Enzymatic synthesis of ascorbyl palmitate in ultrasound-assisted system: Process optimization and kinetic evaluation

Article abstract of DOI:10.1016/j.ultsonch.2010.12.013

This work is focused on the optimization of reaction parameters for the synthesis of ascorbyl palmitate catalyzed by Candida antarctica lipase in different organic solvents under ultrasound irradiation. The sequential strategy of experimental design proved to be useful in determining the optimal conditions for reaction conversion in tert-butanol system using Novozym 435 as catalyst. The optimum production was achieved at 70°C, ascorbic acid to palmitic acid molar ratio of 1:9, enzyme concentration of 5 wt% at 3 h of reaction, resulting in an ascorbyl palmitate conversion of about 27%. Reaction kinetics for ascorbyl palmitate production in ultrasound device showed that satisfactory reaction conversions (～26%) could be achieved in short reaction times (2 h). The empirical kinetic model proposed is able to satisfactorily represent and predict the experimental data.

Full text of DOI:10.1016/j.ultsonch.2010.12.013

Ultrasonics Sonochemistry 18 (2011) 988–996
Contents lists available at ScienceDirect
Ultrasonics Sonochemistry
journal homepage: www.elsevier.com/locate/ultsonch
Enzymatic synthesis of ascorbyl palmitate in ultrasound-assisted system:
Process optimization and kinetic evaluation
Lindomar A. Lerin a, Miriam C. Feiten b, Aline Richetti b, Geciane Toniazzo b, Helen Treichel b,
Marcio A. Mazutti b, J. Vladimir Oliveira b, Enrique G. Oestreicher a, Débora de Oliveira b,
⇑
a Instituto de Química – IQ/UFRJ, CT, Bloco A, Rio de Janeiro 21945-900, RJ, Brazil
b Universidade Regional Integrada do Alto Uruguai e das Missões, URI – Campus de Erechim, Av. Sete de Setembro, 1621, 99700-000 Erechim, RS, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 June 2010
Received in revised form 27 September 2010
Accepted 23 December 2010
Available online 4 January 2011
This work is focused on the optimization of reaction parameters for the synthesis of ascorbyl palmitate
catalyzed by Candida antarctica lipase in different organic solvents under ultrasound irradiation. The
sequential strategy of experimental design proved to be useful in determining the optimal conditions
for reaction conversion in tert-butanol system using Novozym 435 as catalyst. The optimum production
was achieved at 70 °C, ascorbic acid to palmitic acid molar ratio of 1:9, enzyme concentration of 5 wt% at
3 h of reaction, resulting in an ascorbyl palmitate conversion of about 27%. Reaction kinetics for ascorbyl
palmitate production in ultrasound device showed that satisfactory reaction conversions (ꢀ26%) could be
achieved in short reaction times (2 h). The empirical kinetic model proposed is able to satisfactorily rep-
resent and predict the experimental data.
Keywords:
Biocatalysis
Neural network
Lipase
Ascorbyl palmitate
Experimental design
Ultrasound
Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction
ison to high intensity, which increases mass transfer. On the other
hand, high intensity-ultrasound can lead to disruption of the
Enzymes are increasingly becoming the industrial workhorse
for a range of processes, such as biocatalysis and bioremediation,
with the aim of reducing energy and raw material consumption
and amounts of waste and toxic side products [1]. An ultrasound
wave, which is a periodic pressure fluctuation, can control the
enzyme characteristics by altering its structure in response to a
dynamic perturbation [2].
Ultrasound irradiation, a mechanical rather than an electromag-
netic wave, is an alternative method to reduce mass transfer limi-
tations in enzymatic reactions [3–5]. Ultrasonic action in liquids
can cause effects of cavitation. When cavitation bubbles collapse
near the phase boundary of two immiscible liquids, the resultant
shock wave can provide a very efficient stirring/mixing of the lay-
ers. Because the effects of cavitation can enhance heterogeneous
reactions and readily form transient reactive species, ultrasound
is also a useful tool in enzymatic reactions [2,5]. Ultrasound is also
known to perturb weak interactions and to induce conformational
changes in protein structures [6,7].
enzyme [8].
