Pseudomonas cepacia lipase immobilized onto chitosan-coated activated carbon: An efficient catalyst for transesterification enantiomer resolution of (R,S)-1-Phenyl-3-buten-1-ol

Article abstract of DOI:10.14233/ajchem.2014.18170

Pseudomonas cepacia lipase (PSL) immobilized onto chitosan-coated activated carbon (CS-AC) with specific surface area of 798 (m2/g), average pore diameter of 2.13 nm and pore volume of 0.43 cm3/g was synthesized by a simple adsorption method. The immobilized lipase PSL/CS-AC catalyst exhibited enhanced catalytic activity for resolving racemic 1-phenyl-3-buten-1-ol compared to free PSL and PSL immobilized on sole activated carbon or chitosan. The enantiomeric excess value (ees) of (S)-1-phenyl-3-buten-1-ol and the enantiomeric excess value (eep) of (R)-1-phenyl-3-buten-1-ol acetate reached 99 and 94 %, respectively with the reaction substrate conversion of 51 %. PSL/CS-AC immobilized lipase exposed the more surface active sites resulting in the higher catalytic performances. The preparation of the support for immobilizing lipase is low cost and the reaction conditions are moderate and environmentally friendly. The immobilized lipase PSL/CS-AC catalyst possessed of high thermal stability, reusability and storage stability, it could be a potential chiral catalyst for enantiomer resolution reactions.

Full text of DOI:10.14233/ajchem.2014.18170

Asian Journal of Chemistry; Vol. 26, No. 17 (2014), 5619-5622
ASIAN JOURNAL OF CHEMISTRY
http://dx.doi.org/10.14233/ajchem.2014.18170
Pseudomonas cepacia Lipase Immobilized onto Chitosan-Coated Activated Carbon: An Efficient
Catalyst for Transesterification Enantiomer Resolution of (R,S)-1-Phenyl-3- buten-1-ol†
*
PING XUE, WEIGUANG SU , WEIJING YE, TIANSHENG ZHAO and XIAOYONG LAI
State Key Laboratory Cultivation Base of Natural Gas Conversion, Ningxia University, Yinchuan, P.R. China
*
Corresponding author: Fax: +86 951 2062323; Tel: +86 951 2062835; E-mail: weiguangsu@nxu.edu.cn
AJC-15759
2
Pseudomonas cepacia lipase (PSL) immobilized onto chitosan-coated activated carbon (CS-AC) with specific surface area of 798 (m /g),
3
average pore diameter of 2.13 nm and pore volume of 0.43 cm /g was synthesized by a simple adsorption method. The immobilized lipase
PSL/CS-AC catalyst exhibited enhanced catalytic activity for resolving racemic 1-phenyl-3-buten-1-ol compared to free PSL and PSL
s
immobilized on sole activated carbon or chitosan. The enantiomeric excess value (ee ) of (S)-1-phenyl-3-buten-1-ol and the enantiomeric
excess value (ee ) of (R)-1-phenyl-3-buten-1-ol acetate reached 99 and 94 %, respectively with the reaction substrate conversion of 51 %.
p
PSL/CS-AC immobilized lipase exposed the more surface active sites resulting in the higher catalytic performances. The preparation of
the support for immobilizing lipase is low cost and the reaction conditions are moderate and environmentally friendly. The immobilized
lipase PSL/CS-AC catalyst possessed of high thermal stability, reusability and storage stability, it could be a potential chiral catalyst for
enantiomer resolution reactions.
Keywords: Pseudomonas cepacia lipase, (R,S)-1-Phenyl-3-buten-1-ol, Immobilized lipase, Chitosan-coated activated carbon.
as well as hard recovery and re-use. In this case, immobilization
of lipase can availably overcome these applied limits and it
has been a hotspot in lipase catalysis.
