition, CALB
(particularly the
immobilized form)
exhibited high
catalytic properties in
these ILs.
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. These
immobilized methods generally fall into three categories: binding
to a solid carrier, solgel encapsulation, and protein cross-linking
(a carrier-free technique).104 106
2527
heme-containing proteins/enzymes (myoglobin, cytochrome c,

and horseradish peroxidase) onto SWNTs coated with ILs (such as
[BMIM][BF4 ] or [BMIM][PF6 ]), and observed that the encapsulated
enzymes retained their bioelectrocatalytic activities toward the
reduction of oxygen and hydrogen peroxide.
Another new carrier developed by the Goto group111 is polymerized ionic liquid microparticles (pIL-MP), which can be synthesized
through the polymerization of 1-vinyl-3-ethylimidazolium bromide using the crosslinker N,N -methylenebis(acrylamide) in a
concentrated water-in-oil emulsion; horseradish peroxidase (HRP,
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. Very recently, another new enzyme support named
magnetic nanoparticles supported ILs was constructed by covalent bonding of ILs-silane on magnetic silica nanoparticles;
Candida rugosa lipase immobilized on this support showed
higher catalytic activity and thermal stability.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); 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.113
As an interesting application in electrocatalysis, the Pang
group114 116 designed new glassy carbon electrodes by entrapping heme proteins (hemoglobin, myoglobin, or catalase)
in agarose hydrogel films, and observed high electrocatalytic activities of the heme proteinagarose films in both [BMIM][PF6 ] and

897
a) Immobilization on a solid carrier. The binding to a solid

support is a routine method for improving the enzyme stability
in conventional organic solvents as well as in ILs. One main
reason is that the immobilization techniques (both physical
adsorption and covalent attachment) are straightforward and
easy to accomplish via regular laboratory procedures, and
many immobilized enzymes are commercially available (such as
lipase B from Candida antarctica immobilized on acrylic resin,
known as Novozym 435). A recent comprehensive review16 has
summarized many of these examples; therefore, this report will
not replicate the work. However, it is interesting to highlight
some new carriers for enzyme immobilization, one of which
is carbon nanotubes.107 The high surface area and unique
nanoscopic dimensions of carbon nanotubes enable high protein
loading and low mass-transfer resistance. 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; the enzyme-SWNT conjugates were
well dispersed in ILs.108 Similarly, 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 ].109 Du et al.110 immobilized
23
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[BMIM][BF4 ] solutions containing a small amount of water. The native structures of heme proteins were retained in agarose film
as indicated by UV-visible and Fourier transform infrared (FT-IR)
spectroscopy.
898
b) Solgel encapsulation. Encapsulation is a technique for entrapping biomolecules in a polymer matrix via non-covalent
interactions between the network and the biomolecule. The actively pursued encapsulation method for the enzyme stabilization
in ILs is the solgel technology. It is a relatively simple method
(see protocols117,118 ), and has been widely used for entrapping
a variety of biological molecules such as proteins, enzymes and
antibodies.119 This technique involves the acid- or base-catalyzed
hydrolysis of tetraalkoxysilanes, such as tetraethyl orthosilicate
(TEOS) or tetramethyl orthosilicate (TMOS), in aqueous solutions;
the subsequent cross-linking condensation forms a SiO2 matrix
to encapsulate the biomolecules.120,121 The structural rigidity of
the solgel matrix protects the integrity of encapsulated enzyme
molecules and prevents their leaching; the mesoporous structures
and high pore volume of solgel polymer afford the free diffusion
of small substrate molecules and their effective interactions with
the enzyme.122 However, gel shrinkage and pore collapse have
been major drawbacks of this method; in addition, there are other
issues such as the alcohol release during the hydrolysis of silicon
alkoxide.123 To overcome these hurdles, different additives (such
as sugars, amino acids, and N-methylglycine) have been added to
reduce the gel shrinkage and adjust the protein hydration, and to
further increase the activity and stability of enzymes.123
Recently, there is an increasing interest in utilizing ILs as
additives124 for protein/enzyme solgel immobilization. This is
because room-temperature ILs are non-volatile, thermally stable,
and are tunable to be enzyme-compatible. Earlier studies125,126
focused on the preparation of mesoporous silica through high
dispersion of ILs (such as [BMIM][BF4 ]) in solgel, with the
potential inclusion of metal catalysts. More recently, silica
xerogels were prepared with various morphologies via the
solgel method in the presence of ether-functionalized ILs (such
as 1-triethylene glycol monomethyl ether-3-methylimidazolium
methanesulfonate); these ILs act as both morphology controller
and acid pre-catalyst.127,128 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. Several groups have
clearly demonstrated the advantages of enzyme encapsulation in
IL solgel hybrid carrier. The Li group130 observed improved
activity and thermal stability of horseradish peroxidase after
immobilizing in [BMIM][BF4 ] based solgel. The Koo group131,132
examined a variety of ILs and their mixtures as additives
during the solgel immobilization of Candida rugosa lipase,
and achieved greater hydrolytic and esterification activities of
solgel encapsulated lipase coated with ILs than that without ILs.
However, the solvents used in the above assay reactions catalyzed
by IL solgel immobilized enzymes were either aqueous solutions,
or organic solvents (such as hexane); the use of ILs as solvents
was not demonstrated. Sangeetha et al.133 encapsulated subtilisin
in solgel derived silica glasses using alkoxysilane precursors
carrying different chain lengths; the resulting immobilized enzyme
showed enhanced thermal stability, and high activity toward
peptide synthesis in an IL ([BMIM][PF6 ]). The Kanerva group134
immobilized lipase PS from Burkholderia cepacia in a solgel, and
obtained higher enzyme stability in [EMIM][BF4 ], [EMIM][Tf2 N] and
[BMIM][PF6 ] than the enzyme preparation via the cross-linked
enzyme aggregates (CLEAs) method (see next section).
H Zhao
c) Cross-linked enzyme aggregates (CLEAs ). As an early version

