Ultrasound irradiation accelerates the lipase-catalyzed synthesis of methyl caffeate in an ionic liquid

Article abstract of DOI:10.1016/j.molcatb.2014.11.006

Methyl caffeate is a natural ingredient with several biological activities, but its preparation is generally limited to chemical synthesis. To set up a simple, high-yield and low-cost synthesis process for obtaining methyl caffeate, a novel synthesis meth

Full text of DOI:10.1016/j.molcatb.2014.11.006

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Journal of Molecular Catalysis B: Enzymatic
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Ultrasound irradiation accelerates the lipase-catalyzed synthesis of
methyl caffeate in an ionic liquid
Jun Wanga,b,c,∗, Shasha Wanga,b, Zhongjian Lia,b, Shuangshuang Gua,b, Xiangyang Wuc,∗∗
,
Fuan Wua,b,∗ ∗ ∗
a School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212018, PR China
b Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang 212018, PR China
c School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang 212013, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Methyl caffeate is a natural ingredient with several biological activities, but its preparation is generally
limited to chemical synthesis. To set up a simple, high-yield and low-cost synthesis process for obtaining
methyl caffeate, a novel synthesis method using the lipase-catalyzed esterification of methanol and caffeic
acid in an ionic liquid under ultrasound irradiation was established. A maximum yield of 99.79% was
obtained using ultrasound irradiation with an ultrasound frequency of 25 kHz and ultrasound power
of 150 W under the following optimal conditions: Novozym 435 as a biocatalyst, [Bmim][Tf2N] as the
reaction medium, lipase concentration of 60 g/L, reaction temperature of 75 ◦C, and reaction time of 9 h.
Using the optimal conditions under ultrasound irradiation, the reaction time was reduced 0.75-fold, and
the apparent kinetic parameter (Vm/Km) was increased 2-fold. The lipase could be reused 11 times without
significant loss of activity. The results suggest that ultrasound irradiation can accelerate lipase-catalyzed
synthesis of methyl caffeate in an ionic liquid.
Received 3 September 2014
Received in revised form 31 October 2014
Accepted 14 November 2014
Available online xxx
Keywords:
Biocatalysis
Ionic liquid
Lipase
Methyl caffeate
Ultrasound irradiation
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
streptozotocin [4]. Additionally, MC can be used as an intermediate
for the production of natural medicines or food additives such as
Methyl caffeate (methyl-(E)-3-(3,4-dihydroxyphenyl) prop-2-
enoate, MC) is one of the alkyl caffeates extracted from Polygonum
amplexicaule and the fruit of Solanum torvum. Alkyl caffeates
have obvious biological functions in antimicrobial, antioxidative,
antiviral and antineoplastic activities [1,2]. Among the alkyl caf-
feates, compared with caffeic acid (CA), MC has molecular polarity
comparable to that of CA and has appetite suppressant and lar-
val development inhibitory activities [3]. MC also has strong
anti-inflammatory, anticancer, antioxidant, antiviral, detoxicant,
anti-clotting, and anti-diabetic effects on diabetic rats induced by
propyl caffeate (PC) and caffeic acid phenethyl ester (CAPE), which
are oxidative dimerization products of MC [5].
Currently, preparation strategies for MC typically focus more on
chemical synthesis than on enzymatic synthesis. Several reports
have been published on the synthesis of MC from CA and methanol
using an acidic catalyst. For example, Shin et al. described the ester-
ification of CA and methanol in the presence of sulfuric acid in
10 h with an MC yield of 71.0% [6]. Then, Wang et al. described
a similar process catalyzed by p-toluenesulfonic acid (PTSA) for the
preparation of MC in 4 h, achieving a relatively higher MC yield
of 84.0% [7]. However, acidic catalysts are hazardous, requiring
special energy-inefficient processes to dispose of the waste acid.
Moreover, chemical methods are associated with problems such
as high energy consumption, poor reaction selectivity and high
cost of manufacturing. In contrast, the lipase-catalyzed synthesis
of caffeates has garnered increasing attention due to its relative
advantages. Chen et al. [8] used immobilized lipase as a catalyst
to synthesize CAPE from CA and 2-phenylethanol (PE), achiev-
ing 93.08% molar conversion of CAPE. The results indicated that
enzymatic synthesis could offer several advantages, such as mild
reaction conditions and reagents, minimization of side products
in the reaction, higher activation energy, and better control over
∗
Corresponding author at: School of Biotechnology, Jiangsu University of Science
and Technology, Zhenjiang 212018, PR China. Tel.: +86 511 85635867;
fax: +86 511 85620901.
∗∗
Corresponding author at: School of the Environment and Safety Engineering,
Jiangsu University, Zhenjiang 212013, PR China. Tel.: +86 511 85038750;
fax: +86 511 85038451.
∗ ∗ ∗
Corresponding author at: Sericultural Research Institute, Chinese Academy of
Agricultural Sciences, Zhenjiang 212018, PR China. Tel.: +86 511 85616571;
fax: +86 511 85635850.
E-mail addresses: wangjun@just.edu.cn (J. Wang), wuxy@ujs.edu.cn (X. Wu),
fuword@163.com (F. Wu).
http://dx.doi.org/10.1016/j.molcatb.2014.11.006
1381-1177/© 2014 Elsevier B.V. All rights reserved.
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the esterification reaction. Thus, there is a need to explore more
efficient processes for the lipase-catalyzed synthesis of MC via
esterification.
