Buffer-mediated activation of Candida antarctica lipase B dissolved in hydroxyl-functionalized ionic liquids

Article abstract of DOI:10.1016/j.molcatb.2010.09.013

Ionic-liquid buffer having phosphate anion was synthesized for the development of buffered enzymatic ionic liquid systems. Both the conformation and transesterification activity of Candida antarctica lipase B (CALB) dissolved in the hydroxyl-functionalized ionic liquids were buffer dependent. Intrinsic fluorescence studies indicated that the CALB possessed a more compact conformation in the medium consisted of ionic liquid buffer having phosphate anion and hydroxyl-functionalized ionic liquids like 1-(1-hydroxyethyl)-3- methyl-imidazolium tetrafluoroborate and 1-(1-hydroxyethyl)-3-methyl-imidazolium nitrate. High activity and outstanding stability could be obtained with the CALB enzyme in the buffered ionic liquids for the transesterification.

Full text of DOI:10.1016/j.molcatb.2010.09.013

Journal of Molecular Catalysis B: Enzymatic 68 (2011) 66–70
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Buffer-mediated activation of Candida antarctica lipase B dissolved in
hydroxyl-functionalized ionic liquids
Guangnan Oua,∗, Jing Yangb, Biyan Hea, Youzhu Yuanb,∗
a School of Bioengineering, Jimei University, Xiamen 361021, PR China
b State Key Laboratory of Physical Chemistry of Solid Surfaces and National Engineering Laboratory for Green Chemical Productions of Alcohols-Ethers-Esters, College of Chemistry
and Chemical Engineering, Xiamen University, Xiamen 361005, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 April 2010
Received in revised form 1 August 2010
Accepted 27 September 2010
Ionic-liquid buffer having phosphate anion was synthesized for the development of buffered enzymatic
ionic liquid systems. Both the conformation and transesterification activity of Candida antarctica lipase
B (CALB) dissolved in the hydroxyl-functionalized ionic liquids were buffer dependent. Intrinsic fluores-
cence studies indicated that the CALB possessed a more compact conformation in the medium consisted
of ionic liquid buffer having phosphate anion and hydroxyl-functionalized ionic liquids like 1-(1-
hydroxyethyl)-3-methyl-imidazolium tetrafluoroborate and 1-(1-hydroxyethyl)-3-methyl-imidazolium
nitrate. High activity and outstanding stability could be obtained with the CALB enzyme in the buffered
ionic liquids for the transesterification.
Keywords:
Ionic-liquid buffer
Hydroxyl-functionalized ionic liquid
Lipase
© 2010 Elsevier B.V. All rights reserved.
Fluorescence
1. Introduction
but also cause severe enzyme deactivation. Turner and co-workers
reported that incorporation of a hydroxyl-functionality in the
Enzymes catalyze a wide variety of reactions best in aqueous
environments and at physiological pH with exquisite selectiv-
ity and stereospecificity [1,2]. Over the years, much effort, using
both experimental and theoretical approaches, has been devoted
to the studies of enzyme stability [3–5]. Several parameters such
as hydrophobic interactions [3,6], electrostatic interactions [7,8],
hydrogen bonding [9,10], packing [6,11], and entropic effect [12]
have been identified as major contributions to the stability of an
enzyme. Despite of these, much work is still needed to understand
the dominant forces that stabilize the enzymes, especially in ionic
liquid (IL) media.
The use of ILs as novel reaction media may offer a convenient
solution to both the solvent emission and the catalyst recycling
problem [13,14]. Because active enzymes are charged biomacro-
molecules with a hydrophilic nature, they are usually insoluble in
common ILs. Enzymatic reactions in ILs are basically limited to the
use of suspended enzymes [15–19] or immobilized enzymes forms
[15,16,20]. However, it is of interest to develop ILs that can dis-
solve enzymes because an enzyme solution in IL enables detailed
investigation of enzyme structure and function by various spectro-
scopic methods [21,22]. It was reported that ILs containing nitrate
[23], acetate or lactate [24], and chloride [25], can dissolve enzymes
cation of an imidazolium based IL has the potential to stabilize pro-
teins while keeping them soluble [26]. The potential of the IL with a
cation 1-(1-hydroxypropyl)-3-methyl-imidazolium ([C3OHMIM])
for dissolving enzymes without affecting their catalytic activity
was demonstrated by Walker and Bruce [27,28]. Bermejo et al.
