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Methods For Stabilizing and Activating Enzymes
Methods For Stabilizing and Activating Enzymes
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
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891
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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
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
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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
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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
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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
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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
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[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).
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
[BMIM][BF4 ]
[BMIM][PF6 ]
proteinase K
immobilized onto
single-walled carbon
nanotubes (SWNTs)
[BMIM][BF4 ]
[BMIM][OTf]
[BMIM][PF6 ]
[OMIM][PF6 ]
[HMIM][PF6 ]
[BMIM][PF6 ] (and
hexane)
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) 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
(1) transesterification of
N-acetyl-Lphenylalanine ethyl
ester and 1-propanol;
(2) hydrolysis of
N-succinyl-L-ala-L-alaL-pro-L-phe-pnitroanilide
(1) transesterification of
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
93
Enzyme-SWNT
conjugates exhibited
higher catalytic
turnover and higher
thermal stability than
free form.
108
109
The immobilized
exhibited a higher
catalytic activity and
thermal stability.
112
130
131,132
134
157,158
39
895
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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)
transesterification of
2-phenyl-1-propanol
with vinyl acetate; (3)
transesterification of
N-acetyl-Lphenylalanine ethyl
ester with 1-butanol
84,85,163
Enzyme precipitated
and rinsed with
n-propanol(EPRP)
[BMIM][PF6 ]
[BMIM][BF4 ] (and
n-octane)
(1) esterification of
N-acetylphenylalanine
with ethanol; (2)
transesterification of
N-acetyl-Lphenylalanine ethyl
ester with n-propanol
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
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
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
solvent free
Novozym 435
Lipozyme RM IM
Lipozyme TL IM lipase
PS-D lipase AK20
transesterification of
various alcohols with
vinyl acetate at room
temperature
(1) transesterification of
1-phenylethanol and
vinyl acetate; (2)
transesterification of
() -3hydroxypentanenitrile
and vinyl acetate; (3)
transesterification of
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
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
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.
2527
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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).
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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
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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
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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
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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
Cocos +
N
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Et +
N
OH
MeSO4-
OH
HO
OH
CH3COO-
Et
Tallow
+
N
Et
Et +
N
OH
OH
EtSO4HO
OH
H2PO4-
Et
+
N
R'
MeSO4O
R'
O
n
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-
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901
+
N
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H 3C
O
n
H3C
OAc-
+
N(CH2CH3)3 OAcn
902
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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.
REFERENCES
1 Gordon CM, New developments in catalysis using ionic liquids. Appl
Catal A: Gen 222:101117 (2001).
2 Houlton S, Ionic liquids: the route to cleaner and more efficient fine
chemical synthesis? Chem Week Feb 25:s10s11 (2004).
3 Seddon KR, Ionic liquids for clean technology. J Chem Technol
Biotechnol 68:351356 (1997).
4 Welton T, Room-temperature ionic liquids solvents for synthesis
and catalysis. Chem Rev 99:20712083 (1999).
5 Zhao H and Malhotra SV, Applications of ionic liquids in organic
synthesis. Aldrichimica Acta 35:7583 (2002).
6 Earle M, Forestier A, Olivier-Bourbigou H and Wasserscheid P,
Organic synthesis, in IonicLiquidsinSynthesis, ed by Wasserscheid P
and Welton T. Wiley-VCH Verlag, Weinheim, pp 174288 (2003).
7 Jain N, Kumar A, Chauhan S and Chauhan SMS, Chemical and
biochemical transformations in ionic liquids. Tetrahedron
61:10151060 (2005).
8 Wasserscheid W and Keim W, Ionic liquids new solutions for
transition metal catalysis. AngewChemIntEd 39:37723789 (2000).
9 Wasserscheid P and Welton T, Ionic Liquids in Synthesis, 1&2. WileyVCH, Weinheim (2008).
10 Endres F and Welton T, Inorganic synthesis, in Ionic Liquids in
Synthesis, ed. by Wasserscheid P and Welton T. Wiley-VCH Verlag,
Weinheim, pp 289318 (2003).