2.2. Lipase-catalyzed synthesis of MC in an incubator shaker
The esterification reaction was performed in a 5 mL screw-
capped vial at 75 ◦C for 36 h with a constant stirring speed of
120 rpm. CA (100 mg) was dissolved in methanol (10 mL), then dif-
ferent volumes of CA solution were added to IL, making the total
reaction system to be 0.5 mL. The reaction was initiated by adding
immobilized enzyme. At regular time intervals (3, 6, 12, 24, 36 and
48 h), 20 ␮L aliquots were taken from the well-stirred reaction mix-
tures and diluted using 380 ␮L methanol in preparation for HPLC
analysis. The effects of different reaction temperatures (50–80 ◦C),
different concentrations of substrate (0.25, 0.5, 1, 1.5, 2, 4 and 6 g/L),
and concentration of Novozym 435 (20, 40, 60, 80, 100, and 120 g/L)
on MC yield were investigated. All experiments were carried out in
triplicate.
Enzymatic catalysis in ionic liquids (ILs) has been extensively
studied for its high substrate conversion and product yield [9],
high reaction rates, high activity, high stability, and good enantio-
selectivity [5]. Recently, the lipase-catalyzed synthesis of caffeates
has attracted intense interest, with caffeates having higher solu-
bility than CA in hydrophobic media [10]. The synthesis methods
tested include esterification and transesterification, the latter hav-
ing higher reaction efficiency than the former. As we have reported
earlier [11], the use of MC as a substrate during the synthesis of
propyl caffeate (PC) significantly improves the yield. Thus, biosyn-
thesis of MC could provide greener avenue for the production
of the much needed MC to be used in such process. Moreover,
the method of lipase-catalyzed esterification synthesis of MC had
yet not been tested in an ionic liquid system. Therefore, it was
tested in this study for its effectiveness in obtaining better MC
yields.
2.3. Lipase-catalyzed synthesis of MC under ultrasound
irradiation
Recently, ultrasound irradiation has seen numerous applica-
tions in organic chemistry as an environmentally benign method
[12]. Ultrasound irradiation is a useful tool for enhancing mass
transfer in liquid–liquid heterogeneous systems and increasing the
mass transfer rate of reagents and the active sites of enzymes
[13]. Immobilized enzymes are more resistant than native enzymes
to thermal deactivation caused by ultrasound irradiation [14].
Although the application of ultrasound irradiation to enzymatic
reactions has not been extensively explored, it has been used to
accelerate enzymatic reactions [15], such as esterification of phy-
tosterol and different acyl donors [16], enzymatic esterification of
rutin and naringinto catalyzed by Novozym 435 [17], transesterifi-
cation of glycerol and methyl benzoate in the organic solvent [18],
and synthesis of sugar esters in ILs [19]. To date, no known studies
have investigated the application of ultrasound irradiation to the
synthesis of MC.
The esterification reaction was performed in a 5 mL screw-
capped vial at 75 ◦C for 9 h under ultrasound irradiation using an
ultrasound frequency of 25 kHz and ultrasound power of 150 W.
CA (100 mg) was dissolved in methanol (10 mL), and different vol-
umes of CA solution were added to IL, making the total reaction
system to be 0.5 mL. The reaction was initiated by adding the immo-
bilized enzyme. At regular time intervals (1, 2, 3, 6, 12, 24, 36 and
48 h), 20 ␮L aliquots were taken from the well-stirred reaction mix-
ture and diluted using 380 ␮L methanol in preparation for HPLC
analysis. The effects of different ultrasound power (90, 120, 150,
180, 210 W), frequency (15, 20, 25, 30, 35 kHz), operation mode
(sweep, pulse), reaction temperatures (50–80 ◦C), different concen-
trations of substrate (0.25, 0.5, 1, 2, 4 and 6 g/L), and concentration
of Novozym 435 (20, 40, 60, 80, and 100 g/L) on the MC yield were
investigated. All experiments were performed in triplicate.
Hence, the aim of this study was to explore a new method
for the synthesis of MC using lipase-catalyzed esterification of
CA and methanol in ILs. The effects of the type of IL, the types
of lipase, the concentration of substrate, the concentration of
biocatalysts in reaction system, the reaction time and reaction
temperature on the esterification yield were investigated under
incubator shaking. The experiments were subsequently conducted
with incubation under ultrasound irradiation. Nuclear magnetic
resonance (NMR) and mass spectrometry were used to identify the
product.
2.4. Complexation extraction process of MC
The complexation extraction process was used to extract MC
from enzymatic reaction system using trioctylphosphine oxide
(TOPO)–cyclohexane as an extractant [20], in which mass fraction
of complex agent TOPO in diluter cyclohexane was 100 g/L. MC was
extracted from the mixture to extractant with the ratio of 1:1 (v/v).
After cyclohexane was volatilized from extractant, the residue was
crystallized from methanol, thus MC could be separated from mix-
tures to obtain white product with the method of vacuum drying.
2.5. Kinetic study of lipase-catalyzed synthesis of MC
2. Materials and methods
The kinetics of the esterification reaction was investigated by
studying the effect of the CA concentration on the initial rate of
the reaction. The concentration of CA varied within the range of
1.39–33.32 mM. Initial reaction rates, which expressed as mM CA
per hour, were determined from the time course of the reaction by
using a second order polynomial curve fitting via regression anal-
ysis of the product concentration as well as determining the initial
slope of the tangent to the curve.
2.1. Enzymes and materials
Commercial lipases containing Novozyme 435, Lipozyme TL
IM (immobilized lipase from Thermomyces lanuginosus (previ-
ously Humicola lanuginosa), immobilized on a granulated silica
carrier), and Lipozyme RM IM (Rhizomucor miehei, 275 IUN/g,
where IUN represents Interesterification Units Novo; carrier: phe-
nol formaldehyde), were provided by Novo Nordisk A/S (Bagsvaerd,
Denmark). Eight ILs, including [Hmim][HSO4], [Bmim][Tf2N],
[Bmim][PF6], [Bmin][BF4], [Emim][TfOH], [Toma]Cl, [Toma][Tf2N],
[Omim][BF4] were obtained from Shanghai Cheng-Jie Chemical Co.