recently found that when fresh CALB dissolved in the IL 1-(1-
hydroxypropyl)-3-methyl-imidazolium nitrate, [C3OHMIM][NO3],
35% initial activity lost as compared to its aqueous solution. After
three months of storing the CALB-IL solution, the enzyme main-
tained about 80% of its activity with respect to the fresh solution
[29]. Obviously, not all ILs are suitable for biocatalysis. The devel-
opment of dissolved and active enzymes in IL media still remains a
challenge.
It is well known that buffers affect enzyme activity in aqueous
system. There are, however, few reports concerned with the control
of ionization state of enzymes in non-aqueous media. Some types
of buffers have been used to tune the ionization state of enzymes in
organic solvents [30–32]. Unfortunately, they were found to have
only limited solubility in non-aqueous media. To overcome this
restriction, we have synthesized a new class of ILs with buffering
behaviour that are referred to as IL buffers, which are miscible with
polar solvents such as methanol, DMF, and dichloromethane and
also with ILs like [BMIM][PF6] and [BMIM][BF4] [33]. Remarkable
buffer dependence of the catalytic activities has been observed in
hydrogenation of olefins [33] and selective hydrogenation of unsat-
urated aldehyde [34] in non-aqueous media.
∗
Corresponding authors. Tel.: +86 592 2181659; fax: +86 592 2183047.
E-mail addresses: gnou@jmu.edu.cn (G. Ou), yzyuan@xmu.edu.cn (Y. Yuan).
1381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.09.013

G. Ou et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 66–70
67
Our strategy for solubilizing enzymes in ILs is to select
ILs with hydroxyl group that have high affinity for protein
molecules. We have reported that common ILs such as [BMIM][BF4]
and [BMIM][PF6] showed a weak power to solvate PAMAM
dendrimers, which are known to be good mimics of globular pro-
teins. Whereas the IL 1-(1-hydroxyethyl)-3-methyl-imidazolium
tetrafluoroborate, [C2OHMIM][BF4], could dissolve the dendrimers
well. The charged dendrimer-encapsulated Pd nanoparticles in
this IL showed high activity and reusability for hydrogenation of
styrene [35]. Herein, we report a novel IL system consisting of IL
buffer having phosphate anion and hydroxyl-functionalized IL like
[C2OHMIM][BF4] and 1-(1-hydroxyethyl)-3-methyl-imidazolium
nitrate, [C2OHMIM][NO3], for the dissolvation and activation of
Candida antarctica lipase B (CALB). To understand the activation and
denaturation phenomena of the enzyme in the designed IL media,
the buffer effects on dissolved CALB were investigated and Fluoro-
metric measurements were applied to analyze changes in enzyme
conformation.
[C2OHMIM][NO3], and [BMIM][NO3] were calculated by subtract-
ing that of anhydrous methanol, all of which were less than 0.19%.
The 1H NMR, 13C NMR, and 31P NMR spectra were obtained on a
Brüker AV-400 Fourier transform NMR spectrometer. 1H NMR spec-
tra were referenced to tetramethylsilane in CDCl3. 31P NMR spectra
were referenced to phosphoric acid. Mass spectra were acquired
using a Finnigan LCQ mass spectrometer. Thermal gravimetric anal-
ysis (TGA) was performed on a Netzsch TG 209F1 instrument in N2
flow with a heating speed of 10 K min−1
.