of carrier-free immobilization, the cross-linked enzymes (CLEs)
can be created by reacting glutaraldehyde with NH2 groups on
the protein surface.135,136 However, intermolecular cross-linking
of these biomolecules often results in low enzyme activity, poor
reproducibility and low mechanical stability.104,137 At the same
time, the cross-linking of a crystalline enzyme via glutaraldehyde
was reported,138 eventually leading to a commercial immobilization technology called cross-linked enzyme crystals (CLECs or
CLCs).139,140 This technique offers many advantages such as enhanced thermal, mechanical and pH stability, designable particle
sizes, and ease of recycling.104,137 However, the preparation of
CLECs requires laborious purification and crystallization of enzymes. A more recent development in cross-linking enzymes has
been pioneered by the Sheldon group,104 106,137,141 so called crosslinked enzyme aggregates (CLEAs ). This method involves the addition of salts, organic solvents or non-ionic polymers to precipitate
the enzyme as physical aggregates from aqueous solutions; the enzyme aggregates are then cross-linked by glutaraldehyde. The use
of CLEAs has such advantages as ease of preparation and recycling,
improved enzyme activity, high stability and selectivity in organic
solvents, immobilizing biocatalysts containing more than one enzyme, etc.105,142 147 Higher enzyme stability is expected for CLEAs
in ILs than free enzymes. Toral et al.39 immobilized CALB through
two methods: a CLEA method without a solid support (resulting
in CALB-CL), and adsorbed and cross-linked on a polypropylene
carrier (resulting in CALB-PP). They carried out several reactions
using these two enzyme preparations in denaturing ILs (such as
[BMIM][dca], [BMIM][NO3 ], [BMIM][OAc] and [BMIM][lactate]). During the transesterification between ethyl butyrate and 1-butanol,
CALB-PP exhibited considerably higher catalytic activities in denaturing ILs than CALB-CL and free CALB. In addition, CALB-PP
also showed high enantioselectivities in two resolution reactions
(resolution of 1-phenylethanol in [BMIM][NO3 ], and resolution of
1-phenylethylamine in [BMIM][NO3 ] and [BMIM][dca]), although
the cross-linked lipase was not active in some ILs (such as
[BMIM][OAc] and [BMIM][lactate]). 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 ].
d) Enzyme immobilization with ILs in biosensors/electrodes. The
Li group149 developed an amperometric biosensor by immobilizing horseradish peroxidase on [BMIM][BF4 ]-based solgel;
the biosensor exhibited high stability and sensitivity with linear calibration ranging from 0.02 to 0.26 mmol L1 and a low
detection sensitivity of 1.1 mol L1 . An electrochemical H2 O2
biosensor was constructed by entrapping horseradish peroxidase
in [BMIM][BF4 ] doped DNA network casting on a gold electrode; the
new biosensor exhibited a short response time (2 s), a low detection limit (3.5 mol L1 ), a wide linear range (0.017.4 mmol L1 ),
as well as a high sensitivity and stability.150 A glucose biosensor
was fabricated by immobilizing glucose oxidase in thin films of
polyethylenimine-functionalized IL containing carbon nanotubes
and gold nanoparticles; the novel biosensor showed good electrochemical response to glucose and high enzyme compatibility.151
A disposable biosensor was constructed from the composite material based on N-butylpyridinium hexafluorophosphate, sodium
alginate, and graphite; after optimization, this new biosensor could
detect H2 O2 in a linear calibration range of 1.0 to 6.0 mol L1 with
a detection limit of 0.5 mol L1 at a signal/noise ratio of 3.152 Another peroxidase biosensor was assembled by mixing [BMIM][Tf2 N]

