Imidazolium bromide-based ionic liquid assisted improved activity of trypsin in cationic reverse micelles

Article abstract of DOI:10.1021/la9040419

The present work reports the imidazolium-based ionic liquids (ILs) assisted enhancement in activity of water-pool solubilized enzyme trypsin in cationic reverse micelles of CTAB. A set of imidazolium ILs (1-alkyl-3-methyl imidazolium bromides) were prepared with varying lengths of their side armwhich results in the differential location of these organic salts in the reverse micelles. The different ILs offered varied activating effects on the biocatalyst. The activity of trypsin improved 30-300% in the presence of 0.1-10 mM of different ILs in reverse micelles of CTAB. Trypsin showed 300% (4-fold) increment in its activity in the presence of IL2 (1-ethyl-3-methyl imidazoliumbromide,EMIMBr) compared to that observed in the absence of IL in CTAB reverse micelles. The imidazolium moiety of the IL, resembling the histidine amino acid component of the catalytic triad of hydrolases and itsBr^- counterion, presumably increases the nucleophilicity of water in the vicinity of the enzyme by forming a hydrogen bond that facilitates the enzyme-catalyzed hydrolysis of the ester.However, the ILs with increasing amphiphilic character had little to no effect on the activity of trypsin due to their increased distance from the biocatalyst, as they tend to get localized toward the interfacial region of the aggregates. Dynamic light scattering experimentationwas carried out in the presence of ILs to find a possible correlation between the trypsin activity and the size of the aggregates. In concurrencewith the observed highest activity in the presence of IL2, the circular dichroism (CD) spectrum of trypsin in CTAB reverse micelles doped with IL 2 exhibited the lowest mean residue ellipticity (MRE), which is closest to that of the native protein in aqueous buffer.

Full text of DOI:10.1021/la9040419

pubs.acs.org/Langmuir
©
2010 American Chemical Society
Imidazolium Bromide-Based Ionic Liquid Assisted Improved Activity of
Trypsin in Cationic Reverse Micelles
Sisir Debnath, Dibyendu Das, Sounak Dutta, and Prasanta Kumar Das*
Department of Biological Chemistry, Indian Association for the Cultivation of Science Jadavpur,
Kolkata - 700 032, India
Received March 9, 2009. Revised Manuscript Received January 18, 2010
The present work reports the imidazolium-based ionic liquids (ILs) assisted enhancement in activity of water-pool
solubilized enzyme trypsin in cationic reverse micelles of CTAB. A set of imidazolium ILs (1-alkyl-3-methyl imidazolium
bromides) were prepared with varying lengths of their side arm which results in the differential location of these organic salts
in the reverse micelles. The different ILs offered varied activating effects on the biocatalyst. The activity of trypsin improved
∼
30-300% in the presence of 0.1-10 mM of different ILs in reverse micelles of CTAB. Trypsin showed ∼300% (4-fold)
increment in its activity in the presence of IL 2 (1-ethyl-3-methyl imidazolium bromide, EMIMBr) compared to that observed
in the absence of IL in CTAB reverse micelles. The imidazolium moiety of the IL, resembling the histidine amino acid
-
component of the catalytic triad of hydrolases and its Br counterion, presumably increases the nucleophilicity of water in the
vicinity of the enzyme by forming a hydrogen bond that facilitates the enzyme-catalyzed hydrolysis of the ester. However, the
ILs with increasing amphiphilic character had little to no effect on the activity of trypsin due to their increased distance from
the biocatalyst, as they tend to get localized toward the interfacial region of the aggregates. Dynamic light scattering
experimentation was carried out in the presence of ILs to find a possible correlation between the trypsin activity and the size of
the aggregates. In concurrence with the observed highest activity in the presence of IL 2, the circular dichroism (CD) spectrum
of trypsin in CTAB reverse micelles doped with IL 2 exhibited the lowest mean residue ellipticity (MRE), which is closest to
that of the native protein in aqueous buffer.
Introduction
applications. Among these confined systems, water-in-oil (w/o)
microemulsions (also known as reverse micelles) are very attrac-
tive hosts for enzyme-catalyzed reactions, because they can solu-
Enzymology in organized assemblies has been an area of wide
research interest for the past few decades because of its potential
3-5
1-5
bilize both hydrophobic and hydrophilic substrates/reactants.
in biotechnological applications. Studies on structure and acti-
vity of enzymes particularly in confined media have always shown
considerable significance from basic research to technological
W/o microemulsions are optically transparent, macroscopically
isotropic, thermodynamically stable nanometer scale aggregates
of water and surfactant in apolar solvent. In our previous studies,
we have shown particularly in the case of surface-active enzymes,
lipase and horseradish peroxidase (HRP), how the efficiency of
these enzymes was progressively improved in cationic w/o micro-
emulsions by changing the microstructural parameters of the self-
organized aggregates including headgroup hydrophilicity, size,
geometry, nature of counterion, and hydrophobic tail length of
*
(
To whom correspondence should be addressed. E-mail: bcpkd@iacs.res.in.
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976, 56, 168. (b) Zana, R.; Lang, J. In Solution Behavior of Surfactants, Mittal, K. L.,
Fendler, E. J., Eds.; Plenum: New York, 1982; Vol. 2. (c) Fendler, J. H. Acc. Chem. Res.
976, 9, 153. (d) Silber, J. J.; Biasutti, M. A.; Abuin, E. B.; Lissi, E. Adv. Colloid
Interface Sci. 1999, 82, 189. (e) Biasutti, M. A.; Abuin, E. B.; Silber, J. J.; Correa,
N. M.; Lissi, E. A. Adv. Colloid Interface Sci. 2008, 136, 1. (f) Stenius, P. Reverse
Micelles, Luisi, P. L., Straub, B. E., Eds.; Plenum: New York, 1984; p 1. (g) Shinshi, M.;
Sugihara, T.; Osakai, T.; Goto, M. Langmuir 2006, 22, 5937. (h) Kimura, M.; Michizoe,
J.; Oakazaki, S.; Furusaki, S.; Goto, M.; Tanaka, H.; Wariishi, H. Biotechnol. Bioeng.
