Ultrasound technology and molecular sieves improve the thermodynamically controlled esterification of butyric acid mediated by immobilized lipase from Rhizomucor miehei

Article abstract of DOI:10.1039/c3ra47315e

In this research, the effects of ultrasound stirring and the addition of molecular sieves on esterification reactions between butyric acid and several alcohols catalyzed by immobilized lipase from Rhizomucor miehei (Lipozyme RM-IM) were studied. Among the tested alcohols, 1-propanol and isobutanol allowed the highest activities, whereas Lipozyme RM-IM showed poor activities for esterification using secondary and tertiary alcohols. Different solvents were also tested and n-hexane was selected because of its reaction effects, besides being cheaper, available at low boiling point, and ease of recovery. Using the preselected alcohol and solvent, other reaction parameters (butyric acid concentration, temperature, substrate molar rate, and biocatalyst content) were studied to optimize the reaction conditions. Optimal conditions were acid concentration, 0.7 M; substrate molar ratio, 11 alcohol-acid; temperature 45 °C; biocatalyst content, 14% (by substrate mass). Under these conditions, it was possible to obtain a yield of 86% of butyl butyrate in 2.5 h. When molecular sieves (90 mg mmol^-1 butytic acid) were added to the reaction, the observed yield increased to 96%. The biocatalyst was used in 5 successive reaction cycles keeping 100% of its initial activity. The overall process productivity was improved 2-fold when compared to the traditional mechanical agitation, showing that ultrasound is a promising technology for application in biocatalysis. The Royal Society of Chemistry.

Full text of DOI:10.1039/c3ra47315e

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Ultrasound technology and molecular sieves
improve the thermodynamically controlled
esterification of butyric acid mediated by
immobilized lipase from Rhizomucor miehei
Cite this: RSC Adv., 2014, 4, 8675
a
a
a
a
Lucas P. Fallavena, Fabio H. F. Antunes, Joana S. Alves, Natalia Paludo,
´
a
b
a
Marco A. Z. Ayub, Roberto Fernandez-Lafuente and Rafael C. Rodrigues*
In this research, the effects of ultrasound stirring and the addition of molecular sieves on esterification
reactions between butyric acid and several alcohols catalyzed by immobilized lipase from Rhizomucor
miehei (Lipozyme RM-IM) were studied. Among the tested alcohols, 1-propanol and isobutanol allowed
the highest activities, whereas Lipozyme RM-IM showed poor activities for esterification using secondary
and tertiary alcohols. Different solvents were also tested and n-hexane was selected because of its
reaction effects, besides being cheaper, available at low boiling point, and ease of recovery. Using the
preselected alcohol and solvent, other reaction parameters (butyric acid concentration, temperature,
substrate molar rate, and biocatalyst content) were studied to optimize the reaction conditions. Optimal
ꢀ
conditions were acid concentration, 0.7 M; substrate molar ratio, 1 : 1 alcohol–acid; temperature 45 C;
biocatalyst content, 14% (by substrate mass). Under these conditions, it was possible to obtain a yield of
ꢁ1
8
6% of butyl butyrate in 2.5 h. When molecular sieves (90 mg mmol butytic acid) were added to the
Received 4th December 2013
Accepted 17th January 2014
reaction, the observed yield increased to 96%. The biocatalyst was used in 5 successive reaction cycles
keeping 100% of its initial activity. The overall process productivity was improved 2-fold when compared
to the traditional mechanical agitation, showing that ultrasound is a promising technology for application
in biocatalysis.
DOI: 10.1039/c3ra47315e
www.rsc.org/advances
chemical process and for oil and fats modication, is the one
produced by the fungus Rhizomucor miehei. This enzyme is
1
. Introduction
Esters formed by short chain carboxylic acids and alcohols can commercialized in soluble form as Palatase 20000 L and as an
be used as avors or fragrances, considered as natural avors, immobilized preparation commercially named Lipozyme RM-
1
5,6
with relevant applications in many different industrial sectors.
IM, both supplied by Novozymes.