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with the immobilized peroxidase (tissue from pine nuts) onto chitosan crosslinked with citrate.153 A new composite electrode was
constructed using multiwall carbon nanotubes (MWCNT) and the IL
n-octylpyridinium hexafluorophosphate, which was further developed into a sensitive biosensor for glucose through the inclusion
of glucose oxidase.154 Sun et al.155 designed a glucose microsensor
from a composite paste of mesoporous carbon, glucose oxidase
and [BMIM][PF6 ], and demonstrated its high sensitivity over a
concentration range from 10.0 to 80.0 mol L1 . These biosensor
examples suggest that various immobilized enzymes have a high
compatibility with ILs.
C
CH2
CH2
O
CH
C
O
O(C2H4O)33CH3
CH
C
O
8
Figure 2. Structure of comb-shaped poly(ethylene glycol) PM13 .
showed a high catalytic activity and storage stability in these ionic

solvents.84
However, there are also some disadvantages associated
with PEG-modification. For example, the preparation could be
cumbersome, and the enzyme catalytic properties may vary in
different immobilization batches.
Enzyme precipitated and rinsed with 1-propanol (EPRP)
The propanol rinsed enzyme preparation (PREP) is a method
for stabilizing the immobilized enzymes, which is achieved
through repeatedly rinsing the silica-immobilized enzyme with
dry 1-propanol.166 The principle of this technique is that 1propanol removes water from protein, which minimizes protein
denaturation and keeps the majority of enzyme molecules in
an active conformation; only small amounts of water may be
added during the reaction to activate catalysis.166 The PREPs of
subtilisin Carlsberg,166 -chymotrypsin,166 and papain167 have
shown very high enzymatic activities in organic media (such as
acetonitrile and t-butanol). A further development of PREP is a
preparation method called enzyme precipitated and rinsed with
n-propanol (EPRP).168,169 The EPRP technique is a combination
of several methods: enzyme precipitation by alcohols, rinsing
the precipitate with 1-propanol, and the use of salt tuning
during co-precipitation. The EPRP of -chymotrypsin showed two
orders of magnitude higher esterification activity than lyophilized
powder in n-octane.168 The EPRP of subtilisin exhibited >4000
times higher initial rate (for transesterification) than pH-tuned
lyophilized powder in [BMIM][PF6 ]; pH-tuned lyophilized subtilisin
showed no activity in [BMIM][BF4 ], whereas the EPRP gave a
moderately high initial rate in this IL.170 In another study, Shah and
Gupta148 conducted the enzymatic resolution of 1-phenylethanol
in [BMIM][PF6 ] catalyzed by various lipase preparations; they
observed that the EPRP of Candida rugosa lipase (CRL) was more
active than pH-tuned lyophilized CRL and CLEA preparation, and
was more enantioselective than pH-tuned lyophilized CRL and
PREP preparation. On the other hand, the EPRP of Burkholderia
cepacia lipase (BCL) lost substantial amounts of activity, but two
alternative formulations (i.e. enzyme precipitated and rinsed with
acetone (EPRA) and acetone-rinsed enzyme preparation (AREP))
resulted in high enzyme activity and enantioselectivity.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.
The following several methods, on the other hand, intend to
change the ionic media to improve their enzyme compatibility.
The surfactant formation of micelles in ILs is being actively
studied and has been reviewed.100 Recently, the Goto group171
reported the use of water-in-IL microemulsions as a new medium
for dissolving various enzymes and proteins (such as lipase

