Extraction of an active enzyme by self-buffering ionic liquids: A green medium for enzymatic research

Article abstract of DOI:10.1039/c6ra00607h

In this work, the extraction of a model enzyme α-chymotrypsin in its active conformation from an aqueous solution by using new biocompatible and self-buffering Good's buffer ionic liquids (GBILs)-based aqueous biphasic systems (ABSs) is demonstrated. The experimental liquid-liquid phase boundary data for these new GBIL-based ABSs were measured at 298.2 K. These new ionic liquids are derived from biological Good's buffers as anions and tetra-alkylammonium as cations. The buffering potential of these GBILs was confirmed by measuring their pH profiles and protonation constants (pK_a) in aqueous solution by a potentiometric method at various temperatures. Moreover, these GBILs provide a greater stabilizing effect on the activity of α-chymotrypsin, as compared with the corresponding Good's buffers. Therefore, the new self-buffering GBILs may not only serve as a biocompatible medium for enzymatic research, but also avoid the use of an external buffering agent, which is often needed for pH control in the case of using a conventional ILs-based medium. Therefore, these new biocompatible GBILs are more advantageous over the conventional ILs-based media for enzymatic research.

Full text of DOI:10.1039/c6ra00607h

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Extraction of an active enzyme by self-buffering
ionic liquids: a green medium for enzymatic
Cite this: RSC Adv., 2016, 6, 18567
research†
a
Bhupender S. Gupta,a Mohamed Tahaab and Ming-Jer Lee
*
In this work, the extraction of a model enzyme a-chymotrypsin in its active conformation from an aqueous
solution by using new biocompatible and self-buffering Good's buffer ionic liquids (GBILs)-based aqueous
biphasic systems (ABSs) is demonstrated. The experimental liquid–liquid phase boundary data for these new
GBIL-based ABSs were measured at 298.2 K. These new ionic liquids are derived from biological Good's
buffers as anions and tetra-alkylammonium as cations. The buffering potential of these GBILs was
confirmed by measuring their pH profiles and protonation constants (pKa) in aqueous solution by
a potentiometric method at various temperatures. Moreover, these GBILs provide a greater stabilizing
effect on the activity of a-chymotrypsin, as compared with the corresponding Good's buffers. Therefore,
the new self-buffering GBILs may not only serve as a biocompatible medium for enzymatic research, but
also avoid the use of an external buffering agent, which is often needed for pH control in the case of
Received 8th January 2016
Accepted 5th February 2016
DOI: 10.1039/c6ra00607h
using
a conventional ILs-based medium. Therefore, these new biocompatible GBILs are more
www.rsc.org/advances
advantageous over the conventional ILs-based media for enzymatic research.
emerging as a potential replacement for the conventional volatile
1. Introduction
organic solvents that are frequently used in various indus-
tries.34–36 In the last decades, number of reports37–43 mentioned
that ILs, either in pure or in aqueous state, can be served as
biocompatible media to biomolecules such as deoxyribonucleic
acid (DNA), protein, and enzyme and also can stabilize biomol-
ecules' structures against external or internal impetus. In addi-
tion, the applications of ILs as a potential biocatalyst were also
extensively studied.44,45
Biomolecules are highly sensitive system and can be func-
tionally active only in their optimum pH range. External buff-
ering compound is oen needed to maintain the pH values of
media in a suitable range. In a current practice, phosphate-
based buffer is recommended to maintain the required pH of
biological media due to its protein stabilizing tendency.46
However, the complexation of phosphate ions with metal ions
such as magnesium, zinc, and calcium was reported in litera-
ture.47 Since these metal ions are commonly found in various
proteins and therefore, phosphate-based buffer solutions
possess the possibility to interfere with the proteins.
Many efforts have been made on designing the task specic
ionic liquids for biological research, but very few studies48–53
focused on designing ILs with self-buffering potential. Ou et al.48
reported three imidazolium based ionic liquids, 1-butyl-3-
methylimidazolium hydrogen phosphate [BMIM][HP], 1-octyl-3-
methylimidazolium hydrogen phosphate [OMIM][HP], and 1-
butyl-3-methylimidazolium hydrogen tartrate [BMIM][HT] with
self-buffering potential. These ionic liquids are suitable to
maintain pH values in highly acidic region (pH ꢁ 3–4). Since
The use of enzymes has been proven to achieve higher selectivity
and better yields of the nal products at comparatively milder
conditions of temperature and pressure. However, their high
sensitivity towards external factors, such as biocompatibility and
pH value, still limits their industrial applications. Recently, ionic
liquids (ILs), a new class of environmentally friendly solvents,
have been identied as a favorable biocompatible medium to
maintain the active conformation for enzymes or proteins.1–7
Basically, ionic liquids are entirely composed of ions, but exist as
liquids below the boiling point of water (100 ꢀC).8–14 ILs have
special physical and chemical properties, such as negligible or
very low vapor pressure, low ammability, high inherent
conductivities, high thermal stability, existing as liquid state over
a wide temperature range, easy recycling, and being a good
solvent for a wide range of organic and inorganic chemical
compounds.15–19 At present, ILs are widely used in many research
elds, including electrochemistry,20–22 organic chemistry,23,24
catalysis,25–27 engineering process,28–32 and pharmaceutical
applications.33 Thus, they are regarded as future solvents and
aDepartment of Chemical Engineering, National Taiwan University of Science and
Technology, 43 Keelung Road, Section 4, Taipei 106-07, Taiwan. E-mail: mjlee@
mail.ntust.edu.tw
b
´
Departamento de Quımica, CICECO, Universidade de Aveiro, 3810-193 Aveiro,
Portugal
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c6ra00607h
This journal is © The Royal Society of Chemistry 2016
RSC Adv., 2016, 6, 18567–18576 | 18567

View Article Online
RSC Advances
Paper
most of the biological researches are conducted in the physio- enzyme a-chymotrypsin (a-CT). We chose a-CT as a model
logical pH range of 7 to 9, therefore those ionic liquid buffers enzyme because of its unique structural and functional
cannot be used in the biological researches. MacFarlane et al.49 importance. a-CT is commonly used for exploring the mecha-
investigated
three
ionic
liquid
buffers,
choline nism of protein folding or unfolding in the presence of cosol-
dihydrogenphosphate/choline hydrogen phosphate, choline vents.61,62 Overall, converting Good's buffers into ionic liquids
tartrate, and hydrated lysine, which can help to maintain pH not only provides a suitable biocompatible medium with
values in a variety of applications of ionic liquids. However they comparable self-buffering capability, avoiding the use of
did not systematically determined pKa values of these ionic liquid external buffering compound, but also may help in the extrac-
buffers experimentally. Matias et al.50 synthesized seven choline tion and purication of the enzyme.
derivatives by using of Good's buffers (BES, DIPSO, CAPS, EPPS,
MES, TAPSO, and MOPSO) as anions. It was found that the pH
can be maintained from 5.33 to 11.82 in aqueous solution by
2. Experimental section
2.1 Materials
varying the buffer anions. Since the synthesized choline deriva-
tives appear as salts instead of ionic liquids, the application of
those buffers in enzymatic research may be limited to the only
buffering compounds. Moreover, neither the ATPS forming
tendency nor the extraction efficiency for biomolecules by those
choline salts were studied. Recently, Taha et al.51,52 synthesized
similar self-buffering ionic liquids derived from the biological
buffers TES, MES, HEPES, CHES, and tricine; however, their
GBILs did not cover the whole pH range of enzymatic reactions.
