Biochemical Engineering Journal 97 (2015) 2531

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Kinetics of enzymatic synthesis of monoferuloyl glycerol and

diferuloyl glycerol by transesterication in [BMIM]PF6
Shangde Sun , Xiaowei Chen
Lipid Technology and Engineering, School of Food Science and Engineering, Henan University of Technology, Lianhua Road, Zhengzhou 450001, Henan
Province, PR China

a r t i c l e

i n f o

Article history:
Received 28 October 2014
Received in revised form 26 January 2015
Accepted 1 February 2015
Available online 3 February 2015
Keywords:
Monoferuloyl glycerol
Diferuloyl glycerol
Enzymatic transesterication
Ethyl ferulate
Kinetics
Reaction mechanism
[BMIM]PF6

a b s t r a c t
Feruloyl glycerols (FGs), water-soluble glycerides (monoferuloyl glycerol (MFG) and diferuloyl glycerol
(DFG)) of ferulic acid, can be used as natural ultraviolet (UV) lters and antioxidants in chemical, food,
pharmaceuticals, and drug industries. In order to promote the synthesis of FGs, the effect of process
parameters, optimization, thermodynamic and kinetic properties on the enzymatic transesterication
of ethyl ferulate (EF) with glycerol in [BMIM]PF6 were investigated. The maximum yields of MFG
(63.72 1.26%) and DFG (78.80 2.09%) were achieved at low glycerol concentrations in [BMIM]PF6 .
The activation energies for EF conversion and transesterication to form MFG and DFG were calculated
as 40.16, 31.43 and 85.38 kJ/mol, respectively, based on the Arrhenius law. Reaction kinetics agreed with
the PingPong BiBi mechanism with the inhibitions of EF and glycerol. The enzymatic mechanism of
the transesterication of EF with glycerol in [BMIM]PF6 was also proposed.
2015 Elsevier B.V. All rights reserved.

1. Introduction
Ferulic acid (4-hydroxy-3-methoxy cinnamic acid, FA) is a phenolic component of the cinnamic acid family found ubiquitously
throughout the plant kingdom, which can be used as a potential
all-natural UV absorbent and antioxidant in food, health, cosmetic,
and pharmaceutical industries [14]. Feruloyl glycerols (FGs) are
the natural derivatives of FA, which are widely used as UV absorbers
and antioxidants in many commercial applications (such as, cosmetics, pharmaceuticals, and food industries) [57]. However, it is
difcult to separate FGs from natural raw materials since the contents of monoferuloyl glycerol (MFG) and diferuloyl glycerol (DFG)
are very low (<0.1%) [8], which made FGs synthesis very interesting
[912].
According to the previous reports [7,11,13], FGs can be prepared by chemical and enzymatic methods. Owning to the elevated

Abbreviations: FA, ferulic acid; EF, ethyl ferulate; MFG, monoferuloyl glycerol; DFG, diferuloyl glycerol; FGs, feruloyl glycerols; ILs, ionic liquids; [BMIM]PF6 ,
1-N-butyl-3-methylimidazolium hexauorophosphate; [EMIM]TF2N, 1-ethyl-3methylimidazolium bis (triuoromethylsulfonyl) imide; v0 , initial reaction rate;
vmax , maximum reaction rate; KM , Michaelis constants; Ki , inhibition constant; Ccat ,
enzyme concentration.
Corresponding author. Tel.: +86 371 67758022; fax: +86 371 67758022.
E-mail address: sunshangde@hotmail.com (S. Sun).
http://dx.doi.org/10.1016/j.bej.2015.02.002
1369-703X/ 2015 Elsevier B.V. All rights reserved.

