The combined use of ultrasound and molecular sieves improves the synthesis of ethyl butyrate catalyzed by immobilized Thermomyces lanuginosus lipase

Article abstract of DOI:10.1016/j.ultsonch.2014.05.004

In this work, the combined use of ultrasound energy and molecular sieves was investigated for the synthesis of ethyl butyrate, ester with mango and banana notes, catalyzed by the immobilized lipase from Thermomyces lanuginosus (Lipozyme TL-IM). Initially, the best concentrations of biocatalysts (35%) and butyric acid (0.7 M) were tested using ultrasound as an alternative to mechanical agitation. The amount of acid in the reaction could be increased by 2-fold when compared to previous works where mechanical agitation was used. In the next step, substrate molar ratio and reaction temperature were optimized and the best conditions were at their lowest levels: 1:1 (acid:alcohol), and 30 °C, reaching 61% of conversion in 6 h. Molecular sieves (3 A?) were added to optimized reaction medium in order to remove the formed water and improve the maximum yield. The reaction yield increased 1.5 times, reaching 90% of conversion in 6 h, when 60 mg of molecular sieves per mmol of butyric acid was used. Finally, the reuse of Lipozyme TL-IM for the ultrasound-assisted synthesis of ethyl butyrate was verified for 10 batches, without any appreciable loss of activity, whereas in systems using mechanical agitation, the biocatalyst was completely inactivated after 5 batches. These results suggest that the combined use of ultrasound and molecular sieves greatly improve esterification reactions by stabilizing the enzyme and increasing yields.

Full text of DOI:10.1016/j.ultsonch.2014.05.004

Ultrasonics Sonochemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Ultrasonics Sonochemistry
journal homepage: www.elsevier.com/locate/ultson
The combined use of ultrasound and molecular sieves improves
the synthesis of ethyl butyrate catalyzed by immobilized Thermomyces
lanuginosus lipase
Natalia Paludo a, Joana S. Alves a, Cintia Altmann a, Marco A.Z. Ayub a, Roberto Fernandez-Lafuente b,
Rafael C. Rodrigues a,
⇑
a Biotechnology, Bioprocess and Biocatalysis Group, Institute of Food Science and Technology, Federal University of Rio Grande do Sul, Av. Bento Gonçalves,
9500, P.O. Box 15090, ZC 91501-970 Porto Alegre, RS, Brazil
b Department of Biocatalysis, ICP – CSIC, Campus UAM-CSIC, Cantoblanco, ZC 28049 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
In this work, the combined use of ultrasound energy and molecular sieves was investigated for the syn-
thesis of ethyl butyrate, ester with mango and banana notes, catalyzed by the immobilized lipase from
Thermomyces lanuginosus (Lipozyme TL-IM). Initially, the best concentrations of biocatalysts (35%) and
butyric acid (0.7 M) were tested using ultrasound as an alternative to mechanical agitation. The amount
of acid in the reaction could be increased by 2-fold when compared to previous works where mechanical
agitation was used. In the next step, substrate molar ratio and reaction temperature were optimized and
the best conditions were at their lowest levels: 1:1 (acid:alcohol), and 30 °C, reaching 61% of conversion
in 6 h. Molecular sieves (3 Å) were added to optimized reaction medium in order to remove the formed
water and improve the maximum yield. The reaction yield increased 1.5 times, reaching 90% of conver-
sion in 6 h, when 60 mg of molecular sieves per mmol of butyric acid was used. Finally, the reuse of Lipo-
zyme TL-IM for the ultrasound-assisted synthesis of ethyl butyrate was verified for 10 batches, without
any appreciable loss of activity, whereas in systems using mechanical agitation, the biocatalyst was com-
pletely inactivated after 5 batches. These results suggest that the combined use of ultrasound and molec-
ular sieves greatly improve esterification reactions by stabilizing the enzyme and increasing yields.
Ó 2014 Elsevier B.V. All rights reserved.
Received 4 December 2013
Received in revised form 7 April 2014
Accepted 6 May 2014
Available online xxxx
Keywords:
Esterification reactions
Ultrasound
Molecular sieves
Lipases
Lipozyme TL-IM
Operational stability
1. Introduction
Lipases are among the most industrially important enzymes;
they are versatile biocatalysts, whose natural function is the
The production of short chain esters is an apparently simple,
but very interesting reaction of a direct condensation of a short
chain alcohol and a short chain carboxylic acid [1], being the
resulting ester a volatile compound with flavor and fragrance prop-
erties [2]. These esters are industrially important for food, pharma-
ceutical and cosmetic products [2]. In many countries, the
technical definition of natural flavors states that they must be pre-
pared by either physical processes (extraction from natural
sources), or via biotechnology (enzymatic or microbial processes)
[3], therefore stimulating the research in the field of enzymatic
production of these compounds. However, in order to turn enzy-
matic processes competitive, high enzyme stability, activity, and
high reaction yields are required.
hydrolysis of oils and fats [4]. However, under suitable conditions,
lipases are also capable of catalyzing esterification, transesterifica-
tion, and interesterification reactions [5]. Lipases have been used in
low water activity systems (organic solvents, ionic liquids, super-
critical fluids), reaction conditions that are meant to reverse the
equilibrium in the direction of the synthesis [6–8], which is impor-
tant to increase productivity. Additionally, water plays an
important role in biocatalytic reactions: catalytic activity usually
increases with increasing water activity (aw), due to improve
enzyme mobility [9]. However, the raise in the water content also
favors hydrolysis over synthesis. Thus, to maintain the yield, very
low aw values must be attained. A common practical approach
has been the use molecular sieves [10,11].