The buffering range of our new GBILs will cover pH from 6 to 12,
thus these GBILs can be used in enzymatic reactions although
higher pH values are needed.
In the present study, we attempt to synthesize some new self-
buffering ILs, derived from the commonly used Good's buffers
(GB) as anions and the ammonium ions with various chain
lengths as cations. The selected Good's buffers, N-[tris(hydroxy-
methyl)methyl]-3-aminopropanesulfonic acid (TAPS), 3-(N-mor-
pholino)propanesulfonic acid (MOPS), 4-(2-hydroxyethyl)-1-
piperazinepropanesulfonic acid (EPPS), 3-(cyclohexylamino)-1-
propanesulfonic acid (CAPS), and N,N-bis(2-hydroxyethyl)-
glycine (BICINE) are amino acid derivatives and are widely used
in biological eld. Each of these compounds exists as a zwitterion
in aqueous solution and thus can be used as a cation or anion. In
our previous researches, we have found that GB behave as
a kosmotropic salt in nature and suitable to stabilize the native
All the materials used in this study were analytical grade and
obtained from the reliable commercial sources. TAPS (mass
fraction purity $ 0.995), MOPS (mass fraction purity $ 0.995),
EPPS (mass fraction purity $ 0.995), CAPS (mass fraction purity
$ 0.99), BICINE (mass fraction purity $ 0.99), sodium sulfate salt
(mass fraction purity $ 0.99), a-chymotrypsin (a-CT) from bovine
pancreas type II, essentially salt free and tetra-methylammonium
hydroxide [TMA][OH] (25 wt% in H2O) were obtained from Sigma
Chemical Co. (USA). Tetra-ethylammonium hydroxide [TEA][OH]
(35 wt% in H2O), and tetra-butylammonium hydroxide [TBA][OH]
(40 wt% in H2O) were purchased from Alfa Aesar (USA). The
solvents acetonitrile (mass fraction purity $ 0.998), ethanol
(mass fraction purity $ 0.998), deuterium oxide (mass fraction
purity ¼ 0.990 atom D), and dimethyl sulfoxide-d6 (DMSO-d6,
mass fraction purity ¼ 0.999 atom D) were purchased from the
Sigma Aldrich (USA). All the materials were used as they received,
without further purication. De-ionized water used in the
synthesis process was obtained from NANO pure-Ultra pure water
system with resistivity of 18.3 MU cm. All the synthesis samples
were prepared gravimetrically by using an electronic balance
(R&D, Model GR-200) with a precision of ꢂ0.1 mg.
2.2 Synthesis and characterization of the GBILs
structure of the protein.54–57 Since the GB themselves serve as In the synthesis process, aqueous ammonium-hydroxide solu-
a protein stabilizer, the new self-buffering ionic liquids derived tion of tetramethylammonium (TMA), tetraethylammonium
from these buffers are expected to be highly biocompatible.
(TEA) and tetrabutylammonium (TBA) were added drop-wise to
The properties of these new GBILs are fundamentally a slightly excess of an equimolar aqueous buffer solution in
important for practical uses. In the present study, we measured a round bottom ask, tted with a reux condenser. The reac-
the thermal stability temperature (Td) and melting point (Tm) of tion mixture was stirred uniformly with magnetic stirrer for
the GBILs with thermal gravimetric analysis (TGA) and differ- about 12 hours under ambient conditions to ensure the
ential scanning calorimetry (DSC), respectively. Their structures completion of reaction. The mixture was then evaporated at 50–
were analyzed with NMR spectroscopy and water contents were 60 ꢀC under reduced pressure, by using a rotary evaporator
measured with Karl Fischer titration. To analyze their buffering (Panchum Evap., Chering Huei Co., Ltd). To remove the
potential in aqueous solution, we measured their pH proles unreacted materials or any other impurities presented in the
and then determined the acid dissociation constant (pKa) values obtained viscous liquid, a mixture of ethanol and acetonitrile
for each synthesized GBIL at various temperatures.
(1 : 1) was added to the viscous liquid and stirred uniformly at
Moreover, these GBILs are found to be formed two-liquid room temperature for 1 hour. The solution was then ltered and
phase with sodium sulfate solution. Aqueous bi-phasic system the ltrate solution was dried under reduced pressure for about
(ABS) has been widely applied to the separation of cell 8 to 12 hours to obtain the GBIL with lower water content. The
membrane, organelles, and enzyme from the cell lysates or water content in each of these synthesized GBIL samples was
extracts.58–60 To characterize these new GBILs-based ABS, we analyzed by Karl Fischer titration. The measured values of the
measured the phase boundaries data for each GBIL-based ABS water content in the GBILs are compiled in Table S1 of the ESI.†
at 25 ꢀC and checked their extraction efficiency for a model The structure of these new synthesized GBILs have
18568 | RSC Adv., 2016, 6, 18567–18576
This journal is © The Royal Society of Chemistry 2016

View Article Online
Paper
RSC Advances
characterized by measuring 1H NMR spectra with a Bruker
advance 500 spectrometer using tetramethylsilane (TMS) as an
internal reference. The elemental analysis for the synthesized
GBILs was made with Elementar Vario EL cube (Germany) to
further ensure the structures of these GBILs.
2.7 Activities measurements
The detailed procedure for measuring the activity of a-CT in
aqueous solutions of GBILs has been described in the litera-
ture.65 For the sake of better understanding, the brief procedure
of measuring activity is given here. The progress of the hydro-
lysis reaction in presence or absence of the a-CT is monitored by
estimating theꢀ appearance of the p-nitrophenoxide (PNPꢃ) at
400 nm, at 25 C, and at pH ¼ 8.0, for 3 minutes by using UV-
visible spectrophotometer (JASCO, V-550). The activity is
expressed as millimoles (mM) of PNPꢃ formed per minute, per g
of a-CT.
2.3 Thermal gravimetric analysis (TGA)
The TGA prole were measured by using Pyris Diamond TG-
DTA instrument. The samples were weighted (ꢁ10 mg) and
heated from 50 ꢀC to 600 ꢀCꢃu1nder a dry nitrogen atmosphere at
ꢀ
a heating rate of 10 C min
.