These results are somewhat in compliance with some reports in
the sense that increasing ultrasonic power in an appropriate range
enhances enzymatic reaction rate [9,10] which was considered
probably due to a decrease in substrate inhibition and aggregation
based on hydrogen bonding of molecules. However, too high
ultrasonic intensities were reported to reduce the activity or even
inactivate the enzyme [11,12].
Ultrasound is very effective at dispersing material present in
solution [13]. The application of ultrasound, therefore, will contrib-
ute to a more homogeneous reaction mixture and facilitate disper-
sion of lipase through substrate media, reducing agglomeration so
that the reaction rate does not decrease with the increase of lipase
concentration [14].
The synthesis of esters of ascorbic acid by lipase-catalyzed reac-
tions has become a matter of great current commercial interest due
to the steady growing demand for natural materials. An optimized
enzymatic synthesis of L-ascorbyl esters with improved yield at re-
For reactions under ultrasound irradiation, ultrasonic power is
an important influencing factor. Ultrasonic waves of low intensity
have a small effect on the mass transfer of the solution in compar-
duced cost would be more appealing to the consumer and would
bring benefits to the manufacturers [15]. In this sense, due to the
low miscibility of the substrates, the application of ultrasound irra-
diation to this reaction system may lead to high reaction rates.
The importance of enzymatic synthesis catalyzed by lipases to
⇑
Corresponding author. Fax: +55 54 35209090.
E-mail address: odebora@uricer.edu.br (D. de Oliveira).
produce
L-ascorbyl esters via esterification in water-miscible
1350-4177/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ultsonch.2010.12.013

L.A. Lerin et al. / Ultrasonics Sonochemistry 18 (2011) 988–996
989
organic solvents has been emphasized in several works [16,17].
2.4. Products quantification
Palmitic, caprilic, capric, lauric, and stearic acids have been widely
used as acyl donors. Among the unsaturated acids, one could men-
tion oleic, linolenic, arachidonic, and docosahexaenoic acids [18].
Based on the above mentioned information, the present work is
focused on the optimization of reaction conditions for ascorbyl pal-
mitate synthesis under ultrasound irradiation by a sequential
strategy of experimental planning. The kinetics for this reaction
system was also investigated at the maximized experimental con-
ditions obtained from the execution of the experimental design. To
the best of our knowledge, a systematic study for maximizing the
product synthesis as well as experimental data and kinetic model-
ing concerning the enzymatic esterification of ascorbic acid in
ultrasound-assisted system has not been reported.
Quantitative analyses of the products were conducted using an
HPLC system from Agilent Series, equipped with a refractive index
detector. The following instrumentation and conditions were used:
Zorbax C18 column (4.6 m ꢁ 250 mm, 5
lm), flow rate of 1.0 mL/
min, column temperature of 35° C; the mobile phase, ace-
tone:methanol:H2O with 0.5% of acid acetic (75:25:5, v/v/v). The
mobile phase was used as a sample dissolving solvent, and the
injection volume was 20
lL. Quantification was carried out using
authentic standards of ascorbyl palmitate (6-O-palmitoyl-
L-ascorbic
acid). Calibration curves were built with the product concentra-
tions of 240, 480, 960, 1920, 2880, 3840, and 4800 ppm. Reaction
conversion was calculated based on the content of ascorbyl palmi-
tate in the analyzed sample and the reaction stoichiometry.
2. Materials and methods
2.5. Preliminary tests
2.1. Materials
At first, for all experimental runs, the enzyme was activated in
an oven at 40° C for 60 min. Batch reactions were carried out in
125 cm3 conical flasks. In this initial step, some specific conditions
were tested as following: use of solvent (10 mL of tert-butanol)
without agitation; use of solvent with mechanical stirring
(250 rpm); solvent-free system without agitation and solvent-free
system with mechanical stirring (250 rpm). All experiments were
then carried out under sonochemical irradiation of 132 W, temper-
ature of 70° C, substrates molar ratio of 1:9, enzyme concentration
of 5 wt% [20]. For the evaluated system that presented higher
ascorbyl palmitate yields, a kinetic experiment, at the same exper-
imental conditions cited before, was carried out to determine the
reaction time to be fixed in the experimental design step.
It may be important to emphasize that in all cases, destructive
experiments, without sampling, were carried out. After each exper-
imental run, the enzyme was separated from the reaction medium
and washed twice with 10 mL of n-hexane. The recuperated
enzyme was kept in a desiccator for 24 h and, after this period,
the enzyme activity was determined, following the methodology
proposed above.