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the surfactant and also by addition of nonionic surfactant.
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Biochimie 2000, 82, 1063. (e) Mitra, D.; Chakraborty, I.; Bhattacharya, S. C.; Moulik, S. P.;
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Aires-Barros, M. R.; Cabral, J. M. S. Langmuir 2000, 16, 3082. (g) Bauduin, P.; Touraud,
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Eur. J. Biochem. 1986, 155, 453. (m) Martinek, K.; Levashov, A. V.; Klyachko, N. L.;
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4
080 DOI: 10.1021/la9040419
Published on Web 02/09/2010
Langmuir 2010, 26(6), 4080–4086

Debnath et al.
Chart 1. Chemical Structure of Ionic Liquids
Article
from Spectrochem (India) was recrystallized three times from
methanol/ether, and the recrystallized CTAB was without mini-
ma in its surface tension plot. HPLC-grade isooctane, n-hexanol,
solvents, and all other reagents used in the syntheses were obtain-
ed from SRL (India) and were of the highest analytical grade.
Amberlyst A-26 bromide ion-exchange resin from Lancaster was
used to convert the iodide salts to their corresponding bromide
1
forms. H NMR spectra were recorded on AVANCE 300 MHz
(
BRUKER) spectrometer. Chemical shifts are reported in ppm,
1
proficient host to exhibit superior activity of structurally deprived
using TMS for H NMR as internal standard. Mass spectrometric
(MS) data were acquired by electrospray ionization (ESI) techni-
ques in Q-TOF Micro-Quadruple Mass Spectrometer, Micro-
7
enzyme-carbon nanotube hybrids.
Although several measures were taken to boost the efficiencies
6
mass, UK. The synthetic procedures for different ionic liquids
(
of surface-active enzymes in reverse micelles, attempts to imp-
1
Chart 1) along with their H NMR, elemental analysis, and mass
rove the activity of water-pool solubilized hydrophilic enzyme like
trypsin havebeen really scarce. Very few attempts have beenmade
to either correlate or enhance the activity of trypsin by increasing
the local molar concentration of water and the size of the water-
spectroscopic data are listed below. The UV-visible absorption
spectra were recorded on a Varian Cary-50 spectrophotometer.
Synthesis of Imidazolium ILs (1-4). Synthesis of IL 1
and 2. 1-Methylimidazole (36.5 mmol) was taken in two round-
bottom flasks each, and excess methyl iodide and ethyl bromide
were separately added to the flasks for the synthesis of 1 and 2,
respectively. The reaction mixtures were stirred for 6 h at room
temperature. After completion of the reaction, the excess alkyl
halides were evaporated, and the residue was washed repeatedly
with ethyl acetate, hexane, and ether to get the iodide salt of 1 and
the bromide salt 2. Then, ∼1 g of the ILs were dissolved in HPLC-
grade methanol and treated with activated charcoal and filtered
through a Celite bed under hot conditions. The procedure was
repeated three times to make the ILs colorless. Finally, the ILs
were concentrated from the filtrate and dried in vacuum at 70 °C
for three days. After purification, the iodide salts of IL 1 and 2
were colorless. The iodide salt of 1 was finally passed through
Amberlyst A-26 bromide ion exchange column to get the IL 1
pool with anincrease in W (mole ratio of water to surfactant) and
0
8
by varying the headgroup size of the surfactant. Recently, we
found that the efficiency of lipase and HRP was enhanced by
almost 100-200% in the mixed reverse micelles of cetyltrimethyl-
ammonium bromide (CTAB) and imidazolium-based surfactants
9
that are popularly known as “surfactant ionic liquids (ILs)”. The
observed enhancement in the enzyme activity with the increasing
amphiphilic character (alkyl chain length) of surfactant ILs is
presumably due to the activating effect of the IL by improving the
nucleophilicity of H O and H O in the vicinity of the enzyme
2
2
2
through hydrogen bonding.
It is well-known that the proteolytic enzyme trypsin with its
hydrophilic character would localize itself deep inside the water-
3
k
pool of reverse micelles. To establish the activating effect of the
ILs, it is necessary to investigate the trypsin activity in the
presence of imidazolium-salt-based ionic liquids (ILs) with high
water solubility. The effect of such imidazolium-based ILs on the
activity of trypsin has not yet been explored in the w/o micro-
emulsion, and moreover, trypsin activity has never been tested in
any IL-in-oil microemulsion.
(bromide salt).
H NMR (300 MHz, D O) of 1 (1,3-dimethyl imidazolium
bromide) δ/ppm = 3.78 (s, 6H), 7.30 (s, 2H). Anal. Calcd for
1
2
C
5
H
9
BrN : C, 33.92; H, 5.12; N, 15.82. Found C, 33.55; H, 5.15;
2
þ
N, 15.47. MS (ESI) m/z. calcd (for C H N the 4° ammonium
ion, 100%) 97.0766, found 97.1400 (M ).
5
9
2
þ
1
H NMR (300 MHz, D
bromide) δ/ppm= 1.40 (t, 3H), 3.79 (s, 3H), 4.09-4.14 (q, 2H),
.31 (m, 1H), 7.38 (m, 1H). Anal. Calcd for C H BrN : C, 37.72;
2
O) of 2 (1-ethyl-3-methyl imidazolium
In the present work, we made an attempt to enhance the
activity of trypsin in the CTAB reverse micelles using varying
concentrations of water-soluble ILs (1-alkyl-3-methyl imidazo-
lium bromides, 1-4, Chart 1) with different alkyl chain length
from C-1 to C-4. Maximum activation in the efficiency of trypsin
was found when concentrations of 1 and 2 were around 0.5-1 mM.