2.8 Phase boundaries data
2.4 Differential scanning calorimeter (DSC)
The binodal curve of each GBIL based ABS system was
measured by using the cloud point titration method at 25 (ꢂ1)ꢀ
C and atmospheric pressure. To determine the binodal curve,
similar experimental procedure was used as described in the
previous reports.66,67 The freshly prepared solution of sodium
sulfate was added drop-wise to the aqueous solution of the GBIL
until the cloud point appear and then drop-wise addition of
water carried out to get monophasic region. The procedure was
repeated under the constant stirring as far as the appearance of
the cloud point not observed on addition of any amount of salt
solution. The composition of GBIL, salt, and water in the
mixture determine by weight evaluation with an analytical
balance, with measured uncertainty of ꢂ5 mg.
The DSC measurements performed under inert nitrogen
atmosphere with heating rate of 10 ꢀC minꢃ1. The samples were
weighted (10–15 mg) and loaded into aluminum pans and then
heated to the desired temperature.
2.5 pH proles
The pH prole for each synthesized IL in aqueous solution was
measured by the potentiometric titration method, using an
automatic titrate (Metrohm 888 Titrando, USA) at 25 ꢀC. The
measurement was performed in the closed, jacketed equilib-
rium cell of volume 50 cm3, equipped with a magnetic stirrer for
proper mixing. The temperature of the equilibrium cell was
controlled by water circulating thermostatic bath (FIRSTEK,
B402L, Taiwan). The temperature of the titration solution inside
the cell was measured with a precise digital thermometer
(Model-1560, Hart Scientic Co., USA) with an uncertainty of
ꢂ0.1 ꢀC. The concentration of the stock solution of the ILs,
hydrochloric acid (HCl), and sodium hydroxide (NaOH) was
xed to 0.05 M. A constant volume of 30 cm3 from the stocked
solution of IL was introduced into the equilibrium cell. This
titration solution was titrated with the acid or base in the inert
nitrogen atmosphere to obtain the respective pH prole for the
pH range of 2 to 12.
2.9 Extraction of chymotrypsin
The ternary mixture of desired composition of GBIL (30 ꢂ 1.5
wt%) + salt (13 ꢂ 1.5 wt%) + water (57 ꢂ 1.5 wt%), used in the
partitioning of a-CT, were prepared gravimetrically by using an
electronic balance at room temperature and pressure, and at pH
of 8.0. The calculated amount of a-CT (0.5 g dmꢃ3) were added
to the extraction medium and vortexed for 10 minutes, centri-
fuged for 15 minutes, and then le for about 30 minutes to
ensure the complete partitioning of enzyme between two pha-
ses. Aerwards, the two phases were separated carefully and the
amount of a-CT in each phase was determined by using UV-
visible spectrophotometer. For the UV-spectra measurement
each phase was diluted at 1 : 50 by volume in MOPS buffer
solution. Prior to the analysis, the UV-visible spectrophotometer
was calibrated by using series of standard solution of a-CT in
the range of 0.001 g dmꢃ3 to 1.0 g dmꢃ3 at pH 8.0, at 25 ꢀC, and
at atmospheric pressure. At least three separate biphasic
mixture vials were prepared for each GBIL based ABS and the
amount of a-CT were determined from minimum 3 repeated
measurements. To avoid the interference from the salt, and
GBIL in the calculation of a-CT, the absorption of blank solu-
tions containing IL and salt were measured prior to each sample
of a-CT. The extraction efficiency (EE) of a-CT by using GBIL
based ABS system is ascertain by the following equation:
2.6 Protonation constant (pKa)
The protonation constants of all the GBILs were determined by
direct potentiometric titration using standardized NaOH
titrant. The detail procedure has been given in our previous
articles.63 For each titration, a constant volume of 50 cmꢃ3 with
GBIL concentration of 0.01 mol dmꢃ3 were used. The ionic
strength of the titration solution was maintained to 0.1 mol
dmꢃ3 by adding a calculated amount of sodium nitrate
(NaNO3). Based on the obtained potentiometric data, the
calculation of protonation constants of all the investigated
GBILs were performed by using the HYPERQUAD program
(Version 2008).64 The renement of the equilibrium constants
by this program required the inputs such as the concentration
of each species in solution and the potentiometric data. The
quality of the obtained results decide on the basis of parameters
½CTꢄ
ꢅ wGBIL
GBIL
EEð%Þ ¼
½CTꢄ
ꢅ wGBIL þ ½CTꢄ ꢅ wsalt
GBIL
salt
such as possible low value of the standard deviation (s) and the where [CT], represents the concentration of a-chymotrypsin in g
curve tting between calculated and measured pH.
dmꢃ3, the subscripts, GBIL and salt, represents the Good's
This journal is © The Royal Society of Chemistry 2016
RSC Adv., 2016, 6, 18567–18576 | 18569

View Article Online
RSC Advances
Paper
Table 1 Thermal decomposition temperature (Td) and melting point
(Tm) of the synthesized GBILs
buffer ionic liquid rich-phase and salt-rich phase, respectively.
The symbol w is the weight of each phase. Each experimental
procedure is repeated at least three times in order to get the
good quality of result with minimum error and the provided
value is the average of the repeated measurements.
GBILs
Td (ꢀC)
GBILs
Tm (ꢀC)
[TMA][TAPS]
[TEA][TAPS]
[TBA][TAPS]
[TMA][MOPS]
[TEA][MOPS]
[TBA][MOPS]
[TMA][EPPS]
[TEA][EPPS]
[TBA][EPPS]
[TMA][CAPS]
[TEA][CAPS]
[TBA][CAPS]
[TMA][BICINE]
[TEA][BICINE]
[TBA][BICINE]
218
224
271
228
239
215
195
228
221
205
212
216
173
162
163
[TMA][TAPS]
[TMA][MOPS]
[TMA][EPPS]
[TMA][CAPS]
[TMA][BICINE]
88.4
36.4
80.0
50.9
112.2
2.10 Fluorescence spectra measurements
The emission spectra were recorded at room temperature for
the sample of a-chymotrypsin (about 2.5 g per liter) in 30%
aqueous solution of GBILs and for the samples of a-chymo-
trypsin from the upper and the lower phase of GBILs-based ABS
at pH ¼ 8.0 using an RF-5301PC Spectro-uorophotometer
(SHIMADZU), with a 1.0 cm quartz cell. The spectra were
recorded at excitation wavelengths of 295 nm with an excitation
and emission slit width of 5 nm for the emission range of 300
nm to 450 nm. Prior to the analysis of the sample, uorescence
spectrum for the blank solution was measured.
3. Results and discussion
analysis and the corresponding possible molecular structure of
each synthesized GBILs are given in Table S1 of the ESI.†
To further conrm the structure of the synthesized GBILs, we
have selected [TMA][TAPS], [TEA][TAPS], and [TBA][TAPS] as the
representative systems and measured elemental analysis for
these selected GBILs. The results of the analysis are given in
Table S1 of the ESI.† As can be seen from the tabulated values
(Table S1†), the experimentally obtained individual percentages
of the elements (carbon, hydrogen, and nitrogen) in each GBIL
are found very close to the analytically calculated values for the
corresponding GBIL.
