Ionic liquid co-lyophilized enzyme for biocatalysis in organic solvent: Remarkably enhanced activity and enantioselectivity

Article abstract of DOI:10.1016/j.molcatb.2010.11.017

The room temperature solid-phase ionic liquid (RTSPIL) co-lyophilized enzyme exhibited markedly enhanced activity in organic solvent. The enzyme co-lyophilized with a dodecyl-imidazolium salt was 660-fold more active compared to its RTSPIL-free counterpart. The activity enhancement by RTSPILs was mainly attributable to the reduced particle sizes and improved dispersion of enzymes suspended in organic solvent. Also, the RTSPIL co-lyophilized enzyme displayed significantly enhanced enantioselectivity. Its enantioselectivity was 2.5-fold higher than that of its RTSPIL-free counterpart.

Full text of DOI:10.1016/j.molcatb.2010.11.017

Journal of Molecular Catalysis B: Enzymatic 68 (2011) 275–278
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Ionic liquid co-lyophilized enzyme for biocatalysis in organic solvent:
Remarkably enhanced activity and enantioselectivity
a,∗
b,∗∗
Jae Kwan Lee , Mahn-Joo Kim
a
Department of Green Energy & Research Center for Convergence Technology, Hoseo University, 165 Sechul-ri Baebang-myun, Asan, Chungnam 336-795, Republic of Korea
Department of Chemistry, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 October 2010
Received in revised form
The room temperature solid-phase ionic liquid (RTSPIL) co-lyophilized enzyme exhibited markedly
enhanced activity in organic solvent. The enzyme co-lyophilized with a dodecyl-imidazolium salt was
6
60-fold more active compared to its RTSPIL-free counterpart. The activity enhancement by RTSPILs
2
4 November 2010
was mainly attributable to the reduced particle sizes and improved dispersion of enzymes suspended in
organic solvent. Also, the RTSPIL co-lyophilized enzyme displayed significantly enhanced enantioselec-
tivity. Its enantioselectivity was 2.5-fold higher than that of its RTSPIL-free counterpart.
Accepted 24 November 2010
Available online 2 December 2010
©
2010 Elsevier B.V. All rights reserved.
Keywords:
Ionic liquid
Biocatalysis
Trasesterification
Lyophilization
Room temperature solid-phase
1
. Introduction
that if the enzyme were coated by ionic liquid during lyophiliza-
tion in aqueous medium, it would exhibit better activity. We herein
wish to report a room temperature solid-phase ionic liquid co-
lyophilized enzyme, exhibited remarkably enhanced activity and
enantioselectivity.
The room temperature ionic liquids (RTILs) and solid-phase
ionic liquids (RTSPILs), which become liquid at elevated temper-
ature, are very attractive materials. They are organic salts and their
various properties, such as solubility in solvents, melting point, and
conductivity are tunable. Particularly, RTSPILs led us to envisage
that they might be suitable as lyoprotectants for the lyophilization-
induced activation of enzyme, as well as the coating materials for
operational practicality. RTSPILs are commercially available or eas-
ily synthesized in good yields [17]. We used ionic liquids composed
of imidazolium or pyridinium cation and hexafluorophosphate
anion (Scheme 1).
Nonaqueous biocatalysis provide
a useful component of
methodology in organic synthesis [1]. For example, lipase catalysis
in organic solvents is of great use for the synthesis of opti-
cally active compounds such as chiral alcohols, acids, and their
esters [2]. However, biocatalysis in nonaqueous media often suf-
fer from reduced activity, selectivity or stability of enzyme [3].
To overcome these limitations, many approaches have focused on
the development of more efficient enzymes. One of them is the
lyophilization of enzyme in the presence of lyoprotectant such
as inorganic salt, crown ether, and cyclodextrin, which enhanced
the activity, selectivity, or stability of enzyme [4–7]. Also the use
of ionic liquids as an alternative solvent in the biotransforma-
tions enhanced the activity, selectivity, and stability of enzyme
[
8–15].
Previously, we have reported that ionic liquid-coated enzyme
ILCE), which was readily prepared by mixing enzyme powder with
(
2. Experimental
a room temperature solid phase ionic liquid (RTSPIL) at elevated
temperature, exhibited enhanced enantioselectivity and stability in
organic solvent [16]. This method, however, was unable to enhance
the activity of enzyme. To overcome this limitation, we thought
2.1. General remarks
Burkholderia cepacia lipase as crude enzyme was available from
some commercial suppliers such as Fluka, Roche, and Amano. We
used the one provided by Amano. And all reagents were purchased
from Aldrich. Thin-layer chromatography was performed in Merck
silicagel 69F245 andcolumnchromatography was performed using
a Merck silica gel 60. 1H and C NMR spectra were recorded on a
∗
Corresponding author. Tel.: +82 41 540 9639; fax: +82 41 540 9639.
Corresponding author. Tel.: +82 54 279 2112; fax: +82 54 279 0654.
E-mail addresses: jklee@hoseo.edu (J.K. Lee), mjkim@postech.ac.kr (M.-J. Kim).
∗
∗
13
1
381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.11.017