Ltd. (Shanghai, China). The residual chloride content in these ILs
was less than 50 ppm. CA was from Nanjing Zelang Pharmaceu-
tical Sci. & Tech. Co. Ltd., China. Methanol and acetonitrile were
HPLC-grade (Tedia Co., Fairfield, OH, USA), and other reagents were
analytical grade (Sinopharm Chemical Reagent Co. Ltd., Shanghai,
China).
2.6. Analysis of products by LC–MS and HPLC
As in previous test methods [11], LC-PAD-MS was performed
using a Thermo Fisher system. The LC equipment comprised a
Finnigan MAT Spectra System P4000 pump, a Finnigan AQA mass
spectrometer, and an autosampler with a 50 ␮L loop. LC separation
was performed on a Kromasil C18 column (150 mm × 4.6 mm, i.d.;
5 ␮m, W.R.) and monitored using a UV6000 LP diode array detec-
tor. Isocratic elution was used to run the mobile phase, which was
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a mixture of solvent A (methanol) and solvent B (water) at a ratio
of 65:35, v/v, respectively. The wavelength range for PAD detection
was from 200 to 400 nm. The flow rate was 1.0 mL/min for LC and
PAD detection, and the column was maintained at 30 ◦C. Selected
ion monitoring (SIM) was performed under negative ion mode, with
capillary voltage set at 1.6 kV, and temperatures of the curved des-
olvation line (CDL) and heat block both set at 200 ◦C. Electrospray
ionization (ESI) was performed using nitrogen to assist nebuliza-
tion, with the flow rate set at 1.0 mL/min. The data were processed
using Xcalibur 1.2 software. The intense peaks at m/z 179.1 and m/z
193.1 in ESI-MS spectra under negative ion mode correspond to the
deprotonated ion [M−H]− of CA and MC, respectively.
HPLC analyses were performed using a constant flow pump
(2PB0540, Beijing Satellite Factory., Beijing, China) with a UV–vis
detector (L-7420, Tech comp Co. Ltd., Shanghai, China) and N-2000
workstation (Hangzhou Mingtong S&T Ltd., Hangzhou, China). Sep-
aration was achieved on a HC-C18 column (250 mm × 4.6 mm, i.d.;
5 ␮m, W.R. Grace & Co., Deerfield, Illinois, USA) maintained at 30 ◦C.
CA and MC were detected from the mobile phase, which consisted
of a solvent mixture of methanol/water (65:35, v/v) at a flow rate of
1.0 mL/min and detection wavelength of 325 nm. All experiments
were carried out in triplicate.
Fig. 1. Effect of different lipases on the MC yield in [Bmim][Tf2N]. The CA concen-
tration was 0.5 g/L, and the concentration of lipases in TL was 100 g/L. All tests were
performed at 75 ◦C and 120 rpm in an incubator shaker.
External standard method was adopted in calculating of the
reaction conversions, and the CA conversion and MC yield of the
lipase-catalyzed esterification were calculated as follows:
activities of the three lipases and their highest MC yields were as
follows, in descending order: Novozym 435 (93.18%) > Lipozyme TL
IM (23.59%) > Lipozyme RM IM (20.63%).
Converted molar amount of CA(mol)
CA conversion(%) =
× 100
Initial molar amount of CA(mol)
Novozym 435 is a lipase of high thermostability, used mainly as
a catalyst for the synthesis of esters [21]. It has a maximum enzy-
matic activity in 70–80 ◦C. In order to maintain the optimization of
productivity, several literatures recommended temperature span
between 50 and 60 ◦C [20,22]. But in this experiment, in order to
maintain maximum activity of Novozym 435, the option of the tem-
perature in all process optimizations was according to the highest
MC yield. This lipase displayed the highest activity in the esterifica-
tion of CA with methanol [23]. When compared with Lipozyme RM
IM, Lipozyme TL IM is more advantageous for the synthesis of short
chain alkyl esters [24]; moreover, considering the great difference
in the cost of the two enzymes, Lipozyme TL IM may be considered a
better catalyst than Lipozyme RM IM. However, whereas Lipozyme
TL IM has commonly been reported to catalyze resolution of chiral
alcohol and preparation of biodiesel [25], it has rarely been used
to catalyze the synthesis of alkyl caffeates [20]. In conclusion, the
two Lipozyme enzymes tested were not clearly adapted to catalyze
the synthesis of MC. Therefore it is apparent that Novozym 435 is
the most active catalyst for the esterification of CA and methanol.
Hence, Novozym 435 was chosen as the most suitable catalyst for
the following studies.
(1)
(2)
Molar amount of MC (mol)
Initial molar amount of CA (mol)
MC yield (%) =
× 100
2.7. Structural identification of MC
1H NMR spectra were recorded on a Bruker AVANCE spectrom-
eter 400 (Bruker Biospin Co., Billerica, MA, USA). The samples were
dissolved in DMSO-d6, containing tetramethylsilane (TMS) as an
internal standard. Proton spectra were recorded at 400 MHz using
a solvent field lock (400 MHz), which was shown as Fig. S1 in Sup-
plementary Materials. The identification results are as follows: MC:
1H NMR (400 MHz, DMSO-d6) ı (ppm): 9.60 (1H, s, OH), 9.16 (1H, s,
OH), 7.50 (1H, d, J = 15.9 Hz, ˛-H), 7.07 (1H, d, J = 2.0 Hz, Ph-H), 7.01
(1H, dd, J = 8.1 Hz, Ph-H), 6.77 (1H, d, J = 8.1 Hz, Ph-H), 6.28 (1H, d,
J = 15.9 Hz, ˇ-H), 3.69 (3H, s, CH3). The results were consistent with
previous studies [11].
2.8. Statistical analysis
3.2. Selection of a suitable IL for reaction media
Triplicate experiments were performed for each parameter
investigated. The differences in mean values were evaluated using
the analysis of variance (ANOVA) method, and standard deviations
were calculated to verify the results reliability. Significance was
determined at a 95% level of probability.