Most of the studies on lipase-catalyzed esterification have been
performed using standard mechanical stirring conditions [11–14].
However, ultrasound has emerged as an alternative source
of homogenizing energy with potential applications in enzymatic
reaction control. Ultrasound energy acts by increasing the
⇑
Corresponding author. Tel.: +55 51 3308 7793; fax: +55 51 3308 7048.
E-mail address: rafaelcrodrigues@ufrgs.br (R.C. Rodrigues).
URL: http://www.ufrgs.br/bbb.
http://dx.doi.org/10.1016/j.ultsonch.2014.05.004
1350-4177/Ó 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: N. Paludo et al., The combined use of ultrasound and molecular sieves improves the synthesis of ethyl butyrate catalyzed
by immobilized Thermomyces lanuginosus lipase, Ultrason. Sonochem. (2014), http://dx.doi.org/10.1016/j.ultsonch.2014.05.004

2
N. Paludo et al. / Ultrasonics Sonochemistry xxx (2014) xxx–xxx
interaction between phases in a system by cavitation caused by the
collapse of bubbles, whereas the ultrasonic jet disrupts the bound-
ary phase and causes emulsification [15–17]. When applied in
aqueous solutions or suspensions, ultrasound increases mixing,
shearing, and mass transfer rate of the system, reducing process
time when compared with other conventional mixing techniques
[18,19]. In biotechnological processes, ultrasound has been applied
for some enzymatic reactions, wastewater treatment and biofuels
production, with very good results [18,20–22]. While some reports
have shown the positive influence of ultrasound on the kinetic
parameters of esterification reactions catalyzed by enzymes [23–
25], its application in this field remains scarcely explored, and
few reports for lipase-catalyzed flavor esters syntheses are found
in the literature. In a previous work of our group, the use of ultra-
sounds for the esterification of acetic acid and butanol catalyzed by
immobilized lipase B from Candida antarctica (Novozym 435),
allowed us to use higher acetic acid concentrations when com-
pared to systems where mechanical agitation was employed,
increasing the overall reaction productivity by 7.5-fold [26].
In the present work, we investigate the influence of ultrasound
energy on the esterification reaction between butyric acid and eth-
anol using hexane as solvent, catalyzed by the commercial immo-
bilized preparation of the lipase from Thermomyces lanuginosus,
using as support an ionic-exchange resin (Lipozyme TL-IM). Lipase
from T. lanuginosus has been chosen because it is among the most
used lipases, showing high stability [27]. It was also performed the
optimization of the reaction as well as the analysis of the effect of
the use of molecular sieves on the reaction performance and the
possibilities of the reuse of the biocatalyst.
the optimal conditions for esterification reaction. The variables
and their coded and uncoded values are presented in Table 1,
showing the 11 treatments of the two variables, each at five levels.
The design was constructed of 4 factorial points, 4 axial points (two
axial points on the axis of design variable), and 3 replications at the
central point. The mixtures of butyric acid (0.7 M), ethanol, and
Lipozyme TL-IM were placed in the ultrasonic bath for 5 h. The bio-
catalyst content and temperature were varied according to the
CCD. In each case, the esterification percentage of conversion (or
yield) was determined. The second-order polynomial equation for
the variables was as follows:
X
X
X
Y ¼ b0 þ
biXi þ
bijXiXj þ
biiX2
ð1Þ
i
where Y is the response variable, b0 the constant, bi, bii, bij were the
coefficients for the linear, quadratic, and for the interaction effects,
respectively, and Xi and Xj the coded level of variables xi and xj. The
above quadratic equation was used to plot surfaces for all variables.
2.5. Effect of molecular sieves
The effect of molecular sieves in the reaction was evaluated
varying the concentration from 0 to 120 mg of molecular sieves
per mmol of butyric acid. The mixtures of butyric acid and ethanol
(both at 0.7 M), and Lipozyme TL-IM (35%, by substrates mass)
were placed in the ultrasonic bath at 30 °C for 6 h.
2.6. Enzyme reuse
In order to test the possibility of reusing the biocatalyst several
times, which is important for the economics of enzymatic reac-
tions, repeated reaction batches were devised using the same prep-
aration. After one esterification reaction, the immobilized enzyme
was separated from the reaction medium by vacuum filtration
using a sintered glass funnel and reused in a new fresh reaction
without any further treatment.