temperatures, non-enantioselectivity, and pollution [12,14,15],

chemical synthesis of FGs was limited. Compared with chemical
methods, enzymatic synthesis of FGs showed many performances,
such as, mild reaction conditions, high catalytic efciency, high
enantioselectivity, and low energy consumption [7,9,10]. However, in the previous enzymatic synthesis of FGs, excess glycerol
or organic solvents were required as reactants and solvents to
obtain high FGs yields [9,13,15,16]. To overcome these disadvantages, ionic liquids (ILs), due to their negligible vapor pressure,
high thermal stability for enzyme, and favorable solvating property (molecular sieve role) for particular components [1721],
have been used as an alternative green reaction medium for
many enzymatic reactions. Previously, we developed a novel IL
([EMIM]TF2 N) system for lipase-catalyzed DFG production with
100% EF conversion and 45% DFG yield [11]. However, in these
previous reports, MFG and DFG yields were low. And no information about kinetics, thermodynamics, and reaction mechanism of
FGs preparation catalyzed by lipase in room-temperature IL was
available.
In order to improve MFG and DFG yields, for the rst time,
in [BMIM]PF6 , the effects of reaction variables on the synthesis
of MFG and DFG by the transesterication of EF with glycerol
using Novozym 435 as biocatalyst were investigated in the work
(Scheme 1). The thermodynamics, kinetics, and reaction mechanism in [BMIM]PF6 were also evaluated.

26

S. Sun, X. Chen / Biochemical Engineering Journal 97 (2015) 2531

Scheme 1. Enzymatic synthesis of FGs by the transesterication of EF with glycerol in [BMIM]PF6 .

2. Materials and methods

B for 4 min. The eluate was monitored at 325 nm. The FGs were
identied as described previously [9,22,23].

2.1. Materials
2.5. Statistical analyses
Ethyl ferulate (purity >99%) was purchased from Suzhou Chang
Tong Chemical Co., Ltd. (Suzhou, China). Glycerol (purity >99%) was
from Tianjin Ke Mi Ou Chemical Co., Ltd. (Tianjin, China). Novozym
435 (Candida antarctica lipase immobilized on polyacrylic resin,
with an activity of 7000 U/g solid enzyme, where one unit (U) of
activity is dened as the amount of enzyme, which can catalyze the
transformation of 1 mol substrate per minute under standard conditions) was provided by Novozymes A/S (Bagsvaerd, Denmark).
1-butyl-3-methylimidazolium hexauorophosphate ([BMIM]PF6 )
was obtained from Henan Lihua Pharmaceutical Co., Ltd. (Anyang,
China). Methanol and glacial acetic acid were of HPLC grade. All
other solvents were of analytical grade.
2.2. General procedure for enzymatic reaction in [BMIM]PF6
Reactions were performed in 25-ml round-bottom asks, and
EF (0.33 g), glycerol (0.14 g), immobilized Novozym 435 (225 mg),
and 3.35 mL [BMIM]PF6 were added. The reactants (0.47 g) were
mixed by a magnetic stirrer (200 rpm) at 80 C under 10 mm Hg
vacuum. These conditions were used throughout the experiments
except when otherwise stated in the text.
2.3. Kinetics study
Taking into account the results obtained in the experimental designs, the thermodynamics properties and the Arrhenius
equation were acquired from the plot of reaction rate against
temperature. EF conversion was used to determine the maximum
reaction rate (vmax ), MichaelisMenten constants (KM ), and inhibition constants (Ki ). The initial reaction rates (v0 ), dened as the
initial EF conversion per unit time (v0 , mol/(L min)), were calculated
from six experimental points of the yield-time prole corresponding to the rst 0.5 h of the reaction (20.0% or less EF conversion),
where the proles were found to be approximately linear.
2.4. HPLC analysis
Reactants and products were analyzed by HPLC (Waters 1525)
with a C18 reverse phase column (5 m, 250 mm 4.6 mm) tted
with a dual absorbance detector (Waters 2487) at 35 C, and eluted
with a binary gradient of solvent A (water, 0.5% v/v glacial acetic
acid) and solvent B (methanol) at 1 mL/min. The elution sequence
consisted, consecutively, of a linear gradient from 20% (v/v) B to
85% B (v/v) over 24 min, then to 20% B for 6 min, followed by 20%

Response surface methodology (RSM) was employed in the

work. The mathematical relationship relating the variables to the
responses can be calculated by the quadratic polynomial equation
(from BoxBehnken design):
Y = o +