Fig.
2
shows the MC yield in each IL with the
esterification of CA and methanol. The yields in these
reaction media were as follows, in descending order:
[Bmim][Tf2N] (96.14%) > [Bmim][PF6] (66.64%) > [Toma][Tf2N]
(54.60%) > [Omim][BF4] (48.93%) > [Toma]Cl (41.69%) > [Bmim]
[BF4] (41.01%) > [Emim][TfOH] (39.60%) > [Hmim][HSO4] (39.54%).
This could be due to the report that ILs can increase the substrate
solubility [26], and enhance the synthetic activity of lipase because
ILs increase the polarity of the system [27]. Therefore, usually,
enzymatic media containing ILs possess weakly coordinating
anions, such as [Tf2N] and [PF6]. However, other ILs possess
strongly coordinating anions ([HSO4], [BF4] and [TfOH]) and show
dramatically lower yields because they are more nucleophilic than
[Tf2N]. In addition, the complex nature of the ILs leads to many
possible types of interactions, including hydrogen bonding and
3. Results and discussion
3.1. Selection of a suitable lipase as a biocatalyst
Fig. 1 shows the catalytic efficiency of three commercially
available immobilized lipases (Novozym 435, Lipozyme RM IM
and Lipozyme TL IM) as biocatalysts for MC production in 0.5 mL
[Bmim][Tf2N]. The lipase concentration was 100 g/L, and the
reaction temperature was 75 ◦C. The MC yields varied markedly
between Novozym 435 and the remaining two lipases; the
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time was significantly shorted (by three fourths) under ultrasound
irradiation compared to the reaction using the incubator shaker.
The reason may be that during the enzymatic reaction, ultrasound
irradiation can reduce the particle sizes of the substrate and
enzyme, consequently increasing the enzyme surface area, which
is useful for reducing mass transfer limitations [15]. This concept
was further validated in studies using dehydrated enzyme powders
to catalyze reactions in an organic solvent [34]. Thus, the optimal
temperature and reaction time are 75 ◦C and 9 h, respectively, for
the synthesis of MC under ultrasound irradiation.
3.4. The effect of substrate concentration
Fig. 3C and D shows the effects of different CA concentrations on
the MC yield in the incubator shaker and under ultrasound irradia-
tion, respectively. [Bmin][Tf2N] was chosen as the reaction solvent,
and the Novozym 435 concentration in the reaction system was
100 g/L. Fig. 3C shows that when the optimum CA concentration
was 0.5 g/L, the MC yield increased to 96.54%. Any further increase
or decrease in substrate concentration resulted in a decline of MC
yield. Fig. 3D shows that under ultrasound irradiation, the highest
MC yield achieved was 96.0%. Meanwhile, any further increase or
decrease in substrate concentration led to results similar to those
shown in Fig. 3C. Therefore, mass transfer limited the ability of the
substrate to reach the active center of the enzyme [35], and the
charge viscosity of IL added to the mass transfer limitations, which
resulted in low CA conversion. In addition, this inhibition could also
be due to the production of generated water which led equilibrium
reaction toward to ester hydrolysis, herein, resulting in a lower MC
yield.
Fig. 2. Effects of different ILs on the MC yield. The CA concentration was 0.5 g/L,
and the concentration of lipase was 100 g/L. All tests were performed at 75 ◦C and
120 rpm for 36 h in an incubator shaker.
hydrogen-bonding basicities [28]. Thus, the interactions among
[HSO4], [BF4] and [TfOH] and positively charged sites in the struc-
ture of the enzyme could cause conformational changes in the
enzyme, which then becomes deactivated. These results agreed
with previous findings that the anion in an IL plays an essential
role in determining lipase activity [5]. Therefore, [Bmim][Tf2N]
was selected as the most suitable medium for the esterification
reaction of CA and methanol.
This reaction is the first order reaction equation, and the Thiele
modulus ꢀ was calculated by follows:
ꢀ
ꢁ
1
ꢀ
1
1
3ꢀ
3.3. The effect of temperature
ꢁ =
ꢁ =
−
(3)
(4)
tanh(3ꢀ)
Rs
Fig. 3A and B shows the effect of the reaction temperature
on the yield of MC catalyzed by Novozym 435 in an incubator
shaker and under ultrasound irradiation, respectively. The sub-
strate concentration was 0.5 g/L, the solvent was [Bmin][Tf2N], and
the lipase concentration was 100 g/L. Fig. 3A shows that the MC
yield increased with increasing reaction temperature in the range
of 50–75 ◦C within 24 h. However, the MC yield showed a slight
decline after 24 h at 70 ◦C. After 36 h at 75 ◦C and 80 ◦C, the MC
yield continued to exhibit a declining trend. A maximum MC yield
of 93.18% was achieved with 36 h of incubation at 75 ◦C under ultra-
sound irradiation. In addition, Fig. 3B shows that an increase in
the reaction temperature in the range of 50–80 ◦C led to increas-
ing MC yields within 9 h. Generally, the MC yield declined slightly
after 9 h, except at 70 ◦C. It is possible that an excessive duration
of ultrasound irradiation produces excessive heat, which leads to
perturbation of the lipase tertiary structure, ultimately reducing its
enzymatic activity to some extent [29,30]. The maximum MC yield
of 98.71% was achieved with 9 h of incubation at 75 ◦C under ultra-
sound irradiation, the same optimum temperature as which found
in the experiments using the incubator shaker.