The thermal degradation of the synthesized GBILs buffers
was examined by thermal gravimetric analysis (TGA), by using
Pyris Diamond TG-DTA instrument. The thermal proles for
TAPS based GBILs ([TMA][TAPS], [TEA][TAPS], and [TBA][TAPS])
are presented in Fig. 1 and the thermal proles for the rest of
the synthesized GBILs are given in Fig. S6(a–d) of the ESI.†
Here we are introducing een new, buffer and ammonium
based, Good's buffer ionic liquids (GBILs). These GBILs have
been synthesized by using different combination of Good's
buffer as an anion and ammonium as a cation. The synthesis of
these GBILs is based on the fact that the structure of the
selected biological buffers (TAPS, MOPS, EPPS, CAPS, and
BICINE) contains acidic sulfonic or carboxylic group that can be
easily neutralize by a suitable base. Therefore, all the reported
new GBILs are synthesized by the simple acid–base neutraliza-
tion reaction. The process of synthesis is shown in Scheme 1. All
the TMA-based GB-ILs appeared as solid, while TEA-based
GBILs appear as highly viscous liquids at room temperature.
[TBA][TAPS] appears as solid at room temperature, while [TBA]
[MOPS], [TBA][EPPS], [TBA][CAPS], and [TBA][BICINE] exist as
viscous liquids at room temperature. Since the melting point of
[TMA][BICINE] is found to be higher than the normal boiling
point of water (as seen from Table 1), it would be better named
as Good's buffer ionic salt. However, to keep the discussion
general for all the synthesized ionic liquids or ionic salts, we are
using the same abbreviations “GBILs” for all the synthesized
Good's buffer derivatives.
To characterize the exact structure of these new buffers
based GBILs, we have measured the NMR spectra of each
synthesized IL or ionic salt. The measured 1H NMR spectra for
TAPS, MOPS, EPPS, CAPS, and BICINE-based GBILs are given in
Fig. S1 to S5, respectively, of the ESI.† The outcomes of NMR
Fig. 1 Thermal profiles of the TAPS-based GBILs: ( ), [TMA][TAPS]; ( ),
Scheme 1 Synthesis pathways of the new GBILs.
18570 | RSC Adv., 2016, 6, 18567–18576
[TEA][TAPS]; and ( ), [TBA][TAPS].
This journal is © The Royal Society of Chemistry 2016

View Article Online
Paper
RSC Advances
The decomposition temperature (Td) of each GBIL corre- step, an about 75 to 95 percent mass loss occurs immediately
sponding to the beginning of decomposition (estimated from aer the respective decomposition temperature (Table 1).
the respective decomposition curve) is listed in Table 1. It can Whereas, the rest mass depletion takes places in a slow fashion
be seen clearly from the decomposition curves (Fig. 1 and S6†) during a second degradation step. The results of TGA analysis
and the Td values (Table 1). Each GBIL possesses a unique show that the thermal stability behavior of synthesized GBILs is
characteristic stability behavior against thermal response. In unique and depends on the combination of cation or anion.
general, all these new GBILs are found to be thermally stable up
Those GBILs, appeared solid at room temperature, were also
to 200 ꢀC to 300 ꢀC. At temperatures slightly higher than 100 ꢀC, analyzed with the dynamic scanning calorimeter (DSC). The
a small loss in weight was observed (2 to 7 wt%). Erdemi et al.68 results of DSC measurements are presented in Fig. S7 of the
have mentioned that weight loss of samples above the normal ESI.† The determined melting points (Tm) are listed in Table 1,
boiling point of water represents the removal of the physically indicating that the melting points of these GBILs are higher
bounded water. The degradation of most of these investigated than room temperature but lower than 100 ꢀC, except for [TMA]
GBILs were completed in two steps. In the rst degradation [BICINE]. However we would like to bring into attention that the
aqueous solution of IL is generally used as a biological medium
in various studies instead the viscous IL, therefore these TMA
based ionic salts are of equally important. As can be seen from
Table 1, the melting point of TMA-based GBILs follows the order
of BICINE > TAPS > EPPS > CAPS > MOPS.
To ascertain the buffering action of these new GBILs, we
measured their pH proles in the aqueous media in the pH
range of 2 to 12. The measured pH proles for [TMA][MOPS],
[TEA][MOPS], and [TBA][MOPS] are shown in Fig. 2. The pH
proles for the other investigated GBILs are compiled in Fig. S8
of the ESI.† The region of moderate slope in the pH prole,
before the inection point, represents the buffering region. In
this region, the pH is controlled by the equilibrium between the
deprotonated and protonated forms of the GBILs. Therefore,
from the pH prole curves (Fig. 2 and S8†), it can be seen that
these newly synthesized GBILs are able to maintain their buff-
ering potential in aqueous medium.
To further characterize the buffering nature of these GBILs,
the protonation constants (pKa ) of each GBIL were determined
2
at temperatures of 20 ꢀC, 25 ꢀC, and 35 ꢀC with ionic strength of
Fig. 2 pH profiles of the MOPS-based GBILs: ( ), [TMA][MOPS]; ( ),
[TEA][MOPS]; and ( ), [TBA][MOPS].
0.1 mol dmꢃ3 NaNO3. The experimentally determined pKa
2
Table 2 Protonation constants (pKa ) of the GBILs in water at 20 ꢀC, 25 ꢀC, and 35 ꢀC and ionic strength I ¼ 0.1 mol dmꢃ3 NaNO3, with cor-
2
responding standard deviation (s) and pH range used in pKa determination. The pKa values of the Good's buffer (GB) at 25 ꢀC and I ¼ 0.1 mol
2
2
dmꢃ3
GB
pKa
GBILs
pKa
s
pKa
s
pKa
2
s
2
2
2
25 ꢀ
C
20 ꢀC
25 ꢀ
C
35 ꢀ
C
TAPS
MOPS
EPPS
CAPS
BICINE
8.33a
7.17a
7.87b
10.39b
8.22b
[TMA][TAPS]
[TEA][TAPS]
[TBA][TAPS]
[TMA][MOPS]
[TEA][MOPS]
[TBA][MOPS]
[TMA][EPPS]
[TEA][EPPS]
[TBA][EPPS]
[TMA][CAPS]
[TEA][CAPS]
[TBA][CAPS]
[TMA][BICINE]
[TEA][BICINE]
[TBA][BICINE]
8.53
8.58
8.53
7.24
7.30
7.23
8.08
7.96
8.08
11.04
10.96
11.00
8.33
8.34
8.33
0.02
0.02
0.02
0.02
0.05
0.09
0.04
0.08
0.03
0.04
0.04
0.04
0.03
0.02
0.05
8.36
8.48
8.33
7.18
7.21
7.16
7.90
7.87
7.75
10.56
10.45
10.53
8.26
8.26
8.23
0.01
0.02
0.01
0.02
0.02
0.02
0.05
0.09
0.03
0.02
0.02
0.01
0.02
0.02
0.02
8.24
8.19
8.27
7.07
7.11
7.03
7.76
7.75
7.71
9.96
9.85
9.96
8.08
8.13
8.12
0.02
0.02
0.02
0.02
0.03
0.06
0.05
0.08
0.05
0.02
0.02
0.02
0.02
0.02
0.02
a
Ref. 69. b Ref. 70.
