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P. Guo et al. / Journal of Molecular Catalysis B: Enzymatic 93 (2013) 73–78
mechanical stirrer and microtip probe (diameter of 13 mm) con-
nected to a multi-frequency phonochemistry generator (Chengdu
Jiuzhou Ultrasonic Technology Co., Chengdu China) The ultra-
sonic unit has an operating frequency of 20–40 kHz and power
(Waters, USA) equipped with a Waters Acquity BEH C18 column
(100 mm × 2.1 mm and 1.7 m particles, Waters, Milford, MA, USA).
The monoester and diester products were separated by Agilent
6890Ngas chromatograph (GC) [6].
2.3. Lipase-catalyzed synthesis of 4MCG by ultrasonic
pretreatment
Lipase catalyzed esterification was carried out in 250 mL three-
mouth flask by using 10 mmol 4MCA, 30–70 mmol glycerol and
5–30 mg/mL C. antarctica lipase B in 150 mL of iso-octane. The sol-
˚
vent had, in advance, been dehydrated with molecular sieves 3 A
(10%, w/v) for at least 24 h. The flask was placed in a water bath with
both microtip probe ultrasonic pretreatment (0–2 h) and mechan-
ical stirring or only with mechanical stirring for a certain time.
The mechanical stirring rate was kept at 200 rpm. After comple-
was removed by filtration. The filtrate was concentrated on rotary
evaporator at reduced pressure (0.1 MPa). Concentrated reaction
mass was dissolved in methanol for HPLC analysis and conversions
were calculated on basis of 4MCA [8].
Fig. 1. Ultrasonic pretreatment lipase catalyzed esterification of 4-methoxy cin-
namic acid with glycerol.
less extensively studied [19]. The effect of ultrasound on enzymatic
reactions could be divided into two aspects. Firstly, ultrasound is
used as an enzymatic pre-treatment to reduce particle size and
consequent increase in the surface area of substrate and enzyme,
which is helpful to reduce mass transfer limitations [20,21].
Secondly, ultrasound is also known to perturb weak interactions
and to induce conformational changes in protein structures, thus
the substrate’s access to the active site is increased [22,23].
In most ultrasound-assistant reactions, ultrasound irradiation
was needed throughout the reaction, which was energy-consuming
and difficult to scale-up. Therefore, if ultrasound was only used as
a pretreatment at the beginning of the reaction, the process would
be energy-saving, simple to handle and easy to realize industrial-
ization.
In the present study, we report the lipase-catalyzed synthesis of
4MCG under ultrasonic pretreatment for the first time. The effects
of ultrasonic power, ultrasonic frequency, ultrasonic irradiation
ratio on the reaction conversion were studied. In order to better
understand some transport phenomena, experimental kinetic data
of stirring reaction and ultrasound pretreatment reaction was stud-
ied. The esterification of 4MCA with glycerol to form the monoester
and diester is shown in Fig. 1.
2.4. HPLC
Reaction samples were analyzed by HPLC equipped with an auto
sampler fitted with a 5 L loop was used to inject 3 L of the treated
samples (Waters, Milford, MA, USA), and eluted with methanol as
the mobile phase at a flow rate of 0.07 mL/min using UV detection at
280 nm. The strong UV absorbance of 4MCA and the glycerol ester
products provided a sensitive method of detection. The absorbance
of column effluents was monitored at different absorption wave-
length using the diode array detector with UV spectra collected
under the peaks by scanning from 210 nm to 500 nm. The 4MCA
and the esters strongly absorb from 270 nm to 320 nm with maxima
near 310 nm. Glycerol does not absorb in this region.
2.5. Products and substrates separation
The concentrated residue from 2.3 was derivatized with BSTF (N,
O-bis(trimethylsily)trifluoroacetamide) and pyridine. The deriva-
tized samples were carried out by GC equipped with an auto
sampler, and separations were achieved on the HP-5 column,
30 m × 0.32 mm ID × 0.25 m film thickness. Helium was used as
the carrier gas with a linear velocity of 35 cm/s. The oven temper-
ature was programmed from 120 ◦C to 240 ◦C at 10 ◦C/min with
an initial 2 min hold and a final 10 min hold. The inlet was heated
to 230 ◦C and set for split injections (split rate 1:10) with a 1 L
injection volume. The detector source was heated to 230 ◦C and the
detector quadrupole was heated to 150 ◦C. Date were collected and
processed via Chemstation software. The Chromatogram of BSTFA
derivatives of reaction mixture is shown in Fig. 2.
2. Materials and methods
2.1. Materials
C. antarctica lipase B (Novozym 435, immobilized on acrylic
resin) was purchased from Sigma–Aldrich (St. Louis, USA). 4MCA
(purity >98%) was purchased from Hubei YuanCheng Pharma-
ceutical Co., Ltd. (Wuhan China). Glycerol (purity >98%) and
isooctane were purchased from Sinopharm Chemical Reagent Co.,
Ltd. (Shanghai China). Methanol (HPLC grade) was purchased from
Merk KGaA (64271 Darmstadt, Germany). 4MCA (HPLC grade) was
purchased from J&K Scientific Ltd. (Beijing China). All other sol-
vents used for esterification reaction were obtained commercially
and were of analytical grade.
2.6. Kinetic modeling
In this work, in an attempt to represent the experimental kinet-
ics data obtained from esterification of 4-methoxyl cinnamic acid
and glycerol with an immobilized lipase as catalyst in ultrasound
pretreatment system and only mechanical stirring system, respec-
tively. Although the reaction rate might be controlled by internal
mass transfer in the immobilized enzyme, it was also reported that
2.2. Equipment
Experiments were carried out in a reactor with thermostatic
water bath (temperature accuracy of 0.5 ◦C), an IKA RW-20