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
2
3
4
*
College of Horticulture, Jilin Agricultural University, Changchun 130118, China; [email protected]
College of Resources and Environment, Jilin Agricultural University, Changchun 130118, China;
[email protected]
College of Life Science, Jilin Agricultural University, Changchun 130118, China; [email protected]
Key Laboratory of Molecular Enzymology and Engineering of Ministry of Education, School of Life Sciences,
Jilin University, Changchun 130012, China; [email protected] (L.Z.); [email protected] (L.W.)
Correspondence: [email protected] (Z.W.); [email protected] (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 [2–4]. Among the biocatalytic processes, enzyme-catalyzed kinetic
resolution is one of the most efficient methods [5]. In our previous study [6], we successfully used a
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 [8–14]. However, many immobilized enzymes can
not be used efficiently under high-speed agitation or under ultrasound [15,16]. For example, some
immobilized enzymes are prepared by physical adsorption, but, high-speed agitation or ultrasound
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enzymes can not be used efficiently under high-speed agitation or under ultrasound [15,16]. For
example, some immobilized enzymes are prepared by physical adsorption, but, high-speed agitation
can
destroy the
weak
interactions
the enzyme
the immobilized
support, which
will
or ultrasound
can
destroy
the weakbetween
interactions
between and
the enzyme
and the immobilized
support,
Molecules 2016, 21, 565
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which will
induce
lossreaction
during the
reaction
cycles and
the enzyme
[14]. Some
induce
enzyme
lossenzyme
during the
cycles
and inactivate
theinactivate
enzyme [14].
Some enzymes
are
enzymes
are immobilized
onefficiently
fragile
with poorstability
operational
stability
[17]. Both
high-speed
immobilized
on
withsupports
poor
[17].
Both
high-speed
agitation
enzymes
canfragile
not be supports
used
underoperational
high-speed
agitation
or under
ultrasound
[15,16].
For and
agitation
and
ultrasound
can cause
collapse
of by
the
immobilized
support
and affect
the enzyme
ultrasound
can
cause
the collapse
of the
theare
immobilized
support
and affect
the
performance
of
example,
some
immobilized
enzymes
prepared
physical
adsorption,
but, enzyme
high-speed
agitation
or ultrasound
canimmobilized
destroy
weak
the enzyme
and the immobilized
support,
performance
of the
enzyme.
In 2014,
we prepared
a flower-like
nanobioreactor
which
the
immobilized
enzyme.
In the
2014,
weinteractions
prepared
abetween
flower-like
nanobioreactor
which
was
formed
via
2+ and
which will
enzyme
during
the
cycles
inactivate
[14].
Some
wascoordination
formed
viainduce
the
coordination
between
Cureaction
the
atoms
ofthethe
amide
groups
in The
the
the
between
Cu2+loss
and
the nitrogen
atoms
ofnitrogen
theand
amide
groups
inenzyme
the
enzymes
[18].
enzymes
are
immobilized
on
fragile
supports
with
poor
operational
stability
[17].
Both
high-speed
enzymes
[18].
The simple
and efficient
preparation
does not
requireand
expensive
facilities
simple
and
efficient
preparation
procedure
does not procedure
require expensive
facilities
the preparation
agitation and ultrasound can cause the collapse of the immobilized support and affect the enzyme
and the
preparation
cost is our
low.unpublished
Furthermore,study
our unpublished
study has
demonstrated
this kind
cost
is
low.
Furthermore,
has demonstrated
that
this kind of that
immobilized
performance of the immobilized enzyme. In 2014, we prepared a flower-like nanobioreactor which
of immobilized
enzyme
is very
under
many
extreme
environments
(low
or
high
pH,
high
enzyme
is very stable
under
manystable
extreme
environments
(low
or
high
pH,
high
temperature,
organic
was formed via the coordination between Cu2+ and the nitrogen atoms of the amide groups in the
temperature,
organic
reaction
media,
high-speed
agitation
or
ultrasound,
et
al.).
These
promising
reaction
media,
high-speed
agitation
or
ultrasound,
et
al.).
These
promising
advantages
make
enzymes [18]. The simple and efficient preparation procedure does not require expensive facilities this
advantages
this immobilization
method
more
attractivestudy
for further
study.
immobilization
method
more
attractive
for further
study.
and the make
preparation
cost
is low.
Furthermore,
our
unpublished
has demonstrated
that this kind
study,
we
combined
this
immobilization
method
In
this
study,
we
combined
this
immobilization
method
and
ultrasound
tohigh
improve
of immobilized enzyme is very stable under many extreme environments (lowirradiation
or high pH,
enantioselective
of
the enzyme
performance
in
enantioselective
esterification
ibuprofen
(Scheme
1).