The substrates used in the esterification reactions were com-
mercial palmitic acid (Vetec, 98% purity) and L-(+)-ascorbic acid
(Vetec, 99% purity). Novozym 435, a lipase from Candida antarctica,
purchased from Novozymes S/A (Araucária-PR-Brazil), was used as
a catalyst. This enzyme was immobilized on a macroporous anionic
resin, and presents a water content of 1.4 wt% (Karl Fischer titra-
tion method, DL 50, Mettler-Toledo) and an initial enzyme activity
of around 60 U/g of support, determined as the initial rates in
esterification reactions between lauric acid and propanol at a mo-
lar ratio of 3:1, 60° C and enzyme content of 5 wt% in relation to
the substrates [19].
Acetone, tert-butanol, acetic acid, ethanol, acetone, n-propanol,
and methanol of HPLC grade were tested as organic solvents (Ve-
tec). The standard of 6-J-palmitoyl-L-ascorbic acid was obtained
from Sigma–Aldrich (Fluka, 99% purity).
2.2. Equipment
Experiments were carried out in a reactor with a thermostatic
water bath (temperature accuracy of 0.5° C). The experimental
setup consists of an ultrasonic bath (Unique Inc.-model USC
1800A, Brazil, BR) equipped with a transducer having longitudinal
vibrations. The ultrasonic unit has an operating frequency of
37 kHz and a maximum rated electrical power output of 132 W.
The ultrasonic transducer (surface area of 282.2 cm2) is fitted at
the bottom of the bath horizontally along the length of bath. The
advantage of using such a system is that if offers much larger effec-
tive cavitational area compared to the conventional immersion-
based axial transducers and hence results in uniform cavitational
activity distribution in the ultrasonic bath.
2.6. Sequential strategy of experimental designs
With the objective of determining the best reaction conditions,
in the first experimental design the effects of substrates molar ratio
(1:1–1:6), enzyme concentration (5–15 wt%), solvent volume (5–
15 mL), power ultrasound amplitude (52–132 W) and temperature
(40–70° C), were evaluated in terms of ascorbyl palmitate produc-
tion. For this experimental planning, the levels of variables were
defined from preliminary experiments and the reaction time was
constant at 3 h. Three replicates at the central point of each
experimental planning were carried out in order to determine
the experimental error.
After analyzing the results of the first experimental design, a
central composite rotatable design 22 (CCRD) was carried out
adjusting the substrates molar ratio (1:5–1:13) and temperature
(50–80° C) [21,22].
2.3. Lipase esterification activity
The enzyme activity was determined as the initial rates in ester-
ification reactions between lauric acid and n-propanol at a molar
ratio of 3:1, temperature of 60° C and enzyme concentration of
5 wt% in relation to the substrates. At the beginning of the reaction,
samples containing the mixture of lauric acid and n-propanol were
collected and the lauric acid content was determined by titration
with NaOH (0.04 N). After the addition of the enzyme to the sub-
strates, the mixture was kept at 60° C for 15 min. The lauric acid
consumption was then determined. One unit of activity (U) was
2.7. Kinetic study of enzymatic esterification
Taking into account the results obtained in the experimental
designs, reaction kinetic experiments were performed adopting
ascorbic acid to palmitic acid molar ratios of 1:1, 1:3, 1:6, and
1:9, enzyme concentration of 1, 5, 10, and 20 wt% (based on the
total amount of substrates – ascorbic acid and palmitic acid),
temperature of 50, 60, 70, and 80° C. Samples were taken from
the bulk reactive system at 30 min, and 1, 2, 3, 4, and 5 h.
defined as the amount of enzyme necessary to consume 1 lmol
of lauric acid per minute. All enzymatic activity determinations
were replicated at least three times.

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L.A. Lerin et al. / Ultrasonics Sonochemistry 18 (2011) 988–996
2.8. Hybrid model formulation
0.98, respectively. The hybrid model was developed and imple-
mented in FORTRAN 90 language.