These natural avors can be extracted from plants, which is
Esterication is a thermodynamically controlled synthesis
characteristically a high-cost and low-yield process. Otherwise, (TCS), using unmodied acid and alcohol, forcing the reaction
these esters can be obtained through biotechnology process, conditions to shi the equilibrium in the direction of the
7
produced using enzymes or cells, and they can be labeled as synthesis. Esterication reactions are mainly achieved in low
2
natural.
water activity systems because water, being a product of the
8
Lipases are among the most used enzymes as catalyst for reaction, can shi the equilibrium towards hydrolysis. In this
2,3
esterication reactions. These enzymes catalyze in vivo the sense, the use of molecular sieves to control the excess of water
hydrolysis of oils and fats, however, because they present a in the reaction has been reported as a powerful tool to improve
broad specicity, they also catalyze reactions of esterication, the enzyme performance, for example in the synthesis of iso-
transesterication, and acidolysis, being stable in very different propyl acetate in organic medium and in the synthesis of avor
4
9,10
reaction media Moreover, there is a great variety of lipases with esters in ionic liquids.
varying properties. One of the most used lipase, applied in
Considering the use of immobilized lipases, the adsorption
of water or substrates (carboxylic acid and alcohol) on the
support is one of the most important reasons of biocatalyst
inactivation. This might be solved by suitable washings to
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; Web: http://www.ufrgs.br/ eliminate these compounds during successive batch opera-
11
bbb. E-mail: rafaelcrodrigues@ufrgs.br; Fax: +55 51 3308 7048; Tel: +55 51 3308 7793 tions. Another important fact to keep the lipase stability and
b
Department of Biocatalysis, ICP – CSIC, Campus UAM-CSIC, Cantoblanco, ZC 28049,
activity is to control the enzyme microenvironment conditions,
Madrid, Spain
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avoiding the formation of substrate or products phases in this 2.3. Effects of organic solvents on the enzyme performance
environment. The use of proper mixing operations and an
adequate stirring of the system may be very relevant to have a
n-Hexane, cyclohexane, n-heptane, and n-octane were tested as
solvents in the reactions between butyric acid and 1-propanol or
good performance of the biocatalyst, in most cases performed
isobutanol. The mixtures of butyric acid (0.1 M), alcohol (0.1 M),
9,12–15
by traditional mechanical agitation.
More recently, some
and Lipozyme RM-IM (10%, by substrates mass) were placed in
reports showing the advantages of the use of ultrasound tech-
nology as a better mixing strategy for lipase reaction systems
ꢀ
an ultrasonic bath for 2 h at 40 C.
1
6–18
appeared in the literature.
capability of increasing the interaction between phases by
cavitation caused by the collapse of bubbles, and the ultrasonic performance
jet, that disrupts the boundary phase and causes emulsica-
Ultrasound technology has the
2.4. Effects of substrates concentrations on the enzyme
For this study, the concentrations of the reaction substrates
were varied from 0.1 M to 1.0 M (of each substrate), and
measuring the initial reaction rate. The mixtures of butyric acid,
isobutanol (always keeping the molar ratio 1 : 1 alcohol–acid),
and Lipozyme RM-IM (10%, by substrates mass) were placed in
19,20
tion. Furthermore, when ultrasound is applied to an
aqueous solution or suspension, increasing in mixing, shearing
and mass transfer are observed.
21,22
In spite of these possibili-
ties, ultrasound technology has not been extensively used in
21,23,24
biological reactions.
ꢀ
an ultrasonic bath at 40 C.
In this context, this work aimed at studying the application
of ultrasound technology during the synthesis of esters from
butyric acid and different alcohols in organic solvents using
2.5. Experimental design
Lipozyme RM-IM as the biocatalyst. Furthermore, it was also A central composite design (CCD) with two variables (temper-
evaluated the effect of the main parameters affecting the reac- ature and biocatalyst content) was carried out in order to
tion yield (reaction temperature, biocatalyst content, acid obtain the optimal conditions for esterication reaction. The
concentration) using a central composite design and the variables and their coded and uncoded values as well as the 12
response surface methodology (RSM), which is an effective treatments of the two variables, each at ve levels are presented
statistical technique for the investigation of complex processes. in Table 1. The design was constructed of 4 factorial points, 4
The main advantage of RSM is the reduced number of experi- axial points (two axial points on the axis of design variable),
mental runs needed to provide sufficient information for and 4 replications at the central point. The mixtures of 0.7 M
statistically acceptable results, and it is a faster and less butyric acid, 0.7 M isobutanol, and Lipozyme RM-IM were
expensive method for gathering research data than the classical placed in an ultrasonic bath for 1 h. The biocatalyst content
25
method. It was also investigated the effect of using molecular and temperature were varied according to the CCD. In each
sieves to improve the operational stability of the biocatalyst in case, the percentage of conversion for esterication was
repeated batches.