2. Material and methods
2.1. Materials
Commercial immobilized lipase from T. lanuginosus (Lipozyme
TL-IM) was used in this work and it was kindly supplied by Novo-
zymes (Spain). Molecular sieves (3 Å, beads 4–8 mesh), butyric
acid, ethanol, and other chemicals were of analytical grade and
purchased from Sigma–Aldrich (Sigma, St. Louis, USA).
2.7. Statistical analysis
Experiments were performed in triplicates and mean with the
standard errors were plotted in the figures. The experimental
design and analysis of results were carried out using Statistica
7.0 (Statsoft, USA). The statistical analysis of the model was per-
formed as analysis of variance (ANOVA). The significance of the
regression coefficients and the associated probabilities, p(t), were
determined by Student’s t-test; the second order model equation
significance was determined by Fisher’s F-test. The variance
explained by model is given by the multiple determination coeffi-
cients, R2. For each variable, the quadratic models were repre-
sented as contour plots.
2.2. Ultrasound-assisted esterification
The esterification reactions were carried out in an ultrasonic
bath (Unique Inc., model USC 2880A, 40 kHz, 220 W, Brazil). The
mixture of butyric acid, ethanol and enzymes were placed in the
ultrasonic bath at the desired temperature for different times.
The progress of esterification was monitored by determining the
residual acid content of the reaction by titration of 0.5 mL samples
with NaOH (0.01 M) using phenolphthalein as indicator and 5 mL
of ethanol as quenching agent. The amount of ester produced
was calculated as being equivalent to the amount of consumed
acid.
Table 1
2.3. Effect of substrates concentrations
Coded levels, real values (in the parenthesis) and results of CCD.
In order to evaluate the effect of substrates concentrations on
the reaction, butyric acid and ethanol concentrations (maintaining
a molecular ratio of 1) were varied from 0.1 to 1.0 M, measuring
the initial reaction rate. The mixtures of butyric acid, ethanol,
and Lipozyme TL-IM (35%, by substrates mass) were placed in
the ultrasonic bath at 40 °C.
Run
Temperature (°C)
Substrate molar ratio (alcohol:acid)
Yield (%)
1
2
3
4
5
6
7
8
9
ꢀ1 (34.5)
ꢀ1 (34.5)
1 (55.5)
1 (55.5)
ꢀ1.41 (30)
1.41 (60)
0 (45)
0 (45)
0 (45)
0 (45)
0 (45)
ꢀ1 (1.3:1)
1 (2.7:1)
ꢀ1 (1.3:1)
1 (2.7:1)
0 (2:1)
53.7
34.9
18.7
13.2
62.5
15.1
34.5
36.7
25.3
21.7
24.6
0 (2:1)
ꢀ1.41 (1:1)
1.41 (3:1)
0 (2:1)
0 (2:1)
0 (2:1)
2.4. Experimental design
10
11
A central composite design (CCD) with two variables, tempera-
ture and substrate molar ratio, was carried out in order to obtain
Please cite this article in press as: N. Paludo et al., The combined use of ultrasound and molecular sieves improves the synthesis of ethyl butyrate catalyzed
by immobilized Thermomyces lanuginosus lipase, Ultrason. Sonochem. (2014), http://dx.doi.org/10.1016/j.ultsonch.2014.05.004

N. Paludo et al. / Ultrasonics Sonochemistry xxx (2014) xxx–xxx
3
3. Results and discussion
3.1. Effect of biocatalyst content
Using standard mechanical agitation, the amount of 40% (by
substrate mass) of biocatalyst was found as the optimal using Lipo-
zyme TL-IM in the synthesis of butyl butyrate [28]. Lipozyme
TL-IM presents lower activity in esterification reactions when com-
pared to other immobilized lipases (Lipozyme RM-IM and Nov-
ozym 435) [13,29], requiring a higher amount of biocatalyst to
provide similar activities. However, Lipozyme TL-IM is cheaper
than the other two immobilized lipases, justifying its use. In this
research, it was studied the effect of the amount of biocatalyst
on the initial reaction rate of the ultrasound-assisted ethyl buty-
rate synthesis catalyzed by Lipozyme TL-IM. The concentration of
biocatalyst varied from 5% to 40%, and the results are presented
in Fig. 1. There is a linear increase in the initial reaction rate up
to 30%, slowing down in the range of 30–40%, reducing the global
productivity of the reaction. Therefore, it was selected the amount
of 35% (by substrate mass) of biocatalyst for the next experiments.
This concentration was similar to those used in other works using
Lipozyme TL-IM for flavor ester synthesis [30]; although the reac-
tion rate reported by the authors was 50% lower than the value
obtained in this research.
Fig. 2. Effect substrate concentration on the initial rate of reaction of ultrasound-
assisted ethyl butyrate synthesis. Reaction conditions were: 1:1 alcohol:acid; 40 °C;
biocatalyst content, 35%.