INTRODUCTION
9
,10
Enantiomerically pure alcohols and esters are important
intermediates for synthesizing fine organic chemicals and
widely used in the pharmaceuticals, agrochemicals and chemical
Usually, the properties of immobilized enzyme depend on
11
the nature of the support . Hence, it is important to develop an
efficient support to improve enzymatic properties of immobilized
enzyme. Activated carbon (AC) is one of the most promising
supports for enzyme immobilization because of its large surface
1,2
engineering . Optically active 1-phenyl-3-buten-1-ol and its
substituted derivatives are important precursors for synthesis
of enantiomerically pure natural compounds and biologically
active molecules, such as tetrahydrofuran, α,β-unsaturated
lactones, antitumour macrolides, alkaloids, chiral epoxy
alcohols, β-hydroxy carbonyl compounds and optically pure
12-14
area, high adsorption efficiency . However, the enzyme with
a benign surrounding microenvironment cannot be provided by
simple physical adsorption between enzyme and activated carbon
due to low surface OH groups density on activated carbon. There-
fore, many investigations have focused on the surface modifi-
cation of activated carbon with organic functionalities to improve
3
-5
1
,3-diols, etc. . Efficient transesterification resolution of
racemic alcohols is one of the most important reactions to
produce optically pure alcohols and esters. In comparison with
classical chemical methods such as preferential crystallization,
chromatographic separation, biocatalytic processes are broadly
15,16
enzymatic properties . Chitosan (CS), the amino polysaccha-
ride has received much attention because of its low toxicity, good
biocompatibility, physiological inertness, remarkable affinity to
protein and easy availability of its reactive amino and hydroxyl
6
accepted as good options to prepare optically pure alcohols .
Lipase is one of the important biocatalysts and widely used in
the synthesis and resolution of high optically pure chiral
17,18
groups . Chitosan was used as a modified material to improve
the surface biocompatibility and increase the surface functional
groups density of the activated carbon.
7,8
compounds , which is mainly based on its high efficiency,
stereoselectivity, mild reaction condition and environment
friendly, etc. However, free lipase is difficult to be widely used
in industry because of its insufficient stability, easy deactivation
In the present work, the surface of activated carbon was
modified by the natural polymer chitosan with a physical
†Presented at 2014 Global Conference on Polymer and Composite Materials (PCM2014) held on 27-29 May 2014, Ningbo, P.R. China

5
620 Xue et al.
Asian J. Chem.
OH
coating method, which was denoted as CS-AC. The lipase
Pseudomonas cepacia lipase, PSL) was successfully immo-
O
Immobilized lipase
(
O
bilized on the CS-AC and used for transesterification resolution
of (R,S)-1-phenyl-3-buten-1-ol to obtain high-purity chiral
single enantiomers. The immobilized lipase PSL/CS-AC
showed the highest catalytic activity and enantioselectivity
compared with free PSL and PSL immobilized on sole acti-
vated carbon or chitosan. The enantiomeric excess value of
(
R,S)-1-phenyl-3-buten-1-ol
OAc
OH
O
H
(
R)-1-phenyl-3-buten-1-ol acetate (S)-1-phenyl-3-buten-1-ol
(
S)-1-phenyl-3-buten-1-ol and (R)-1-phenyl-3-buten-1-ol
acetate reached 99 and 94 %, respectively at 51 % substrate
conversion. Furthermore, the PSL/CS-AC catalyst possessed
of higher thermal stability, re-usability and storage stability.
Reaction process and enantiomeric excess (ee) were moni-
tored by periodic withdrawal of clear liquid samples from the
reaction mixture which were analyzed by high performance
gas chromatography (HPGC) (FL9790II, FuliAnalysis Instru-
EXPERIMENTAL
ment Co., Ltd., China) equipped with Cyclosil-B (Agilent
technologies, China). The conversion (C, %) was calculated
Activated carbon, obtained from Aladdin (Shanghai,
China). Chitosan (DACe ≥ 90 %) was obtained from Haidei
Bei Marine Biological Engineering Co., Ltd. (Jinan, China).
Pseudomonas cepacia Lipase (PSL) was supplied by Amano
from the enantiomeric excess of the substrate (ee , %) and
s
product (ee , %) based on the following formulas:
p
eeS
C (%) =
(
USA). (R,S)-1-Phenyl-3-buten-1-ol was purchased from
(1)
(2)
ee + eeP
S
Sigma-Aldrich Shanghai Pvt. Ltd. (Shanghai, China). Heptane,
isooctane, cyclohexane, toluene, benzene, acetonitrile, vinyl
acetate and other analytical reagents were purchased from
Kelong Chemicals (Chengdu, China).All chemicals and enzymes
were used without any further purification.
Preparation and characterization of chitosan coating-
modified activated carbon:The activated carbon was modified
by chitosan in order to improve its surface biocompatibility.
Chitosan was dissolved in 1 % (w/w) aqueous acetic acid
solution at a concentration of 0.1 g/100 mL at 50 ºC. Dry
activated carbon particles (0.5, 1.0 and 1.5 g) were added to
chitosan solution and the resulting mixture was shaken for
[
[
S] −[R]S
[R] −[S]P
P
S
ee (%) =
, ee (%) =
S
P
S
S] +[R]S
P
[R] +[S]P
In the formulae, [S]
of (S)-1-phenyl-3-buten-1-ol and (R)-1-phenyl-3-buten-1-ol,
respectively, also [R] and [S] denotes the concentration of
S
and [R] denotes the concentration
S
P
P
(R)-1-phenyl-3-buten-1-ol ester and (S)-1-phenyl-3-buten-1-
ol ester, respectively.