determined. The second-order polynomial equation for the
variables was as follows:
P
P
P
2
. Materials and methods
2
i
Y ¼ b
0
+
b
i
X
i
+
b
ij
X
i
X
j
+
b
ii
X
(1)
2.1. Materials
where Y is the response variable, b
0
the constant, b
i
, bii, bij were
Lipase from R. miehei immobilized in Duolite ES 562, a weak
anion-exchange resin based on phenol–formaldehyde copoly-
mers (Lipozyme RM-IM) was kindly provided by Novozymes
the coefficients for the linear, quadratic, and for the interaction
effects, respectively, and X and X the coded level of variables x
and x . The above quadratic equation was used to plot surfaces
for the variables.
i
j
i
j
˚
Spain. Molecular sieves (3 A, beads 4–8 mesh), butyric acid,
ethanol, 1-propanol, 2-propanol, 1-butanol, isobutanol, tert-
butanol, 1-pentanol, and other chemicals were of analytical
grade and purchased from Sigma-Aldrich (Sigma. St, Louis,
USA). Ultrasonic bath (Unique Inc., model USC 2880A, 40 kHz,
Table 1 Coded levels, real values, and the yields of esterification
obtained in the CCD
2
20 W, Brazil) with temperature control was used in all
ꢀ
experiments.
Run
Temperature ( C)
Biocatalyst content (%)
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 (3.8)
1 (17.2)
ꢁ1 (3.8)
1 (17.2)
0 (10.5)
0 (10.5)
ꢁ1.41 (1)
1.41 (20)
0 (10.5)
0 (10.5)
0 (10.5)
0 (10.5)
9.88
61.67
21.59
59.53
46.02
66.02
0.89
58.44
42.34
46.25
31.42
52.93
2
.2. Effects of alcohols on the enzyme performance
The different alcohols (ethanol, 1-propanol, 2-propanol,
-butanol, isobutanol, tert-butanol, 1-pentanol) were mixed
1
with butyric acid (0.1 M) at a molar ratio of 1 : 1 in 50 mL
Erlenmeyer asks (working volume of 10 mL), followed by the
addition of enzyme (10%, by substrates mass), using n-hexane
as solvent. The mixtures of butyric acid, alcohol, and Lipozyme
RM-IM were placed in an ultrasonic bath (Unique Inc., model
1
1
12
0
1
ꢀ
USC 2880A, 40 kHz, 220 W, Brazil) for 2 h at 40 C.
0 (45)
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.6. Effects of the presence of molecular sieves on the
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2
enzyme performance
The effect of molecular sieves on the reaction was evaluated by
varying the amounts of sieves from 0 to 120 mg of molecular
sieves per mmol of butyric acid. The mixtures of 0.7 M butyric
acid, 0.7 M isobutanol, and Lipozyme RM-IM (14%, by substrates
ꢀ
mass) were placed in the ultrasonic bath at 45 C for 2.5 h.
2.7. Operational stability of Lipozyme RM-IM over repeated
batches
Lipozyme RM-IM was tested in repeated batches under the
optimal reaction conditions dened in our study. Aer the
esterication reaction, the immobilized enzyme was separated
from the reaction medium by vacuum ltration using a sintered
glass funnel. The recovered biocatalyst was then used in a new
batch reaction without any treatment.
2.8. Reaction analysis
The progress of esterication was monitored by determining
the residual acid content by titration of 0.5 mL samples with
Fig. 1 Effects of the nature of alcohol on the ultrasound-assisted
esterification of butyric acid catalyzed by Lipozyme RM-IM. Reaction
NaOH (0.01 M) using phenolphthalein as indicator and 5 mL of conditions: butyric acid, 0.1 M; substrate molar ratio, 1 : 1 (alcohol–
ꢀ
ethanol as quenching agent. The amount of ester was calculated acid); biocatalyst content, 10% (by substrate mass); 40 C, 2 h.
as being equivalent to the amount of consumed acid. A cali-
bration curve was constructed to ensure the reliability of this
acid determination using laboratory-made mixtures of acid,
alcohol, and commercial ester, and conrmed by GC analysis in
some samples.
were higher than n-butyl acetate in the reaction catalyzed by the
lipase from Rhizopus oligosporus. Isobutanol allowed a slightly
higher activity than n-butanol, and lower than the obtained
using 1-propanol, suggesting than the short branches has not a
2
.9. Statistical analysis
relevant effect in the enzyme recognition. These results suggest
that the length and the ramication of the aliphatic chain, in the
case of secondary and tertiary alcohols, are both important
factors for the enzyme activity. From these results, 1-propanol
and isobutanol were selected for the next experiments.