This study was concerned on the development of a hybrid mod-
el to predict the ascorbyl palmitate (AP) production. The model
consists of a mass balance for the ascorbyl palmitate production
in the batch reactor with no density and volume changes according
to the following equation:
2.9. Statistical analysis
The statistical analysis related to the estimated effects of each
variable and process optimization was performed using the global
error and the relative standard deviation between experimental
data and calculated values. It may be important to mention that
the kinetic results subsequently presented in this work are in fact
mean values of triplicate runs, which resulted in an overall abso-
lute deviation of reaction conversion of around 2%. The quality of
the hybrid model formulation was verified using the correlation
coefficient (r). All analysis was performed using the software
Statistica version 6.0 (Statsoft Inc., USA).
d½APꢂ
¼
v
ð1Þ
dt
The main difficult in the above model is to determine the
reaction rate ( ), since there no data concerning the kinetics of
v
the substrates employed. As an alternative, a feed-forward neural
network (ANN) is used to predict the reaction rate in Eq. (1). The
ANN was employed with the following topology: the inputs for
ANN were reaction time, temperature, initial enzyme concentra-
tion, palmitic acid and ascorbic acid concentrations; only one hid-
den layer was used and the number of nodes in this layer was
determined by trial and error; the transfer function employed
was the hyperbolic tangent in the output and hidden layer; the
output layer was composed of one node related to the reaction rate
of ascorbyl palmitate production.
3. Results and discussion
3.1. Preliminary tests
Table 1 presents the results obtained in the preliminary tests.
One can observe that the highest conversion (25.07%) was obtained
using organic solvent without agitation. A comparable conversion
(20.86%) was obtained from run two, after 2 h of reaction, using
tert-butanol as solvent and 250 rpm. In the reaction systems with-
out solvent, no conversion of substrates was observed. Taking into
The above inputs were combined to minimize the objective
function F defined according to Eq. (2).
j¼NPE
X
exp
calc 2
F ¼
ð½APꢂj ꢃ ½APꢂj
Þ
ð2Þ
J¼1
Table 1
Results of ascorbyl palmitate yield (%) for the preliminary tests in ultrasound device
(37 kHz and 132 W) using 5 wt% of Novozym 435, ascorbic acid to palmitic acid molar
ratio of 1:9, 70 °C and 2 h of reaction.
where NPE is the number of experimental data points where the F is
exp
calc
calculated, and ½APꢂj and ½APꢂj represent the experimental and
calculated ascorbyl palmitate concentrations, respectively.
Reaction condition
Ascorbyl palmitate yield (%)
During the training procedure, the weights and the bias were
optimized using the Simulated Annealing algorithm combined
with the Nelder and Mead [23] algorithm. The parameters adopted
for the Simulated Annealing algorithm as the initial artificial
Tert-butanol without agitation
Tert-butanol and 250 rpm
Without solvent
25.07
20.86
0
Without solvent and 250 rpm
0
annealing temperature (TA) and cooling rate (a) were 10.0 and
Fig. 1. Kinetic experiment in ultrasound assisted system in organic medium in terms of ascorbyl palmitate conversion and residual enzyme activity. Experimental condition
of ascorbic acid to palmitic acid molar ratio of 1:9, 10 mL of solvent, 5 wt% of Novozym 435 and 70° C.

L.A. Lerin et al. / Ultrasonics Sonochemistry 18 (2011) 988–996
991
Table 2
Matrix of the Plackett–Burman experimental design (coded and real values) for ascorbyl palmitate production under sonochemical irradiation after 2 h of reaction.