RESULTS AND DISCUSSION
Characterizations of CS-AC: N adsorption-desorption
2
isotherms of activated carbon and CS-AC with different mass
ratios were showed in Fig. 1. With increasing chitosan amount,
m (chitosan): m (activated carbon) from 1:15 to 1:5, the adsor-
ption capacity of CS-AC composite gradually decreased (1:5,
1-2 h. After added 0.1 N sodium hydroxide, flocculent preci-
pitate appeared from chitosan solution, the precipitate was
obtained by filtration and washed with distilled water to remove
residual sodium hydroxide. The chitosan-coated activated
carbons CS-AC were dried overnight in a vacuum oven at 60 ºC.
The surface area and pore size distribution were derived
1
:10, 1:15 mean the mass ratio of chitosan and activated
carbon). The surface morphologies of activated carbon with
and without coating(s) were obtained by SEM (Fig. 2). The
surface of uncoated activated carbon had a rough and highly
porous structure. However, the surface of chitosan-coated acti-
vated carbon was greatly improved and much smoother than
uncoated activated carbon.
from the N adsorption-desorption isotherms (Micromeritics
2
ASAP-2010). Surface morphology of CS-AC was observed
under a scanning electron microscope (SEM, JSM-7500F/
QUANTAX).
Pseudomonas cepacia lipase (PSL) immobilization on
CS-AC: Pseudomonas cepacia lipase was immobilized onto
the CS-AC as follows: 0.1 g CS-AC was dispersed into 10 mL
isooctane followed by the addition of 30 mg of PSL in solution.
The mixture was shaken in a water bath for 4 h at 30 ºC. The
resulting products were separated, washed with 10 mL of
isooctane and then dried under vacuum, denoted as PSL/CS-
AC.
Comparing the catalytic activity of immobilized lipase
and free lipase: The catalytic activities of transesterification
resolution of (R,S)-1-phenyl-3-buten-1-ol as a function of
reaction time over the free and immobilized lipase were shown
in Fig. 3. It indicated that immobilized lipase PSL/CS-AC
exhibited much higher catalytic activity comparing with the
free lipase. The conversion of (S)-1-phenyl-3-buten-1-ol
reached 50 % on PSL/CS-AC after 12 h, however the conver-
sion was only 6.5 % on free lipase. It was worthy to mention
that the enantioselectivity of (S)-1-phenyl-3-buten-1-ol was
very high on PSL/CS-AC.After reaction 18 h, the enantiomeric
Transesterification resolution of (R,S)-1-phenyl-3-
buten-1-ol: 100 mg of immobilized lipase PSL/CS-AC or
30 mg of free lipase was firstly placed in a 25 mL apparatus,
followed by the addition of 10 mL of heptane, 92 µL of vinyl
acetate and 0.25 mmol of (R,S)-1-phenyl-3-buten-1-ol. Then
the apparatus was oscillated in a 130 rpm water bath shake
bed at the reaction temperature. The principle of transesteri-
fication resolution of (R,S)-1-phenyl-3-buten-1-ol catalyzed
by lipase is shown in the following equation:
excesses (ee
at 99 %, thereby higher optical purity substrate (S)-1-phenyl-
-buten-1-ol could be obtained. However, the enantiomeric
excesses (ee ) was only 9.0 % on free lipase after 24 h.
Free PSL was well dispersed during immobilization,
which decreases the chance of free enzyme convergence in
s
) of (S)-1-phenyl-3-buten-1-ol were maintained
3
s

Vol. 26, No. 17 (2014)
An Efficient Catalyst for Transesterification Enantiomer Resolution of (R,S)-1-Phenyl-3- buten-1-ol 5621
3
3
2
2
50
00
50
00
enhancing the activity of immobilized enzyme.A second possi-
bility was that the conformation of lipase changed during the
course of the CS-AC preparation, which possibly made the
lipase adopt a more active conformation.