The temperature usually has an important influence on enzy-
matic reactions, including esterification and transesterification,
because it can influence the enzymatic enantioselectivity [31] and
the reaction rate [32]. Increasing the temperature within certain
limits could improve the enzyme activity, reduce the ionic liquid
viscosity, and increase the mass transfer speed, thus improving the
yield of the product. However, when the temperature increases
beyond optimal, the yield decreases. It can be inferred that higher
temperatures cause partial thermal inactivation of the lipase,
which enables the enzyme thermodynamic parameters to be
determined but delays reaction times [33]. As a whole, the reaction
Rs
0
where ꢁ is internal diffusion factor effectively, Rs is actual reaction
rate in granular (mol/(s g)), and Rs is reaction rate when concen-
tration in granular is the same as s0urface (mol/(s g)).
Therefore, effective diffusion coefficients of both processes were
calculated as follows:
rmax
kv
=
(5)
Km
ꢂ
R
kv1
De
ꢀ =
(6)
3
where coefficient Km is the reaction kinetic parameter (mM), and
rmax is reaction rate (mol/m3 s); R is enzyme radius (cm), kv1 is reac-
tion rate constant (s−1), and De is the effective diffusion coefficient.
De is in proportion to the corresponding porosity and pore size,
increased De can indicate the enlarging on corresponding porosity
and pore size. The micro-porous diffusion coefficient of immobi-
lized enzyme between incubator shaker and ultrasound irradiation
could be measured by Di/Du (Di was the diffusion coefficient in
incubator shaker; Du was the diffusion coefficient in ultrasound
irradiation), and Di/Du was 0.33 in this two conditions, it illustrated
that porosity and pore size were increased under the ultrasound
process.
The highest MC yield in ultrasound irradiation condition was
slightly lower than that an in incubator shaker. When CA concen-
tration was 0.5 g/L, the best reaction time was decreased greatly
from 36 h to 9 h. Therefore, some certain errors were inevitable
existing. In addition, the results showed that ultrasound irradia-
tion had little impacts on substrate concentration than reaction
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Fig. 3. Effect of temperature (A and B) and substrate concentration (C and D) on the MC yield in [Bmim][Tf2N]. The lipase used was Novozym 435, CA concentration was
0.5 g/L, and the concentration of lipase was 100 g/L. The reactions analyzed in (A) and (C) were performed at 120 rpm in an incubator shaker; those analyzed (B) and (D) were
10 W under ultrasound irradiation.
time. These results suggest that the interaction between the sub-
strate molecules and the enzyme was maximized at a substrate
concentration of 0.5 g/L. Therefore, 0.5 g/L was treated as the opti-
mum CA concentration in [Bmim][Tf2N], and further experiments
on concentration of substrate were carried out at various lipase
concentration levels.
MC yield (96.42%) occurred when the CA concentration was 0.5 g/L
and Novozym 435 concentration was 60 g/L. Excessive amounts of
enzyme lead to a marginal decrease in yield. When the substrate
concentration was 0.15 g/L and Novozym 435 concentration was
20 g/L, the MC yield reached 88.10%. Further increases in enzyme
concentration led to a marginal decrease in MC yield. The most
likely reason was that the excess enzyme particles have the poten-
tial to attract each other and form enzyme aggregates, which may
reduce the accessibility of substrate to enzyme particles. When the
lipase concentration exceeded 20 g/L, the probability of collisions
between substrate and enzyme diminished [36].
3.5. The effect of lipase concentration
Fig. 4A and B shows the effect of varying lipase concentration
on MC yield in the incubator shaker and under ultrasound irradi-
ation, respectively. Varying Novozym 435 concentrations (20, 40,
60, 80, 100 and 120 g/L) and CA concentrations (0.15, 0.25, 0.5, 1,
1.5 and 2 g/L) were used under the two different incubation con-
ditions. Fig. 4A shows that with the incubator shaker, the highest
As shown in Fig. 4B, under ultrasound irradiation with ultra-
sound frequency of 25 kHz and ultrasound power of 150 W, the
highest MC yield (96.44%) was obtained when the substrate con-
centration was 0.5 g/L and Novozym 435 concentration was 60 g/L,
Fig. 4. Effect of lipase concentrations on the MC yield at different CA concentrations in [Bmim][Tf2N]. The lipase was Novozym 435. The reactions analyzed in (A) were
performed at 120 rpm for 36 h in an incubator shaker and those analyzed in (B) were performed at 150 W for 9 h under ultrasound irradiation.
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Table 1
Comparative results for the lipase-catalyzed synthesis of MC (in incubator shaker and ultrasound irradiation) at 75 ◦C in [Bmim][Tf2N].
Methods
Substrate concentration (g/L)
Lipase concentration (g/L)
Reaction time (h)
MC yield (%)
Incubator shakera
0.5
0.5
60
60
36
9
96.54
99.79
Ultrasound irradiationb
a
The experiment was conducted 120 rpm in an incubator shaker.
The experiment was conducted with ultrasound power set to 150 W and ultrasound frequency of 25 kHz under ultrasound irradiation.
b
conditions similar to those for the reactions performed with the
incubator shaker. The obvious difference between the results
shown Fig. 4B and A for the two different incubation conditions
was the effect of the two conditions on the MC yield. Performing
the reaction under ultrasound irradiation can obtain a higher MC
yield than using the incubator shaker. One possible reason for the
improved yield is that ultrasound irradiation may aid the forma-
tion of micro bubbles in the solution, which can result in drastic
changes to local conditions, such as enhanced mixing that could
contribute to the achievement of higher reaction rate. Thus, it is
the first time to report a study concerning the biosynthesis of MC
from CA and methanol using lipase-catalyzed esterification under
ultrasound irradiation.
and ultrasound frequency (15, 20, 25, 30 and 35 kHz) on MC yield
at different reaction time when the CA concentration was 0.5 g/L,
reaction temperature was 75 ◦C, and Novozym 435 concentration
was 60 g/L. Different powers were optimized by controlling ultra-
sound frequency of 25 kHz, which was chosen as an optimum
parameter on esterification reaction catalyzed by Novozym 435
[39]. The results indicated that the MC yield increased with the
increase of ultrasound power, and reached to the highest value
when ultrasound power was 150 W. Continued increasing power
led to decrease of MC yield, which was possibly because excessive
powerful of ultrasound could result in deactivation of the lipase. It
clearly proved that a suitable sonication (power of 150 W and fre-
quency of 25 kHz) has a significant effect on the enzymatic activity
[17].