Furthermore,
temperature, organic reaction media, high-speed agitation or ultrasound, et al.). These promising the
advantages
make this of
immobilization
method more attractive
for further under
study.
stability
reusability
of the
the APE1547-incorporated
APE1547-incorporated
nanobioreactor
under ultrasound
ultrasound irradiation
irradiation
stability
and reusability
nanobioreactor
In thisstudied.
study, we combined this immobilization method and ultrasound irradiation to improve
had also been
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.
+ C8H17-OH
immobilized APE1547
+
+H2O
ultrasound
COOH + C8H17-OH
immobilized APE1547
ultrasound
(R,S)-ibuprofen
COOH
H
+
H
COOC8H17
+H2O
COOH
H
(R)-ibuprofen
COOCester
8H17
H
(S)-ibuprofen
COOH
Scheme 1. Schematic diagram of
the resolution
of ibuprofen.
(R)-ibuprofen
ester
1. Schematic diagram of
the resolution
of(S)-ibuprofen
ibuprofen.
(R,S)-ibuprofen
Scheme
2. Results and DiscussionScheme 1. Schematic diagram of the resolution of ibuprofen.
2. Results and Discussion
2. Results and Discussion
2.1. The Morphologies of the Immobilized Samples
2.1. The Morphologies of the Immobilized Samples
2.1.large
The Morphologies
the ImmobilizedAPE1547
Samples have been successfully prepared (Figure 1A,B). The
A
number ofofimmobilized
A large number of immobilized APE1547 have been successfully prepared (Figure 1A,B). The
shape of the
immobilized
looked
like ahave
natural
with prepared
dozens of(Figure
separate
petals
A large
number of enzyme
immobilized
APE1547
beenflower,
successfully
1A,B).
The with
shape of the immobilized enzyme looked like a natural flower, with dozens of separate petals with
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
high
high surface (Figure 1C). Its diameter was about 4–5 µm. The excellent hierarchical structure with high
high
(Figure
Its diameter
was about
μm. The
excellent hierarchical
structure withenzyme.
high
surface
tosurface
volume
ratios1C).
is likely
to improve
the4–5
enzyme
performance
of the immobilized
surface
to volume
ratios
is likely
totoimprove
performance
immobilized
enzyme.
surface
to volume
ratios
is likely
improvethe
the enzyme
enzyme performance
ofof
thethe
immobilized
enzyme.
Figure 1. The morphologies of the immobilized samples. (A–C): SEM images of the immobilized
Figure 1. The morphologies of the immobilized samples. (A–C): SEM images of the immobilized
samples
with
different enlargement
factors.
Figure
1. The
morphologies
of the immobilized
samples. (A–C): SEM images of the immobilized
samples with different enlargement factors.
samples with different enlargement factors.
Molecules 2016, 21, 565
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2.2. FTIR
2.2. FTIR
FTIR
The FTIR
FTIR spectraofof
immobilized
samples
recorded
and
the results
are in
shown
thethe
immobilized
samples
werewere
recorded
and the
results
shown
Figurein
2.
The FTIRspectra
spectra of
the
immobilized
samples
were
recorded
and
the are
results
are shown
in
´
Figure
2.characteristic
Several characteristic
peaks
could be in
observed
inAmong
curve c.
Among
of peaks
them, at
the
peaks
at1
Several
peaks
could
be
observed
curve
c.
of
them,
the
1053
cm
Figure 2. Several characteristic peaks could be observed in curve c. Among of them, the peaks at
−1
´1 556 assigned
´1 cm−1 1521
´1cm
1053
cm−1−1
and
cm−1 are assigned
to the vibrations
PO43−3−
[19].
The peaks
1652
and 1521
and
to the vibrations
of PO4 3´of
peaks
1652at
are
−1
1053556
cmcmand are
556 cm−1 are assigned
to the vibrations
of[19].
PO4The
[19].
The at
peaks
atcm
1652 and
cm−1 and cm
1521 cm
are
ascribed
to the
vibrations
of
the
amide
I and
II
bands
ofthe
theenzyme
enzyme[20].
[20].These
Theseresults
resultssuccessfully
successfully
ascribed
to
the
vibrations
of
the
amide
I
and
II
bands
of
are 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
immobilized samples.
verified the presence of enzyme in the immobilizedsamples.
samples.
Figure
2. The
FTIR spectrum
spectrum of Cu
Cu3(PO44))22matrices
b) and
the immobilized
Figure
matrices(curve
(curvea),
a),APE1547
APE1547 (curve
(curve
Figure 2.
2. The
The FTIR
FTIR spectrumof
of Cu33(PO
(PO4)2 matrices
(curve
a),
APE1547
(curve b)
b) and
and the
the immobilized
immobilized
APE1547
(curve
c).
APE1547
(curve
c).
APE1547 (curve c).
2.3.
Effect of
Ultrasound Power
2.3.