Trypsin showed ∼300% (4-fold) activation in the presence of IL
7
6
11
2
H, 5.80; N, 14.66. Found C, 37.51; H, 6.01; N, 14.52. MS (ESI)
the 4° ammonium ion, 100%) 111.0922,
þ
m/z. calcd (for C
found 111.0523 (M ).
6
H
11
þ
N
2
Synthesis of IL 3. 1-Methylimidazole and propyl bromide
were taken in a round-bottom flask and stirred for 6 h at 60 °C
with a reflux condenser. After the reaction, the excess propyl
bromide was evaporated and the product was purified in similar
ways as described for IL 1 and 2. The IL was dried in vacuum at
2
(1-ethyl-3-methyl imidazolium bromide, EMIMBr) compared to
that observed in only CTAB reverse micelle. However, the activa-
tion effect of imidazolium bromide was not prominent in the case
of ILs 3 (1-propyl-3-methyl imidazolium bromide, PMIMBr) and
7
0 °C for three days.
1
H NMR (300 MHz, D O) of 3 (1-propyl-3-methyl imidazo-
2
lium bromide) δ/ppm = 0.8 (t, 3H), 1.74-1.79 (m, 2H), 3.79
s, 3H), 4.05 (t, 2H), 7.33 (m, 1H), 7.37 (m, 1H). Anal. Calcd for
C H BrN : C, 40.99; H, 6.39; N, 13.66. Found C, 40.71; H, 6.45;
4
(1-butyl-3-methyl imidazolium bromide, BMIMBr). Dynamic
(
light scattering experimentation was carried out over the entire W0
range to find out the influence of ILs having varying side chain
lengths on the size of the aggregates and consequently on the
trypsin activity. Circular dichroism (CD) experiments were carried
out to see the change of secondary structure of the enzymes in the
microemulsions in the presence of different ILs and its correlation
with the trypsin activity.
7
13
2
þ
N, 13.52. MS (ESI) m/z. calcd (for C
ion, 100%) 125.1079, found 125.0568 (M ).
Synthesis of IL 4. IL 4 was synthesized according to a pre-
7
H
13
N
2
the 4° ammonium
þ
1
0
viously published protocol. Briefly, 36.5 mmol of 1-methylimi-
dazole and 40.15 mmol of 1-bromobutane were taken in a round-
bottom flask, and the reaction mixture was heated intermittently
in a microwave oven at 320 W (20 s irradiation with 10 s mixing),
until a clear single phase is obtained. The resulting IL was cooled
and purified in similar ways as described for IL 1 and 2. The IL
Experimental Section
Materials. Trypsin (EC 3.4.21.4) and N-R-benzyloxycarbo-
nyl-L-lysine-p-nitrophenyl ester hydrochloride were purchased
from Sigma, USA, and used as received. Analytical-grade CTAB
was dried in vacuum at 70 °C for three days.
1
H NMR (300 MHz, CDCl ) of 4 (1-butyl-3-methyl imidazo-
lium bromide) δ/ppm=0.97 (t, 3H), 1.40 (m, 2H), 1.91 (m, 2H),
3
4.1 (s, 3H), 4.34 (t, 2H), 7.45 (m, 1H), 7.57 (m, 1H), 10.38 (s, 1H).
(
(
(
7) Das, D.; Das, P. K. Langmuir 2009, 25, 4421.
8) Dasgupta, A.; Das, D.; Das, P. K. Biochimie 2005, 87, 1111.
9) Das, D.; Das, D.; Das, P. K. Biochimie 2008, 90, 820.
(10) Das, D.; Dasgupta, A.; Das, P. K. Tetrahedron Lett. 2007, 48, 5635.
Langmuir 2010, 26(6), 4080–4086
DOI: 10.1021/la9040419 4081

Article
Debnath et al.
Anal. Calcd for C
8
H
15BrN
2
: C, 43.85; H, 6.9; N, 12.78. Found C,
against the substrate concentrations (20-250 μM) and the kcat
(s ) values were determined by fitting that experimental data to a
linearized Michaelis-Menten plot.
þ
-1
4
4
3.55; H, 7.15; N, 12.47. MS (ESI) m/z. calcd (for C
° ammonium ion, 100%) 139.1235, found 139.0744 (M ).
Preparation of Microemulsions. CTAB (910 mg) was taken
8
H
15
N
2
the
þ
UV-vis Spectroscopy. Solubility of ILs in different media
was investigated using UV-vis spectroscopy (Figure S1, Support-
ing Information). ILs 1 (17.7 mg) and 4 (21.9 mg), respectively,
were added to separate volumetric flasks each containing 5 mL of
isooctane, water, and w/o microemulsion of 0.1 M CTAB. All the
solutions were vigorously shaken to ensure the maximum solubi-
lization of ILs in the respective systems. Aliquots from different
media were taken in a 10 mm quartz cell, and the absorbance was
measured in the range 230-400 nm.
Size Determination. Dynamic light scattering (DLS) measure-
ment was performed using a fixed angle apparatus (Brookhaven
Instruments Corporation, USA) equipped with Melles Griot
HeNe-laser (model BI-200SM Goniometer VER-2.0 plus particle
sizeanalyzeroperating at633 nm) with 35 mWpower. The sample
solutions were filtered through 0.22 μm Millipore filters three to
four times before each experiment. The scattering intensity data
collected at different conditions were computed by a data pro-
cessorwith necessary software (Figures S2-S4, Supporting Infor-
mation). The hydrodynamic diameter were obtained for CTAB
w/o microemulsion and CTAB microemulsions in the presence of
in 25 mL (required to prepare 0.1 M solution) isooctane/n-hexa-
nol solution ([n-hexanol]=0.8 M and z=8, where z=[n-hexanol]/
[surfactant]) and allowed to disperse by vortexing in a volumetric
flask. During preparation of IL containing microemulsion, the
required amount of IL was either added directly with CTAB or
added via aqueous buffer solution when the concentration of IL
was too low. To the dispersed suspension of surfactant and IL, the
desired volume of aqueous phosphate buffer (pH=6.0, 20 mM)
was added to obtain a particular W . Finally, the volume was
0
increased up to the mark of the volumetric flask by the isooctane/
n-hexanol and vigorously vortexed to get a macroscopically homo-
geneous solution. The concentrations of the ILs, enzyme, sub-
strate, the cosurfactant (n-hexanol), and CTAB are referred to as
the overall concentration with respect to the total volume of the
reverse micellar solution.