Experiments were performed in triplicates and mean with the
standard errors were plotted in the gures. The experimental
design and analysis of results were carried out using Statistica
7.0 (Statso, USA). The statistical analysis of the model was
performed as analysis of variance (ANOVA). The signicance of
the regression coefficients and the associated probabilities, p(t),
3.2. Effects of organic solvents on the enzyme performance
were determined by Student's t-test; the second order model Esterication reactions are usually performed in low water
equation signicance was determined by Fisher's F-test. The activity medium, and organic solvents, ionic liquids, or super-
10,26,27
variance explained by model is given by the multiple determi- critical uids are used for this purpose.
In the case of
nation coefficients, R . For each variable, the quadratic models organic solvents, it is convenient to use the one producing the
2
were represented as contour plots (2D).
lowest deleterious effects on enzyme activity and stability. In
this work, four different organic solvents were evaluated for the
esterication reactions catalyzed by Lipozyme RM-IM under
ultrasound energy. The results of esterication yields using
each solvent as well as their melting points and log P are pre-
3. Results and discussion
3.1. Effects of alcohols on the enzyme performance
In order to verify the effect of alcohol in the esterication of sented in Table 2. Good enzyme activities were obtained using
butyric acid catalyzed by Lipozyme RM-IM under ultrasound anyone of the solvents, for both alcohols, except for cyclohexane
energy, 7 different alcohols were tested, and the results are pre- when isobutanol was used as substrate. According to Laane
28
sented in Fig. 1. The enzyme presented good activity in the et al. the higher the log P (log P > 4.0), the better the perfor-
esterication reactions with most alcohols, except for 2-propanol mance of the biocatalysts in organic solvents. Since there were
and tert-butanol, secondary and tertiary alcohols, respectively. no important differences among the solvents, n-hexane was
Lipozyme RM-IM presented a negligible esterication activity chosen for the following experiments because it is the cheapest
with these alcohols. The ester conversion improved from ethanol and it has a lower boiling point than the other solvents and
ꢀ
to 1-propanol, and then decreased with the alcohol chain increase butyl butyrate (165 C). Concerning the ester, isobutyl butyrate
(
n-butanol and pentanol). A similar behavior was reported by is more important as avor ester in food industries than propyl
13
Mahapatra et al., who showed that the yields of n-propyl acetate butyrate, thus, it was chosen for the sequence of the work.
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Table 2 Effects of the nature of the organic solvents on the ultra-
sound-assisted synthesis of isobutyl butyrate
3.4. Experimental design and model tting
Aer dening the target product, the organic solvent and the
substrates concentration, it was performed an experimental
design to optimize the temperature and biocatalyst content of
the reaction, both important variables in esterication reac-
Yield (%)
Boiling
point ( C)
ꢀ
Solvent
log P
1-Propanol
Isobutanol
33–36
tions.
Experimental data obtained for the ultrasound-assis-
Cyclohexane
n-Hexane
n-Heptane
Isooctane
3.2
3.5
4.0
4.5
81.0
69.0
98.0
99.0
80.3
87.7
85.2
82.7
57.6
84.4
83.4
83.9
ted lipase-catalyzed synthesis of isobutyl butyrate are shown in
Table 1. The highest yield of conversion in 1 h was 66% in
treatment 6, the highest temperature, while the less effective,
with yields lower than 1%, was the treatment 7, the lowest
biocatalyst content. In general, using ultrasound energy, most
of treatments presented yields of conversion higher than 40% in
only 1 h of reaction, much faster than using mechanical agita-
tion. For instance, during the optimization of butyl butyrate
3
.3. Effects of substrates concentrations on the enzyme
performance
In order to assess the effect of butyric acid and isobutyl catalyzed by Lipozyme RM-IM, the yields were around 60% aer
29
alcohol on the enzymatic activity, experiments were carried 24 h of reaction.