3.2. Effect of substrates concentrations
explained by the distribution of substrates and products inside
the porous matrix of the support, which would avoid the formation
of substrates/products gradients on the support and enzyme sur-
face that could decrease the enzyme activity [17,33]. Based in these
results, it was selected the substrate concentration of 0.7 M for the
following experiments.
For the application of a biocatalyst in an industrial process, it is
important to use the highest possible substrate concentration to
maximize the productivity. However, both acid and alcohol might
produce some negative effects on the enzyme activity and stability
[28,31,32]. Therefore, it was performed a study varying the sub-
strates concentrations from 0.1 to 1.0 M. The results, in terms of
initial reaction rate, are shown in Fig. 2. The reaction rate increased
linearly up to 0.7 M and remained constant up to 1.0 M. In a previ-
ous work of our group, the reaction of butyl butyrate synthesis
using Lipozyme TL-IM under conventional mechanical agitation,
produced the highest reaction rate with 0.3 M butyric acid [28].
The positive effect of ultrasound on the reaction might be
3.3. Experimental design
A central composite design testing temperature and the sub-
strate molar ratio was used to optimize the ultrasound-assisted
ethyl butyrate synthesis catalyzed by Lipozyme TL-IM, with results
presented in Table 1. The highest yield was obtained with the low-
est reaction temperature (run 5).
Statistical testing of the model was done by the Fisher’s statis-
tical test for ANOVA. The computed F value (10.24) was significant
(p = 0.0116). The determination coefficient (R2 = 0.9110) implies
that the sample variation of 91.10% for ethyl butyrate synthesis
is attributed to the independent variables and can be explained
by the model; the correlation coefficient (R = 0.9544) suggests a
highly satisfactory representation of the process model and a good
correlation between the experimental results and the theoretical
values predicted by the model equation. The experimental data
have been adjusted to the proposed model by the second-order
polynomial Eq. (1), and the second-order polynomial model is pre-
sented in Eq. (2):
Y ¼ 23:88 ꢀ 15:47X1 þ 5:71X2 þ 4:10X2
ð2Þ
1
2
where Y is the percentage yield, and X1 and X2, are the coded values
of reaction temperature and substrate molar ratio, respectively.
3.4. Effect of parameters and optimal conditions
The two variables presented negative linear effects on the ultra-
sound-assisted ethyl butyrate synthesis catalyzed by Lipozyme TL-
IM, ꢀ30.94 and ꢀ5.31 for reaction temperature and substrate
molar ratio, respectively. Only reaction temperature presented a
linear effect statistically significant at a confidence level of 95%.
The relationship between the variables and the response can be
Fig. 1. Effects of biocatalyst content on the initial rate of the reaction of ultrasound-
assisted ethyl butyrate synthesis. Reaction conditions were: 1:1 alcohol:acid; 40 °C;
acid concentration 0.1 M.
Please cite this article in press as: N. Paludo et al., The combined use of ultrasound and molecular sieves improves the synthesis of ethyl butyrate catalyzed
by immobilized Thermomyces lanuginosus lipase, Ultrason. Sonochem. (2014), http://dx.doi.org/10.1016/j.ultsonch.2014.05.004

4
N. Paludo et al. / Ultrasonics Sonochemistry xxx (2014) xxx–xxx
better observed in the contour plot of Fig. 3. The optimal conditions
for the synthesis of ethyl butyrate catalyzed for Lipozyme TL-IM
under ultrasonic energy were: temperature, 30 °C; substrate molar
ratio, 1:1 (alcohol:acid); and biocatalyst content, 35% (by substrate
mass). For model validation, an experiment was performed at the
optimal conditions and the results for the reaction time course
are presented in Fig. 4 (filled squares). A yield of 61% after 6 h of
reaction was obtained, close to the predicted model value of
65.2%, showing a good correlation between experimental and the-
oretical results. It is interesting to point out that for the synthesis
of butyl butyrate (i.e., using butanol instead of ethanol) catalyzed
by the same lipase, the optimal temperature and substrate molar
ratio were quite different using conventional mechanical agitation
(48 °C and 3:1 alcohol:acid) [28]. While in ultrasound-assisted
reactions an equimolecular concentration of both substrates could
be used, improving the global productivity of the process and sim-
plifying the purification steps, under mechanical agitation higher
amounts of alcohol, biocatalyst and temperature were needed to
reach to similar reaction rates.
3.5. Effect of molecular sieves on the esterification reaction
Fig. 4. Effect of molecular sieves (mg mmolꢀ1 of butyric acid) in the ultrasound-
assisted ethyl butyrate synthesis. (j) 0; (s) 30; (4) 60; (Ç) 90; (h) 120. Reaction
conditions: Butyric acid, 0.7 M; Substrate molar ratio, 1:1 (alcohol:acid); biocatalyst
content, 35%; 30 °C.