Substrate Stock Solution in Reverse Micelles. Substrate
(N-R-benzyloxycarbonyl-L-lysine-p-nitrophenyl ester hydrochlo-
ride) stock solution (5-62.5 mM) was prepared in HPLC-grade
DMSO. The reaction concentration of substrate (20-250 μM)
was achieved by taking 6 μL of the stock solution to 1.5 mL of the
reacting solution in the cuvette.
0
all the ILs (5 mM) in their entire W range by the DLS measure-
ments.
-3
Enzyme Solution. Trypsin stock solution (1.2ꢀ10 ) M was
prepared in pH=3 buffer solution containing 1 mM CaCl in a
mL volumetric flask. This stock solution of enzyme was diluted
0 times with pH = 6 (20 mM phosphate buffer) solution just
Circular Dichroism. The CD spectra of trypsin in reverse
micelles were taken in Jasco J-815 using a 1 mm path length cell at
wavelength 220-300 nm with a scan speed of 20 nm/min and
accumulated 32 times. All the spectra were corrected by subtract-
ing the corresponding blank spectrum (without enzyme) of 1-4,
respectively, in the reverse micelles to correct any addition effect
originating from the particular IL. Results were expressed in
2
1
1
before the experiment. The final concentration of trypsin (0.32 μM,
.62 μg/mL) was reached by the addition of 4 μL of diluted solu-
tion of enzyme to 1.5 mL of the reaction volume in the cuvette.
7
2
-1
Measurement of Trypsin Activity. The kinetic para-
terms of mean residue ellipticity, MRE (deg cm dmol ). Trypsin
(20 μL, from a stock of 2 mg/mL at pH = 6, 20 mM phosphate
buffer) was added for the preparation of 2 mL reverse micelles at
meters (kcat) for the hydrolysis of N-R-benzyloxycarbonyl-
p-nitrophenylester hydrochloride by the compartmentalized trypsin
0.32 μM) inreversemicellesorinaqueous bufferweredetermined
L-lysine-
(
W = 16 in isooctane/n-hexanol at z = 8 in the absence and
0
spectrophotometrically (on a Cary 50, Varian UV-visible spectro-
photometer) following the formation of p-nitrophenol at the
isosbestic points. The isosbestic points (λiso) and the molar
extinction coefficients (∈) at λiso of the p-nitrophenol/p-nitrophe-
nolate couple in w/o microemulsions of CTAB (0.1 M)/water/
isooctane/n-hexanol at z = 8 were determined spectrophoto-
presence of ILs (5 mM). However, when the CD experiment was
performed at higher concentrations of ILs (>5 mM) in the reverse
micelles, off-scale noise in the spectra was observed. Conse-
quently, the spectra could not be run below 230 nm, as the
imidazolium ions at these concentrations absorb strongly in this
region (below 230 nm). Again, when a lower concentration of the
ILs was used, no significant effect of the ILs on the CD spectra of
enzyme was observed. This is the reason for using a 5 mM
concentration of ILs in the CD experiments so that the spectra
of the enzyme can be measured up to 220 nm without the problem
of off-scale signals. The final overall concentration of protein is
20 μg/mL. This is the lowest possible concentration of the enzyme
used for the CD experiments to get measurable signal intensity.
However, ataneven lower concentration of trypsin, the CD signal
intensitywastooweakto measure. Also, thisconcentration isonly
∼2.5-fold higher than the concentrations of protein used in the
activity measurement study.
-
1
metrically. The λiso and ∈ were found to be 340.6 nm, 4370 M
-1
-1
-1
cm and 340 nm, 6500 M cm , respectively, in CTAB/(water/
isooctane/n-hexanol) in the presence and absence of different
imidazolium ILs and in aqueous solution of pH=6.0. In a typical
experiment, 6 μL of the different substrate stock solutions pre-
pared in HPLC dimethyl sulfoxide were added to 1.5 mL micro-
emulsion or aqueous solution in a cuvette at pH = 6 (here,
pH refers to the pH of the aqueous phosphate buffer solutions
used for the preparation of w/o microemulsions, as the pH within
the water-pool of w/o microemulsions does not vary significantly,
1
1
-4
<
in pH = 6 buffer solution) was added to the cuvette, and the
1 unit). Then, 4 μL of the diluted enzyme stock (1.2ꢀ10 M,
activity of the enzyme was checked in a UV-visible spectro-
Results and Discussion
photometer at λ . The trypsin activity was monitored at pH =
iso
Enzyme and biomolecules solubilized in reverse micelles are
protected from bulk organic solvent by a layer of surfactant mole-
6
rather than at optimum pH (around 8) primarily to avoid the
spontaneous chemical hydrolysis of the substrate, N-R-benzyloxy-
carbonyl- -lysine-p-nitrophenylester hydrochloride in CTAB
3
n
cule without a significant loss of its activity. Enzymes entrapped
in such reverse micelles can be treated as an excellent biomimetic
model for intracellular enzymes. Improvement in the activity of
enzymes entrapped in the w/o microemulsions is always demand-
L
reverse micelles. Experiments at pH=6 also facilitate the opera-
tional stability of the enzyme, as the formation of p-nitrophenol is
most convenient to follow at the steady state. The overall reaction
concentration of trypsin was 0.32 μM (7.62 μg/mL), and the
concentration of substrate was varied from 20 to 250 μM. The
initialvelocitiesoftrypsin-catalyzedhydrolysisofN-R-benzyloxy-
ing because of its extensive potential in technological and bio-
1-5
technological applications.