11 Husum TL, Jorgensen CT, Christensen MW and Kirk O, Enzyme
catalysed synthesis in ambient temperature ionic liquids. Biocatal
Biotransform 19:331338 (2001).
12 Kragl U, Eckstein M and Kaftzik N, Enzyme catalysis in ionic liquids.
Curr Opin Biotechnol 13:565571 (2002).
13 Park S and Kazlauskas RJ, Biocatalysis in ionic liquids advantages
beyond green technology. Curr Opin Biotechnol 14:432437
(2003).
14 Sheldon RA, Lau RM, Sorgedrager MJ, van Rantwijk F and Seddon KR,
Biocatalysis in ionic liquids. Green Chem 4:147151 (2002).
15 van Rantwijk F, Madeira Lau R and Sheldon RA, Biocatalytic
transformations in ionic liquids. Trends Biotechnol 21:131138
(2003).
16 van Rantwijk F and Sheldon RA, Biocatalysis in ionic liquids. Chem
Rev 107:27572785 (2007).
17 Kubisa P, Application of ionic liquids as solvents for polymerization
processes. Prog Polymer Sci 29:312 (2004).
18 Carmichael AJ and Haddleton DM, Polymer synthesis in ionic liquids,
in Ionic Liquids in Synthesis, ed. by Wasserscheid P and Welton T.
Wiley-VCH Verlag, Weinheim, pp 319335 (2003).
19 Brennecke JF and Maginn EJ, Purification of gas with liquid ionic
compounds. US 6,579,343 (2003).
20 Zhao H, Xia S and Ma P, Use of ionic liquids as green solvents for
extractions. J Chem Technol Biotechnol 80:10891096 (2005).
www.soci.org
63 Schroder
U, Wadhawan JD, Compton RG, Marken F, Suarez PAZ,
Consorti CS et al, Water-induced accelerated ion diffusion:
voltammetric studies in 1-methyl-3-[2,6-(S)-dimethylocten-2yl]imidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium
tetrafluoroborate and hexafluorophosphate ionic liquids. New
J Chem 24:10091015 (2000).
64 Dupont J, On the solid, liquid and solution structural organization of
imidazolium ionic liquids. J Brazil Chem Soc 15:341350 (2004).
65 Rejasse B, Besson T, Legoy M-D and Lamare S, Influence of microwave
radiation on free Candida antarctica lipase B activity and stability.
Org Biomol Chem 4:37033707 (2006).
66 Zhao H, Effect of ions and other compatible solutes on enzyme
activity, and its implication for biocatalysis using ionic liquids.
J Mol Catal B: Enzym 37:1625 (2005).
67 Zhao H, Campbell S, Jackson L, Song Z and Olubajo O, Hofmeister
series of ionic liquids: kosmotropic effect of ionic liquids on the
enzymatic hydrolysis of enantiomeric phenylalanine methyl ester.
Tetrahedron: Asymmetry 17:377383 (2006).
68 Zhao H, Jackson L, Song Z and Olubajo O, Enhancing protease
enantioselectivity by ionic liquids based on chiral- or -amino
acids. Tetrahedron: Asymmetry 17:15491553 (2006).
69 Zhao H, Jackson L, Song Z and Olubajo O, Using ionic liquid
[EMIM][CH3 COO] as an enzyme-friendly co-solvent for resolution
of amino acids. Tetrahedron: Asymmetry 17:24912498 (2006).
70 Zhao H, Are ionic liquids kosmotropic or chaotropic? An evaluation
of available thermodynamic parameters for quantifying the
www.interscience.wiley.com/jctb
903
30 Kohler
S and Heinze T, Efficient synthesis of cellulose furoates in 1-Nbutyl-3-methylimidazolium chloride. Cellulose 14:489495 (2007).
31 Heinze T, Schwikal K and Barthel S, Ionic liquids as reaction medium
in cellulose functionalization. Macromol Biosci 5:520525 (2005).