Trial
Temperature (°C)
Enzyme concentration (wt%)
Solvent volume (mL)
Substrates molar ratioa
Ultrasonic power (W)
Yield (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
70 (1)
70 (1)
5 (ꢃ1)
15 (1)
15 (1)
5 (ꢃ1)
15 (1)
15 (1)
15 (1)
5 (ꢃ1)
5 (ꢃ1)
5 (ꢃ1)
15 (1)
5 (ꢃ1)
10 (0)
10 (0)
10 (0)
15 (1)
5 (ꢃ1)
15 (1)
15 (1)
5 (ꢃ1)
15 (1)
15 (1)
15 (1)
5 (ꢃ1)
5 (ꢃ1)
5 (ꢃ1)
5 (ꢃ1)
10 (0)
10 (0)
10 (0)
1:1 (ꢃ1)
1:6 (1)
1:1 (ꢃ1)
1:6 (1)
1:6 (1)
1:1 (ꢃ1)
1:6 (1)
1:6 (1)
1:6 (1)
1:1 (ꢃ1)
1:1 (ꢃ1)
1:1 (ꢃ1)
1:3 (0)
52 (ꢃ1)
52 (ꢃ1)
132 (1)
52 (ꢃ1)
132 (1)
132 (1)
52 (ꢃ1)
132 (1)
132 (1)
132 (1)
52 (ꢃ1)
52 (ꢃ1)
92 (0)
7.28
22.68
7.66
24.66
22.99
12.07
7.63
16.42
13.58
10.23
2.40
40 (ꢃ1)
70 (1)
70 (1)
70 (1)
40 (ꢃ1)
40 (ꢃ1)
40 (ꢃ1)
70 (1)
40 (ꢃ1)
40 (ꢃ1)
55 (0)
0.37
13.68
13.94
16.64
55 (0)
50 (0)
1:3 (0)
1:3 (0)
92 (0)
92 (0)
a
Ascorbic acid:palmitic acid.
account the results obtained here, further studies were carried out
in tert-butanol system without agitation.
define the reaction time to be fixed in the experimental design
step. The experimental condition of ascorbic acid to palmitic acid
molar ratio of 1:9, 10 mL of solvent, 5 wt% of Novozym 435 and
70° C was tested here. From this figure one can see that the conver-
sion improves as a function of reaction time, reaching a maximum
(25.11%) after 4 h. Related to the residual esterification activity, it
can be observed that no significant activity losses were verified,
with a maximum loss of 20.75%. Further studies for optimization
of process conversion were performed at 3 h of reaction.
Liu et al. [14] observed in the hydrolysis of soybean oil in a sol-
vent-free system in an ultrasound device that a reaction yield of
94% at 5 h was achieved. The kinetic evaluation of hydrolysis of
soybean oil catalyzed by Lipozyme TL IM in ultrasound system
afforded a diglyceride yield of 40% after 1.5 h [24].
Previous literature relates enzymatic synthesis using
ultrasound devices in organic and solvent-free systems [14,24].
In general, these works show the influence of ultrasound on reac-
tion conversion. For example, Babicz et al. [24] verified that
mechanical agitation is an important factor for the production of
mono- and diglycerides by enzymatic hydrolysis of soybean oil,
but that this variable can also damage the enzyme support or pro-
voke the adherence of the enzyme in the reactor. The authors
obtained higher conversions at 700 rpm using Lipozyme TL IM
and Lipozyme 87 IM as catalysts.
Xiao et al. [25] obtained conversions of 98% after 2 h and 120 W
of ultrasound, and only 48% of conversion was observed without
agitation under the same experimental conditions. The authors
also observed that the ultrasound did not alter the regio-selectivity
of the catalyst.
3.2. Experimental design
Fig. 1 presents the kinetic experiment in ultrasound-assisted
system in organic medium in terms of ascorbyl palmitate yield
and residual enzyme activity. This experiment was carried out to
After determining the preliminary experimental condition that
afforded the highest conversion, a Plackett–Burman design for
ascorbyl palmitate production was carried out (Table 2) keeping
Fig. 2. Pareto chart of the effects of ascorbic acid to palmitic acid molar ratio and temperature on the ascorbyl palmitate production (p < 0.05). Experimental data and
conditions shown in Table 2.

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L.A. Lerin et al. / Ultrasonics Sonochemistry 18 (2011) 988–996
Table 3
Matrix of the second experimental design (coded and real values) with responses in terms of ascorbyl palmitate conversion.
ꢀ
ꢀ
ꢀ
ꢀ
ꢀY
ꢀ
exp ꢃYmodel
RED (%)b RED
ꢁ 100
Trial
Temperature (°C)
Molar ratioa
Experimental conversion (%)
Predicted conversion (%)
Yexp
1
2
3
4
5
6
7
8
9
ꢃ1 (54.4)
1 (75.6)
ꢃ1 (54.4)
1 (75.6)
ꢃ1.41 (50)
1.41 (80)
0 (65)
0 (65)
0 (65)
0 (65)
0 (65)
ꢃ1 (1:6)
ꢃ1 (1:6)
1 (1:12)
1 (1:12)
0 (1:9)
11.08
19.85
9.08
11.81
21.81
11.39
21.75
11.66
26.01
14.73
14.47
22.18
22.18
22.18
ꢃ6.58
ꢃ9.87
ꢃ25.44
ꢃ1.87
11.97
4.20
1.85
14.48
5.41
21.35
13.25
27.16
15.01
16.93
23.45
21.34
21.74
0 (1:9)
ꢃ1.41 (1:5)
1.41 (1:13)
0 (1:6)
0 (1:6)
0 (1:6)
10
11
ꢃ3.93
ꢃ2.02
a
Ascorbic acid:palmitic acid.