In this context, we mentioned in
the Introduction that the efficiency of surface-active enzymes was
modulated by the alteration of microstructural parameters of the
self-organized aggregates and also by using surfactant ILs.
Herein, we have tried to improve the activity of water-pool
solubilized hydrophilic enzyme trypsin in cationic reverse micelles
carbonyl-L-lysine-p-nitrophenyl ester hydrochloride were plotted
6-9
(
11) (a) Fletcher, P. D. I.; Robinson, B. H.; Freedman, R. B.; Oldfield, C.
J. Chem. Soc. Faraday Trans. 1985, 81, 2667. (b) Das, P. K.; Chaudhuri, A. Langmuir
999, 15, 8771.
1
4
082 DOI: 10.1021/la9040419
Langmuir 2010, 26(6), 4080–4086

Debnath et al.
Article
by using highly water-soluble imidazolium bromide-based ILs as
activators.
It is expected that the catalytic efficiency of the encapsulated
trypsin would be influenced by the change in the microstructural
parameters of the water-pool in the reverse micelles. The imida-
zole moiety was found to influence the activity of many enzymes
1
0,12,13
by helping in the respective catalytic processes.
To investi-
gate the prospect of the imidazolium bromide ILs as activators for
water-pool solubilized enzyme, the activity of trypsin was exami-
ned in the w/o microemulsions and (water þ IL)-in-oil micro-
emulsions of CTAB (0.1 M)/isooctane/n-hexanol/water in the
presence of 0.1-10 mM ILs (1-4) at z = 8, pH 6.0 (20 mM
phosphate), and room temperature across a varying range of W0
at which isotropic solutions were formed. The solubility of the ILs
in the water phase of the isotropic solutions was examined by
UV-vis spectroscopy (Figure S1, Supporting Information). For
this investigation, the most polar IL having the methyl side chain
(
(
1) and comparatively less polar IL having the n-butyl side chain
4) were employed. These ILs (20 mM) were taken in isooctane,
water, and the w/o microemulsion containing 100 mM CTAB. All
the solutions were vigorously shaken to ensure the maximum
solubilization of ILs in the respective systems. An indistinguish-
able peak of very low intensity at ∼280 nm was observed for the
insoluble ILs in isooctane. However, in the macroscopically
homogeneous solution of water and microemulsions, a peak of
substantial intensity was observed at ∼280 nm. This confirms the
localization of ILs in the water phase of w/o microemulsions.
Initially, we have started the experiment with IL 1, which is
highly soluble in water and expected to be solubilized deep inside
the water-pool. In the absence of any IL, the activity of trypsin
Figure 1. Trypsin activity profile (kcat) in CTAB (0.1 M)/water/
isooctane/n-hexanol (z=8) w/o microemulsions in the presence of
varying concentration of IL 1 across the W range at room tem-
0
perature and pH = 6.0 (20 mM phosphate buffer). In duplicate
experiments, observed kcat values are variable within (5%.
(
IL þ water). With increasing concentration of IL, accommoda-
tion of more water in the same pool is not possible. In fact, in the
presence of more than 10 mM IL there was no formation of w/o
microemulsion (Table S1, Supporting Information).
At this point, we were curious to know the reasons for the
activation. The imidazole moiety is part of the histidine amino
acid, which is an important residue of the catalytic triad of trypsin
and known to participate inhydrogen bonding with water. To this
end, the bromide counterion of the imidazolium IL also has
(
9
kcat) in CTAB reverse micelles was found to improve from 1.7 to
.5 s (Figure 1) with increasing W from 8 to 40, which is
0
-1
consistent with the previous report, as the size of water-pool
increases and the local molar concentration of water reaches the
14
-
strong hydrogen bond-accepting capability. So, Br being the
counterpart of the imidazolium cation presumably helps in the
enhancement of nucleophilicity of water present in the vicinity of
the enzyme through hydrogen bonding, which consequently
8
concentration of bulk water. In the presence of 0.05 and 0.1 mM
of IL 1, trypsin showed an enhancement in the k from ∼3.9 to
cat
-1
8
.9 s (Figure 1) across the similar range of W . Interestingly, the
0
enzyme showed notable enhancement in the activity particularly
13d
facilitates the enzyme-catalyzed hydrolysis of ester. To inves-
-
1
in the lower W range, 8-24. The k increased from 1.7 to 5.3 s
0
-
cat
tigate the role of the imidazolium moiety on the activation of the
enzyme, we checked the trypsin activity in the presence of 1 mM
and 5 mM NaBr keeping all other experimental conditions
identical. No activation of the protein was observed, which
consequently proved the essential presence of the imidazolium
moiety along with the bromide counterion in the improvement of
trypsin activity. Because of its high solubility in water, IL 1 also
possibly participated in the formation of the hydrophilic pool
(water þ IL) of the microemulsion at a concentration of g1 mM.
The IL at such a high concentration in the hydrophilic pool can
interact with the active site of the enzyme through hydrogen
1
and 5.8 to 8.9 s for W =8 and 16, respectively, in the presence
0
of IL 1 compared to that observed in only CTAB microemulsions.
Hence, the activation in the trypsin efficiency was ∼50-200% in
the presence of 0.05 and 0.1 mM of 1. However, in W >24, the
0
kcat was almost comparable in the presence or absence of IL 1 in
CTAB reverse micelles. At a slightly higher concentration of 1
(
0.5 and 1.0 mM), the activation effect of the IL was still
prominent in lower W . However, activity of trypsin decreased
0
a little at W >24. Surprisingly, with further increase in the con-
0
centration of 1 (5 mM), significant deactivation was observed in
the catalytic efficiency of trypsin in CTAB microemulsions
13a
bonding in addition to its hydrogen bonding ability with water.