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 has both hydrophilic and hydrophobic
properties, therefore, modified enzymes become soluble in some
organic solvents (such as benzene, toluene and chlorinated
hydrocarbons)156 and ILs,85 and show an increased stability
in these solvents. By strict definition, PEG-modification is one
type of enzyme immobilization method. The most common PEGmodification is achieved through physical adsorption because of
its simple procedures, mild conditions, and the unchanged protein
structures. The Goto group157,158 applied PEG with different
molecular weights (average Mn = 400035 000) as the enzymecoating amphiphile for the preparation of PEGlipase complexes.
They investigated the PEGlipases in several alcoholysis reactions
in hydrophobic ILs (PF6 and Tf2 N ), and observed higher
enzyme activity (as high as 14-fold) of the PEGlipase complex
than that of the free form,157,158 as well as comparable or
higher enantioselectivity of the enzyme complex than that of
free lipase.158 Turner et al.38 reported higher enzyme activity of
PEGcellulase complex (PEG average Mn = 1000) in aqueous
solution and [BMIM]Cl solutions than that of free cellulase. On
the other hand, the PEG-modified -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.159
These differences could be due to a number of factors such as
different preparation conditions, different PEG molecular weights,
and different enzymes involved.
The second strategy for associating enzymes with PEG is through
covalent attachment. For example, PEG p-nitrophenyl carbonate
is commonly used to connect PEG units with amino residues
of proteins to form stable carbamates.160,161 Alternatively, Kaar
et al.48 used PEG-monoisocyanate to link PEG with lysine residues
of the protein to form a PEGlipase complex, however, this
complex showed no improvement in lipase activity in [BMIM][PF6 ],
[BMIM][NO3 ], and [MMEP][OAc] compared with the free form.
PEG-modification of cytochrome c (cyt.c) allowed the protein
to become soluble in [EMIM][Tf2 N] without denaturation.162
Meanwhile, the Goto group adopted an unusual comb-shaped
PEG, PM13 , (SUNBRIGHT AM-1510K from NOF Co., Ltd., Japan)
for covalently conjugating PEG with subtilisin Carlsberg85,163 and
Candida rugosa lipase,84 respectively. As shown in Fig. 2, PM13 is
a copolymer of PEG derivatives with maleic anhydride (molecular
weight 15, 000); the acid anhydrides react with amino groups
of the enzyme.164 The PM13 -lipase complex showed a higher
activity and stability in benzene than its free form.165 In Tf2 N
based ILs, PM13 -subtilisin is soluble and exhibited much higher
transesterification activity and stability than its free enzyme.85,163
Similarly, PM13 -lipase is also soluble in Tf2 N based ILs, and
www.soci.org
PS, Candida antarctica lipase B, -chymotrypsin, horseradish
peroxidase and enhanced green fluorescent protein). 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), followed
by the addition of aqueous buffer to prepare a microemulsion.
The lipase PS showed a higher hydrolytic activity in water-in-IL
microemulsions than in water-saturated IL and in water-inisooctane microemulsions.171 The same group optimized the
oxidation of pyrogallol in water-in-IL microemulsions catalyzed
by horseradish peroxidase, and found that the reaction in
IL microemulsions was much more effective than that in a
conventional AOT/water/isooctane microemulsion.172
Zhou et al.173 formed the water-in-[BMIM][PF6 ] microemulsions
using nonionic surfactant Triton X-100, 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 ]. Similarly, in waterin-[BMIM][PF6 ] microemulsions containing Triton X-100, alcohol
dehydrogenase from yeast maintained a high catalytic activity,
while in homogeneous solutions, the enzymatic activity rapidly
decreased with the increase of IL concentration.174 More recently,
the Stamatis group175 developed water-in-IL microemulsions
through using non-ionic surfactants (Tween 20 or Triton X-100)
in [BMIM][PF6 ]. The catalytic properties of lipases from Candida
rugosa, Chromobacterium viscosum and Thermomyces lanuginosa
in these novel microemulsions were investigated through the
esterification of natural fatty acids with various aliphatic alcohols,
and the hydrolysis of p-nitrophenyl butyrate. The operational
stability of these lipases in water-in-IL microemulsions was much
higher than that in other microheterogeneous media. 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.
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;
Co (II) salt was chosen as the probe because of different colors
of the hexa-coordinated and tetra-coordinated complexes of the
cation, depending on the solvating environment; the color change
can be determined by UV-Visible absorbance spectra.
900
Coating enzymes with ILs