Water is a product in lipase-mediated esterification reactions
and its accumulation can shift the equilibrium towards ester
hydrolysis. If a water phase is formed in the environment of the
enzyme, this should have a very low pH value due to the concen-
tration of acid and have some deleterious effects on enzyme activ-
ity/stability. Both reasons make convenient the removal of the
formed water. To this purpose, we tested the addition of molecular
sieves (3 Å) to the reaction mixture varying its amount from 0 to
120 mg per mmol of butyric acid, and the obtained reaction
courses are shown in Fig. 4. For the initial stages of the reactions
there are no significant effects caused by the molecular sieves, pre-
sumably because most of water needs to move out from inside of
the support. As the reaction progress, the water content of the
reaction increases and the effect of the addition of molecular sieves
is clearly observed. After 6 h of reaction, the final yield is around of
60% for the control without molecular sieves compared to 90%
when they were present. There were no appreciable differences
among the tested amounts of molecular sieves; thus, in further
experiments, an amount of 60 mg/mmol butyric acid was used.
3.6. Operational stability of Lipozyme TL-IM over repeated batches
under optimal conditions
As stated before, for industrial applications of enzymes, charac-
teristics of high operational activity and good stability are neces-
sary. We tested these characteristics by submitting Lipozyme
TL-IM to several batches of synthesis reactions under ultrasonic
energy and in the presence of molecular sieves (condition a), and
the results were compared to the synthesis under standard condi-
tions (mechanical agitation and absence of sieves, condition b)
(Fig. 5). While it was possible to reuse the biocatalyst for at least
10 batches keeping 100% of its initial activity under condition a,
the biocatalyst was rapidly losing activity and it was fully inacti-
vated after 4 batches under condition b. The combined use of ultra-
sound and molecular sieves allowed to improve the overall
productivity of the process, because it was possible to use higher
concentrations of substrates and, more important, to reuse the
enzyme for more batches than under conventional mechanical agi-
tation. Both strategies are very important and have shown syner-
gistic effects: while ultrasound promotes a better mixing of
substrates and reduces layers formation or substrates/products
concentration gradients inside the porous matrix of the support
[17,25], the molecular sieves remove the produced water improv-
ing the yield of reaction, since eliminating water from the reaction
medium, the hydrolysis reaction is avoided [34].
3.7. Discussion
The use of ultrasound in enzymatic reactions, especially using
lipases, is very recent and scarce information has been reported
about the effects of ultrasound in these reactions. Thus, in this dis-
cussion section, we tried to analyze and better understand the
mechanisms involved in the ultrasound-assisted esterification.
The main mechanism involved using ultrasound is the cavita-
tion. Cavitation is defined as the generation, consequent growth
Fig. 3. Contour plots of yields (%) for ultrasound-assisted ethyl butyrate synthesis
as a function of substrate molar ratio (alcohol:acid) and temperature (°C). Reaction
conditions were: Butyric acid, 0.7 M; biocatalyst content, 35%.
Please cite this article in press as: N. Paludo et al., The combined use of ultrasound and molecular sieves improves the synthesis of ethyl butyrate catalyzed
by immobilized Thermomyces lanuginosus lipase, Ultrason. Sonochem. (2014), http://dx.doi.org/10.1016/j.ultsonch.2014.05.004

N. Paludo et al. / Ultrasonics Sonochemistry xxx (2014) xxx–xxx
5
and thus the highest interfacial area is likely to form for reaction
mixture with the lowest amount of alcohol [43]. This can explain
that in our work, the optimal substrate molar ratio is 1:1 alco-
hol:acid, which is the stoichiometric relation and the lowest
amount of alcohol possible to have good yields conversions of
the acids.
Furthermore, using a heterogeneous catalyst, Choudhury et al.
[39] observed, also for the transesterification reaction, that
although the methanol vapor undergoes thermal dissociation at
collapse resulting in numerous species, the temperature peak at
collapse is too small to result in generation of radical species. Thus,
the cavitation bubbles were not capable of inducing any sono-
chemical effect [39], and probably in our case, due to the similarity
between the reactions, the effects produced by cavitation are only
physical, i.e. micro-convection and shock waves, adding to the
physical effect induced by ultrasound irradiation, i.e. micro-
streaming.
4. Conclusions
The performance of Lipozyme TL-IM for esterification reactions
was greatly improved by the combined use of molecular sieves and
ultrasound technology in substitution of the conventional mechan-
ical agitation. It was possible to increase the concentrations of sub-
strates, as well as to increase the enzyme activity. The optimal
conditions of temperature and substrate molar ratio were deter-
mined for the ethyl butyrate synthesis and the overall reaction
yield could be improved by 50% when all studied variables were
optimized. These conditions allowed the reuse of the enzyme for
10 batches without any loss of activity, greatly improving the per-
formance of the biocatalyst during ester synthesis. The combined
use of ultrasound technology and molecular sieves seems to be a
promising alternative for the synthesis of flavor esters catalyzed
by lipases.