4

i=1

i Xi +

4


ii Xi2 +

i=1

4
3 


ij Xi Xj

(1)

i=1 j=i+1

where Y is one of the two responses; Xi and Xj represent the independent variables; o is the constant, i the linear term coefcient,
ii the quadratic term coefcient, and ij the cross term coefcient.
Triplicate experiments were carried out for each parameter
investigated. Results were expressed as average S.E.M. The differences in mean values were evaluated by analysis of variance
(two-way ANOVA) method. Statistical signicance was considered
as p < 0.05.
3. Results and discussion
3.1. Effect of reaction temperature
In Fig. 1A, MFG and DFG yields slightly increased with the
increasing of temperature (<90 C), as well as EF consumption. The
time to reach equilibrium at higher temperatures was shorter than
that of lower temperatures, for example, at 90 C, the time (8 h)
to reach equilibrium was four sevenths that (14 h) of 80 C. However, when reaction temperature increased up to 100 C, MFG and
DFG yields and EF conversion all decreased (Fig. 1A and B). These
results were due to the fact that high temperature can reduce mixture viscosity, enhance enzyme activity, and decrease mass transfer
limitations. However, excessive high temperatures result in the
deactivation of Novozym 435.
In all experiments, MFG yields all exceeded those of DFG
(Fig. 1A), which can be explained by the fact that the activation
energy for DFG formation (85.38 kJ/mol) was almost three times
that of MFG formation (31.43 kJ/mol) (Fig. 1C). These results also
suggested that MFG was rstly formed by the transesterication of
EF with glycerol, and then reacted with another EF to form DFG.
3.2. Effect of enzyme concentration
EF conversion increased with the increase of enzyme concentration up to 60 mg/mL (Fig. 2A), which was ascribed to the fact
that the increase of enzyme concentration can provide more active

S. Sun, X. Chen / Biochemical Engineering Journal 97 (2015) 2531

27

Fig. 2. Effect of enzyme concentration on EF conversion (A) and initial reaction rate
(B). Reaction conditions: molar ratio of glycerol to EF was 1:1, EF concentration
0.40 mol/L, 80 C, vacuum pressure 10 mmHg, 200 rpm, and 14 h.

Fig. 1. Effect of reaction temperature on yields of MFG and DFG (A), and EF conversion (B), and the relationship between the initial reaction rate (v0 ) and reaction
temperature (C). The activation energy was calculated by multiplying the slope of the
Arrhenius plots with the gas constant (8.314 J/(mol K)). Reaction conditions: molar
ratio of glycerol to EF was 1:1, EF concentration 0.40 mol/L, Novozym 435 60 mg/ml,
vacuum pressure 10 mmHg, 200 rpm, and 14 h.

of MFG and DFG decreased rapidly to 32.32 2.48%, 29.37 0.98%,

and 2.58 0.83%, respectively. The results were attributed to the
inhibition of EF at high EF concentration [26].
Among the tested molar ratio of glycerol to EF, the maximum
EF conversion (99.13 0.87%) and MFG yield (61.0 2.39%) were
achieved at 0.8:1 (molar ratio of glycerol to EF) and 1:1 (molar
ratio of glycerol to EF), respectively (Fig. 4). Reaction selectivity

sites for acyl-enzyme complex formation, increase the probability of enzyme-substrate collision and subsequent reaction, and
enhance higher reaction rate [24]. However, when the enzyme
concentration was above 60 mg/mL, the excessive enzyme particles were clustered, which reduced the accessibility of enzyme
particles to reactants and enhanced the external mass transfer
resistance [25]. A linear relationship between the initial reaction
rate (v0 ) and enzyme concentration was achieved (Fig. 2B), and
can be expressed as: v0 = 0.1149Ccat . Where Ccat is enzyme concentration. The results indicated that the transesterication was
kinetically controlled.
3.3. Effect of substrate concentration
The effects of EF concentration on EF conversion and FGs yields
were shown in Fig. 3. At lower EF concentrations (<0.4 mol/L), EF
conversion and yields of MFG and DFG remained at high levels
(93.42 2.09%, 63.06 1.48%, and 35.01 3.1%). However, at high
EF concentrations (from 0.4 to 1.0 mol/L), EF conversion and yields

Fig. 3. Effect of EF concentration on EF conversion and yields of MFG and DFG.

Reaction conditions: molar ratio of glycerol to EF was 1:1, Novozym 435 60 mg/ml,
80 C, vacuum pressure 10 mmHg, 200 rpm, and 14 h.