Lee and Kim177 coated an ionic liquid, [PPMIM][PF6 ] ([PPMIM]
= 1-(31 -phenylpropyl)-3-methylimidazolium), onto lipase from
Pseudomonas cepacia, and achieved high enantioselectivity and
activity from this enzyme preparation. The Itoh group178,179
prepared an imidazolium IL carrying anions of cetyl-PEG10-sulfate
(Fig. 3), and coated it on lipases to stabilize the enzymes in
organic solvents (such as diisopropyl ether). The IL-coated lipases
exhibited high enantioselectivity and high reaction rates in several
resolution reactions (up to 500- to 1000-fold acceleration for
some substrates). The IL-coated lipase PS is also commercially
available.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 1.81,
1.66, 1.56 and 1.60 for [BMIM][PF6 ], [EMIM][Tf2 N], [BMIM][BF4 ],
and [EMIM][BF4 ] respectively.180 Lozano et al.181 carried out the
enzymatic synthesis of citronellyl esters mediated by Novozym
435, and observed that synthetic activity could be improved up to
H Zhao
O
N + N
O S O
O
10
n-C15H33
O
Figure 3. Structure of 1-butyl-2,3-dimethylimidazolium cetyl-PEG10sulfate.
two-fold by using the IL coating onto lipase; they also found the
increase in activity followed the hydrophobicity of ILs ([OMIM][PF6 ]
> [HMIM][PF6 ] > [BMIM][PF6 ]). Mutschler et al.47 coated various ILs
onto Novozym 435 beads, which resulted in higher conversions
in lipase-mediated esterification of methyl--D-glucopyranoside
with fatty acids than uncoated lipase.
Manipulation/design of IL structures
As mentioned previously, the structures of ILs (as defined by the
types of cations and anions, and their combinations) considerably
influence the IL physical properties that are crucial for ILenzyme interactions and enzyme stabilization. These properties
include the polarity, H-bond basicity, anion nucleophilicity, IL
network, kosmotropicity, viscosity and hydrophobicity. Therefore,
it is important to customize the structures of ILs for particular
biocatalytic applications based on current knowledge of IL
structure and enzyme activity relationship. Several studies have
tailored the IL structures to dictate the compatibility of enzymes.
The Xu group182 187 judiciously selected a group of commercial
tetraammonium-based ILs as reaction media for the enzymatic
glycerolysis. As shown in Fig. 4, each of these ILs is an ionic mixture
containing multiple alkyloxy groups, which have both hydrophilic
and hydrophobic properties like PEG. In particular, 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;183,184 trioctylmethylammonium
bis(trifluoromethylsulfonyl)imide ([TOMA][Tf2 N]) and its mixture
with Ammoeng 102 have also been demonstrated to be suitable
solvents for enzymatic glycerolysis.186 188 De Diego et al.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; however, 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. Xu and co-workers183,185 utilized the conductorlike screening model for real solvents (COSMS-RS) to derive
various parameters (such as misfit, H-bonding and van der Waals
interaction energy) to understand the multiple interactions in ILs;
the model also provides guidance in designing the structures of
cations and anions.190 The Kragl group191 found that an IL in
the Ammoeng family Ammoeng 110 (Fig. 5) is quite effective
in forming aqueous two-phase (ATP) for the purification of
active enzymes (two different alcohol dehydrogenases); the IL
is capable of stabilizing the enzymes and enhancing the solubility
of hydrophobic substrates. It is interesting to mention that oxygencontaining ILs (such as Ammoeng series, and [C2 OHmim]Cl) were
used as additives in the enantioselective hydrolysis of diester
malonates by pig liver esterase (PLE), 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.192
Based on the lyoprotectant effect of tris(hydroxymethyl)
aminomethane (Tris) as excipient in horseradish peroxidase
From the name cocosalkyl pentaethoxy methylammonium methylsulfate.