2.5. General procedures of enzymatic transesterification
CALB (1.2 mg) was dissolved in 500 ␮L of IL medium (30 mg of
IL buffer having phosphate anion was added in the case of buffered
medium) in a 5 mL flask. 110 ␮L (0.83 mmol) ethyl butyrate and
110 ␮L (1.21 mmol) n-butanol and 50 ␮L nonane (internal stan-
dard) were added. The reaction mixture was stirred at 40 ◦C in oil
bath for 3 h. After the reaction was complete, the products were
decanted from [C2OHMIM][BF4]. In the cases of [BMIM][NO3] and
[C2OHMIM][NO3] as reaction media, the products were extracted
with cyclohexane. The organic phase was analyzed with a gas
chromatograph equipped with an FID and a capillary column (SE-
30, 30 m × 0.32 mm × 0.25 ␮m). The residual reactant mixture in IL
phase was removed in vacuum at 40 ◦C for more than 1 h. The new
cycle was restarted by addition of fresh substrate. This process was
repeated 9 times.
2. Materials and methods
2.1. Materials
C. antarctica lipase B (CALB, ∼9 units mg−1) was purchased
from Fluka. Tryptophan and tyrosine was purchased from
Sigma–Aldrich. Ethyl butyrate and n-butanol were analytical
reagents and were dried by 3A molecular sieves before use. All other
chemicals and reagents were of analytical grade.
2.6. Fluorescence spectroscopy
Fluorescence spectra were monitored on a fluorescence spec-
trophotometer (FP-6200, Jasco, Japan) at room temperature (20 ◦C).
Enzyme samples were excited at 290 nm and emission registered
from 300 to 450 nm using 5 nm band width in both the excitation
and emission path. The final concentration in all the media was
0.5 mg mL−1 of CALB or Trp. The fluorescence spectra of CALB were
obtained by subtracting the spectra of the ILs without the enzyme or
Trp to discount the influence of the imidazolium ring fluorescence
on the fluorescence spectrum.
2.2. Synthesis of ionic liquids
[BMIM][BF4] [36], [BMIM][PF6] [36], [C2OHMIM][BF4] [37],
[C2OHMIM][NO3] and [BMIM][NO3] [21] were synthesised accord-
ing to published procedures and checked for the absence of chloride
and acid. ILs were passed through a neutral alumina column, dried
at 50 ◦C under reduced pressure for more than 18 h, and stored
under dry N2.
2.3. Synthesis of IL buffer having phosphate anion
3. Results and discussion
An aqueous solution of [BMIM]OH was prepared by passing
the corresponding imidazolium halide ([BMIM]Cl) through a col-
umn filled with anion exchange resin, as described in the literature
[33,34]. The aqueous [BMIM]OH was then neutralized with phos-
phoric acid in a beaker and the pH of the solution was adjusted
to 6.86. The solution was evaporated at 50 ◦C under reduced pres-
sure to give a viscous liquid, which was then vacuum dried at 50 ◦C
for 18 h to afford the ionic liquid buffer product. mp 48–53 ◦C.
NMR spectra of IL buffer having phosphate anion were shown in
Fig. S1. 1H NMR (400 MHz, D2O) ı: 8.555 (s, 1H, im-H), 7.306 (s,
1H, im-H), 7.265 (s, 1H, im-H), 4.022 (t, 2H, –CH2), 3.721 (s, 3H,
im-CH3), 1.651–1.688 (m, 2H, –CH2), 1.108–1.164 (m, 2H, –CH2),
0.722–0.759 (t, 3H, –CH3); 13C NMR (400 MHz, D2O) ı: 135.754,
123.392, 122.098, 49.147, 35.534, 31.161, 18.632, 12.538. 31P NMR
(400 MHz, D2O) ı: 1.324. Mass spectra of IL buffer having phosphate
anion were shown in Fig. S2. MS (m/z) calcd for [BMIM]+ of the IL
3.1. Synthesis and characterization of IL buffer having phosphate
anion
The synthesis pathway of IL buffer having phosphate anion is
shown in Scheme 1. The IL buffer was synthesized as previously
reported [33,34] and characterized by NMR, MS and TGA. All the
characterization data are consistent with the expected composi-
tions and structures (Figs. S1–S3). Buffer value (number of moles
of strong base required to change the pH of one liter of solution
by one unit) and dilution value (change of pH on dilution with an
equal volume of water) of the aqueous solution of mixed phosphate
IL buffer at 0.025 mol L−1 and 20 ◦C are 0.026 and 0.10, respectively,
which buffering behaviors are exactly in agreement with those of
the inorganic counterpart, mixed phosphate buffer [38].