Fig. 5. Operational stability of Lipozyme TL-IM in the ethyl butyrate synthesis. (j)
ultrasound-assisted; (s) mechanical agitation. Reaction conditions: Butyric acid,
0.7 M; Substrate molar ratio, 1:1 (alcohol:acid); biocatalyst content, 35%; 30 °C;
molecular sieves, 60 mg mmolꢀ1 of butyric acid.
and collapse of cavities, resulting in very high local energy densi-
ties [35]. The acoustic cavitation is the phenomenon occurred
when the ultrasounds pass through a liquid medium promoting
the interaction between the ultrasound, dissolved gas and liquid.
Conditions of very high temperatures and pressures are locally
generated during milliseconds by cavitation, while the overall
environment remains equivalent to ambient conditions [36]. More-
over, cavitation results in the formation of an intense turbulence
and the development of vibrations in the liquid system, which
leads to a significant increase in the mass-transfer rates [37].
Additionally, both ultrasound and cavitation induce physical
and chemical effects in the reaction medium. The physical effects
promoted by the ultrasound are related to the intense mixing in
the medium, mainly due to the oscillatory motion of fluid, known
as micro-streaming [38]. The chemical effect is promoted by the
cavitation, and are related to the generation of radicals; due to
extreme conditions reached in the bubble, the solvent as well as
gas molecules undergo dissociation to form numerous chemical
species, which also react among themselves [39]. Also, the radial
motion of cavitation bubbles induces physical effects by micro-
convection and shock wave [40]. Moreover, ultrasounds influence
enzymatic reactions in other ways, including probable effects on
the formation and dissociation of enzymeꢀsubstrate complex.
These effects lead to an increase in enzyme activity, as well as an
increase in the overall rate of the enzymatic reactions [41].
Considering the esterification reaction, none information was
found about the physical and chemical effects of ultrasound and
cavitation. Nevertheless, it is possible to draw a parallel with the
transesterification reaction. This is the reaction between an alcohol
and a triglyceride (i.e., an oil), while the esterification is between
and alcohol and a carboxylic acid. Although the esterification is a
more homogeneous system than transesterification, some interfa-
cial area is formed and is necessary for lipase activity, since this
enzyme acts on interfaces [42]. According to Parkar et al. [43], in
the transesterification reaction under sonication, the interfacial
area is determined by the intensity generated by cavitation. The
authors demonstrated that for acid-catalyzed transesterification
between oil and methanol, the intensity of convection generated
by cavitation bubbles in oil is much weaker than that in methanol,
Acknowledgments
This work was supported by grants and scholarships (first and
second authors) from FAPERGS (Fundação de Amparo a Pesquisa
do Rio Grande do Sul – ARD/2011), CNPq (Brazilian Bureau of
Science and Technology), and CTQ2009-07568 (Ministerio de
Ciencia y Innovación, Spain). The authors wish to thank Mr. Ramiro
Martínez (Novozymes, Spain) for kindly supplying the enzymes
used in this research.
References
[1] R. Lortie, Enzyme catalyzed esterification, Biotechnol. Adv. 15 (1997) 1–15.
[2] K.P. Dhake, D.D. Thakare, B.M. Bhanage, Lipase: a potential biocatalyst for the
synthesis of valuable flavour and fragrance ester compounds, Flavour
Fragrance J. 28 (2013) 71–83.
[3] R.J. Marriott, Greener chemistry preparation of traditional flavour extracts and
molecules, Agro Food Ind. Hi-Tech 21 (2010) 46–48.
[4] S. Nagarajan, New tools for exploring old friends-microbial lipases, Appl.
Biochem. Biotechnol. 168 (2012) 1163–1196.
[5] A. Rajendran, A. Palanisamy, V. Thangavelu, Lipase catalyzed ester synthesis for
food processing industries, Braz. Arch. Biol. Technol. 52 (2009) 207–219.
[6] T. Kobayashi, Lipase-catalyzed syntheses of sugar esters in non-aqueous
media, Biotechnol. Lett. 33 (2011) 1911–1919.
[7] Z. Yang, Z.L. Huang, Enzymatic synthesis of sugar fatty acid esters in ionic
liquids, Cat. Sci. Technol. 2 (2012) 1767–1775.
[8] M. Habulin, S. Šabeder, Z. Knez, Enzymatic synthesis of sugar fatty acid esters
in organic solvent and in supercritical carbon dioxide and their antimicrobial
activity, J. Supercrit. Fluids 45 (2008) 338–345.
[9] N. Fontes, J. Partridge, P.J. Halling, S. Barreiros, Zeolite molecular sieves have
dramatic acid–base effects on enzymes in nonaqueous media, Biotechnol.
Bioeng. 77 (2002) 296–305.
[10] H. Yang, Y. Mu, H. Chen, Z. Xiu, T. Yang, Enzymatic synthesis of feruloylated
lysophospholipid in a selected organic solvent medium, Food Chem. 141
(2013) 3317–3322.