out varying the substrates concentrations from 0.1 to 1.0 M,
Statistical testing of the model was done using Fisher's
but keeping the molar ratio of 1 : 1 constant (Fig. 2). An statistical test for ANOVA. The computed F value (30.62) was
increase in the initial reaction rate was observed up to 0.7 M of highly signicant (p ¼ 0.0009). The determination coefficient
2
substrates, above that remaining constant up to 1.0 M. (R ¼ 0.9683) implies that the sample variation of 96.83% for
Comparatively, in a previous study using mechanical agita- ultrasound-assisted isobutyl butyrate synthesis is attributed to
2
9
tion, the initial reaction rate increased linearly up to 1.0 M, the independent variables and can be explained by the model.
but was 3 times lower than that obtained in this work. The correlation coefficient (R ¼ 0.9840) suggests a highly
Therefore, ultrasound technology produced much higher satisfactory representation of the process model and a good
yield in shorter reaction times. The maximum substrate correlation between the experimental results and the theoretical
concentration of 0.7 M found in this work is higher than that values predicted by the model equation. The experimental data
reported in many studies using this enzyme and carboxylic have been adjusted to the proposed model by the second-order
3
0–32
acid,
and was selected in the following experiments. The polynomial eqn (1). and the second-order polynomial model is
possibility of using high butyric acid concentration may be presented in eqn (2):
interesting, since it might increase the industrial feasibility of
2
2
the process.
Y ¼ 47.19 + 21.42X
2
– 9.98X
(2)
where Y is the percentage yield, X
1
and X
2
, are the coded values
of biocatalyst content.
3.5. Effect of parameters and optimal conditions
Only biocatalyst content presented a statistically signicant
effect on the reaction performance (95% condence level),
4
2.84, considering the two variables. These effects and their
interactions can be better understood observing the contour
plot in Fig. 3. The effect for reaction temperature was positive
(
9.47) but not statistically signicant. The biocatalyst content,
however, positively affected reaction rate up to 14%, with
yields remaining constant above this value. Therefore, we
propose the optimal conditions for ultrasound-assisted iso-
butyl butyrate synthesis catalyzed by Lipozyme RM-IM as
ꢀ
temperature of 45 C and biocatalyst content of 14% (by
substrate mass). Under these conditions, the theoretical value
for the yield of reaction, predicted by the model, was 55.4%. In
order to validate the model, an experiment was carried out
under the optimal conditions and the kinetics of ultrasound-
assisted isobutyl butyrate synthesis is presented in Fig. 4 (lled
squares). Aer 1 h, the reaction yield was 60.9%, which is close
to the theoretical value, showing a good correlation between
the model and the experimental data. Aer 2.5 h, a yield of
86% was reached.
Fig. 2 Effects of butyric acid concentration on the initial reaction rate.
Reaction conditions: butyric acid, 0.1 M; substrate molar ratio, 1 : 1
ꢀ
(
alcohol–acid); biocatalyst content, 10% (by substrate mass); 40 C.
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caused by the accumulation of water in the enzyme environ-
11
˚
ment. The mass of molecular sieves (3 A) varied from 0 to
20 mg per mmol of butyric acid, and the results for the reac-
1
tion yields aer 2.5 h are shown in Fig. 4. The yields were
progressively improved with the addition of sieves from 86 to
ꢁ
1
ꢁ1
9
6% up to 90 mg mmol . The use of 120 mg mmol of
molecular sieves reduced the observed yields, possibly because
the capture of the water molecules necessary to keep the enzyme
7
activity. The positive effect of molecular sieves was also
9,10,37
observed by other authors.
In our conditions, this was
possible using 90 mg of sieves per mmol of carboxylic acid.
In order to compare and measure the effects of ultrasound
and molecular sieves, an experiment was performed under the
optimal conditions using traditional mechanical agitation and
the ultrasound energy, in the presence and absence of 90 mg of
sieves per mmol of carboxylic acid. The results presented in the
Fig. 5 show that the use of molecular sieves improved the yield
in both agitation methods and, the combined use of ultrasound
and molecular sieves improved the yield in almost 1.5-times
compared to mechanical agitation without sieves. It is impor-
tant to remark that the conditions used for mechanical agita-
Fig. 3 Contour plots of yields (%) of isobutyl butyrate synthesis as a
function of biocatalyst content (%) and temperature ( C). Reaction
conditions: butyric acid, 0.7 M; substrate molar ratio, 1 : 1 (alcohol–
acid), 1 h.
29
tion here were different from that previously optimized. In the
previous study, lower temperature and biocatalyst content were
used and yielded 90% of conversion only aer 16 h.