2
76
J.K. Lee, M.-J. Kim / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 275–278
Scheme 1. Structures of room temperature solid-phase ionic liquids (RTSPILs).
Bruker AM-300 instrument with peak referenced to tetramethyl
2.4. RTSPIL co-lyophilized enzyme catalyzed transesterification
for reactivity
silane in CDCl . Mass spectroscopy was recorded using a Kratos
3
Ms 25RFA (70 eV, EI). HPLC from SpectraSYSTEM (P2000) and GC
from Hewlett Packard (HP6890) were used for determining the
enantioselectivity and reactivity of enzymes.
The lipase (1 mg/mmol), 1-phenethyl alcohol (0.2 mmol), vinyl
acetate (0.6 mmol) were mixed with anhydrous toluene (0.2 M),
and the resulting mixture was shaken at 170 rpm and 25 C. The
◦
2
.2. Synthesis of room-temperature solid-phase ionic liquids
reaction mixture was sampled several times to determine the reac-
tivity using GC analysis.
(
RTSPILs)
The RTSPILs used in this study were prepared according to
the following procedure reported previously [16]: alkyl halide
2.5. RTSPIL co-lyophilized enzyme catalyzed transesterification
for enantioselectivity
(
0.125 mol) was dissolved in 1-methylimidazole (or 3(or 4)-methyl
◦
pyridine) (0.125 mol) and then refluxed for 24 h at 70 C. Water
The bulky secondary alcohol substrate (20 mg, 0.1 mmol), vinyl
acetate (28 ml, 0.3 mmol), and lipase (0.5 mg/mmol) were added
in toluene (0.5 ml), and the resulting semi-homogeneous mixture
(
100 ml) and HPF6 (0.15 mol) were added to the reaction mixture
and stirred vigorously at room temperature. The reaction mix-
ture was diluted with CH Cl2 (200 ml) and washed with saturated
◦
2
was shaken at 170 rpm and 25 C for 8 h. Then, the enantiomeric
NaHCO (2× 100 ml). The organic layer was dried and concentrated
purities were determined by HPLC using a chiral column.
3
in vacuo. Various RTSPILs containing PF6 anion were obtained
in good yields (>90%): 1-ethyl-3-methylimidazolium ([EMIM], 1,
mp: 60 C), 1-(3 -phenylpropyl)-3-methylimidazolium ([PPMIM],
3
. Results and discussion
◦
ꢀ
◦
ꢀ
2
(
(
(
6
1
,
mp: 52 C), 1-(3 -phenylpropyl)-2,3-dimethylimidazolium
3.1. Catalytic activity
◦
[PPDMIM], 3, mp: 116 C), 1-dodecyl-3-methylimidazolium
[C12MIM], 4, mp: 50 C), 1-dodecyl-2,3-dimethylimidazolium
◦
As a representative enzyme for lyophilization in presence of
◦
[C12DMIM], 5, mp: 69 C), 1-ethyl-4-methylpyridinum ([E4MPy],
ionic liquid, the crude Burkholderia cepacia lipase was chosen since
◦
◦
, mp: 78 C), 1-butyl-4-methylpyridinum ([B4MPy], 7, mp: 43 C),
it had been frequently used for biotransformations in organic
ꢀ
◦
-(3 -phenylpropyl)-4-methylpyridinum ([PP4MPy], 8, mp: 80 C),
and 1-(3 -phenylpropyl)-3-methylpyridinum ([PP4MPy], 9, mp:
solvents. The ionic liquid co-lyophilized enzyme samples1 were
ꢀ
prepared by lyophilizing the THF-buffer solution (pH 7.0) con-
taining different amounts of enzyme, ionic liquid and sodium
phosphate buffer for 48 h. The enzymatic activities of these sam-
ples were determined as the initial rates of the transesterification
reaction between sec-phenethyl alcohol and vinyl acetate in anhy-
drous organic solvents, and compared with those of crude, salt-free,
and KCl co-lyophilized enzymes. The results were described in
Table 1.
The data from Table 1 indicate that the enzymatic activities
were dependent on the ionic liquid contents of lyophilized enzyme
preparations. Because the ionic liquids having hexafluorophos-
phate anion are hydrophobic and insoluble in water, THF was
employed as the cosolvent with aqueous buffer. It was observed
◦
7
4 C).
2.3. Preparation of RTSPIL co-lyophilized enzyme
An enzyme solution was prepared by dissolving crude lipase
(
1.25 g) in a 10 mM sodium phosphate buffer (5 ml) and filtered to
remove insoluble contaminant. The RTSPIL (wt% IL/enzyme) in THF
was added to the enzyme solution, then the homogeneous solu-
◦
tion was titrated to pH 7.0 by HCl and frozen at −78 C, followed
◦
by lyophilization at around 60 Pa and −53 C for 48 h to provide.
The buffer content in final preparation was 0.1 wt%. We determined
the particle sizes of IL-colyophilized enzyme using a particle size
analyzer (Malvern Instrument) according to the following method;
the IL-colyophilized enzyme (1 g) was suspended in toluene (1 L),
which was used as organic solvent for the biocatalytic reaction
in this study, and then analyzed by means of laser diffraction to
determine the average particle sizes.
1
The water concentration of lyophilized enzyme was determined by the
Karl–Fisher coulometer. All ionic liquid and KCl co-lyophilized enzymes contained
0.4–0.5 wt% of water and both crude and salt-free enzymes contained 5 wt% of water.