Please cite this article in press as: N. Paludo et al., The combined use of ultrasound and molecular sieves improves the synthesis of ethyl butyrate catalyzed
by immobilized Thermomyces lanuginosus lipase, Ultrason. Sonochem. (2014), http://dx.doi.org/10.1016/j.ultsonch.2014.05.004

6
N. Paludo et al. / Ultrasonics Sonochemistry xxx (2014) xxx–xxx
[11] P. Lozano, J.M. Bernal, A. Navarro, A clean enzymatic process for producing
flavour esters by direct esterification in switchable ionic liquid/solid phases,
Green Chem. 14 (2012) 3026–3033.
[12] H.S.G. Tan, B. Yu, P. Curran, S.Q. Liu, Lipase-catalysed synthesis of natural
aroma-active 2-phenylethyl esters in coconut cream, Food Chem. 124 (2010)
80–84.
[13] A.B. Martins, N.G. Graebin, A.S.G. Lorenzoni, R. Fernandez-Lafuente, M.A.Z.
Ayub, R.C. Rodrigues, Rapid and high yields of synthesis of butyl acetate
catalyzed by Novozym 435: reaction optimization by response surface
methodology, Process Biochem. 46 (2011) 2311–2316.
[28] A.B. Martins, J.L.R. Friedrich, R.C. Rodrigues, C. Garcia-Galan, R. Fernandez-
Lafuente, M.A.Z. Ayub, Optimized butyl butyrate synthesis catalyzed by
Thermomyces lanuginosus lipase, Biotechnol. Prog. 29 (2013) 1416–1421.
[29] A.S.G. Lorenzoni, N.G. Graebin, A.B. Martins, R. Fernandez-Lafuente, M.A.
Záchia, M.A. Záchia Ayub, R.C. Rodrigues, Optimization of pineapple flavour
synthesis by esterification catalysed by immobilized lipase from Rhizomucor
miehei, Flavour Fragrance J. 27 (2012) 196–200.
[30] A. Anschau, V.C. Aragão, B.D.A. Porciuncula, S.J. Kalil, C.A.V. Burkert, J.F.M.
Burkert, Enzymatic synthesis optimization of isoamyl butyrate, J. Braz. Chem.
Soc. 22 (2011) 2148–2156.
[14] C. Shu, J. Cai, L. Huang, X. Zhu, Z. Xu, Biocatalytic production of ethyl butyrate
from butyric acid with immobilized Candida rugosa lipase on cotton cloth, J.
Mol. Catal. B Enzym. 72 (2011) 139–144.
[15] D. Yu, L. Tian, H. Wu, S. Wang, Y. Wang, D. Ma, X. Fang, Ultrasonic irradiation
with vibration for biodiesel production from soybean oil by Novozym 435,
Process Biochem. 45 (2010) 519–525.
[16] R. Konwarh, S. Pramanik, D. Kalita, C.L. Mahanta, N. Karak, Ultrasonication – a
complementary ‘green chemistry’ tool to biocatalysis: a laboratory-scale study
of lycopene extraction, Ultrason. Sonochem. 19 (2012) 292–299.
[17] H. Fazlena, S. Norsuraya, S.N. Nadiah, Ultrasonic assisted enzymatic reaction:
An overview on ultrasonic mechanism and stability-activity of the enzyme, in:
2013 IEEE Business Engineering and Industrial Applications Colloquium,
Langkawi, 2013, pp. 85–90.
[18] B. Kwiatkowska, J. Bennett, J. Akunna, G.M. Walker, D.H. Bremner, Stimulation
of bioprocesses by ultrasound, Biotechnol. Adv. 29 (2011) 768–780.
[19] M.M. Zheng, L. Wang, F.H. Huang, L. Dong, P.M. Guo, Q.C. Deng, W.L. Li, C.
Zheng, Ultrasonic pretreatment for lipase-catalyed synthesis of phytosterol
esters with different acyl donors, Ultrason. Sonochem. 19 (2012) 1015–1020.
[20] J.V. Sinisterra, Application of ultrasound to biotechnology: an overview,
Ultrasonics 30 (1992) 180–185.
[31] N.G. Graebin, A.B. Martins, A.S.G. Lorenzoni, C. Garcia-Galan, R. Fernandez-
Lafuente, M.A.Z. Ayub, R.C. Rodrigues, Immobilization of lipase B from Candida
antarctica on porous styrene–divinylbenzene beads improves butyl acetate
synthesis, Biotechnol. Prog. 28 (2012) 406–412.
[32] A.B. Martins, J.L.R. Friedrich, J.C. Cavalheiro, C. Garcia-Galan, O. Barbosa, M.A.Z.
Ayub, R. Fernandez-Lafuente, R.C. Rodrigues, Improved production of butyl
butyrate with lipase from Thermomyces lanuginosus immobilized on styrene-
divinylbenzene beads, Bioresour. Technol. 134 (2013) 417–422.