ꢀ
3.7. Operational stability of Lipozyme RM-IM over repeated
batches
For a cost-competitive application, immobilized enzymes must
be reused several times. The downstream processing and
ꢁ1
Fig. 4 Effect of the addition of molecular sieves (mg mmol butyric
acid) in the ultrasound-assisted isobutyl butyrate synthesis catalyzed by
ꢁ1
Lipozyme RM-IM. (-) 0; (B) 30; (O) 60; (A) 90; (,) 120 mg mmol
.
Reaction conditions: butyric acid, 0.7 M; substrate molar ratio, 1 : 1
ꢀ
(
alcohol–acid); biocatalyst content, 14% (by substrate mass); 45 C.
Fig. 5 Comparison of ultrasound and mechanical agitation in the
isobutyl butyrate synthesis catalyzed by Lipozyme RM-IM. (C,B)
mechanical agitation; (-,,) ultrasound energy. Closed symbols:
without molecular sieves; open symbols: addition of 90 mg mmol of
molecular sieves. Reaction conditions: butyric acid, 0.7 M; substrate
3
.6. Effect of molecular sieves on the esterication reaction
In order to improve the equilibrium yield, it was tested the effect
of the addition of molecular sieves to the reaction mixture for
removing the formed water, therefore shiing the equilibrium
ꢁ1
molar ratio, 1 : 1 (alcohol–acid); biocatalyst content, 14% (by substrate
ꢀ
towards synthesis, also preventing the loss of enzyme activity mass); 45 C.
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product purication could be performed by a simple ltration
and distillation to obtain a pure product. In order to test the that creates cavitation in the liquid solution, reducing the
Ultrasound mechanism is based on the high-energy waves
38
reuse of Lipozyme RM-IM, several batches were run at the diffusion mass limitations of substrates and products in the
19
optimal conditions, without any washing treatments in between porous matrix of enzyme supports. Another advantage of
them, and the results are shown in Fig. 6. It was possible to using ultrasound is that it requires only 30 to 50% of the energy
20
reuse the biocatalyst for 4 cycles keeping 100% of its initial that is consumed during mechanical agitation.
activity, fast loosing activity in the following cycles. Considering
that the molecular sieves were also reused, the result may be a
consequence of the saturation of the molecular sieves with 4. Conclusions
released water. In the synthesis of butyl butyrate using
Ultrasound technology has shown to be very useful to improve
the productivity of the esterication process catalyzed by Lip-
ozyme RM-IM. Under the tested conditions, this biocatalyst
exhibited good esterication activity using different primary
alcohols. In addition, the use of molecular sieves, to capture the
released water, increased the observed yield aer 2.5 h from 86
to 96%. Moreover, the combined use of ultrasound technology
and molecular sieves allowed improving the enzyme stability.
Furthermore, the process productivity was improved by two
factors: washing steps were avoided and enzyme stability was
higher.
mechanical agitation, the recovery of activity was only possible
29
by performing an n-hexane washing between every batch. Even
performing this washing, enzyme stability decreased since the

rst reuse, which did not happen using ultrasound energy. For
industrial purposes, the elimination of this unit operation
would be very important for reducing costs. Moreover, the
process productivity was also improved using the ultrasound.
Under mechanical agitation, a conversion yield of 96% was
reached aer 20 h, using 1.0 M butyric acid, which represents a
ꢁ
1
ꢁ1 29
productivity of 50 mmol L
h
.
In contrast, under ultra-
sound energy the conversion yield of 96% was reached in 2.5 h.
using 0.7 M butyric acid, which represents a productivity of
ꢁ
1
ꢁ1
250 mmol L
h , 5-fold higher. If we compare both agitation
Acknowledgements
methods under the same conditions, such as presented in
Fig. 5, the overall productivity is 2-times higher using ultra-
sound and molecular sieves than using mechanical agitation. A
similar effect was observed for the synthesis of butyl acetate
catalyzed by Novozym 435, where reaction productivity and
enzyme stability were improved by the use of ultrasound
This work was supported by grants and scholarships (JA Silva
and N Paludo) from FAPERGS (Fundaç ˜a o 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 Innovaci ´o n, Spain). The authors wish to thank Mr
Ramiro Mart ´ı nez (Novozymes, Spain) for kindly supplying the
enzymes used in this research.
18
technology.
