Ultrasound-assisted enantioselective esterification of ibuprofen catalyzed by a flower-like nanobioreactor

Article abstract of DOI:10.3390/molecules21050565

A flower-like nanobioreactor was prepared for resolution of ibuprofen in organic solvents. Ultrasound irradiation has been used to improve the enzyme performance of APE1547 (a thermophilic esterase from the archaeon Aeropyrum pernix K1) in the enantioselective esterification. Under optimum reaction conditions (ultrasound power, 225 W; temperature, 45°C; water activity, 0.21), the immobilized APE1547 showed an excellent catalytic performance (enzyme activity, 13.26 μmol/h/mg; E value, 147.1). After ten repeated reaction batches, the nanobioreactor retained almost 100% of its initial enzyme activity and enantioselectivity. These results indicated that the combination of the immobilization method and ultrasound irradiation can enhance the enzyme performance dramatically.

Full text of DOI:10.3390/molecules21050565

molecules
Article
Ultrasound-Assisted Enantioselective
Esterification of Ibuprofen Catalyzed by a
Flower-Like Nanobioreactor
Baiyi An 1, Hailin Fan 2, Zhuofu Wu 3,4, Lu Zheng 4, Lei Wang 4, Zhi Wang 4,* and Guang Chen 3,
*
1
College of Horticulture, Jilin Agricultural University, Changchun 130118, China; swabyswaby@163.com
College of Resources and Environment, Jilin Agricultural University, Changchun 130118, China;
2
fanhl@jlau.edu.cn
3
College of Life Science, Jilin Agricultural University, Changchun 130118, China; wuzhuofu@163.com
4
Key Laboratory of Molecular Enzymology and Engineering of Ministry of Education, School of Life Sciences,
Jilin University, Changchun 130012, China; zhengl14@mails.jlu.edu.cn (L.Z.); w_lei@jlu.edu.cn (L.W.)
Correspondence: wangzhi@jlu.edu.cn (Z.W.); chg61@163.com (G.C.);
*
Tel.: +86-431-8518-2281 (Z.W.); +86-431-8453-2942 (G.C.)
Academic Editor: Derek J. McPhee
Received: 17 March 2016; Accepted: 25 April 2016; Published: 28 April 2016
Abstract: A flower-like nanobioreactor was prepared for resolution of ibuprofen in organic
solvents. Ultrasound irradiation has been used to improve the enzyme performance of APE1547
(a thermophilic esterase from the archaeon Aeropyrum pernix K1) in the enantioselective esterification.
Under optimum reaction conditions (ultrasound power, 225 W; temperature, 45 ˝C; water activity,
0.21), the immobilized APE1547 showed an excellent catalytic performance (enzyme activity,
13.26 µmol/h/mg; E value, 147.1). After ten repeated reaction batches, the nanobioreactor retained
almost 100% of its initial enzyme activity and enantioselectivity. These results indicated that the
combination of the immobilization method and ultrasound irradiation can enhance the enzyme
performance dramatically.
Keywords: ibuprofen; APE1547; resolution; ultrasound; nanobioreactor
1. Introduction
Ibuprofen is a nonsteroidal drug and has an asymmetric carbon in its second position. It’s known
that its bioactivity mainly resides in its S-enantiomer [1]. Therefore, the synthesis of (S)- ibuprofen is
strongly recommended. It is well known that chemical synthesis is the classical method for preparing
the chiral compounds, but it is usually conducted using asymmetric synthesis catalyzed by expensive
catalysts, which are inefficient, laborious and toxic. Compared with the chemical synthesis, many
biocatalytic processes have been developed and turned out to be more attractive for obtaining the chiral
compounds with high enantiopurity [
resolution is one of the most efficient methods [
2
–
4
]. Among the biocatalytic processes, enzyme-catalyzed kinetic
]. In our previous study [ ], we successfully used a
5
6
thermophilic esterase (APE1547) from the archaeon Aeropyrum pernix K1 to carry out the enantioselective
esterification of ibuprofen. We also found that ultrasound can be adopted to improve the biocatalytic
properties of APE1547 in the resolution of ibuprofen [7]. However, the enzyme performance of
thermophilic esterase (APE1547) is still not satisfacctory and the used enzyme could not be recycled.
It’s known that immobilization is a powerful tool to avoid the enzyme aggregation in organic
solvents and can enhance the reusability. Furthermore, immobilization may also improve other enzyme
properties, such as stability, selectivity or specificity [
not be used efficiently under high-speed agitation or under ultrasound [15
immobilized enzymes are prepared by physical adsorption, but, high-speed agitation or ultrasound
8–
14]. However, many immobilized enzymes can
,16]. For example, some
Molecules 2016, 21, 565; doi:10.3390/molecules21050565
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can destroy the weak interactions between the enzyme and the immobilized support, which will
induce enzyme loss during the reaction cycles and inactivate the enzyme [14]. Some enzymes are
immobilized on fragile supports with poor operational stability [17]. Both high-speed agitation and
ultrasound can cause the collapse of the immobilized support and affect the enzyme performance of
the immobilized enzyme. In 2014, we prepared a flower-like nanobioreactor which was formed via
the coordination between Cu2+ and the nitrogen atoms of the amide groups in the enzymes [18]. The
simple and efficient preparation procedure does not require expensive facilities and the preparation
cost is low. Furthermore, our unpublished study has demonstrated that this kind of immobilized
enzyme is very stable under many extreme environments (low or high pH, high temperature, organic
reaction media, high-speed agitation or ultrasound, et al.). These promising advantages make this
immobilization method more attractive for further study.
In this study, we combined this immobilization method and ultrasound irradiation to improve
the enzyme performance in enantioselective esterification of ibuprofen (Scheme 1). Furthermore, the
stability and reusability of the APE1547-incorporated nanobioreactor under ultrasound irradiation
had also been studied.
immobilized APE1547
+
+H2O
+ C8H17-OH
ultrasound
H
COOH
COOC8H17
COOH
H
(R,S)-ibuprofen
(R)-ibuprofen ester
(S)-ibuprofen
Scheme 1. Schematic diagram of the resolution of ibuprofen.
2. Results and Discussion
2.1. The Morphologies of the Immobilized Samples
A large number of immobilized APE1547 have been successfully prepared (Figure 1A,B). The
shape of the immobilized enzyme looked like a natural flower, with dozens of separate petals with
high surface (Figure 1C). Its diameter was about 4–5
µm. The excellent hierarchical structure with high
surface to volume ratios is likely to improve the enzyme performance of the immobilized enzyme.
Figure 1. The morphologies of the immobilized samples. (A–C): SEM images of the immobilized
samples with different enlargement factors.