[33] R.C. Rodrigues, C. Ortiz, A. Berenguer-Murcia, R. Torres, R. Fernandez-Lafuente,
Modifying enzyme activity and selectivity by immobilization, Chem. Soc. Rev.
42 (2013) 6290–6307.
[34] H.A. Choudhury, P. Srivastava, V.S. Moholkar, Single-step ultrasonic synthesis
of biodiesel from crude Jatropha curcas oil, AIChE J. (2014), http://dx.doi.org/
10.1002/aic.14371.
[35] K.S. Suslick, W.B. McNamara III, Y. Didenko, Hot spot conditions during multi-
bubble cavitation, in: L. Crum, T. Mason, J. Reisse, K. Suslick (Eds.),
Sonochemistry and Sonoluminescence, Springer, Netherlands, 1999, pp. 191–
204.
[36] T.T. More, M.M. Ghangrekar, Improving performance of microbial fuel cell with
ultrasonication pre-treatment of mixed anaerobic inoculum sludge, Bioresour.
Technol. 101 (2010) 562–567.
[37] P.B. Subhedar, P.R. Gogate, Intensification of enzymatic hydrolysis of
lignocellulose using ultrasound for efficient bioethanol production: a review,
Ind. Eng. Chem. Res. 52 (2013) 11816–11828.
[21] L. Batistella, L.A. Lerin, P. Brugnerotto, A.J. Danielli, C.M. Trentin, A. Popiolski, H.
Treichel, J.V. Oliveira, D. De Oliveira, Ultrasound-assisted lipase-catalyzed
transesterification of soybean oil in organic solvent system, Ultrason.
Sonochem. 19 (2012) 452–458.
[22] E.V. Rokhina, P. Lens, J. Virkutyte, Low-frequency ultrasound in biotechnology:
state of the art, Trends Biotechnol. 27 (2009) 298–306.
[23] V.G. Deshmane, P.R. Gogate, A.B. Pandit, Process intensification of synthesis
process for medium chain glycerides using cavitation, Chem. Eng. J. 145 (2008)
351–354.
[24] V.G. Deshmane, P.R. Gogate, A.B. Pandit, Ultrasound assisted synthesis of
isopropyl esters from palm fatty acid distillate, Ultrason. Sonochem. 16 (2009)
345–350.
[25] M.M. Zheng, L. Wang, F.H. Huang, P.M. Guo, F. Wei, Q.C. Deng, C. Zheng, C.Y.
Wan, Ultrasound irradiation promoted lipase-catalyzed synthesis of flavonoid
esters with unsaturated fatty acids, J. Mol. Catal. B Enzym. 95 (2013) 82–88.
[26] A.B. Martins, M.F. Schein, J.L.R. Friedrich, R. Fernandez-Lafuente, M.A.Z. Ayub,
R.C. Rodrigues, Ultrasound-assisted butyl acetate synthesis catalyzed by
Novozym 435: enhanced activity and operational stability, Ultrason.
Sonochem. 20 (2013) 1155–1160.
[38] R.S. Malani, S. Khanna, V.S. Moholkar, Sonoenzymatic decolourization of an azo
dye employing immobilized horse radish peroxidase (HRP): A mechanistic
study, J. Hazard. Mater. 256–257 (2013) 90–97.
[39] H.A. Choudhury, S. Chakma, V.S. Moholkar, Mechanistic insight into
sonochemical biodiesel synthesis using heterogeneous base catalyst,
Ultrason. Sonochem. 21 (2014) 169–181.
[40] R. Patidar, S. Khanna, V.S. Moholkar, Physical features of ultrasound assisted
enzymatic degradation of recalcitrant organic pollutants, Ultrason. Sonochem.
19 (2012) 104–118.
[41] S. Jian, T. Wenyi, C. Wuyong, Ultrasound-accelerated enzymatic hydrolysis of
solid leather waste, J. Cleaner Prod. 16 (2008) 591–597.
[42] R. Verger, ‘Interfacial activation’ of lipases: facts and artifacts, Trends
Biotechnol. 15 (1997) 32–38.
[43] P.A. Parkar, H.A. Choudhary, V.S. Moholkar, Mechanistic and kinetic
investigations in ultrasound assisted acid catalyzed biodiesel synthesis,
Chem. Eng. J. 187 (2012) 248–260.
[27] R. Fernandez-Lafuente, Lipase from Thermomyces lanuginosus: uses and
prospects as an industrial biocatalyst, J. Mol. Catal. B Enzym. 62 (2010) 197–
212.
Please cite this article in press as: N. Paludo et al., The combined use of ultrasound and molecular sieves improves the synthesis of ethyl butyrate catalyzed
by immobilized Thermomyces lanuginosus lipase, Ultrason. Sonochem. (2014), http://dx.doi.org/10.1016/j.ultsonch.2014.05.004