Table 1 shows the comparative results of the lipase-catalyzed
synthesis of MC in an incubator shaker or under ultrasound irra-
diation at 75 ◦C in [Bmim][Tf2N]. The product yield is known to be
related to the reaction time: generally, the longer the reaction time,
the more products obtained [37]. However, the yield can suffer
from instability and oxidation at high temperatures. Therefore, it is
important to reduce the reaction time. In this reaction, the reaction
time was shortened to 9 h under ultrasound irradiation, whereas
it required 36 h in an incubator shaker. In other words, the reac-
tion time under ultrasound irradiation was less than half that of the
reaction performed using the incubator shaker. Moreover, the max-
imum yield reached 99.79% under ultrasound irradiation, which
was higher than that of the reaction performed using the incuba-
tor shaker. Therefore, the reaction under ultrasound irradiation was
more favorable than in the incubator shaker for the lipase-catalyzed
synthesis of MC. To further verify these results, the kinetics of
lipase-catalyzed esterification was next determined by monitoring
the changes in reaction rate with different substrate concentra-
tions. After the optimum reaction conditions were determined,
further ultrasound parameters (ultrasound power, frequency, and
operation mode) were explored in the following experiments.
3.7. Kinetic constants analysis
The enzymatic kinetics in batch reaction systems were
determined using the Michaelis–Menten model [40], and the
experimental data were fitted to the Lineweaver–Burk equation
(Eq. (7)):
1
V
1
Vmax
Km
1
=
+
Vmax [S]
(7)
where [S] is the initial substrate concentration, Vmax is the maxi-
mum velocity, and Km is the Michaelis constant. The value 1/V can
be plotted against 1/[S].
The esterification of CA and methanol was accorded with
ordered mechanism, namely, two substrates combined with
enzyme one after another, it means CA was first coupled on enzyme,
later it turns to methanol. In the end, the products were broke away
from the enzyme. CA concentrations ranged in control, but the con-
centration of substrate [S] of methanol far exceeded the enzyme
concentration [E] ([S] ꢀ [E]), such that all enzyme could be assumed
to form into enzyme–substrate complexes (ES), and the diminish-
ing [S] could be ignored. Under these conditions, the intermediate
ES complex concentration remains stable after its initial formation;
in other words, the rates of ES formation and its disappearance
remain equal, thus achieving a steady state. Furthermore, when all
the enzyme complexes with substrate, [E] = [ES], in which case, the
3.6. The effect of ultrasound power and frequency
Using pulse mode of ultrasound operation [38], Fig. 5A and B
shows the effects of different power (90, 120, 150, 180 and 210 W)
Fig. 5. Effect of ultrasound power (A) and frequency (B) on the MC yield at different reaction time in [Bmim][Tf2N]. Reaction conditions: temperature – 75 ◦C, concentration
of lipase – 60 g/L, CA concentration – 0.5 g/L. All tests were performed under ultrasound irradiation using pulse mode.
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Table 2
7
Kinetic parameters for the lipase-catalyzed synthesis of MC (in an incubator shaker
and ultrasound irradiation) at 75 ◦C in [Bmim][Tf2N].
Parametera
Value
Incubator shaker
Ultrasound irradiation
Km (app) (mM)
Vmax (g/L h)
Vm/Km (g/mmol h)
6.32
0.26
0.04
13.15
1.60
0.12
a
The kinetic assay was performed with a CA concentration that varied within the
range of 1.39–33.32 mM.
incubator shaker. One possible reason for the increase of Km (app) is
that ultrasound irradiation led to a little disruption of the enzyme.
Ultrasound irradiation has impact of reducing enzyme activity and
even inactivating the enzyme [41]. However, the initial reaction
rate improved greatly under ultrasound irradiation compared to
the incubator shaker, potentially due to the dispersion effect of
ultrasound irradiation, which could have increased the frequency
of collisions between enzyme and substrate [42]. The Vm/Km of
0.12 g/mmol h calculated for the reaction performed under ultra-
sound irradiation was greater than that for the reaction performed
in the incubator shaker (0.04 g/mmol h). Therefore, the process of
esterification under ultrasound irradiation was demonstrated to be
a more effective method for the synthesis of MC compared to that
without ultrasound irradiation.
Fig. 6. Kinetic plots of the lipase-catalyzed synthesis of MC in [Bmim][Tf2N] at differ-
ent CA concentrations (1.39–33.32 mM). Reaction conditions: temperature – 75 ◦C,
concentration of lipase – 60 g/L, CA concentration – 0.5 g/L. All tests were performed
at 120 rpm for 3 h in an incubator shaker and 150 W for 0.5 h under ultrasound
irradiation.
parameters of the kinetics of the enzyme could only be described
and presented by considering the substrate of CA.
Fig. 6 illustrates Eq. (2) double reciprocal plot obtained for the
esterification reaction by studying the effect of the concentration of
CA on the initial rate of the reaction. The kinetic parameter values
and the corresponding correlation coefficient (r2) for the lipase-
catalyzed synthesis of MC are summarized. It can be observed that
r2 was 0.9502 for the reaction in the incubator shaker, and it was
0.9331 for the reaction under ultrasound irradiation. The kinetic
parameters in this study were calculated. The intercept of Km/Vmax
and slope of 1/Vmax were fit-plotted in Fig. 6. The linear fitting
agrees well with the experimental data. The initial rate increased
with increasing CA concentration, and the reaction mechanism
exhibited Michaelis–Menten kinetics at the low substrate concen-
tration. This result indicates a strong affinity between substrate and
enzyme binding sites in this reaction.