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2.2. FTIR
The FTIR spectra of the immobilized samples were recorded and the results are shown in Figure 2.
Several cha´ra1cteristic peaks could be observed in curve c. Among of them, the peaks at 1053´c1m´1
3´ ´1
[19]. The peaks at 1652 cm and 1521 cm are
and 556 cm are assigned to the vibrations of PO4
ascribed to the vibrations of the amide I and II bands of the enzyme [20]. These results successfully
verified the presence of enzyme in the immobilized samples.
Figure 2. The FTIR spectrum of Cu3(PO4)2 matrices (curve a), APE1547 (curve b) and the immobilized
APE1547 (curve c).
2.3. Effect of Ultrasound Power
The effect of ultrasound power on the enantioselective esterification was examined and the results
are shown in Figure 3. The highest enantioselectivity was obtained at an ultrasound power of 225 W
and the maximum enzyme activity could be observed at 200 W. The enhancement of enzyme activity
obtained at suitable ultrasound power (225 W) was probably attributed to the better mixing of the
reactants [21]. Furthermore, the improvement of the mass transfer processes in the nanobioreactor can
also increase the enzyme activity considering the complexity and heterogeneity of the immobilized
enzyme. However, the mechanism for the ultrasound-induced alteration in the enantioselectivity is
still unclear. Our previous study has demonstrated that ultrasound can induce a slight change of
the secondary structure of APE1547 and then influence its enantioselectivity [7]. Further research is
needed to clarify the mechanism and will be published in due course.
Figure 3. Effect of ultrasound power on the enzymatic esterification of ibuprofen. Reaction conditions:
the reactions were carried out in n-heptane (10 mL, aw 0.21) with racemic ibuprofen (0.2 mmol),
1-octanol (0.2 mmol) and the immobilized APE1547 powder (160 mg) at 45 ˝C.