2.3. Effect
Effect of
of Ultrasound
Ultrasound Power
Power
The
effectofofultrasound
ultrasound
power
the
enantioselective
esterification
was examined
the
The
power
on on
the
enantioselective
esterification
was examined
and theand
results
The effect
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
are
shown
in
Figure
3.
The
highest
enantioselectivity
was
obtained
at
an
ultrasound
power
of
225
W
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
and
theWmaximum
enzyme activity
be could
observed
at 200 W.atThe
of enzyme
activity
of 225
and the maximum
enzymecould
activity
be observed
200enhancement
W. The enhancement
of enzyme
activity
obtained
at suitable
ultrasound
power
was probably
attributed
to the mixing
better mixing
obtained
at suitable
ultrasound
power (225
W)(225
wasW)
probably
attributed
to the better
of the
activity obtained
at suitable
ultrasound
power
(225
W)
was probably
attributed
to the better mixing
of
the reactants
[21]. Furthermore,
the improvement
ofmass
the mass
transfer
processes
innanobioreactor
the nanobioreactor
reactants
[21].
Furthermore,
the
improvement
of
the
transfer
processes
in
the
can
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
also
increase
the enzyme
activity
considering
thethe
complexity
and
heterogeneity
can also
increase
the enzyme
activity
considering
complexity
and
heterogeneityofofthe
theimmobilized
immobilized
enzyme.
However, the
mechanism for
the ultrasound-induced
ultrasound-induced alteration in
the enantioselectivity
enantioselectivity is
enzyme.
enzyme. However,
However, the
the mechanism
mechanism for
for the
the ultrasound-induced alteration
alteration in
in the
the enantioselectivity is
is
still
unclear.
Our
previous
study
has
demonstrated
that
ultrasound
can
induce
aa slight
change
of
still
unclear.
Our
previous
study
has
demonstrated
that
ultrasound
can
induce
slight
change
of
still unclear.
has demonstrated
ultrasound
induce a slight change of
the
secondary structure
of APE1547
and then
influence its
its enantioselectivity [7].
Further research
is
the
the secondary
secondary structure
structure of
of APE1547
APE1547 and
and then
then influence
influence its enantioselectivity
enantioselectivity [7].
[7]. Further
Further research
research is
is
needed
to
clarify
the
mechanism
and
will
be
published
in
due
course.
needed
to
clarify
the
mechanism
and
will
be
published
in
due
course.
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:
Figure 3. Effect of ultrasound power on the enzymatic esterification of ibuprofen. Reaction conditions:
Figure
3. Effect
of ultrasound
power
on the enzymatic
ibuprofen.
Reaction(0.2
conditions:
ibuprofen
mmol),
the
reactions
were
carried out
in n-heptane
(10 mL, esterification
aw 0.21) withofracemic
the reactions were carried out in n-heptane (10 mL, aw 0.21) with racemic ibuprofen (0.2 mmol),
the
reactions
were
carried
out
in
n-heptane
(10
mL,
a
0.21)
with
racemic
ibuprofen
(0.2
mmol),
w
1-octanol (0.2 mmol) and the immobilized APE1547 powder
(160 mg) at 45 °C.
1-octanol (0.2 mmol) and the immobilized APE1547 powder (160 mg) at 45 °C.
1-octanol (0.2 mmol) and the immobilized APE1547 powder (160 mg) at 45 ˝ C.
Molecules 2016, 21, 565
4 of 8
Molecules 2016,
565of Temperature
2.4.21,
Effect
4 of 8
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
2.4. Effect of Temperature
temperature. The possible explanation is that high temperature may destroy the conformation of the
enzymeof
byultrasound
heat-induced destruction
of non-covalent
interactions
and decreasehave
the enantioselectivity
The effects
temperature
on the enzyme
performance
also been investigated
[22,23]. The enzyme activity exhibited a bell shaped curve with the changing ultrasound temperature,
in this study.
The
result
in
Figure
4
showed
that
higher
enantioselectivity
could be obtained
and the optimal temperature was 45 °C.
at lower temperature.
possiblemay
explanation
that probability
high temperature
may and
destroy the
High reactionThe
temperatures
elevate the is
collision
between enzyme
substrate
molecules
to by
form
enzyme-substrate
complexes and
then enhance the
enzyme activity.
conformation
of the
enzyme
heat-induced
destruction
of non-covalent
interactions
and decrease
The activity significantly
decreased
at higher
temperatures,
which
might
be due to
the denaturation
Molecules 2016, 21, 565
of 8 changing
the enantioselectivity
[22,23]. The
enzyme
activity
exhibited
a bell
shaped
curve
with 4the
of the enzyme, especially under ultrasound irradiation [23]. Considering
both
the
enzyme activity
˝
ultrasoundand
temperature,
and the optimal temperature was 45 C.
enantioselectivity,
2.4. Effect
of Temperature45 °C was selected as the optimal reaction 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.