(
Figure 1). Thus, IL 1 at its lower concentration served as an
Such interaction with the active site of the trypsin by the IL
activator for hydrophilic enzyme trypsin in CTAB reverse micel-
les. Interestingly, as the concentration of the IL increases toward
probably inhibited the catalytic process resulting in poor effi-
13a,d,15
ciency of the enzyme.
On the other hand, an activation
5
mM, shrinkage of the W range was observed. The W range
0 0
effect of imidazolium bromide at the lower concentration was not
decreased from 4 to 40 in the presence of 0.05 mM 1 to 8-24 in the
observed in the higher W range possibly due to its unrestricted
0
presence of 5 mM 1. The decrease in the W range strongly
0
mobility in a bigger water-pool that reduces the hydrogen bond-
ing between the IL and water in the vicinity of the enzyme. Hence,
the nucleophilicity of water around the active site of the enzyme
did not increase.
indicates that the ILs reside in the water phase of the reverse
micelles and participate in the formation of the hydrophilic pool
(
12) (a) Newmyer, S. L.; Ortiz de Montellano, P. R. J. Biol. Chem. 1996, 271,
4891. (b) Newmyer, S. L.; Ortiz de Montellano, P. R. Biochemistry 1996, 35, 12788.
13) (a) Liu, B. K.; Wang, N.; Chen, Z. C.; Lin, X. F. Bioorg. Med. Chem. Lett.
006, 16, 3769. (b) Ganske, F.; Bornscheuer, U. T. Org. Lett. 2005, 14, 3097. (c) Itoh,
T.; Han, S. H.; Matsushita, Y.; Hayase, S. Green Chem. 2004, 6, 437. (d) Liu, B. K.; Wu,
Q.; Xu, J. M.; Lin, X. F. Chem. Commun. 2007, 295.
1
(
(14) (a) Swatloski, R. P.; Spear, S. K.; Holbrey, J. D.; Rogers, R. D. J. Am.
Chem. Soc. 2002, 124, 4974. (b) Fukaya, Y.; Hayashi, K.; Wadab, M.; Ohno, H. Green
Chem. 2008, 10, 44.
2
(15) Baker, G. A.; Heller, W. T. Chem. Eng. J. 2009, 147, 6.
Langmuir 2010, 26(6), 4080–4086
DOI: 10.1021/la9040419 4083

Article
Debnath et al.
Figure 3. Trypsin activity profile (kcat) in CTAB (0.1 M)/water/
isooctane/n-hexanol (z=8) w/o microemulsions in the presence of
varying concentration of IL 3 across the W range at room tem-
0
perature and pH = 6.0 (20 mM phosphate buffer). In duplicate
experiments, observed kcat values are variable within (5%.
Figure 2. Trypsin activity profile (kcat) in CTAB (0.1 M)/water/
isooctane/n-hexanol (z=8) w/o microemulsions in the presence of
varying concentration of IL 2 across the W range at room tem-
0
perature and pH = 6.0 (20 mM phosphate buffer). In duplicate
experiments, observed kcat values are variable within (5%.
Interestingly, trypsin showed remarkable enhancement in its
activity in the CTAB reverse micelles in the presence of the next
higher homologue IL, 2, under identical experimental conditions
as those used for 1 (Figure 2). Here, also, the activating effect is
more prominent in the lower W range where we have found
0
∼
200-300% (3-4-fold) enhancement in the enzyme efficiency as
-1 -1
the k increased from 1.7 to 7.2 s and 5.8 to 16.3 s at W =
cat
0
8
and 16, respectively, at concentrations of 0.5 and 1.0 mM of 2.
Importantly, trypsin also showed higher activity at W >24 in the
0
presence of varying concentration of 2 compared to that observed
for only CTAB reverse micelles. For example, at W =40, in the
0
-
1
presence of 2, the k varied from 10.3 to 15.7 s , which was
9
cat
-1
.5 s in the absence of any IL (Figure 2). Interestingly, the acti-
vation effect in higher W range offered by the IL 2 is sufficiently
0
high in contrast to the observed comparable or lowered activity of
trypsin in the presence of 1 (Figure 1). This activation effect
increased with increasing concentration of 2 and became satu-
rated at ∼1 mM, after which no remarkable improvement in the
catalytic efficiency of trypsin was observed. Grossly, trypsin
activity improved ∼30-300% in the presence of 0.1-10 mM of
Figure 4. Trypsin activity profile (kcat) in CTAB (0.1 M)/water/
isooctane/n-hexanol (z = 8) w/o microemulsions in the presence of
varying concentration of IL 4 across the W range at room tem-
0
perature and pH = 6.0 (20 mM phosphate buffer). In duplicate
experiments, observed kcat values are variable within (5%.
2
(Figure 2). The activation effect of this IL is obviously caused by
the same mechanism discussed in the preceding paragraph.
However, surprisingly no deactivation in the efficiency of trypsin
was noted at a higher concentration of 2. A very minute increase
in the dimension of the ILs from 1,3-dimethyl imidazolium (1) to
comparable to or even lower than that observed in only CTAB
reverse micelles (Figure 3). Similar results were also found for IL 4
having the longest chain length in our study. IL 4 also showed no
1
-ethyl-3-methyl imidazolium (2) probably hinders the hydrogen
bonding of the latter with the active site of enzyme. Additionally,
activation of trypsin at W > 8 (Figure 4). In most of the cases
0
the water-pool localization of 2 was also evident from a similar
with varying concentrations of 4, the activity profile of trypsin
remained almost comparable to the activity profile in CTAB
(Figure 4). Only in higher concentration of 4 and at higher range
shrinkage of the W range as observed for 1 (Table S1, Supporting
0
Information). The W0 range decreased from 4 to 40 in the
presence of 0.1 mM 1 to 8-20 in the presence of 20 mM 2.