Cocos +
N
www.soci.org
Et +
N
OH
MeSO4-
OH
HO
OH
CH3COO-
Et
(b) Ammoeng 111

m = 50-60
(a) Ammoeng 100

Cocos, C14 alkyl group; m + n = 4-14
Tallow
+
N
Et
Et +
N
OH
OH
EtSO4HO
OH
H2PO4-
Et
(d) Ammoeng 112

m = 50-60
(c) Ammoeng 102

Tallow, C18 acyl group; m + n = 14-25
O
R
+
N
R'
MeSO4O
R'
O
n
(e) Ammoeng 120

R, R', C18 acyl group; m, n, unavailable
Figure 4. Structures of tetraammonium-based ILs (AmmoengR series).
OH
O
CH3CH2 +
N
H3C CH2CH3
Figure 5. Structure
of Ammoeng
n = 5-15
OH
HO
Cl
CF3SO3OH
110.
HO
Figure 6. Structure
of
flouromethanesulfonate.
tetrakis(2-hydroxyethyl)ammonium
tri-
et al.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, and achieved high conversion yields (72.5% and 76.7%, respectively, in two ILs under
optimal conditions). These two ILs are considered as amphiphilic
(hydrophilic cation and hydrophobic anion), and have relatively
low viscosities.
Recently, 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, so called deep eutectic ILs
(DEILs).198 200 A good example is the mixture of choline chloride
(m.p. = 302 C) and urea (m.p. = 133 C) in a 1 : 2 molar ratio
resulting in a room-temperature IL (Tf = 12 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

901
lyophilization,193 Das et al.194 mimicked the structure of Tris

and rationally designed a new IL known as tetrakis(2hydroxyethyl)ammonium triflouromethanesulfonate (Fig. 6); they
reported that horseradish peroxidase in this new IL
was 10 times more active than in methanol and
at least 30240-fold more active than in conventional
ILs. Abe et al.23 synthesized an alkyloxy-containing hydrophobic IL named 2-methoxyethyl(tri-n-butyl)phosphonium
bis(trifluoromethane)sulfonamide ([MEBu3 P][Tf2 N]), and observed
a faster reaction rate (lipase PS-catalyzed transesterification of secondary alcohols) in this IL than in diisopropyl ether. Lourenco
et al.195 measured little Novozym 435 activity in denaturing
[BMIM][dca], but high activity and enantioselectivity in [aliq][dca]
(aliq = trioctylmethylammonium (Aliquat 336 )). The possible
explanation is that the denaturing anions molar concentration in [aliq][dca] is much lower than that in [BMIM][dca] due
to much higher molar mass of the former IL.25,26,196 Vafiadi
+
N
www.soci.org
H 3C
O
n
H3C
OAc-
+
N(CH2CH3)3 OAcn
Figure 7. Imidazolium and ammonium based ILs consisting of

alkyloxyalkyl-substituted cation and acetate anion (abbreviated to
[Me(OEt)n -Et-Im][OAc] and [Me(OEt)n -Et3 N][OAc], respectively) (n=2, 3,
. . .).
902
be finely tuned with different combinations of organic salts and