This journal is © The Royal Society of Chemistry 2016
RSC Adv., 2016, 6, 18567–18576 | 18571

View Article Online
RSC Advances
Paper
Table 3 Catalytic activities of a-CT in 0.05 M, MOPS, [TMA][MOPS],
[TEA][MOPS], and [TBA][MOPS], at 400 nm, 25 ꢀC, and pH 7.8
Compound
Activity
MOPS
5.689
8.684
8.965
[TMA][MOPS]
[TEA][MOPS]
[TBA][MOPS]
10.918
to make it certain that the addition of these ammonium cations
(TMA, TEA, and TBA) to the GBs does not change its inherent
biocompatible nature, we selected MOPS based ionic liquids,
[TMA][MOPS], [TEA][MOPS], and [TBA][MOPS], as representative
compounds and measured the biological activities of the enzyme
a-chymotrypsin (a-CT) in the presence of these selected GBILs.
The basic idea is, if the GBILs are non-biocompatible for bio-
logical system, enzyme a-CT will be denatured and lose its bio-
logical activity. The enzyme a-CT is highly reckoned protease
enzyme and known for its capability to catalyze the hydrolysis of
p-nitrophenylacetate (PNPA).72,73 Thus, this enzyme catalyzed
hydrolysis reaction serves as the basis for measuring the bio-
logical activity of the enzyme a-CT because the enzyme can work
as a catalyst only in its active or re-nature form. Any deviation
from the native or active conformation induced by co-solvent can
be directly detected by monitoring the progress of the hydrolysis
reaction. Therefore, we measured the activity of a-CT in 0.05 mol
mdꢃ3 MOPS, [TMA][MOPS], [TEA][MOPS], and [TBA][MOPS] to
catalyze the hydrolysis of PNPA at 25 ꢀC and pH ¼ 7.8. The results
of activity measurements are shown in Table 3. From the tabu-
lated values of the enzyme activity, it can be seen that the activ-
ities of a-CT in the aqueous solutions of [TMA][MOPS], [TEA]
[MOPS], and [TBA][MOPS] is greater than that in the aqueous
buffer solution of MOPS.
Fig. 3 Representative titration curves for [TMA][MOPS] ( ), [TEA]
[MOPS] ( ), and [TBA][MOPS] ( ) at 25 ꢀC and at 0.1 mol dmꢃ3 of
NaNO3. The smooth lines represent the corresponding best-fit from
the Hyperquad 2008.
values of all the GBILs from pH-metric data by using HYPER-
QUAD 2008 are reported in Table 2. Fig. 3 shows a representa-
tive titration curves and the corresponding best t for [TMA]
[MOPS], [TEA][MOPS], and [TBA][MOPS] at 25 ꢀC with ionic
strength of 0.1 mol dmꢃ3 of NaNO3.
From the Fig. 3, it can be seen that the agreement between
the experimental and correlated values is excellent. Similar
curves were obtained for the rest of the studied GBILs. For the
sake of clarity, we are not presenting all the titration curves. The
zwitterionic Good's buffers TAPS, MOPS, EPPS, MOPS, CAPS,
and BICINE known to have two protonation constants. The rst
protonation constant (pKa ) corresponds to the dissociation of
1
sulphonic or carboxylic group. The second protonation
It indicates that the synthesized GBILs are biocompatible in
nature and provide more active conformation to the a-CT in
comparison with the conventional buffer. These results of activity
measurement show that it is advantageous to convert Good's
buffers into ionic liquids. Moreover, the activity of a-CT in
aqueous solutions of GBILs follows the order of [TBA][MOPS] >
[TEA][MOPS] > [TMA][MOPS]. It indicates that the activity of a-CT
or biocompatibility of GBILs increases with the increase in alkyl
chain length of the counter cation part in the GBILs. Some
conventional ILs were also found to be biocompatible for the
enzyme a-CT, but the activity testing always required an addi-
tional buffer compounds to maintain the pH value. However, in
the present case, GBILs themselves can also be used as a buffer to
maintain the pH of the system. Thus these new GBILs are more
useful in comparison with the conventional non-buffering
biocompatible ILs used in biological or biochemical research
because they are not only biocompatible but also can avoid using
an extra buffering agent to the biological or enzymatic system.
The separation or extraction of novel compounds by using
aqueous biphasic systems (ABSs) is not only highly valuable but
also very economical because the process usually occurs at
ambient condition. Many reports are available in the litera-
ture74–76 emphasizing the scope of IL-based ABS for the recovery
constant (pKa ) corresponds to the dissociation of the amino
group, which is responsible for their observed buffering action.
2
As can be seen from Table 2, the pKa values of the each
2
investigated GBILs decreasing with the increase of temperature.
This trend was expected because with the increase of tempera-
ture extent of dissociation increases. For the comparison
purpose, the pKa values of the Good's buffer (GB), TAPS, MOPS,
2
EPPS, CAPS, and BICINE in aqueous solutions under the similar
experimental condition at 25 ꢀC taken from the literature69,70
and presented in the Table 2. The order of pKa for these buffers
2
follows the order of CAPS > TAPS > BICINE > EPPS > MOPS. The
same trend in the pKa's of GBILs has been observed for the
respective GB (Table 2). Moreover, the presence of ammonium
cations (TMA, TEA, and TBA) in the solutions increase the pKa's
of GBILs in comparison with the corresponding GBs (CAPS,
TAPS, BICINE, EPPS, and MOPS).
The selected biological buffers (TAPS, MOPS, EPPS, CAPS, and
BICINE) are considered as biocompatible, nontoxic, and green
compounds. Especially, TAPS, MOPS, and EPPS buffers were
found to protect the native structure of the protein BSA against
thermal denaturation, in aqueous solutions containing high
concentration of the respective buffer.54,55,71 Thus, the synthesized
GBILs could be biocompatible and green compounds. However,
18572 | RSC Adv., 2016, 6, 18567–18576
This journal is © The Royal Society of Chemistry 2016

View Article Online
Paper
RSC Advances
Table
4
Initial mixture compositions in weight percentage (w),
extraction efficiency (EE) of the a-chymotrypsin in the GBIL based ABS
systems and activities of a-chymotrypsin in GBIL phase after extraction
at 25 ꢀC, at atmospheric pressure and pH 8.0
GBIL
wIL (%)
wsalt (%)
wwater (%)
EE (%)
Activity
[TBA][TAPS]
[TBA][MOPS]
[TBA][EPPS]
[TBA][BICINE]
29.67
30.24
29.95
30.35
13.47
13.66
13.63
13.70
56.86
56.10
56.42
55.95
100
100
100
100
12.842
17.095
12.026
17.382
solution. These results indicate toward the excellent capability of
these GBIL based ABS systems for completely recovering the
precious biomolecule through a single step.