J.K. Lee, M.-J. Kim / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 275–278
277
Table 1
Initial activity of lipase for transesterification reaction of secondary alcohol in
a
toluene .
Entry
Excipient
Initial activity ␮mol
Activation
factor
Total activation
factor
−1
−1
d
e
(mg enzyme)
h
)
1
2
3
4
5
6
7
8
9
0
1
2
3
Crudeb
None
None
KCl
1
2
3
4
5
6
0.8
0.8
1.0
37
151
50
–
1.0
1.3
46
189
63
1
0.1
0.1
5
19
6
c
60
75
8
530
384
347
237
306
234
663
480
434
396
384
293
66
48
43
30
38
29
1
1
1
1
7
8
9
Fig. 1. Correlation between particle size (ꢀ) and initial activity (᭹) of RTSPIL (4)
co-lyophilized enzyme in toluene.
a
The reactions were performed using PCL (1 mg/mmol), 1-phenethyl alcohol
(
2
0.2 mmol), vinyl acetate (0.6 mmol) in anhydrous toluene (0.2 M) at 170 rpm and
◦
5
C, and analyzed by GC equipped with a chiral capillary column (Chiraldex B-PH,
Altech).
data from Fig. 1 indicate that the average particle sizes of ionic
liquid-enzyme preparations decrease with increasing the ionic liq-
uid content. This observation therefore suggests that the enhanced
enzymatic activity should result from the decreased particle sizes
of insoluble enzyme aggregates. Consequently, we note that the
ionic liquid-induced activation is mainly the size effect rather than
the matrix effect.
For the preparation of ionic liquid-colyophilzed enzymes, we
used water-soluble enzymes (about 10% of total weight) prepared
from the commercially available crude enzymes. It was found that
the water-soluble enzymes had the same specific activity as that of
the crude enzymes. But the total activity of soluble enzymes was
only 10% of that of crude enzymes used (entry 2, Table 1). This
implies that the purification of soluble enzymes from the crude
enzymes is not practical from a total activity point of view. This
problem can be overcome by the lyophilization of soluble enzymes
in the presence of ionic liquids, which enhances dramatically
the enzymatic activity. The total activitiy of ionic liquid-activated
enzymes was 6–66-fold higher than that of the crude enzyme
(entries 4–13). On the basis of the results, we believe that the
lyophilization of soluble enzymes in presence of ionic liquid pro-
vide a very practical method for the activation of enzymes for use
in organic solvent.
b
Crude PCL before purified.
Lyophilization in THF-buffer medium.
Activation factor is (initial activity)excipient/(initial activity)none.
Total activation factor is (total activity)excipient/(total activity)crude.
c
d
e
that the co-medium had little effect on the activity of lyophilized
enzymes (entry 3).
Previously, it was reported that the enzymatic activities were
dramatically enhanced in case proteases such as subtilisin were
colyophilized with 98% (w/w) KCl salts [6]. In this work, 98% (w/w)
KCl co-lyophilized lipase displayed 40-fold enhanced activity com-
pared to its salt-free counterpart. Surprisingly, 98% (w/w) ionic
liquid co-lyophilized enzyme preparations were significantly more
active than the 98% (w/w) KCl-lipase preparation. The former dis-
played 63–663-fold enhanced activities (entry 4–13). It was notable
that the largest activity enhancement was achieved with ionic liq-
uid 4. All the data from Table 1 thus indicate that ionic liquids are
more efficient lyoprotectant than KCl for lipase. We could not find
any dependency of activation on the structure or side chain length
of ionic liquid. Until now, there is no conclusive explanation for the
activationof enzyme bylyoprotectants, ligands, orinorganic saltsas
KCl. However many researcher have typically explained the activa-
tion of enzyme by co-lyophlization as lyoprotectant, imprinting, or
matrix effects. For example, Dordick and Clark have hypothesized
that the activation of protease by KCl salts is due to a protective
effect afforded by the salt matrix against deactivation by direct
contact with the organic solvent [6]. Initially, we also assumed that
ionic liquid matrix might protect enzyme because most ionic liq-
uids are insoluble in toluene. However, it was observed that the
enzyme colyophilized with the organic soluble ionic liquids, 4 and
3.2. Enantioselectivity
The enantioselectivity of ionic liquid (4)-co-lyophilized enzyme
was examined in the transesterification reaction of a bulky sec-
◦
ondary alcohol in the presence of vinyl acetate at 25 C (Scheme 2).
For comparison, the same reactions were carried out with both
crude and salt-free lyophilized enzymes. The results were summa-
rized in Table 2. The ionic liquid co-lyophilized enzyme displayed
better enantioselectivity than the crude and salt-free enzymes. The
enantioselectivity enhancement by the use of RTSPIL is about 2.5-
fold (compare entries 1–2 and 3). The results are comparable to
those reported previously for the ionic liquid coated enzyme (ILCE)
[16], suggesting that the RTSPIL enhances the enantioselectivity of
enzyme in a similar fashion to the ILCE. However, it is not clear why
5, exhibited the best activities, which is against the matrix effect.
Accordingly, other factor should be responsible for the ionic liquid-
induced activation of enzyme. Because the activity of enzyme is
dependent on the ionic liquid content of lyophilized preparation,
we investigated the correlation between the ionic liquid contents
and the particle sizes of ionic liquid 4 co-lyophilized enzymes in
toluene. The ionic liquid content varied from 0 to 98 (w/w%). The
Scheme 2. Lipase-catalyzed transesterification of secondary alcohol in toluene.