28

S. Sun, X. Chen / Biochemical Engineering Journal 97 (2015) 2531

Fig. 4. Effect of molar ratio of glycerol to EF on EF conversion and yields of MFG

and DFG. Reaction conditions: Novozym 435 60 mg/ml, 80 C, vacuum pressure
10 mmHg, 200 rpm, and 14 h.

for DFG increased with the increase of EF concentration, and the

maximum DFG yield (76.98 2.83%) was obtained at 0.5:1 (molar
ratio of glycerol to EF) (Fig. 4).
3.4. Model tting and verication
Response surface methodology (RSM) was employed in the
work to obtain empirically signicant (p < 0.05) models for the synthesis of MFG and DFG. According to the statistical method, the data
were analyzed employing a multiple regression technique to evaluate the true relationship between the factors and EF conversions,
MFG and DFG yields (Tables 1 and 2). And quadratic regression
models are given as follows.

Fig. 5. Relationship between the initial reaction rate and substrates (EF and glycerol) concentrations, and the LineweaverBurk plot for Novozym 435 catalyzed the
transesterication of glycerol (dotted line a) with EF (dotted line b) in [BMIM]PF6 .
Reaction conditions: Novozym 435 60 mg/ml, 80 C, vacuum pressure 10 mmHg, and
200 rpm.

In the work, the DFG yield (78.80 2.09%) was much higher than
that (45%) of previous report [11]. These results can be ascribed to
the molecular sieve role of [BMIM]PF6 , which can drag out DFG
from hydration layer of Novozym 435 into [BMIM]PF6 . Compared
with free-solvent (10:1, mole ratio of glycerol and EF) and organic
solvent (85:1, mole ratio of glycerol and FA) [9,10], a higher EF conversion (100%) was obtained in [BMIM]PF6 at the lower molar
ratio (1:1) of glycerol to EF, which may be attributed to the solvation of [BMIM]PF6 as solvents. Compared with p-toluenesulfonic
acid, a high DFG yield (78.80 2.09%) was obtained using Novozym

(I) For MFG preparation

EF conversion(%) = 11.25X 1 + 5.90X 2 12.60X 3 + 8.62X 4 4.56X 1 X 2 7.22X 1 X 3 3.41X 1 X 4 7.45X 2 X 3 4.16X 2 X 4 + 4.86X 3 X 4
7.47X 1 2 2.67X 2 2 24.47X 3 2 5.24X 4 2 + 92.61R2 = 0.9404

(2)

MFG yield(%) = 1.26X 1 + 0.86X 2 + 8.31X 3 + 3.90X 4 2.05X 1 X 2 + 4.95X 1 X 3 1.06X 1 X 4 1.83X 2 X 3 + 0.72X2 X 4 + 7.41X 3 X 4
4.86X 1 2 1.04X 2 2 27.62X 3 2 5.19X 4 2 + 62.68R2 = 0.9353

(3)

(II) For DFG preparation

EF conversion(%) = 2.20X 1 2.48X 2 + 35.16X 3 0.93X 1 X 2 + 0.77X 1 X 3 + 0.84X 2 X 3 2.86X 1 2 1.95X 2 2 31.71X 3 2 + 95.04R2 = 0.9901
(4)
2

DFG yield(%) = 4.74X 1 0.023X 2 + 9.16X 3 1.12X 1 X 2 + 2.36X 1 X 3 + 2.38X 2 X 3 5.50X 1 5.70X 2 42.93X 3 + 77.53R = 0.9919
(5)
where X1 , X2 , X3 , and X4 represent reaction time (h), temperature ( C), molar ratio of glycerol to EF, and enzyme concentration
(mg/mL), respectively.
For MFG preparation, the maximum MFG yield (63.72 1.26%)
and EF conversion (98.26 1.05%) were achieved under the optimal conditions: 8.6 h, 68.5 mg/mL enzyme concentration, 1:1 molar
ratio of glycerol to EF, and 70 C, which were agreed with the predicted values (63.38% MFG yield and 97.91% EF conversion). For
DFG preparation, the maximum DFG yield (78.80 2.09%) and EF
conversion (99.24 0.60%) were achieved at 0.62:1 mole ratio of
glycerol to EF, 90.6 C, 60.0 mg/mL enzyme concentration for 13.4 h,
which were also agreed with the predicted values (79.03% DFG
yield and 99.85% EF conversion). These results indicated that the
validation of the quadratic regression model.