3.2. Enzymatic transesterification in the presence of IL buffer
having phosphate anion
−
buffer 139.1 obsd 139.1; MS (m/z) calcd for H2PO4 of the IL buffer
97.0 obsd 97.2.
To test the activity of CALB lipase in dissolved form in ILs, we
examined the CALB-catalyzed transesterification of ethyl butyrate
with n-butanol. All the reactions were performed under the same
conditions, 40 ◦C and 300 rpm, in this study to eliminate any tem-
perature or mixing effects. The transesterification efficiencies of
CALB in dried water-miscible ILs, [BMIM][NO3], [C2OHMIM][NO3],
and [C2OHMIM][BF4], are shown in Table 1. It can be found
2.4. Characterizations
Each dried IL was diluted with anhydrous methanol to reduce
viscosity, then its water content was measured by Karl–Fischer
titration (Metrohm Ltd., model 787 KF Titrino). Water con-
tents of the dry [BMIM][BF4], [BMIM][PF6], [C2OHMIM][BF4],

68
G. Ou et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 66–70
Me
Bu
Bu
BuCl
Me
Me
N
N
N
N
Cl
N
N
OH
Reflux
Ionic exchange
[BMIM]OH
Bu
H3PO4
Bu
Me
N
[HPO24-
]
Me
-
[H2PO4 ]
N
+
N
N
pH 6.86
2
IL buffer having phosphate anion
Scheme 1. Synthesis of IL buffer having phosphate anion.
that CALB was inactive in pure and buffered [BMIM][NO3], and
also afforded lower substrate conversion in pure [C2OHMIM][BF4]
(8.7%) and [C2OHMIM][NO3] (6.8%). In contrast, CALB exhib-
ited high levels of transesterification activity in the buffered
[C2OHMIM][BF4] and [C2OHMIM][NO3], indicating that the buffer
was responsible for the rate enhancement.
the introduction of hydroxyl group into ILs to obtain higher dielec-
tric constant and salvation ability to protein molecules. And an
appropriate buffer is needed to closely regulate the ionization state
of ionizable groups of the enzyme. One can conclude that enzyme
activity in ILs is also buffer dependent as in aqueous systems.
The recycling ability of the CALB solubilized in buffered
[C2OHMIM][BF4] under the above reaction conditions is illustrated
in Fig. 1. After each reaction, the reactant mixture in the upper layer
and CALB-IL in the lower layer were separated by decantation. The
CALB-IL phase was then evacuated to remove residual reactant mix-
ture and charged for the next reaction without adding any new
enzyme, IL and buffer. The reaction conversion can maintain 75%
of its initial value after 10 runs, indicating that the dissolved CALB
in the buffered [C2OHMIM][BF4] is a kind of very stable biocatalyst
system.
The above results showed that the enzyme activity is greatly
affected by the solvent and buffer. It has long been know that ion-
ization equilibrium of an acid or a base is affected by a solvent
change, not only because of the acicity or basicity of the solvent,
but also because of its dielectric constant, and the ability of the
solvent to solvate the various species of the equilibrium equation.