2
78
J.K. Lee, M.-J. Kim / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 275–278
Table 2
The enantioselectivities in the lipase-catalyzed transesterifications in toluene , .
electivity than the ionic liquid-free enzymes. Further studies to
broaden the scope of ionic liquid co-lyophilized enzymes as well
as to apply them in synthetic transformations are in progress at
this laboratory.
a
b
Entry
Lipase
ees
eep
Ec
1
2
3
Crude
Salt-free lyophilized
4 co-lyophilized
0.459
0.214
0.549
0.971
0.977
0.987
107
106
266
Acknowledgment
a
Experimental procedure: the ionic liquid co-lyophilized PCL-catalyzed reaction
is described as a representative procedure. Substrate, 11 (20 mg, 0.1 mmol), vinyl
acetate (28 ␮L, 0.3 mmol), and enzyme (0.5 mg/mmol) were mixed with toluene
This work was supported by the National Research Foundation
of Korea (grant numbers, 2009-0074357 and 2010-00165519).
(
2
0.5 mL), and the resulting semi-homogeneous mixture was shaken at 170 rpm and
◦
5
C for 8 h.
b
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.0 ml/min, UV: 217 nm), acetate, 11 (Whelk-O1, hexane/2-propanol: 96/4, flow
1
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may be due to more favorable structural adaptation of enzyme in
polar ionic environment.
[
2
2
[
[
4
. Conclusion
(
1
In conclusion, this work has demonstrated that the RTSPIL
[
co-lyophilized enzyme is an efficient and practical catalyst for
use in organic solvents. In case using a dodecyl immidazolium
salt as RTSPIL, it is 660-fold more active compared to the ionic
liquid-free enzyme. Also, it is superior to its KCl-activated counter-
part. The enhancement of enzymatic activity by RTSPILs is mainly
attributable to the better dispersion of enzymes in organic solvent
because the average particle sizes of enzymes suspended in organic
solvent are significantly reduced by co-lyophilization with RTSPIL.
The RTSPIL co-lyophilized enzymes also display better enantios-
4
[
[
[
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