To ascertain the complete extraction of the tested enzyme in
the GBIL phase and to analyze the structure of enzyme aer
extraction, we have relied on the uorescence technique. The
uorescence method is highly recognized for the exact analysis
of the structural conformation of the enzymes in solvent.77,78
Since the uorescence intensity of the uorophore molecule is
very sensitive to the molecular environment, it serves as a basis
to analyze the structural and conformational change in the
molecule brought by the surrounding solvent.79 The aromatic
amino acid residues like tryptophan (Trp), phenylalanine (Phe),
and tyrosine (Tyr) are commonly found uorophores in the
enzyme or proteins and are responsible for their respective
uorescence behavior in polar or non-polar solvent. Moreover,
it is recognized that, at the excitation wavelength of the 295 nm,
the emission spectra of enzyme appears mainly due to the Trp
residue and the contribution of Tyr or Phe became minor.80 On
the basis of these facts, Park et al.81 suggested that the uo-
rescence spectra of protein using Trp as a uorescence probe
can provide an exact idea of protein conformation. Fortunately,
the crystal structure of enzyme a-CT was noticed to have eight
Fig. 4 Liquid–liquid phase boundaries of the systems composed of
GBIL + Na2SO4 + H2O at 25 ꢀC and at atmospheric pressure: (
),
), [TBA][MOPS]; ( ), [TBA][EPPS]; ( ), [TBA][CAPS];
), [TBA][BICINE].
[TBA][TAPS]; (
(
of a broad range of valuable materials including precious drugs,
metal ions, alkaloids, amino acids, proteins, and enzymes.
Therefore, we attempt to test the possibility of inducing the
liquid–liquid phase splitting of the aqueous solution of sodium
sulfate by adding these new GBILs. Interestingly, all the TBA-
based GBILs can induce ABS for the aqueous solution of
sodium sulfate, while GBILs with short alkyl chain (TMA and
TEA) fail to form ABS. In these new GBIL-based ABSs, the top is
GBIL-rich phase and the lower is aqueous phase containing
electrolytic compound.
To further characterize the new GBIL-based ABSs, we
measured the phase boundaries data (binodal curve) for each
reported ABS system by using well-reckoned cloud point titra-
tion method under ambient condition. The results of
measurements are presented in Table S3 of the ESI.† The phase
diagram for all the TBA-based GBILs are graphically shown in
Fig. 4. The region just above the curve represents the two phase
regions.
In addition, to check the extraction efficiency of these GBIL-
based ABSs especially for the biomolecule, we have selected
enzyme a-chymotrypsin (a-CT) as a model enzyme. The ternary
mixture composed of GBIL (30 ꢂ 1.5 wt%) + salt (13 ꢂ 1.5 wt%) +
water (57 ꢂ 1.5 wt%), used in the partitioning of a-CT, were
prepared gravimetrically with an electronic balance at pH of 8.0.
Here, we would like to bring into notice that the pH (about 8.0) of
the extraction medium in each case maintain by the self-buffering
action of these GBILs, without using any external buffering agent.
Since the required experimental pH is away from the suitable pH
range of the [TBA][CAPS], we did not test the extraction of a-CT in
the [TBA][CAPS] based ABS system. The results of measurement
including, initial mixture compositions and the percentage
extraction efficiency (EE) of a-CT are given in Table 4. From the
tabulated values of the percentage of EE in Table 4, it can be seen
that GBIL completely extract enzyme a-CT from the aqueous
Fig. 5 The fluorescence spectra of a-chymotrypsin in [TBA][TAPS]
before and after extraction: ( ); a-chymotrypsin in aqueous [TBA]
[TAPS] (before extraction), ( ); a-chymotrypsin in upper phase after
extraction, ( ); a-chymotrypsin in lower phase after extraction.
This journal is © The Royal Society of Chemistry 2016
RSC Adv., 2016, 6, 18567–18576 | 18573

View Article Online
RSC Advances
Paper
tryptophan residues (Trp27, Trp29, Trp51, Trp141, Trp172, Trp buffer systems, which possess strong complexation tendency
207, Trp215, and Trp 237).82
with various metallo-proteins.
Therefore, to analyze the structural changes in the enzyme a-
CT in GBIL-rich phase, we have measured emission uores-
cence spectra for the a-CT samples taken from upper and lower
4. Conclusion
phases of the GBIL-based ABS system at an excitation wave- The main objective of this study is to synthesize and charac-
length of 295 nm. The obtained emission spectra of a-CT in terize the new ionic liquids that solve the most of problems
aqueous solution of [TBA][TAPS] (before extraction), a-CT in encountered in enzymatic research, including biocompatibility
upper phase aer extraction, and a-CT in lower phase aer and pH control. In the present study, een new self-buffering
extraction are presented in Fig. 5 and the results for the rest of GBILs have been produced from Good's biological buffers as
GBILs are given in Fig. S9 of the ESI.† From these gures, it can anions and ammonium as cations. The structure and purity of
be seen that no uorescence peak observed for a-CT in the lower these new IL buffers have been checked with NMR technique.
phase of the ABS, which further conrms the complete extrac- The thermal decomposition and melting point of these new
tion of a-CT from the aqueous solution to the upper phase of ionic liquids analyzed by the thermal gravimetric analysis (TGA)
the ABS. In addition, the uorescence intensity of the peak of a- and dynamic scanning calorimetry (DSC) techniques, respec-
CT aer extraction is almost comparable with that of a-CT tively. These new compounds are found to be thermally stable
before extraction (Fig. 5 and S9†), indicating that enzyme over wide range of temperature. The reported GBILs not only
remains in its native conformation aer extraction. However, provide suitable biocompatible medium for conducting enzy-
the peak of a-CT aer extraction was noticed to shi slightly matic or biological research but also can avoid using an external
toward a higher wavelength. From the study of the crystal buffering agent. The buffering potential of these new ILs
structure of a-CT,83 it reveals that two tryptophan amino acid conrmed by measuring their pH prole and protonation
residues (Trp 207 and Trp 237) of the a-CT are easily available constants in aqueous solutions, at different temperatures (20
for the external solvents. Moreover, it is observed that80 the ꢀC, 25 ꢀC, and 35 ꢀC). The results indicate that these new GBILs
increase in the hydrophobic environment around Trp residues are promising towards maintaining the required pH of the
may cause the shiing in the emission peak towards lower medium. Moreover, the enhanced enzyme activity and the
wavelength and the increase in hydrophillic environment excellent extraction efficiency of a-chymotrypsin in the presence
around Trp residues results in the shiing of the emission peak of these new GBILs indicate towards the suitability of using
towards the higher wavelength. Probably the observed slightly GBILs based medium for the enzymatic research. Thus, in
blue shi in the peak of a-CT in upper phase is the result of comparison with the conventional ionic liquids or buffer itself,
comparatively hydrophillic microenvironment around Trp these new ionic liquid buffers can serve as a better biological
residue provided by the GBIL and the phase forming sodium medium as well as a buffering agent and thus can help in
sulphate in the upper phase. The outcomes of the uorescence conducting various biological processes in more effective, and
measurements suggest that a-CT remains in its native confor- green way.