complexing agents. Gorke et al.201 reported that several hydrolases
(CALB, CALA and epoxide hydrolase) maintained high activities
in DEILs based on choline chloride or ethylammonium chloride
(H-bond donors include acetamide, ethylene glycol, glycerol, urea,
and malonic acid). Although some DEILs contain alcohols (such
as ethylene glycol or glycerol), ethylene glycol was 9-fold less
reactive than 1-butanol, and glycerol was >600-fold less reactive
than 1-butanol in CALB-catalyzed transesterifications of butyl
valerate. When being used as co-solvents, DEILs were capable
of improving the reaction rate and/or conversion of hydrolases
(esterases and epoxide hydrolase). The polarity data suggest these
DEILs are more polar than common imidazolium-based ILs. The
H-bond network in DEILs is suspected responsible for reducing the
chemical potential of the components of DEILs and making them
less reactive.
Recently, the authors group synthesized a series of
alkyloxyalkyl-containing ILs based on acetate (Fig. 7).25 It was
found that ILs can be designed to dissolve many substrates (such
as cellulose, sugars, glucose, ascorbic acid, amino acids, betulinic
acid, fatty acid, and triglycerides) that are not readily soluble in
common organic solvents.25 27,202,203 In addition, these ILs can be
optimized to be compatible with CALB, and thus become ideal
solvents for producing the derivatives of many substrates via enzymatic reactions.25 27 Therefore, it is possible to manipulate the
structures of ILs to make them more compatible with enzymes,
and to be able to dissolve a variety of compounds. This could
significantly improve the catalytic efficiency of these reactions
as some substrates may not be soluble in conventional organic
media.
In summary, 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; (2) they often contain multiple ether and/or hydroxyl
groups so that the solvent properties (such as IL viscosity, H-bond
basicity and water affinity) are optimized for mild ILenzyme
interactions.
As shown by discussions above and the illustration in
Table 1, the types of ILs used in biocatalysis are usually limited to quaternary alkyl-substituted ammonium, phosphonium,
imidazolium, pyridinium, and pyrrolidinium salts. It is important to realize that new ILs based on other cations (such as
triazolium, pyrazinium, pyridazinium, pyrimidinium, pyrazolium,
piperazinium, thiazolium, oxazolium, oxazolidinium and morpholinium cations204 206 ) have been prepared, and their physical
properties have been characterized.207 212 It will be interesting to
expand our scope of media to these new ILs for biocatalysis, and we
may find new opportunities to design more enzyme-compatible
solvents.
H Zhao
SUMMARY
In addition to the methods discussed above, other techniques
have also been explored to improve the enzymes adaptability
to ionic media including three-phase partitioning (TPP) of
subtilisin,170 the use of additives (such as NaHCO3 , Na2 CO3 or
triethylamine),45,213 enzyme/protein-coated micro-crystals,148,214
and lipase lyophilization with cyclodextrins.215 At present, many
of these methods have not been investigated in-depth in terms of
the variety of enzymes and different types of ILs, therefore, more
systematic studies on these subjects are necessary to explore the
full potentials of ILs. Those ILs that are enzyme-compatible and can
dissolve substrates that are not soluble in conventional organic
solvents, will be extremely valuable to the biocatalysis field.
ACKNOWLEDGEMENTS
The Donors of the American Chemical Society Petroleum Research
Fund (46776-GB1) are thanked for partial support of this research.
Financial support by the Research Fund Grant from Royal Society
of Chemistry is also acknowledged.
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Methods For Stabilizing and Activating Enzymes

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Methods For Stabilizing and Activating Enzymes

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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

J Chem Technol Biotechnol 2010; 85: 891907

Correspondence to: Hua Zhao, Chemistry Program, Savannah State

c 2010 Society of Chemical Industry

Ionic liquids (ILs) consist of ions and are liquid at temperatures

of these unique properties, ILs have been widely recognized

Figure 1. Structures of some typical cations and anions in ILs.

Recently, some ILs have been shown capable of dissolving many

MAJOR FACTORS THAT AFFECT ENZYME

Basicity refers to the ability of a base to accept a proton, and is a matter

c 2010 Society of Chemical Industry

J Chem Technol Biotechnol 2010; 85: 891907

Stabilizing and activating enzymes in ionic liquids

that the decreasing stability was, in general, consistent with the

J Chem Technol Biotechnol 2010; 85: 891907

c 2010 Society of Chemical Industry

the enzyme activity and stability. The above preliminary studies

STABILIZATION AND ACTIVATION METHODS

c 2010 Society of Chemical Industry

J Chem Technol Biotechnol 2010; 85: 891907

Stabilizing and activating enzymes in ionic liquids

lipase B from Candida

Candida rugosa lipase

Candida rugosa lipase

(1) alcoholysis between

Reaction rates were

The immobilized lipase

Enhanced activity and

Lower initial activity but

They observed a higher

J Chem Technol Biotechnol 2010; 85: 891907

c 2010 Society of Chemical Industry

In Tf2 N based ILs,

EPRP of subtilisin and

The EPRP of subtilisin

lipases from Candida

The lipase PS and

lipases coated with an

Novozym 435 beads

tetraammoniumbased ILs such as

Higher conversions and

c 2010 Society of Chemical Industry

J Chem Technol Biotechnol 2010; 85: 891907

Stabilizing and activating enzymes in ionic liquids

transesterification of (E) 4-phenylbut-3-en-2-ol

Modifying the enzymes

J Chem Technol Biotechnol 2010; 85: 891907

heme-containing proteins/enzymes (myoglobin, cytochrome c,

c 2010 Society of Chemical Industry

a) Immobilization on a solid carrier. The binding to a solid

c) Cross-linked enzyme aggregates (CLEAs ). As an early version

c 2010 Society of Chemical Industry

J Chem Technol Biotechnol 2010; 85: 891907

Stabilizing and activating enzymes in ionic liquids

J Chem Technol Biotechnol 2010; 85: 891907

Figure 2. Structure of comb-shaped poly(ethylene glycol) PM13 .

showed a high catalytic activity and storage stability in these ionic