435 as catalyst in [Bmim]PF6 , which may be attributed to the 1,3selectivity of Novozym 435. Similar result was also found in the
synthesis of diacylglycerols using Novozym 435 as catalyst [27].
3.5. Kinetics and reaction mechanism of the transesterication
As observed in Fig. 5, when EF concentration (A) increased, by
keeping glycerol concentration (B) constant, the initial reaction
rate (v0 ) increased proportionally and reached a maximum at a
critical concentration. A subsequent increase in EF concentration
led to a decrease of v0 . The effect of glycerol concentration on v0
was similar to that of EF concentration. The LineweaverBurk plots
revealed that the enzymatic transesterication of EF with glycerol was accorded with classical MichaelisMenten kinetics, and

S. Sun, X. Chen / Biochemical Engineering Journal 97 (2015) 2531

29

Table 1
Experimental design and results of EF conversion and MFG yield as affected by reaction time, reaction temperature, substrate ratio and enzyme concentration.
Treament no.a

Reaction time X1
(h)

Reaction temperature
X2 ( C)

Substrate ratiob
X3 (mol/mol)

Enzyme concentration
X4 (mg/mL)

EF conversion (%)

MFG yield (%)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29

4
4
8
8
8
8
12
12
8
8
8
8
12
12
4
8
8
8
4
12
8
8
4
8
4
8
8
8
12

90
80
80
80
90
90
70
80
80
80
80
80
90
80
80
90
80
70
80
80
70
90
80
80
70
80
70
70
80

1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.5
1.0
1.0
1.0
0.5
1.5
1.5
1.0
1.0
0.5
0.5
0.5
1.5
0.5
1.0
0.5
1.0
1.5
1.5

60
40
60
60
80
40
60
40
60
60
80
60
60
80
60
60
40
40
80
60
60
60
60
80
60
40
80
60
60

83.45 2.01
58.62 1.29
93.42 3.0
92.03 1.69
96.85 2.01
90.14 4.70
90.75 2.58
90.08 1.60
92.46 2.01
92.62 2.58
63.50 3.10
92.52 2.31
99.20 3.11
98.02 1.99
56.85 1.82
49.87 0.79
31.84 1.07
60.44 2.91
80.20 2.16
89.31 1.08
69.83 1.58
81.17 1.22
42.68 0.90
84.36 1.58
56.77 1.02
72.16 2.66
83.80 1.02
68.32 2.00
46.24 1.68

60.91 1.93
46.10 1.37
63.06 2.99
62.98 1.39
63.38 1.37
53.56 3.01
58.19 1.93
55.39 2.38
62.26 1.38
62.15 2.86
51.06 1.37
62.94 0.39
61.65 1.82
59.90 2.12
30.27 1.26
39.35 0.37
27.84 1.28
46.67 2.68
54.84 2.30
15.91 1.02
27.90 1.52
20.79 0.38
30.28 2.09
18.50 1.92
49.24 0.97
24.91 1.57
53.59 1.58
53.78 0.79
35.74 1.08

a
b

Numbers were run in random order.

Glycerol/EF (mol/mol).

Table 2
Experimental design and results of EF conversion and DFG yield as affected by reaction time, reaction temperature, and substrate ratio.
Treament no.a

Reaction time X1
(h)

Reaction temperature
X2 ( C)

Substrate ratiob X3
(mol/mol)

EF conversion (%)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17

8
16
12
12
12
12
16
12
12
16
12
12
8
12
8
8
16

90
90
80
90
100
90
80
80
90
90
90
90
100
100
80
90
100

1.0
0.1
0.1
0.55
1.0
0.55
0.55
1.0
0.55
1.0
0.55
0.55
0.55
0.1
0.55
0.1
0.55

97.58
21.82
34.83
94.92
89.61
95.26
95.47
97.45
94.78
99.72
95.42
94.83
86.85
23.63
85.39
22.78
93.21

a
b

3.10
2.79
0.28
1.26
0.38
1.36
1.99
3.01
2.81
1.78
2.08
0.98
1.67
2.81
0.87
1.99
1.72

DFG yield (%)

35.20
18.26
23.28
76.89
39.28
78.51
75.11
33.01
76.76
45.14
78.89
76.58
59.78
20.02
59.13
17.77
71.27

1.92
1.08
0.28
1.00
0.28
1.62
2.91
0.79
1.93
1.82
1.03
2.80
0.93
0.01
1.98
1.72
1.07

Numbers were run in random order.