mation aer extraction. This expectation is in accordance with
our previous reports,54,55,71 in which we found that the selected
GBs (TAPS, MOPS, and EPPS) are highly biocompatible and are
Acknowledgements
suitable for protecting the native structure of protein against The authors gratefully acknowledge the nancial support from
thermal denaturation and maintain the stable and compact Ministry of Science & Technology, Taiwan, through NSC102-
native conformation to the protein. However, to further conrm 2811-E011-014 and NSC 102-2221-E011-137-MY3.
our expectation that enzyme remains in its active conformation
in GBIL-rich phase, we have performed the activities measure-
ment for the sample of a-CT in GBIL-rich upper phase at 25 ꢀC
Notes and references
and pH 8.0. The results of the measurement are compiled in
Table 4. The tabulated values of the activities in Table 4 suggest
that the GBILs assist the enzyme maintains in its active
conformation and are suitable for extracting the enzyme in
active conformation from aqueous solutions. Since the
complete extraction of a-CT in its active form was attained in
a single step and the self-buffering nature of these GBILs avoids
using external buffer compounds in the extractive medium, it
suggest that these new GBIL-based biological media can be
more promising and economical for the enzymatic research.
The conventional ionic liquids have a tendency to interfere with
external buffering compounds, but the use of our synthesized
self-buffering GBILs can avoid such a situation from happening
and can be more effective over the conventional phosphate
1 A. Kumar and P. Venkatesu, Chem. Rev., 2012, 112, 4283–
4307.
2 N. Byrne, L. M. Wang, J. P. Belieres and C. A. Angell, Chem.
Commun., 2007, 2714–2716.
3 N. Byrene and C. A. Angell, Chem. Commun., 2009, 1046–
1048.
¨
4 H. Weingartner, C. Cabrele and C. Herrmann, Phys. Chem.
Chem. Phys., 2012, 14, 415–426.
5 D. Constatinescu, C. Herrmann and H. Weingartner, Phys.
Chem. Chem. Phys., 2010, 12, 1756–1763.
6 M. V. Quental, M. Caban, M. M. Pereira, P. Stephnowski,
J. A. P. Cutinho and M. G. Freire, Biotechnol. J., 2015, 10,
1457–1466.
7 S. A. Forsyth, D. R. MacFarlane, R. J. Thomson and
M. V. Itzstein, Chem. Commun., 2002, 714–715.
18574 | RSC Adv., 2016, 6, 18567–18576
This journal is © The Royal Society of Chemistry 2016

View Article Online
Paper
RSC Advances
8 R. D. Rogers and K. R. Seddon, Science, 2003, 302, 792–793.
9 T. Welton, Chem. Rev., 1999, 99, 2071–2084.
818, American Chemical Society, Washington, DC, 2002,
pp. 2–14.
10 K. R. Seddon, J. Chem. Technol. Biotechnol., 1997, 68, 351– 36 A. J. Carmichael, M. Deetlefs, M. J. Earle, U. Frohlich and
356.
K. R. Seddon, Ionic Liquids as Green Solvents: Progress and
Prospects, ACS Symposium Series 856, American Chemical
Society, Washington, DC, 2003, pp. 14–31.
11 K. R. Seddon, A. Stark and M. J. Toress, Pure Appl. Chem.,
2000, 72, 2275–2287.
12 R. Sheldon, Chem. Commun., 2001, 2399–2407.
13 D. R. MacFarlane, M. Forsyth, E. I. Izgorodina, A. P. Abbott,
37 C. A. Summers and R. A. Flowers II, Protein Sci., 2000, 9,
2001–2008.
G. Annata and K. Fraser, Phys. Chem. Chem. Phys., 2009, 11, 38 P. Lozano, T. D. Diego, J. P. Guegan, M. Vaultier and
4962–4967.
J. L. Iborra, Biotechnol. Bioeng., 2001, 75, 563–569.
¨
14 H. Weingartner, Angew. Chem., Int. Ed., 2008, 47, 654–670.
15 Y. Chauvin, Actual. Chim., 1996, 44, 0151–9093.
39 D. Constantinescu, H. Weingartner and C. Herrmann,
Angew. Chem., Int. Ed., 2007, 46, 8887–8889.
16 Y. Chauvin, L. Mussmann and H. Olivier, Angew. Chem., Int. 40 N. Byrne and C. A. Angell, J. Mol. Biol., 2008, 378, 707–714.
Ed. Engl., 1995, 34, 2698–2700.
17 L. Magna, G. P. Niccolai, Y. Chauvin and J. M. Basset,
Organometallics, 2003, 22, 4418–4425.
18 C. A. Angell, Y. Ansari and Z. Zhao, Faraday Discuss., 2012,
154, 9–27.
41 I. Khimji, K. Doan, K. Bruggeman, P. J. Huang, P. Vajha and
J. Liu, Chem. Commun., 2013, 49, 4537–4539.
42 S. Cantone, U. Hanefeld and A. Basso, Green Chem., 2007, 9,
954–971.
43 A. Kumar and P. Venkatesu, RSC Adv., 2013, 3, 362–367.
´
´
19 A. Garcıa, L. C. Torres-Gonzalez, K. P. Padmasree, 44 U. Kragl, M. Eckstein and N. Kazik, Curr. Opin. Biotechnol.,
´
M. G. Benavides-Garcia and E. M. Sanchez, J. Mol. Liq.,
2002, 13, 565–571.
2013, 178, 57–62.
45 R. F. Van and R. A. Sheldon, Chem. Rev., 2007, 107, 2757–
20 ACS Symposium Series 818, ed. R. D. Rogers and K. R.
2785.
Seddon, Ionic Liquids: Industrial Applications for Green 46 M. G. Freire, A. F. M. Claudio, J. M. M. Araujo,
Chemistry, American Chemical Society, Washington, DC,
J. A. P. Coutinho, I. M. Marrucho, J. N. C. Lopes and
2002.
L. P. N. Rebelo, Chem. Soc. Rev., 2012, 41, 4966–4995.
21 W. W. Tu, J. P. Lei and H. X. Ju, Chem.–Eur. J., 2009, 15, 779– 47 N. E. Good, G. D. Winget, W. Winter, T. N. Connolly, S. Izawa
784. and R. M. M. Singh, Biochemistry, 1966, 5, 467–477.
22 H. Xu, H. Y. Xiong, Q. X. Zeng, L. Jia, Y. Wang and S. F. Wang, 48 G. N. Ou, M. X. Zhu, J. R. She and Y. Z. Yuan, Chem.
Electrochem. Commun., 2009, 11, 286–289. Commun., 2006, 4626–4628.
23 M. J. Earle and K. R. Seddon, Pure Appl. Chem., 2000, 72, 49 D. R. MacFarlane, R. Vijayaraghavan, H. N. Ha, A. Izgorodin,
1391–1398.
K. D. Weaver and G. D. Elliott, Chem. Commun., 2010, 46,
7703–7705.
24 S. Ikegami and H. Hamamoto, Chem. Rev., 2009, 109, 583–
593.