Comparing the catalytic activity of immobilized lipase
on different supporters: Fig. 4 displayed the supporter effect
on catalytic activity of immobilized lipase. The results showed
that the enzyme activity of PSL/CS-AC was higher than that
of PSL/CS and PSL/AC. For the immobilized lipase PSL/CS-
AC catalyzing transesterification resolution of racemic 1-phenyl-
3
-buten-1-ol, when reaction temperature was 40 ºC and reaction
time was 6 h, the ee values of (S)-1-phenyl-3-buten-1-ol and
R)-1-phenyl-3-buten-1-ol acetate reached, respectively 90.1
CS-AC(1:5)
CS-AC(1:10)
CS-AC(1:15)
AC
(
and 98.1 % with the reaction substrate conversion of 47.9 %.
It demonstrated that chitosan could improve the surface
biocompatibility of activated carbon and provide a suitable
surface for enzyme immobilization. Therefore, chitosan may
have a widely potential application in preparation of the
required functional carriers.
0
.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Relative pressure (P/P0)
2
Fig. 1. N adsoption-desorption isotherms of activated carbon and CS-AC
ee (%)
100
p
ee (%)
S
C (%)
80
60
Fig. 2. SEM images of activated carbon (a) and CS-AC (1:10) (b)
(
a)100
40
8
6
4
2
0
0
0
0
0
2
0
PSL/CS-AC
free PSL
0
PSL/CS
PSL/AC
Immobilized lipase
PSL/CS-AC
Fig. 4. Compared catalytic performance of immobilized lipase on different
supporters
Recycle reusability of immobilized lipases PSL/CS-AC:
The major and important purpose of immobilized lipases is in
the design of reusable biocatalysts. Hence, we also investigated
the durability of PSL/CS-AC in the repeated catalyzed trans-
esterification of (R,S)-1-phenyl-3-buten-1-ol. After each run,
the immobilized lipase was washed several times with heptane
and was reintroduced into fresh reaction medium for another
assay run. These procedures were repeated up to several times.
0
4
8
12
16
20
24
Reaction time (h)
(
b)100
PSL/CS-AC
free PSL
80
60
40
20
0
Fig. 5 showed that the ee
s
and ee values were only slightly
p
decreased during 10 consecutive operations at 40 ºC under
the same reaction time of 18 h. These results indicated that
PSL/CS-AC could retain high enantioselectivity and catalytic
performance in the process of reuse and the recycle reusability
of PSL/CS-AC was good. However, it was still not ideal because
the activity of immobilized lipase was decreased with incre-
asing the recycle number, which may be due to the leakage
and deactivation of partial lipase resulting from long-playing
mechanical stirring under the continuous reactions.
0
4
8
12
16
20
24
Reaction time (h)
Fig. 3. Compared catalytic performance of PSL/CS-AC and free PSL
organic solvents. Comparing with the free PSL, the exposed
active sites on immobilized PSL were much more, increasing
the accessibility for substrate to the active site of enzyme and
Comparing the thermal stability of free PSL and
immobilized lipases PSL/CS-AC: The transesterification of
(
R,S)-1-phenyl-3-buten-1-ol catalyzed by PSL/CS-AC in a

5
622 Xue et al.
Asian J. Chem.
TABLE-2
1
00
STORAGE STABILITY OF PSL/CS-AC
ee (%)
S
a
s
ee (%)
Storage days (d)
1
ee
(%)
99.9
94.1
ee
94.9
99.7
p
(%)
C (%)
51.3
47.4
p
C (%)
8
0
0
0
0
0
90
a
Reaction conditions: reaction temperature 40 ºC, reaction time 18 h.
6
4
2
Conclusion
In summary, we have successfully developed an efficient
method for the transesterification resolution of racemic 1-
phenyl-3-buten-1-ol by PSL immobilized on chitosan-coated
activated carbon. Chitosan was used as a modified material to
improve the surface biocompatibility and increase the surface
functional groups density of the activated carbon. The prepared
immobilized lipase PSL/CS-AC catalyst exhibited much
enhanced catalytic activity for resolving racemic 1-phenyl-3-
buten-1-ol to the corresponding optically pure (S)-1-phenyl-
0
1
2
3
4
5
6
7
Recycle numbers
8
9
10
11
12
Fig. 5. Reusability of PSL/CS-AC
temperature range from 40 to 85ºC was investigated in Table-1
and the highest activity of PSL/CS-AC was obtained at 40 ºC.
The thermal stability of the free and immobilized lipase was
studied under the same temperature range. The enantiomeric
excess of (S)-1-phenyl-3-buten-1-ol and (R)-1-phenyl-3-buten-
3
-buten-1-ol compared with the free lipase PSL and PSL
immobilized on single activated carbon or chitosan. The ee
and ee reached 99 and 94 %, respectively with the reaction
s
p
substrate conversion of 51 %. Moreover, the immobilized lipase
PSL/CS-AC catalyst possessed of high thermal stability, reusa-
bility and storage stability.