Glycerol/EF (mol/mol).

enzyme activity was inhibited by both EF and glycerol at their

higher concentrations. Therefore, the enzymatic transesterication can be modeled using the PingPong BiBi kinetic mechanism
with the inhibitions of EF and glycerol at higher concentrations
[25,28,29]. The initial reaction rate can be expressed as follows:

v0 =

vmax [A][B]
KMA [B](1+([B]/K iB )) + K MB [A](1+([A]/K iA ))+[A][B]

where, v0 is the initial reaction rate; vmax is the maximum reaction

rate; [A] and [B] are the initial concentrations of EF and glycerol,
respectively; KMA and KMB are Michaelis constants for EF and glycerol, respectively; KiA and KiB are the inhibition constants for EF and
glycerol, respectively.

Initial reaction rates were calculated from the linear portion of the concentrationtime proles, and the kinetic constants
can be obtained by non-linear regression analysis for the above
model [30]. Results showed that vmax , KMA , and KMB were
12.01 mol/(L min), 0.23 mol/L, and 0.42 mol/L, respectively, which
indicated that the afnity between enzyme and EF is higher
than that of enzyme and glycerol. KiA and KiB were 1.20 mol/L
and 0.48 mol/L, respectively, which were attributed to a glycerol
hindrance layer formed in the enzyme micro-environment and
electron-donating and steric hindrance of EF [28,31].
Based on PingPong BiBi mechanism with the inhibitions of EF
and glycerol, the transesterication mechanism for the synthesis of
FGs was proposed as follows (Fig. 6): (i) the acyl donor, EF, is rstly

30

S. Sun, X. Chen / Biochemical Engineering Journal 97 (2015) 2531

Fig. 6. Reaction mechanism for the transesterication of EF with glycerol to form MFG and DFG.

binded to enzyme (E) to form a non-covalent enzymeester complex (EFE); and EFE complex is transferred to the acylenzyme
complex (FE) with the release of ethanol, or EFE is hydrolyzed
to form FA and ethanol; (ii) glycerol is combined with FE to form
another complex ([FE]glycerol), and [FE]glycerol is isomerized
to the esterenzyme complex (MFGE), and then MFGE complex
releases E to form the rst product MFG (1); (iii) MFG is binded with
another FE to form the MFG[FE] complex, and the MFG[FE]
complex is isomerized to the DFGE complex, and then the DFGE
complex releases E to form the second product DFG (2). However,
at higher glycerol concentrations, the dead-end binary complex
(glycerolE) between glycerol and enzyme is formed instead of the
EFE (3). In contrary, at higher EF concentrations, another dead-end
complex (FEEF) between EF and enzyme is formed instead of the
[FE]glycerol (4).
4. Conclusions
Enzymatic synthesis of FGs by the transesterication of EF with
glycerol catalyzed by Novozym 435 were successfully performed in
[BMIM]PF6 . Under the optimized conditions, the maximum yields
of MFG and DFG were 63.72 1.26% and 78.80 2.09% at 1:1 and
0.62:1 molar ratio of glycerol to EF, respectively. Due to the 1,3selectivity of Novozym 435 and the molecular sieve effect of the
[BMIM]PF6 , it was found to be benecial to reduce the amount of
glycerol in the substrates. The two-step transesterication reaction
kinetics, rst to form MFG, and then to form DFG, was agreed with
PingPong BiBi mechanism with the inhibitions of EF and glycerol.
Compared with glycerol, EF showed a higher afnity to Novozym
435.
Acknowledgements
The authors gratefully acknowledge nancial support from
National Natural Science Foundation of China (31101301), Funding
Scheme for Young Teachers in Colleges and Universities in Henan
Province (2012GGJS-83), and Plan for Scientic Innovation Talent
of Henan University of Technology (11CXRC03).
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