50 S. C. Matias, A. Rocha, R. Teixeira, L. J. P. Fonseca and
N. M. T. Lourenço, RSC Adv., 2014, 4, 15597–15601.
51 M. Taha, F. A. E. Silva, M. V. Quental, S. P. M. Ventura,
M. G. Freire and J. A. P. Coutinho, Green Chem., 2014, 16,
3149–3159.
25 V. I. Parvulescu and C. Hardacre, Chem. Rev., 2007, 107,
2615–2665.
26 M. Singh, R. S. Singh and U. C. Banerjee, J. Mol. Catal. B:
Enzym., 2009, 56, 294–299.
27 A. Karout and A. C. Pierre, Catal. Commun., 2009, 10, 359– 52 M. Taha, M. R. Almeida, F. A. E. Silva, P. Domingues,
361.
S. P. M. Ventura, J. A. P. Coutinho and M. G. Freire,
Chem.–Eur. J., 2015, 21, 4781–4788.
53 L. Xu, G. Ou and Y. Yuan, J. Organomet. Chem., 2008, 693,
3000–3006.
54 B. S. Gupta, M. Taha and M. J. Lee, Process Biochem., 2013,
48, 1686–1696.
55 M. Taha, B. S. Gupta, I. Khoiroh and M. J. Lee,
Macromolecules, 2011, 44, 8575–8589.
56 M. Taha and M. J. Lee, J. Chem. Phys., 2013, 138, 244501–
244513.
28 S. P. Verevkin, S. A. Kozlova, V. N. Emel'yanenko, P. Goodrich
and C. Hardacre, J. Phys. Chem. A, 2008, 112, 11273–11282.
29 J. C. Lassegues, J. Grondin, C. Aupetit and P. Johansson, J.
Phys. Chem. A, 2009, 113, 305–314.
30 T. M. Chang and L. X. Dang, J. Phys. Chem. A, 2009, 113,
2127–2135.
31 K. Hayamizu, S. Tsuzuki and S. Seki, J. Phys. Chem. A, 2008,
112, 12027–12036.
32 J. D. Oxley, T. Prozorov and K. S. Suslick, J. Am. Chem. Soc.,
2003, 125, 11138–11139.
57 B. S. Gupta, M. Taha and M. J. Lee, RSC Adv., 2014, 4, 51111–
51116.
58 T. Agasoster, J. Chromatogr. B: Biomed. Sci. Appl., 1998, 716,
293–298.
33 I. M. Marrucho, L. C. Branco and L. P. Rebelo, Annu. Rev.
Chem. Biomol. Eng., 2014, 5, 527–546.
34 C. J. Adams, Ionic Liquids: Industrial Applications to Green
Chemistry, ACS Symposium Series 818, American Chemical 59 P. A. Albertsson, Nature, 1956, 177, 771–774.
Society, Washington, DC, 2002, pp. 15–29.
35 J. D. Holbrey and R. D. Rogers, Ionic Liquids: Industrial
60 M. Rito-Palomares, J. Chromatogr. B: Anal. Technol. Biomed.
Life Sci., 2004, 807, 3–11.
Applications to Green Chemistry, ACS Symposium Series 61 V. Y. Levitsky, A. A. Panova and V. V. Mozhaev, Eur. J.
Biochem., 1994, 219, 231–236.
This journal is © The Royal Society of Chemistry 2016
RSC Adv., 2016, 6, 18567–18576 | 18575

View Article Online
RSC Advances
Paper
62 P. Attri, P. Venkatesu and A. Kumar, Phys. Chem. Chem. Phys., 73 P. Viparelli, F. Alfani and M. Cantarella, J. Mol. Catal. B:
2011, 13, 2788–2796. Enzym., 2001, 15, 1–8.
63 M. Taha, B. S. Gupta and M. J. Lee, J. Chem. Eng. Data, 2011, 74 S. P. M. Ventura, R. L. F. de Barros, J. M. de Pinho Barbosa,
56, 3541–3551.
C. M. F. Soares, A. S. Lima and J. A. P. Coutinho, Green
Chem., 2012, 14, 734–740.
64 P. Gans, A. Sabatini and A. Vacca, Talanta, 1996, 43, 1739–
1753.
75 C. M. S. S. Neves, J. F. O. Granjo, M. G. Freire, A. Robertson,
N. M. C. Oliveira and J. A. P. Coutinho, Green Chem., 2011,
13, 1517–1526.
65 P. Attri, P. Venkatesu and M. J. Lee, J. Phys. Chem. B, 2010,
114, 1471–1478.
´
66 M. G. Freire, C. M. S. S. Neves, J. N. Canongia Lopes, 76 A. F. M. Claudio, A. M. Ferreira, C. S. R. Freire,
I. M. Marrucho, J. A. P. Coutinho and L. P. N. Rebelo, J.
Phys. Chem. B, 2012, 116, 7660–7668.
A. J. D. Silvestre, M. G. Freire and J. A. P. Coutinho, Sep.
Purif. Technol., 2012, 97, 142–149.
˜
´
67 T. Mourao, A. F. M. Claudio, I. Boal-Palheiros, M. G. Freire 77 M. R. Eink and M. C. R. Shastry, Methods Enzymol., 1997,
and J. A. P. Coutinho, J. Chem. Thermodyn., 2012, 54, 398–
278, 258–286.
405.
78 H. Morawetz, J. Polym. Sci., Part A: Polym. Chem., 1999, 37,
1725–1735.
¨
68 H. Erdemi, U. Akbey and W. H. Meyer, Solid State Ionics,
2010, 181, 1586–1595.
69 B. S. Gupta, M. Taha and M. J. Lee, J. Solution Chem., 2013,
42, 2296–2309.
79 N. C. Stellwagen, A. Bossi, C. Gel and P. G. Righetti, Anal.
Biochem., 2000, 287, 167–175.
80 P. Attri and P. Venkatesu, Process Biochem., 2013, 48, 462–
470.
70 R. N. Roy, L. N. Roy, J. G. Grant, M. P. Cummins, B. J. Tabor
III, S. J. Richard, C. A. Himes, B. R. Lively, P. L. Blackwell and 81 S. H. Park, M. C. R. Shastry and H. Roder, Nat. Struct. Biol.,
A. N. Simon, J. Solution Chem., 2002, 31, 861–873. 1999, 6, 943–947.
71 B. S. Gupta, M. Taha and M. J. Lee, Phys. Chem. Chem. Phys., 82 R. A. Blevins and A. Tulinsky, J. Biol. Chem., 1985, 260, 4264–
2015, 17, 1114–1133. 4275.
72 N. Spetri, F. Alfani, M. Cantarella, F. D. Amico, R. Germani 83 P. D. Boyer, The enzymes, Academic Press, New York, 3rd edn,
and G. Savelli, J. Mol. Catal. B: Enzym., 1999, 6, 99–110. 1970.
18576 | RSC Adv., 2016, 6, 18567–18576
This journal is © The Royal Society of Chemistry 2016