Table 2 shows the kinetic parameter values comparing the
lipase-catalyzed synthesis of MC incubated in the incubator shaker
and under ultrasound irradiation. From these parameters, the min-
imum values of the apparent Km (Km (app)) and Vmax were 6.32 mM
and 0.26 g/L h, respectively, in the incubator shaker. The results
indicate that the affinity of the Novozym 435 toward CA is greater
than it is toward PE. A Km (app) of 13.15 mM and Vmax of 1.60 g/L h
were obtained in the ultrasound irradiation process, increases of
1.08-fold and 5.15-fold, respectively, compared to those from the
3.8. Study of lipase reusability
To investigate the reusability of the Novozym 435 for synthesis
of MC under ultrasound irradiation, selected reaction conditions
were used to test its stability. After each batch, the lipase was fil-
tered by a filter funnel, and then washed the lipase using a little
[Bmim][Tf2N], thus the lipase was recovered. A total of 16 batch
cycles were conducted. Fig. 7A shows the reusability of Novozym
435. The activity of lipase was stable during the first 11 batches
in [Bmim][Tf2N], with MC yields more than 82.0%; the highest
MC yield was 99.79%. Moreover, Novozym 435 could be reused 15
times while maintaining MC yield more than 50% under ultrasound
irradiation, matching the levels of activity and stability reported
previously [11]. Immobilized enzymes were more stable compared
to soluble enzymes and resisted the deactivating effect of ultra-
sound [43]. The protein concentration in the reaction medium was
measured according to previously explained [44], which increased
rarely after each reaction batch (Fig. 7B) in spite of the increase of
porosity and pore size in ultrasound irradiation. The results indi-
cated that Novozym 435 was not deactivated or denatured under
ultrasound irradiation [45], and the given lipase loading was still
Fig. 7. Reusability (A) and protein leakage (B) of Novozym 435 used in the synthesis of MC in [Bmim][Tf2N]. Reaction conditions: temperature – 75 ◦C, concentration of lipase
– 60 g/L, CA concentration – 0.5 g/L. All tests were performed at 150 W for 9 h under ultrasound irradiation.
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8
meet the economic feasibility [46]. In addition, by calculating, the
overall PC yield (82.74%) [8] could be increased to 98.29%. The
results indicated that the developed lipase-catalyzed esterification
method of MC under ultrasound irradiation could greatly improve
the overall yield of product using lipase-catalyzed transesterifica-
tion of an intermediate MC and an alcohol in ionic liquid.
[5] A. Kurata, Y. Kitamura, S. Irie, S. Takemoto, Y. Akai, Y. Hirota, T. Fujita, K. Iwai,
M. Furusawa, N. Kishimoto, J. Biotechnol. 148 (2010) 133–138.
[6] K. Shin, I. Kim, Y. Park, J. Ha, J. Choi, H. Park, Y.S. Lee, K. Lee, Biochem. Pharmacol.
68 (2004) 2327–2336.
[7] J. Wang, S. Gu, N. Pang, F. Wang, F. Wu, J. Serb. Chem. Soc. 78 (2013) 1023–1034.
[8] H. Chen, J. Chen, C. Chang, C. Shieh, Ultrason. Sonochem. 18 (2011) 455–459.
[9] M. Erbeldinger, A.J. Mesiano, A.J. Russell, Biotechnol. Progr. 16 (2000)
1129–1131.
[10] K. Uwai, Y. Osanai, T. Imaizumi, S. Kanno, M. Takeshita, M. Ishikawa, Bioorg.
Med. Chem. 16 (2008) 7795–7803.
[11] N. Pang, S. Gu, J. Wang, H. Cui, F. Wang, X. Liu, X. Zhao, F. Wu, Bioresour. Technol.
139 (2013) 337–342.
[12] J. Li, S. Wang, G. Chen, T. Li, Chem. Inform. 36 (2005) 415–436.
[13] P.B. Subhedar, P.R. Gogate, J. Mol. Catal. B-Enzym. 101 (2014) 108–114.
[14] J.V. Sinisterra, Ultrasonics 30 (1992) 180–185.
[15] P. Guo, C. Zheng, F. Huang, M. Zheng, Q. Deng, W. Li, J. Mol. Catal. B-Enzym. 93
(2013) 73–78.
[16] M. Zheng, L. Wang, F. Huang, L. Dong, P. Guo, Q. Deng, W. Li, C. Zheng, Ultrason.
Sonochem. 19 (2012) 1015–1020.
[17] M. Zheng, L. Wang, F. Huang, P. Guo, F. Wei, Q. Deng, C. Zheng, C. Wan, J. Mol.
Catal. B-Enzym. 95 (2013) 82–88.
[18] G. Ceni, P.C.D. Silva, L. Lerin, J.V. Oliveira, G. Toniazzo, H. Treichel, E.G. Oestre-
icher, D. de Oliveira, Enzyme Microb. Technol. 48 (2011) 169–174.
[19] S.H. Lee, H.M. Nguyen, Y. Koo, S.H. Ha, Process Biochem. 43 (2008)
1009–1012.
[20] J. Wang, S. Gu, H. Cui, X. Wu, F. Wu, Bioresour. Technol. 158 (2014) 39–47.
[21] J.K. Poppe, C. Garcia-Galan, C.R. Matte, R. Fernandez-Lafuente, R.C. Rodrigues,
M.A.Z. Ayub, J. Mol. Catal. B-Enzym. 94 (2013) 51–56.