32 Schlufter K, Schmauder H-P, Dorn S and Heinze T, Efficient
homogeneous chemical modification of bacterial cellulose in the
ionic liquid 1-N-butyl-3-methylimidazolium chloride. Macromol
Rapid Commun 27:16701676 (2006).
33 Barthel S and Heinze T, Acylation and carbanilation of cellulose in
ionic liquids. Green Chem 8:301306 (2006).
34 Biswas A, Shogren RL, Stevenson DG, Willett JL and Bhowmik PK,
Ionic liquids as solvents for biopolymers: acylation of starch and
zein protein. Carbohydr Polym 66:546550 (2006).
35 Remsing RC, Swatloski RP, Rogers RD and Moyna G, Mechanism
of cellulose dissolution in the ionic liquid 1-n-butyl-3methylimidazolium chloride: a 13 C and 35/37 Cl NMR relaxation
study on model systems. Chem Commun 12711273 (2006).
36 Remsing RC, Hernandez G, Swatloski RP, Massefski WW, Rogers RD
and Moyna G, Solvation of carbohydrates in N,N dialkylimidazolium ionic liquids: a multinuclear NMR spectroscopy
study. J Phys Chem B 112:1107111078 (2008).
37 Novoselov NP, Sashina ES, Petrenko VE and Zaborsky M, Study of
dissolution of cellulose in ionic liquids by computer modeling.
Fibre Chem 39:153158 (2007).
38 Turner MB, Spear SK, Huddleston JG, Holbrey JD and Rogers RD, Ionic
liquid salt-induced inactivation and unfolding of cellulase from
Trichoderma reesei. Green Chem 5:443447 (2003).
39 Toral AR, de los Ros AP, Hernandez FJ, Janssen MHA, Schoevaart R,
van Rantwijk F et al, Cross-linked Candida antarctica lipase B
is active in denaturing ionic liquids. Enzyme Microb Technol
40:10951099 (2007).
40 de los Ros AP, Hernandez-Fernandez FJ, Martnez FA, Rubio M and
Vllora G, The effect of ionic liquid media on activity, selectivity
and stability of Candida antarctica lipase B in transesterification
reactions. Biocatal Biotransform 25:151156 (2007).
41 Yang Z and Pan W, Ionic liquids: green solvents for nonaqueous
biocatalysis. Enzyme Microbiol Technol 37:1928 (2005).
42 Carmichael AJ and Seddon KR, Polarity study of some 1-alkyl-3methylimidazolium ambient-temperature ionic liquids with the
solvatochromic dye, Nile Red. J Phys Org Chem 13:59195 (2000).
43 Aki SNVK, Brennecke JF and Samanta A, How polar are roomtemperature ionic liquids? Chem Commun 413414 (2001).
44 Reichardt C, Solvatochromic dyes as solvent polarity indicators. Chem
Rev 94:23192358 (1994).
45 Park S and Kazlauskas RJ, Improved preparation and use of roomtemperature ionic liquids in lipase-catalyzed enantio- and
regioselective acylations. J Org Chem 66:83958401 (2001).
46 Lozano P, de Diego T, Guegan J-P, Vaultier M and Iborra JL,
Stabilization of -chymotrypsin by ionic liquids in
transesterification reactions. BiotechnolBioeng 75:563569 (2001).
www.soci.org
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
904
www.interscience.wiley.com/jctb
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
H Zhao
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
www.interscience.wiley.com/jctb
905
141
www.soci.org
www.soci.org
906
www.interscience.wiley.com/jctb
H Zhao
www.soci.org
211 Forsyth SA, Batten SR, Dai Q and MacFarlane DR, Ionic liquids based
on imidazolium and pyrrolidinium salts of the tricyanomethanide
anion. Austr J Chem 57:121124 (2004).
212 Anderson JL, Ding R, Ellern A and Armstrong DW, Structure and
properties of high stability geminal dicationic ionic liquids.
J Am Chem Soc 127:593604 (2005).
907
www.interscience.wiley.com/jctb