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2.4. Effect of Temperature
The effects of ultrasound temperature on the enzyme performance have also been investigated
in this study. The result in Figure 4 showed that higher enantioselectivity could be obtained
at lower temperature. The possible explanation is that high temperature may destroy the
conformation of the enzyme by heat-induced destruction of non-covalent interactions and decrease
the enantioselectivity [22,23]. The enzyme activity exhibited a bell shaped curve with the changing
˝
ultrasound temperature, and the optimal temperature was 45 C.
Figure 4. Effects of temperature on the enzymatic esterification of ibuprofen. Reaction conditions: the
reactions were carried out in n-heptane (10 mL, aw 0.21) with racemic ibuprofen (0.2 mmol), 1-octanol
(0.2 mmol) and the immobilized APE1547 powder (160 mg) at different temperature. The ultrasound
power was 225 W.
High reaction temperatures may elevate the collision probability between enzyme and substrate
molecules to form enzyme-substrate complexes and then enhance the enzyme activity. The activity
significantly decreased at higher temperatures, which might be due to the denaturation of the
enzyme, especially under ultrasound irradiation [23]. Considering both the enzyme activity and
˝
enantioselectivity, 45 C was selected as the optimal reaction temperature.
2.5. Effect of Water Activity
Water may influence the performance of the enzymes in organic solvents [24]. The results in
Figure 5 showed that the enzyme activity increased as water activity increased from 0.09 to 0.21, then
decreased at higher water activity. The maximum enantioselectivity (E value) could also be observed
at aw = 0.21. Water activity may influence the reaction rates of the two isomers and then induce the
alteration of enantioselectivity of APE1547 [24]. Since the E value (147.1) was found to be highest at
aw =0.21 while maintaining the highest enzyme activity (13.26 µmol/h/mg), the water activity of 0.21
was selected for further study.
Figure 5. Effect of water activity on the enzymatic esterification of ibuprofen. Reaction conditions:
the reactions were carried out in n-heptane (10 mL) with racemic ibuprofen (0.2 mmol), 1-octanol
(0.2 mmol) and the immobilized APE1547 powder (160 mg) at 45 ˝C. The ultrasound power was 225 W.

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2.6. Reusability and Stability
For checking the reusability, the immobilized enzyme was used in ten continuous batches for
the enzymatic esterification of ibuprofen under the optimal reaction conditions (ultrasound power,
225 W; temperature, 45 ˝C; water activity, 0.21). It could be found from Table 1 that the loss of the
enzyme activity and enantioselectivity of the immobilized enzyme was almost negligible even after
ten reaction cycles.
Table 1. Reusability of the nanobioreactor under ultrasound.
Reaction Cycle
Relative Activity (%)
E value
1
2
3
4
5
6
7
8
9
100
99.6
99.5
98.7
99.2
99.6
98.8
98.7
99.1
99.5
147.1
150.9
148.4
146.3
151.4
147.2
148.5
151.3
146.5
147.7
10
Furthermore, no change of the morphologies could be observed for the recycled immobilized
nanobioreactor (Supplementary Figure S2). All these phenomena indicated that the prepared
nanobioreactor was very stable under low power ultrasound conditions and exhibited an
excellent reusability.
3. Experimental
3.1. Materials
(S)-( )-1-(1-naphthyl) ethylamine ((S)-NEA), (R)-fenoprofen and racemic ibuprofen were
´
purchased from Sigma-Aldrich-Fluka Chemical Co. (St. Louis, MO, USA). All other commercial
reagents were from Shanghai Chemical Reagent Company, Shanghai, China. The (S)-NEA solution
(0.02 M) was prepared in triethylamine-acetonitrile solution (10 mM). APE1547 was prepared in our
lab [7,25].
3.2. Ultrasound Equipment
The ultrasound bath (KQ-250DE) was purchased from Kunshan Ultrasound Co. Ltd., Kunshan,
China. Its maximum power is 250 W (scale, 40%–100%) and its temperature (
circulating water.
˘
1 ˝C) is controlled by
3.3. Preparation of the Nanobioreactor
The preparation of nanobioreactor was carried out according to our previous work [18]. The
APE1547 solution (60 mL, 1 mg/mL) and CuSO4 solution (20 mL, 120 mmol/L) was mixed into a 3 L
of PBS solution (50 mmol/L, pH 7.4). After incubation at 25 ˝C for three days, the blue precipitate
at the bottom of the flash was collected by centrifugation (12,000 rpm for 20 min) and washed by
deionized water for three times. The protein concentration in the supernatant was quantified by the
Bradford protein assay [26] and the collected sample was about 960 mg. As a result, the immobilization
yield of protein was determined. Since no protein in the pooled supernatant and washing solutions
was detected, the immobilization yield of APE1547 was nearly 100% and the loading capacity of the
nanoflowers was calculated to be 62.5 mg of protein per gram of nanoflowers.