4. Conclusions
A
novel synthesis method for MC was established using
lipase-catalyzed esterification of CA and methanol in an IL using
incubation shaking or under ultrasound irradiation, and the
effects of various parameters on the yield of MC were investi-
gated. Novozym 435 was found to be most active for catalysis
in [Bmim][Tf2N]; the optimum Novozym 435 concentration was
60 g/L; optimum CA concentration was 0.5 g/L at a temperature of
75 ◦C. In comparison to incubator shaking, under ultrasound irradi-
ation when ultrasound frequency of 25 kHz and ultrasound power
of 150 W, the reaction time reduced from 36 h to 9 h, the kinetic
parameter (Vm/Km) increased from 0.04 g/mmol h to 0.12 g/mmol h,
and the maximum MC yield increased from 96.54% to 99.79%. The
enzyme could be reused 11 times while maintaining MC yields
greater than 82% under ultrasound irradiation. These results indi-
cate that ultrasound irradiation is an efficient way to enhance
lipase-catalyzed synthesis of MC in an IL.
[22] J. Wang, S. Gu, H. Cui, L. Yang, X. Wu, Bioresour. Technol. 149 (2013) 367–374.
[23] Z. Duan, W. Du, D. Liu, J. Mol. Catal. B-Enzym. 89 (2013) 1–5.
[24] R. Rodrigues, G. Volpato, K. Wada, M. Ayub, J. Am. Oil Chem. Soc. 85 (2008)
925–930.
[25] H. Wang, M. Zong, H. Wu, W. Lou, J. Biotechnol. 129 (2007) 689–695.
[26] J. Wang, G. Sun, L. Yu, F. Wu, X. Guo, Bioresour. Technol. 128 (2013) 156–163.
[27] P. Lozano, T. De Diego, D. Carrié, M. Vaultier, J.L. Iborra, Biotechnol. Lett. 23
(2001) 1529–1533.
Acknowledgments
[28] J.L. Kaar, A.M. Jesionowski, J.A. Berberich, R. Moulton, A.J. Russell, J. Am. Chem.
Soc. 125 (2003) 4125–4131.
[29] E.C.S. Brenelli, J.L.N. Fernandes, Tetrahedron-Asymmetr. 14 (2003)
This work was supported by the National Natural Science
Foundation of China (21206061), the China Postdoctoral Science
Foundation Funded Projects (2012M510124, 2013T60505), the
Postdoctoral Science Foundation Funded Project of Jiangsu Univer-
sity (1143002085), the Qing Lan Project of Jiangsu Province (2014),
and the Postgraduate Innovation Project of Jiangsu Province (497).
1255–1259.
[30] L. Ge˛bicka, J.L. Ge˛bicki, J. Enzyme Inhib. Med. Chem. 12 (1997) 133–141.
[31] X. Jin, B. Liu, Z. Ni, Q. Wu, X. Lin, Enzyme Microb. Technol. 48 (2011) 454–457.
[32] P. You, E. Su, X. Yang, D. Mao, D. Wei, J. Mol. Catal. B-Enzym. 71 (2011) 152–158.
[33] M. Noel, D. Combes, J. Biotechnol. 102 (2003) 23–32.
[34] B. Castillo, Y. Pacheco, W. Al-Azzam, K. Griebenow, M. Devi, A. Ferrer, J. Mol.
Catal. B-Enzym. 35 (2005) 147–153.
[35] W.Y. Lou, M.H. Zong, Y.Y. Liu, J.F. Wang, J. Biotechnol. 125 (2006) 64–74.
[36] G.D. Yadav, S. Devendran, Process Biochem. 47 (2012) 496–502.
[37] H. Mu, X. Xu, C.E. Hoy, J. Am. Oil Chem. Soc. 75 (1998) 1187–1193.
[38] D. Rueter, T. Morgenstern, Ultrasonics 54 (2014) 2141–2150.
[39] S.R. Bansode, V.K. Rathod, Process Biochem. 49 (2014) 1297–1303.
[40] L.E. Rasmussen, C. Xu, J.F. Sørensen, M.K. Nielsen, A.S. Meyer, Biochem. Eng. J.
69 (2012) 8–16.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.molcatb.
2014.11.006.
[41] A. Vercet, J. Burgos, S. Crelier, P. Lopez-Buesa, Innov. Food Sci. Emerg. 2 (2001)
139–150.
[42] M. Galesio, J. Lourenc¸ o, D. Madeira, M. Diniz, J.L. Capelo, J. Mol. Catal. B-Enzym.
74 (2012) 9–15.
[43] A.M. Gumel, M.S.M. Annuar, Y. Chisti, Ultrason. Sonochem. 20 (2013) 937–947.
[44] M.M. Bradford, Anal. Biochem. 72 (1976) 248–254.
[45] D.H. Yu, L. Tian, H. Wu, S. Wang, Y. Wang, D.X. Ma, X.X. Fang, Process Biochem.
45 (2010) 519–525.
[46] C.M. Trentin, R.P. Scherer, C. Dalla Rosa, H. Treichel, D. Oliveira, J.V. Oliveira,
Bioproc. Biosyst. Eng. 37 (2014) 841–847.
References
[1] P. Padaratz, M. Fracasso, D.C. Buzzi, R. Corrêa, R. Niero, F.D. Monache, V.C. Filho,
Basic Clin. Pharmacol. 105 (2009) 257–261.
[2] A. Kalogeropoulos, V. Georgiopoulou, S.B. Kritchevsky, B.M. Psaty, N.L. Smith,
A.B. Newman, N. Rodondi, S. Satterfield, D.C. Bauer, K. Bibbins-Domingo, Arch.
Intern. Med. 169 (2009) 708–715.
[3] P.U. Rani, P. Devanand, J. Pest Sci. 86 (2013) 579–589.
[4] G.R. Gandhi, S. Ignacimuthu, M.G. Paulraj, P. Sasikumar, Eur. J. Pharmacol. 670
(2011) 623–631.
Please cite this article in press as: J. Wang, et al., J. Mol. Catal. B: Enzym. (2014), http://dx.doi.org/10.1016/j.molcatb.2014.11.006