As the enzyme activity was found to increase from IL 1 (C-1) to
IL 2 (C-2), we were very curious to see whether the higher
homologue ILs with C-3 (3) and C-4 (4) chain length could
further increase the activity of trypsin. However, the results were
not as encouraging as observed for 1 and 2. There was ∼2-3-fold
of W
did activity of trypsin drop to a certain extent. However, the
=8 was possibly due
0
observed activation effect only at lowest W
0
to the confinement of IL and enzyme in a very small-sized water-
pool. This compelled the BMIMBr and water to come into
vicinity of the enzyme resulting in enhanced nucleophilicity of
water through hydrogen bonding. In general, ILs 3 and 4 did not
exhibit notable activation effect on the activity of trypsin pre-
sumably due to their increasing amphiphilic character with inc-
rease in chain length. Accordingly, they prefer to locate themselves
enhancement in the trypsin activity only at W = 8 in varying
0
concentrations (0.1-10 mM) of 3 and 4. In other W range up to
0
4
0, with increasing concentration of 3, trypsin activity was either
4
084 DOI: 10.1021/la9040419
Langmuir 2010, 26(6), 4080–4086

Debnath et al.
Article
Figure 5. Pictorialrepresentationofthe activating effectofimidazoliumbromide ILontrypsin activityatthe water poolofthe CTABreverse
micelles.
toward the interfacial region of the aggregates instead of deep
inside the water-pool as expected for ILs 1 and 2 (Figure 5). This
possible partition of ILs 3 and 4 between the water-pool and the
interfacial region of w/o microemulsions might have increased the
distancebetween the IL and the waterin the vicinity of the trypsin,
which reduces the hydrogen bonding among them (Figure 5). As a
result, no improvement in the nucleophilicity of the water in the
vicinity of the enzyme is possible. The localization of these ILs
(3 and 4) toward the interfacial region of the water phase was fur-
ther confirmed from their minimized shrinkage effects on the W0
range of the CTAB microemulsion. The formation of isotropic
solutions was possible even when the ILs 3 and 4 are present at
concentrations of 30 and 50 mM, respectively (Table S1, Support-
ing Information).
Overall among all the imidazolium-based ILs, trypsin showed
-1
its highest activity (kcat=16.3 s ) in the presence of 2 at its con-
centration of 1 mM and W =16. Also in other concentrations
0
and W , IL 2 always had a better activation effect than others. IL
0
2
probably possesses the perfect balance between size and water
solubility that is suitably fitted for hydrogen bonding with the
water in the vicinity of enzyme, but not with the active site of
trypsin. Hence, the activity of trypsin was found to be maximum
in the presence of IL 2.
h 0
Figure 6. Variationofthehydrodynamicdiameter(D ) withW in
different CTAB-based w/o microemulsions in the presence of
5 mM IL 1-4.
To investigate the effects of the ILs with varying side chain
lengths on the hydrophilic (water þ IL) pool size, we have
becomes more significant as the side chain lengths of the ILs
decreases. The D values of the systems containing the larger side
h
calculated the hydrodynamic diameter (D ) of the reverse micelles
chain analogues (for C-3 and C-4, Figure 6), showed a compara-
tively lower increase with respect to the CTAB systems alone. On
the other hand, for the smaller ILs the size of the aggregate inc-
h
through dynamic light scattering (DLS) experiments. Initially, the
aggregated sizes of the CTAB-based reverse micelles in the
absence of ILs were investigated throughout its W range from
reases sharply reaching up to a maximum value of D =15.5 nm
0
h
8
to 40. The D was found to increase with the rise in the water
at W =24 for IL 1. Strikingly, this value is even higher than the
h
0
content of the system (D increased from 4.1 to 13.0 nm, Figure 6).
dimension of the CTAB reverse micellar aggregates at the highest
h
This is in agreement with the works reported by other authors,
which has shown that the increase in the water content in the
reverse micellar media, keeping the concentration of the surfac-
tants fixed, results in a concomitant increase of the size of the aggre-
gate. In the presence of ILs in the reverse micelles of CTAB,
there was an overall increase of the aggregate sizes for all the ILs
used compared to that of only CTAB reverse micelles at compa-
W value of 40. These results conclusively prove that size of
0
reverse micelles as a whole increases in the presence of ILs, and
particularly, the smaller-sized ILs (C-1 and C-2 analogue) have
significant influence in modulating the dimension of the CTAB-
5
d
reverse micellar aggregates. Therefore, the highest D observed
h
for ILs 1 and 2 throughout their W range strongly implies that
0
these ILs are the most polar and localized inside the water-pool.
Consequently, the hydrophilic pool of water and IL was formed,
rable W (Figure 6). However, the increase in the aggregate size
0
Langmuir 2010, 26(6), 4080–4086
DOI: 10.1021/la9040419 4085

Article
Debnath et al.
reverse micelles of CTAB with IL 2. Thus, in addition to its
activating effect by means of hydrogen bonding with the water in
the vicinity of the enzyme, the secondary structure of trypsin was
also preserved in the larger reverse micelles in the presence of 2
(
Figure 6). This is in complete concurrence with the observed
highest activity of trypsin in the reverse micelles of CTABþIL 2
Figure 2). However, the spectra of the protein in systems
(
CTABþIL 3, CTABþIL 4, and only CTAB are close to each
other but have less negative value compared to the native
conformation of trypsin in aqueous buffer (Figure 7). This is also
in nice agreement with the activity of trypsin, as the observed
kcat is comparable in all three (CTAB, CTAB þ IL 3, and CTAB þ
IL 4) w/o microemulsions (Figures 3 and 4) and also did not have
a notable activation effect as observed in the case of 2. In the
presence of 5 mM 1, the trypsin was found to be deactivating,
which is reflected by its less negative MRE value in the CD spectra
(
Figure 7). Interestingly, in aqueous buffer solution the MRE
values of trypsin were almost the same in the presence of any of
the ILs 1-4 or in the absence of IL. This also corresponds to the
activity of trypsin in aqueous solution that was found to be
unaffected by the presence of ILs 1-4 even up to 20 mM
concentration (data not shown). This result clearly indicates that
the imidazolium bromide-assisted activation effect on trypsin
takes place only when they are in close proximity in a confined
system like reverse micelles.