1
-ol acetate reached, respectively 63.2 and 96.8 % with the
reaction substrate conversion of 40.0 % when the reaction
temperature was 85 ºC. However, both conversion and enantio-
ACKNOWLEDGEMENTS
meric excesses (ee
concluded that the thermal stability of PSL could be greatly
improved through the immobilization on CS-AC.
s
) were only 0.8 % on the free lipase. It was
This work was financially supported by the National
Natural Science Foundation of China ( No.21263020) and the
National Basic Research Program 973 (No. 2012CB723106).
REFERENCES
TABLE-1
1
2
.
.
G.W. Zheng and J.H. Xu, Curr. Opin. Biotechnol., 22, 784 (2011).
R.N. Patel, Food Technol. Biotechnol., 42, 305 (2004).
THERMAL STABILITY OF FREE PSL AND PSL/CS-AC
Reaction
temperature (ºC)
a
Lipase
ee
s
(%)
ee
p
(%)
C (%)
3. A. Arefolov and J.S. Panek, Org. Lett., 4, 2397 (2002).
T.P. Loh, Q.Y. Hu and J.J. Vittal, Synlett., 523 (2000).
5. S. Singh, S. Kumar and S.S. Chimni, Tetrahedron Asymm., 13, 2679
2002).
A. Ghanem and H.Y. Aboul-Enein, Tetrahedron, 15, 3331 (2004).
7. M. Nagarl, S.K. Dwivedi and D. Shrivastava, Int. J. Pharm. Res. Sci.,
2, 631 (2013).
8. Q. Wu, P. Soni and M.T. Reetz, J. Am. Chem. Soc., 135, 1872 (2013).
4
.
Free PSL
PSL/CS-AC
Free PSL
PSL/CS-AC
free PSL
PSL/CS-AC
Free PSL
PSL/CS-AC
1.7
90.1
1.1
79.1
0.9
70.0
0.8
99.9
98.1
99.9
95.8
99.9
97.7
99.9
96.8
1.7
47.9
1.1
45.2
0.9
41.6
0.8
40.0
40
70
80
85
(
6
.
9
.
R.C. Rodrigues, C. Ortiz, Á. Berenguer-Murcia, R. Torres and R. Fernández-
Lafuente, Chem. Soc. Rev., 42, 6290 (2013).
10. S. Cantone, V. Ferrario, L. Corici, C. Ebert, D. Fattor, P. Spizzo and L.
63.2
a
Reaction conditions: reaction time 6 h.
Gardossi, Chem. Soc. Rev., 42, 6262 (2013).
1
1. G. Yang, J. Wu, G. Xu and L. Yang, J. Mol. Catal. B, 57, 96 (2009).
12. A.S. Rani, M.L.M. Das and S. Satyanarayana, J. Mol. Catal. B, 10,
71 (2000).
Storage stability of immobilized lipases PSL/CS-AC:
4
Enzyme is generally stored at 0-4 ºC to maintain the stability
of its nature conformation. However, enzyme is inevitably
exposed to external environment and loses activity rapidly in
industrial application. Thus the storage stability of immobilized
enzyme has always been of interest. As shown in Table-2,
immobilized lipase PSL/CS-AC could remain high catalytic
performance and enantioselectivity after storing for 90 days
at room temperature, the enantiomeric excesses of (S)-1-phenyl-
1
1
1
1
3. A.G. Kumar, S. Swarnalatha, P. Kamatchi and G. Sekaran, Biochem.
Eng. J., 43, 185 (2009).
4. J.C. Naranjo, A. Córdoba, L. Giraldo, V.S. García and J.C. Moreno-
Piraján, J. Mol. Catal. B, 66, 166 (2010).
5. J. Miao, F. Zhang, M. Takieddin, S. Mousa and R.J. Linhardt, Langmuir,
2
8, 4396 (2012).
6. K. Ramani, S. Karthikeyan, R. Boopathy, L.J. Kennedy, A.B. Mandal
and G. Sekaran, Process Biochem., 47, 435 (2012).
17. B. Krajewska, Enzyme Microb. Technol., 35, 126 (2004).
1
8. V.R. Sinha, A.K. Singla, S. Wadhawan, R. Kaushik, R. Kumria, K.
3
-buten-1-ol and (R)-1-phenyl-3-buten-1-ol acetate were still
sustained at 94.1 and 99.7 %, respectively with the reaction
substrate conversion of 47.4 %.
Bansal and S. Dhawan, Int. J. Pharm., 274, 1 (2004).