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.
Figure 4. Effects of temperature on the enzymatic esterification of ibuprofen. Reaction conditions:
Figure 4. Effects
of temperature
enzymatic
of ibuprofen.
Reaction
conditions: the
the reactions
were carried on
outthe
in n-heptane
(10 esterification
mL, aw 0.21) with
racemic ibuprofen
(0.2 mmol),
reactions were
carried
out inand
n-heptane
(10 mL,
aw 0.21)
with(160
racemic
(0.2 mmol),
1-octanol
(0.2 mmol)
the immobilized
APE1547
powder
mg) at ibuprofen
different temperature.
The 1-octanol
power was 225 W.
(0.2 mmol)ultrasound
and the immobilized
APE1547 powder (160 mg) at different temperature. The ultrasound
power was 225 W.
2.5. Effect of Water Activity
Water may influence the performance of the enzymes in organic solvents [24]. The results in
High Figure
reaction
temperatures
may elevate
the collision
probability
between
enzyme
and substrate
5 showed
that the enzyme
activity increased
as water
activity increased
from
0.09 to 0.21,
decreased
at higher water activity.
The maximum
enantioselectivity
(E value) could
also The
be activity
moleculesthen
to form
enzyme-substrate
complexes
and then
enhance the enzyme
activity.
observed
at aw = 0.21.
Water activity
may influence
the reaction
the two
isomers
and then
significantly
decreased
at higher
temperatures,
which
mightrates
be ofdue
to the
denaturation
of the
induce the alteration of enantioselectivity of APE1547 [24]. Since the E value (147.1) was found to be
enzyme, especially
under ultrasound irradiation [23]. Considering both the enzyme activity and
highest at aw =0.21 while maintaining the highest enzyme activity (13.26 μmol/h/mg), the water
Figure 4. Effects
of temperature on the enzymatic esterification of ibuprofen. Reaction conditions:
˝ C was
enantioselectivity,
selected
as the
optimal reaction temperature.
activity of 45
0.21 was
selected
for further
study.
2.5. Effect of
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
Water
Activity
ultrasound power was 225 W.
Water2.5.
may
influence the performance of the enzymes in organic solvents [24]. The results in
Effect of Water Activity
Figure 5 showed that the enzyme activity increased as water activity increased from 0.09 to 0.21, then
Water may influence the performance of the enzymes in organic solvents [24]. The results in
decreased Figure
at higher
water
activity.
Theactivity
maximum
enantioselectivity
(E value)from
could
be observed
5 showed
that
the enzyme
increased
as water activity increased
0.09also
to 0.21,
at aw = 0.21.
activity
maywater
influence
of the two (E
isomers
and then
thenWater
decreased
at higher
activity.the
Thereaction
maximumrates
enantioselectivity
value) could
also beinduce the
at aw = 0.21. Water
may[24].
influence
thethe
reaction
rates(147.1)
of the two
isomers
and
alteration observed
of enantioselectivity
of activity
APE1547
Since
E value
was
found
tothen
be highest at
induce the alteration of enantioselectivity of APE1547 [24]. Since the E value (147.1) was found to be
aw =0.21 while
maintaining the highest enzyme activity (13.26 µmol/h/mg), the water activity of 0.21
highest at aw =0.21 while maintaining the highest enzyme activity (13.26 μmol/h/mg), the water
was selected
for further
study.
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.
Figure 5. Effect of water activity on the enzymatic esterification of ibuprofen. Reaction conditions:
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
the reactions
were and
carried
out in n-heptane
(10 mL)
with
racemic
ibuprofen
(0.2was
mmol),
(0.2 mmol)
the immobilized
APE1547 powder
(160 mg)
at 45
°C. The ultrasound
power
225 W. 1-octanol
(0.2 mmol) and the immobilized APE1547 powder (160 mg) at 45 ˝ C. The ultrasound power was 225 W.
Molecules 2016, 21, 565
5 of 8
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
10
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
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 (˘1 ˝ C) is controlled by
circulating water.
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.
Molecules 2016, 21, 565
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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 µL). After 3 min, the derivatization was stopped by adding 100 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 (µmol/h/mg) was defined as the amount (µmol) of ibuprofen ester produced per
milligram of APE1547 containing in the immobilized sample per hour. According to our previous
study [6], APE1547 favored the (R)-ibuprofen. The ee of the un-reacted (S)-ibuprofen and the
enantiomeric ratio (E value) were calculated by the formula suggested by Chen et al. [27]:
rSs´r Rs
enantiomeric excesses, eep%q “ rSs`rRs ˆ 100%
lnrp1´Cqp1´eeqs
enantioselectivity. E “ lnrp1´Cqp1`eeqs
(1)
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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Sample Availability: Samples of the compounds ((S)-ibuprofen, 99% ee) are available from the authors.
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