Figure 7. CD spectra of trypsin in CTAB (0.1 M)/water/isooctane/
n-hexanol w/o microemulsions at W
phosphate) in the presence of different ILs at the concentration of
mM.
0
= 16, z=8,pH=6.0(20mM
5
which obviously has an impact on altering the size of the reverse
micelles. As a result, their concurrent effect on the activation of
tryspin, which resides in the deep of the water pool, also increases.
Additionally, these results also prove that the location of the ILs is
in the water phase of the reverse micelles, as there was a greater or
lesser increment of the water-pool dimension in the case of all the
ILs. This observation corroborateswellwith the literature that the
size of the reverse micellar aggregates increases with the increase
Conclusion
In this work, we have exploited the activating effect of the
water-soluble imidazolium bromide ILs on hydrophilic enzyme
trypsin in the reverse micelles of CTAB. The presence of water-
soluble IL in CTAB-based w/o microemulsions makes it a better
host of trypsin over the microemulsions formed by CTAB only.
The side-chain lengths of the ILs were varied, and the best
performance of trypsin was observed in the system having the
IL with C-2 tail length (2). Optimum activation from the
imidazolium bromide-assisted enzyme hydrolysis was observed
in ∼0.5-1 mM concentration of the organic salts. The activity of
trypsin in the presence of 2 was significantly higher (3-4-fold)
compared to that observed in only CTAB w/o microemulsion.
The IL 2 is the best activator of trypsin because of its perfect
balance in size and water-pool solubility. 1-Ethyl-3-methyimida-
zolium bromide (2) can suitably offer its activating effect to the
trypsin by improving the nucleophilicity of water in the vicinity of
the enzyme through hydrogen bonding and also by preserving the
secondary structure in the larger-sized reverse micelles. Thus, our
present investigation leads to the first use of water-soluble
imidazolium-bromide IL as activator for trypsin, maximizing its
efficiency in CTAB reverse micelles.
1
6
of the polar components. This was also found to be true for the
1
7
different IL-in-oil microemulsions reported in the literature.
The smaller size obtained for 3 and 4 is presumably because of the
partitioning of these ILs between water-pool and interface, and
consequently, the ILs move away from the enzyme (Figure 5) as
the size of the arm length increases. Hence, these two ILs have
little impact on the activity of trypsin in w/o microemulsion.
At this point, we were interested in understanding the influence
of the synergistic effect of imidazolium ILs (1-4) and the size of
the hydrophilic pool on the secondary structure of trypsin, if any,
and finding a correlation between conformational change and
activity. The CD signal in the far-UV region and the mean residue
1
8
ellipticity (MRE) indicate the secondary structure of the protein.
The concentration of enzymes used in CD experimentation was
0 μg/mL at W =16 to get a reasonable CD signal, and 5 mM of
2
0
ILs was used. To compare the observed secondary structural
1
8d
changes with the native structure of trypsin, the spectra of the
protein was also measured in 20 mM (pH=6) phosphate buffer.
The spectrum in buffer had the most negative MRE value of all
the systems (Figure 7). Interestingly, among all four ILs, the MRE
value of trypsin decreased to a maximum extent in the presence of
IL 2 in CTAB reverse micelles (Figure 7) and reached closest to
the value observed for protein in buffer. This result strongly
indicates that trypsin possesses native-like conformation in the
Acknowledgment. P.K.D. is thankful to Department of
Science and Technology, India, for financial assistance through
the Ramanna Fellowship (No. SR/S1/RFPC-04/2006). S.D.,
D.D., and S.D. acknowledge Council of Scientific and Industrial
Research, India, for their Research Fellowships.
(
16) Gao, Y.; Li, N.; Zheng, L.; Zhao, X.; Zhang, J.; Cao, Q.; Zhao, M.; Li, Z.;
Zhang, G. Chem.;Eur. J. 2007, 13, 2661.
17) (a) Zech, O.; Thomaier, S.; Bauduin, P.; Ruck, T.; Touraud, D.; Kunz, W.
J. Phys. Chem. B 2009, 113, 465. (b) Gao, Y.; Voigt, A.; Hilfert, L.; Sundmacher, K.
ChemPhysChem 2008, 9, 1603.
Supporting Information Available: W range of CTAB in
0
(
the presence of different ILs and the UV spectrum of the IL
in the various solvents. Representative DLS data of the
aggregate size distribution in different w/o microemulsions
in the presence and absence of ILs. This material is available
free of charge via the Internet at http://pubs.acs.org.
(
18) (a) Walde, P.; Peng, Q.; Fandavis, W. N.; Battistel, E.; Luisi, P. L. Eur. J.
Biochem. 1988, 173, 401. (b) Peng, Q.; Luisi, P. L. Eur. J. Biochem. 1990, 188, 471.
c) Naoe, K.; Takeuchi, C.; Kawagoe, M.; Nagayama, K.; Imai, M. J. Chromatogr. B
007, 850, 277. (d) Ghosh, S.; Banerjee, A. Biomacromolecules 2002, 3, 9.
(
2
4
086 DOI: 10.1021/la9040419
Langmuir 2010, 26(6), 4080–4086