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3.4. Control and Measurement of Water Activity
All the reaction mixtures were previously dried under vacuum at 1 mm Hg for 12 h. Then, each
reaction mixture with specific water activity (aw) was prepared through adding a specific volume of
water. The resulting samples were pre-equilibrated at the desired temperature for 24 h in a sealed vial
before being subjected to measurement of water activity (aw) with the Hygrolab Humidity Detector
(Rotronic, Bassersdorf, Switzerland).
3.5. Enzyme-Catalyzed Esterification of Ibuprofen
Racemic ibuprofen (0.2 mmol), 1-octanol (0.2 mmol) and n-heptane (10 mL, aw 0.21) were mixed in
˝
a 25-mL bottle. The bottle was incubated in the ultrasound (45 C, 225 W). The immobilized APE1547
powder (160 mg) was added into the bottle to begin the enantioselective esterification. During the
reaction, 50
µL of the reaction mixture was withdrawn from the bottle and derivatized with (S)-NEA
(100 L). After 3 min, the derivatization was stopped by adding 100
µ
µL of ethanolamine solution
(0.02 M in acetonitrile). Finally, the sample (about 20 µL) was withdrawn for HPLC analysis.
3.6. Characterization of the Prepared Nanobioreactor
The SEM of the samples was observed by a JSM-6700F electron microscope (JEOL, Tokyo, Japan)
with an acceleration voltage of 30 kV. The FTIR spectra of the samples were surveyed using a 5700
FTIR spectrometer (Nicolet, Madison, WI, USA) with a resolution of 4 cm´1 through KBr method.
3.7. Recycling the Enzyme
After each batch (2.5 h), the reaction mixture was centrifuged at 10,000 rpm and 4 ˝C for 5 min.
The precipitate (the immobilized APE1547) was washed at least three times with the reaction media
and dried at room temperature.
3.8. High Performance Liquid Chromatography (HPLC) Analysis
An Agilent 1200 HPLC (Agilent, Santa Clara, CA, USA) equipped with a YMC C18 column
(150 mm
ˆ
4.6 mm; Greenherbs Co. Ltd., Beijing, China) was used for HPLC detection (flow
rate, 1.6 mL/min; 280 nm). The mixture of acetonitrile-water-acetic acid-triethylamine (volume
ratio: 60:40:0.1:0.02; pH 5.0) was used as mobile phase. (R)-fenoprofen was used as an internal
standard (retention time was 9.8 min). The retention time of the produced ibuprofen ester was
6.3 min. The retention time of the (S)-ibuprofen and (R)-ibuprofen was 15.1 and 16.8 min, respectively
(Supplementary Figure S1).
The degree of conversion (C) was calculated from the reduction of ibuprofen. One unit of the
enzyme activity (
milligram of APE1547 containing in the immobilized sample per hour. According to our previous
study [ ], APE1547 favored the (R)-ibuprofen. The ee of the un-reacted (S)-ibuprofen and the
µmol/h/mg) was defined as the amount (µmol) of ibuprofen ester produced per
6
enantiomeric ratio (E value) were calculated by the formula suggested by Chen et al. [27]:
rSs´rRs
enantiomeric excesses, eep%q “
ˆ 100%
rSs`rRs
(1)
lnrp1´Cqp1´eeqs
lnrp1´Cqp1`eeqs
enantioselectivity. E “
In the above equation, [S] and [R] represent the concentration of the S and R isomer of
ibuprofen, respectively.
4. Conclusions
In summary, we prepared a flower-like nanobioreactor for resolution of ibuprofen in
organic solvents under ultrasound irradiation. After optimizing the reaction conditions, the
nanobioreactor exhibited an excellent enzyme performance (enzyme activity, 13.26 µmol/h/mg;

Molecules 2016, 21, 565
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E value, 147.1). Compared with the native APE1547 in conventional reaction [
6], the enzyme
activity of the nanobioreactor under ultrasound was significantly enhanced about 61.25-fold and
the enantioselectivity was enhanced about 3.86-fold. The most important finding is that the prepared
nanobioreactor retained almost 100% of its initial enzyme activity and enantioselectivity, even after ten
repeated reaction batches under ultrasound. These results demonstrated that ultrasound can enhance
the enzyme performance dramatically and the prepared nanobioreactor can improve the reusability
and stability of the used enzyme under ultrasound, which make this combination more attractive for
further study.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
5/565/s1.
Acknowledgments: The authors are grateful for the financial support from the National Natural Science
Foundation of China (No. 21172093 and 31070708), the Scientific and Technological Project of Education
Department of Jilin Province of China (No. 2015.182), the China Postdoctoral Science Foundation (No.
2014M561287), the Jilin Province Innovation Platform of Straw Comprehensive Utilization Technology (Technology
and Innovation Platform of Colleges and Universities of Jilin Province (2014) C-1)) and the Natural Science
Foundation of Jilin Province of China (No. 20140101141JC).
Author Contributions: Baiyi An wrote the paper and measured the enzymatic activities. Hailin Fan contributed
to SEM images. Zhuofu Wu contributed to FTIR spectra. Lu Zheng and Lei Wang analyzed the data. Zhi Wang
and G.C. conceived and designed the experiments.
Conflicts of Interest: The authors declare no conflict of interest.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
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