The ionization constant of ionizable groups is greatly affected by
the solvent [39]. Thomazeau et al. reported that the acid HNTf2
is more acidic in [BMIM][NTf2] and [BMIM][BF4] than in water
[40]. In aqueous systems, enzymes have an optimum pH or pH
range in which their activity is maximal, and at higher or lower
pH their activity decreases. Drastic changes in pH often lead to
denaturation. Hydrogen ion concentration affects enzymes in sev-
eral ways. First, catalytic activity is related to the ionic state of active
site. Changes in hydrogen ion concentration can affect the ioniza-
tion of active site groups. Secondly, changes in ionizable groups
may change the tertiary structure of the enzyme. Ionic interac-
tions occur between charged amino and carboxyl groups in the side
chains of some amino acids in the folded protein. If the ionization
state of the enzyme is changed, some of those functional groups
will gain or lose a proton and, therefore, will lose their charge or
become charged, depending on which way the ionization state of
the enzyme is changed and by how much. That will eliminate some,
perhaps many, of the ionic interactions that were necessary for
maintenance of the active enzyme conformation. Russell and Fersht
reported that changing the surface charge of subtilisin by site-
directed mutagenesis produces enzymes with significantly shifted
pH-activity profiles, higher catalytic activities and altered specifici-
ties [7]. Based on the above analyses, enzyme-benign ILs needs to
have water-like physicochemical properties, one of which involves
3.3. Intrinsic fluorescence studies
Structural analyses of the enzyme had provided much insight
into the enzyme activity and the mechanism of enzymatic
reactions. The solubility of CALB in hydroxyl-functionalized ILs pro-
vided the opportunity to investigate the enzyme conformation by
various spectroscopic methods. To better understand the effects
of the IL buffer on the conformation of CALB, fluorescence emis-
50
40
30
20
10
0
Table 1
Buffer dependence of transesterification activities of CALB in different ILs.a
Reaction medium (IL)
Conversion/%b
In pure IL medium
In buffered IL medium
1
2
3
4
5
6
7
8
9
10
[BMIM][NO3]
[C2OHMIM][NO3]
[C2OHMIM][BF4]
0
6.8
8.7
0.3
15.6
44.5
Run number
Fig. 1. Stability of CALB in buffered [C2OHMIM][BF4] during recycling. Reaction
conditions: 1.2 mg of CALB; 30 mg of mixed phosphate IL buffer; 500 ␮L of IL;
110 ␮L of ethyl butyrate (0.83 mmol); 110 ␮L of n-butanol (1.21 mmol); stirring
speed = 300 rpm; temperature = 40 ◦C; reaction time = 3 h.
a
The reaction conditions are the same as Fig. 1.
b
Conversion refers to ethyl butyrate. In the absence of CALB, no conversion was
observed in the IL solvent systems studied.

G. Ou et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 66–70
69
140
15
10
5
426
120
426
100
424
80
60
40
20
0
A
B
C
A
C
D
425
0
B
300
350
400
450
Wavelength / nm
300
350
400
450
Wavelength / nm
Fig. 2. Fluorescence emission spectra of CALB in several ILs. (A) Buffered
C2OHMIM][NO3], (B) pure [C2OHMIM][NO3], (C) pure [BMIM][NO3], and (D)
buffered [BMIM][NO3].
Fig. 4. Fluorescence emission spectra of CALB in [C2OHMIM][BF4]. (A) Buffered IL,
(B) pure IL, and (C) buffered IL containing [C2OHMIM][NO3].
sion spectra of CALB are obtained under dilute conditions where
protein–protein interactions are minimal. Fig. 2 indicated that
[BMIM][NO3] completely quenched the fluorescence of the tryp-
tophan (Trp) residues in CALB while a maximum emission at near
425 nm was observed in both pure and buffered [C2OHMIM][NO3].
Compared with the emission contours of the ‘naked’ Trp in bulk
[C2OHMIM][BF4] (Fig. 3), the peak at wavelength ca. 425 nm
in Fig. 2 could be assigned to the fluorescence of Trp residues
in CALB.
partially quenched, suggesting that CALB still preserves its compact
conformation.