RED = relative error deviation.
b
the reaction time fixed at 3 h, evaluating the effect of temperature,
enzyme concentration, volume of solvent, substrates molar ratio
and ultrasound power by means the Pareto chart on product yield.
From this table one can observe that the highest conversions were
obtained at 70° C and substrates molar ratio of 1:6.
Experimental data appearing in Table 2 were statistically trea-
ted and Fig. 2 presents the standard Pareto chart [22], which shows
the effects of reaction variables (substrates molar ratio, tempera-
ture, ultrasonic power, enzyme concentration, and solvent volume)
on ascorbyl palmitate conversion. An inspection of this figure
shows that the substrates molar ratio and temperature present a
positive significant effect (p < 0.05) on the ascorbyl palmitate yield,
making it possible to conclude that an increase in this variable
leads to higher conversions. The volume of solvent, enzyme con-
centration and ultrasound power did not affect the ascorbyl palmi-
tate conversion.
Taking into account the results obtained in the first experimen-
tal design, a central composite rotatable design (CCRD) 22 was car-
ried out, keeping constant the enzyme concentration (5 wt%),
volume of solvent (5 mL), ultrasound power (132 W) and reaction
time (3 h), varying the temperature and substrates molar ratio.
Table 3 presents the matrix of the experimental design with the
responses in terms of ascorbyl palmitate production. The highest
conversion (27.16%) was obtained in experiment four, correspond-
ing to a temperature of 80° C and substrates molar ratio of 1:9,
showing the positive effect of these variables on process
conversion.
Results obtained in the second experimental design were also
statistically treated and permitted the validation of an empirical
coded model for ascorbyl palmitate conversion in terms of sub-
strates molar ratio and temperature. The empirical model was val-
idated by variance analysis (ANOVA). The R-value and F-test for
regression showed that the model (Eq. (1)) was able to well repre-
sent the experimental data of ascorbyl palmitate conversion in the
range of variables evaluated, allowing the development of the re-
sponse surface presented in Fig. 3. This provides a satisfactory rep-
resentation of the process by the empirical model, as illustrated by
the predicted conversion (column five of Table 3) and the standard
deviation (RED) (column six of Table 3) [22]:
Fig. 3. Response surface (a) and contour curve (b) for ascorbyl palmitate conversion
in function of temperature and ascorbic acid to palmitic acid molar ratio in
ultrasound system.
This may be explained in that an excess of palmitic acid in organic
phase may induce conformational changes in the enzyme structure,
limiting the access of ascorbic acid to the active site of the enzyme
[20].
Lerin et al. [20] evaluated the same experimental system pre-
sented here, without the use of ultrasonic irradiation. The opti-
mum production was achieved at an ascorbic acid to palmitic
acid mole ratio of 1:9, a stirring rate of 150 rpm, a temperature
of 70° C, an enzyme concentration of 5 wt%, and 17 h of reaction,
resulting in an ascorbyl palmitate conversion of about 67%.
Although no work was found in the literature regarding the
production of ascorbyl palmitate under ultrasonic irradiation by a
systematic methodology of experimental design, several studies
exist concerning the use of ultrasound in the improvement of ester-
ification and hydrolysis reactions catalyzed by lipases [9,10,24–27].
Ascorbyl palmitate ¼ 22:18 þ 5:09 ꢄ T ꢃ 1:68 ꢄ T2 ꢃ 0:09
ꢄ RM ꢃ 3:81 ꢄ RM2 þ 0:12 ꢄ T ꢄ RM
ð3Þ
where T denotes system temperature (°C) and MR the substrates
molar ratio.