In the presence of mixed phosphate IL buffer, the fluorescence
emission spectra of CALB showed an increase in fluorescence inten-
sity (curve A in Figs. 2 and 4) compared with the spectra in pure
hydroxyl-functionalized ILs (curve B in Figs. 2 and 4). A decrease
in fluorescence intensity at ꢀmax is generally observed when pro-
teins unfold. The observed changes in the Trp intensity are most
likely due to Förster-resonance energy transfer (FRET). The most
commonly observed resonance energy transfer in proteins is from
tyrosine (Tyr) to Trp. The rate of energy transfer kT(r) is given by
Eq. (1),
Indole, Trp, and their derivatives are uniquely sensitive to
quenching by electron scavengers such as protons, NO3−, and
fumarate, which are collisional quenchers [41]. For collisional
quenching to occur, the quencher must collide with the fluorophore
during the lifetime of the excited state, causing the fluorophore to
return to the ground state without emission of a photon. Conse-
quently, if the Trp residue is buried inside the protein, quenching
is not expected to occur. If the Trp residue is on the protein sur-
face, then quenching is expected. Native CALB has two surface Trp
residues and three buried Trp residues [42]. The Trp residues in
CALB are almost completely quenched in [BMIM][NO3]. This sug-
gests that under this condition all the Trp residues are accessible
ꢀ
ꢁ
6
R
r
1
D
0
kT (r) =
(1)
ꢁ
where ꢁD is decay time of donor in absence of the acceptor, R0
is the Förster distance, and r is the distance between donor and
acceptor. Any changes in donor-acceptor distance will affect the
rate of energy transfer. If a suitable acceptor fluorophore is nearby,
then non-radiative energy transfer between the donor and accep-
tor can occur. CALB possesses 5 Trp residues (2 on the surface and
3 in the interior) and 9 Tyr residues. Tyr 61, Tyr 91, and Tyr 234
make a “cage” around Trp 52, whereas Tyr 82, Tyr 135, and Tyr
300 surround Trp 113 [42]. Efficient FRET may occur if the distance
−
to NO3 of the solvent, indicating the complete unfolding of the
protein. While in the case of [C2OHMIM][NO3], the Trp residues are
˚
between Tyr and Trp is below the Förster distance 9–18 A [41,43].
100
It was observed that addition of Tyr in Trp [C2OHMIM][BF4] solu-
tion resulted in intensity increase of the Trp (Fig. 3). It is likely that
a significant part of the observed intensity increase at wavelength
425 nm in buffered hydroxyl-functionalized ILs is due to a smaller
Tyr–Trp distance in CALB and thus a more efficient energy transfer,
which may reflect a more compact enzyme conformation. Changes
in the activity of CALB caused by the buffer were found to corre-
late well with the compaction of the specific three-dimensional
structure or conformation of CALB.
426
80
425
60
40
B
Fluorescence quenching experiments have been proved to give
precise information about local conformational fluctuation around
Trp residues. We thus used this approach to investigate the con-
formation of CALB in buffered [C2OHMIM][BF4]. The selective ion
20
A
0
−
NO3 was used as a quencher to preferentially probe Trp residues
near the surface of the protein. The results are shown in Fig. 4. In the
presence of quencher, [C2OHMIM][NO3], the fluorescence emis-
sion spectrum showed a decrease in fluorescence intensity (curve
C in Fig. 4) compared with the spectrum of CALB in the absence of
[C2OHMIM] [NO3] (curve A in Fig. 4). In this case, the surface Trp
300
350
400
450
Wavelength / nm
Fig. 3. Fluorescence emission spectra of Trp. (A) Trp in [C2OHMIM][BF4], (B) addition
of Tyr to Trp IL solution.

70
G. Ou et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 66–70
residues are quenched and the buried ones are not. The water sol-
uble quencher does not readily penetrate the hydrophobic regions
of CALB. This suggests that CALB keeps the compact conformation
in buffered [C2OHMIM][BF4].
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In summary, we have, for the first time, found that both
the conformation and transesterification activity of CALB solubi-
lized in the hydroxyl-functionalized ILs like [C2OHMIM][BF4] and
[C2OHMIM][NO3] were buffer dependent. Intrinsic fluorescence
studies showed that CALB possessed a more compact conformation
in the buffered [C2OHMIM][BF4] or [C2OHMIM][NO3], resulting
in an enhancement of transesterification activity with highly sta-
bility during recycles. The knowledge gained in this study might
be applied to homogeneous enzymatic catalysis in organic sol-
vents with hydroxyl group. The research is currently underway of
progress and will be reported in due course.
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We acknowledge financial supports from the NSFC (20873108
and 20923004), the Science Foundation of Jimei University, the 973
program (2009CB939804) and the Key Scientific Project of Fujian
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Appendix A. Supplementary data
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