3.3. Kinetic study of enzymatic production of ascorbyl palmitate
An analysis of Fig. 3 permits us to verify the positive effect of
temperature and substrates molar ratio on reaction conversion
(maximum of 27.16%). However, increase in the substrate molar
ratio (>1:9) led to a reduction in the process conversion (Table 3).
The effects of ascorbic acid to palmitic acid molar ratio, temper-
ature and enzyme concentration were investigated on the kinetics
of ascorbyl palmitate production in the ultrasound-assisted
system.

L.A. Lerin et al. / Ultrasonics Sonochemistry 18 (2011) 988–996
993
Fig. 4. Kinetics of ascorbyl palmitate production at (a) ascorbic acid to palmitic acid molar ratio of 1:1 and 1:3, (b) ascorbic acid to palmitic acid molar ratio of 1:6 and 1:9. The
reaction was carried out at 70° C, 5 mL of solvent and ultrasound power of 132 W, except for when the kinetic study of the referred variable was performed.
3.4. Effect of acid to alcohol molar ratio
The substrate molar ratio is usually one of the most important
parameters in enzymatic esterification reactions. Since the reaction
is reversible, an enhancement in the concentration of one substrate
(particularly, the palmitic acid) can displace the chemical equilib-
rium to products formation, resulting in higher conversions. On
the other hand, high palmitic acid concentrations may reduce the
reaction rate due to the inhibition effect [15,28–30].
No data could be found in the literature regarding the effect
of substrate molar ratio on enzymatic esterification for ascorbyl
palmitate production under an ultrasound-assisted system. The
kinetic behavior obtained here can be directly compared to that of
Lerin et al. [20] for the same experimental system under mechan-
ical agitation. Similar results were observed by Burham et al. [28],
where higher conversions (about 70%) in palm-based ascorbyl
esters were obtained at a molar ratio of 1:8. However, for a molar
ratio value of palm oil higher than eight, reduction in production of
To evaluate the effect of ascorbic acid to palmitic acid molar
ratio on ascorbyl palmitate yield, the temperature was fixed at
70° C, the enzyme concentration was set at 5 wt%, 5 mL of solvent
were used, and the ultrasound power was set at 132 W, making it
possible to build the experimental curves of reaction conversion
versus reaction time, as presented in Fig. 4a and b. These figures
also present the empirical kinetic model fitting obtained using
the proposed hybrid neural network approach.
From this figure one can observe that similar conversions were
obtained at higher molar ratios. The molar ratio of 1:1 produced
lower conversions. These results are in agreement with those ob-
tained in the experimental design that pointed out the molar ratio
of 1:9 as being the most appropriate for this system after 3 h of
reaction.

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L.A. Lerin et al. / Ultrasonics Sonochemistry 18 (2011) 988–996
Fig. 5. Kinetics of ascorbyl palmitate production at (a) enzyme concentration of 1 and 5 wt%, (b) enzyme concentration of 10 and 20 wt%. The reaction was carried out at
70° C, ascorbic acid to palmitic acid mole ratio of 1:9, 5 mL of solvent and ultrasound power of 132 W.
palm based ascorbyl esters was observed. This system was also
investigated under mechanical agitation. Another result using the
same procedure of agitation is due to Chang et al. [15], who studied
the enzymatic esterification of ascorbyl laurate and observed that
an enhancement on enzyme concentration and substrate molar
ratio produced a conversion of about 80% (180 rpm, 45° C, 3 mL of
acetonitrile, 6 h). Lv et al. [29] obtained the maximum conversion
in ascorbyl benzoate (79.48%) at a molar ratio of 10:1, and higher
molar ratios were not tested due to the solubility limitations. These
3.5. Effect of enzyme concentration
The effect of enzyme concentration on ascorbyl palmitate yield
in an ultrasound-assisted system was evaluated at 70° C keeping
the ascorbic to palmitic acid molar ratio constant at 1:9, 5 mL of
solvent and ultrasound power of 132 W, varying the enzyme
concentration of 1, 5, 10, and 20 wt% (based on the substrates
amount). Fig. 5a and b shows the experimental data and kinetic
modeling results obtained in this step. When using these enzyme
concentrations it can be observed that similar initial reaction rates
were obtained, leading to high conversions in short reaction times.
The use of 5, 10, and 20 wt% of Novozym 435 does not present a
significant difference with regard to ascorbyl palmitate production.
On the other hand, at the experimental condition with 1wt% of
enzyme, lower conversions were achieved, reaching 16.10%
results indicate that an excess of L-ascorbic acid may have a stron-
ger promotion effect on esterification than an excess of benzoic
acid. Hsieh et al. [30] also evaluated the effect of the substrates
molar ratio for the synthesis of ascorbyl palmitate by surfactant-
coated lipase and observed higher conversions (47%) at mole ratios
of 1:6, 24 h and 50° C.

L.A. Lerin et al. / Ultrasonics Sonochemistry 18 (2011) 988–996
995
Fig. 6. Kinetics of ascorbyl palmitate production at temperatures of 50, 60, 70, and 80° C. The reaction was carried out at ascorbic acid to palmitic acid molar ratio of 1:9, 5 mL
of solvent and ultrasound power of 132 W.
conversion after 3 h. From Fig. 5 one can also observe that the high-
est conversion was obtained using 20 wt% of enzyme and 4 h of
reaction, resulting in a conversion of about 33%. Fig. 5b shows that
the use of 10 and 20 wt% of enzyme do not affect significantly the
reaction conversion. A possible explanation might be related to the
fact that an excess of enzyme in the reaction medium could not
contribute to the conversion enhancement, since high enzyme
concentration may form aggregates, thus not making the enzyme
active site available to the substrate. The enzyme molecules on
the external surface of such particles are exposed to high substrate
concentrations but the mass transport could drastically limit the
substrate concentration inside the particles. Lower activities of
the biocatalyst reduce the efficiency of the enzyme, not enhancing
the reaction conversion [31].
Babicz et al. [24] evaluated the effect of enzyme concentration
(1 and 2 wt%) on the hydrolysis of soybean oil in ultrasound system
(47 kHz and 125 W) and obtained conversions in diglycerides of
40% using 1 wt% of Lipozyme TL IM after 1.5 h of reaction. The
use of 2 wt% led to conversions of 32% in 3 h.
Liu et al. [14] studied the effect of enzyme concentration on the
hydrolysis of soybean oil at 0.1, 0.55, 1, 2, and 3 wt% of lipase from
C. lipolytica in a solvent-free system using ultrasound-assisted sys-
tem. Reaction rates showed a slight enhancement as a function of
enzyme concentration. The authors observe a reduction in reaction
rate with an increase in enzyme concentration. Based on the re-
sults obtained, the authors believe that there is a critical enzyme
concentration (0.55 wt%, in this case), above that the reaction
interface becomes saturated.
From this figure one can observe that at 80° C, after 3 h of reac-
tion, an ascorbyl palmitate yield of 35.06% was obtained. At 50, 60,
and 70° C similar behavior was verified. This result seems to be
coherent with those obtained in the experimental design step.
Liu et al. [14] evaluated the influence of temperature on hydro-
lysis of soybean oil catalyzed by a lipase from C. lipolytica in sol-
vent-free system using ultrasonic energy. The authors reported
that good hydrolytic activities were reached from 30 to 55° C; at
65° C the enzyme lost all its activity.
4. Conclusions
New experimental data on ultrasound-assisted enzymatic ester-
ification for ascorbyl palmitate production is reported in this work,
showing a perspective of the technique to overcome mass transfer
limitations arising from the use of ascorbic acid as substrate. The
use of tert-butanol without mechanical agitation in an ultrasound-
assisted system led to satisfactory conversions in ascorbyl palmi-
tate. The optimum production (27.15%) was achieved at ascorbic
acid to palmitic acid molar ratio of 1:9, ultrasound power of
132 W, 80° C, Novozym 435 concentration of 5 wt% at 3 h of reac-
tion. The reaction kinetics for ascorbyl palmitate production showed
that good reaction conversions (ꢀ26%) could be achieved in a short
reaction time (2 h). The empirical kinetic model proposed proved to
be capable of satisfactorily representing the experimental data.
Acknowledgements
The authors thank CNPq and CAPES for the financial support
and scholarships.
3.6. Effect of temperature
References
To evaluate the effect of temperature (50, 60, 70, and 80° C) on
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making it possible to follow the course of the reaction conversion,
as presented in Fig. 6. This figure also shows good agreement with
the results obtained using the proposed empirical kinetic model.
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