References
1
2
R. G. Berger, Biotechnol. Lett., 2009, 31, 1651–1659.
K. P. Dhake, D. D. Thakare and B. M. Bhanage, Flavour
Fragrance J., 2013, 28, 71–83.
3
4
5
6
7
8
9
S. Nagarajan, Appl. Biochem. Biotechnol., 2012, 168, 1163–
1
196.
U. T. Bornscheuer, OCL: Oleagineux, Corps Gras, Lipides,
013, 20, 45–49.
2
R. C. Rodrigues and R. Fernandez-Lafuente, J. Mol. Catal. B:
Enzym., 2010, 64, 1–22.
R. C. Rodrigues and R. Fernandez-Lafuente, J. Mol. Catal. B:
Enzym., 2010, 66, 15–32.
A. L. Paiva, V. M. Balc ˜a o and F. X. Malcata, Enzyme Microb.
Technol., 2000, 27, 187–204.
S. Hari Krishna and N. G. Karanth, Catal. Rev. Sci. Eng., 2002,
4
4, 499–591.
M. L. Verma, W. Azmi and S. S. Kanwar, Enzyme Res., 2011,
011, 919386.
0 P. Lozano, J. M. Bernal and A. Navarro, Green Chem., 2012,
4, 3026–3033.
2
1
1
Fig. 6 Operational stability of Lipozyme RM-IM in repeated batches of
ultrasound-assisted isobutyl butyrate synthesis. Reaction conditions:
butyric acid, 0.7 M; substrate molar ratio, 1 : 1 (alcohol–acid); bio-
1
1 A. B. Martins, N. G. Graebin, A. S. G. Lorenzoni,
R. Fernandez-Lafuente, M. A. Z. Ayub and R. C. Rodrigues,
Process Biochem., 2011, 46, 2311–2316.
ꢀ
catalyst content, 14% (by substrate mass); 45 C; molecular sieves,
ꢁ1
9
0 mg mmol butyric acid.
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12 N. G. Graebin, A. B. Martins, A. S. G. Lorenzoni, C. Garcia- 25 P. Xu, Z.-Y. Ding, Z. Qian, C.-X. Zhao and K.-C. Zhang,
Galan, R. Fernandez-Lafuente, M. A. Z. Ayub and
R. C. Rodrigues, Biotechnol. Prog., 2012, 28, 406–412.
3 P. Mahapatra, A. Kumari, V. Kumar Garlapati, R. Banerjee
and A. Nag, J. Mol. Catal. B: Enzym., 2009, 60, 57–63.
Enzyme Microb. Technol., 2008, 42, 325–331.
26 M. D. R. Diaz, J. M. G ´o mez, B. D ´ı az-Suelto and A. Garc ´ı a-
Sanz, Eng. Life Sci., 2010, 10, 171–176.
27 G. E. Jeromin and A. Zoor, Biotechnol. Lett., 2008, 30, 925–
928.
1
1
4 A. B. Martins, J. L. R. Friedrich, J. C. Cavalheiro, C. Garcia-
Galan, O. Barbosa, M. A. Z. Ayub, R. Fernandez-Lafuente 28 C. Laane, S. Boeren, K. Vos and C. Veeger, Biotechnol. Bioeng.,
and R. C. Rodrigues, Bioresour. Technol., 2013, 134, 417–422. 1987, 30, 81–87.
5 J. Sun, J. H. Chin, W. Zhou, B. Yu, P. Curran and S. Q. Liu, 29 A. S. G. Lorenzoni, N. G. Graebin, A. B. Martins,
1
1
1
1
J. Am. Oil Chem. Soc., 2012, 89, 1991–1998.
6 K. M. Gonçalves, F. K. Sutili, S. G. F. Leite, R. O. M. A. De Souza
and I. C. R. Leal, Ultrason. Sonochem., 2012, 19, 232–236.
R. Fernandez-Lafuente, M. A. Z ´a chia Ayub and
R. C. Rodrigues, Flavour Fragrance J., 2012, 27, 196–
200.
7 R. Konwarh, S. Pramanik, D. Kalita, C. L. Mahanta and 30 G. Bayramoglu, B. Karagoz, B. Altintas, M. Yakup Arica and
N. Karak, Ultrason. Sonochem., 2012, 19, 292–299. N. Bicak, Bioprocess Biosyst. Eng., 2011, 34, 735–746.
8 A. B. Martins, M. F. Schein, J. L. R. Friedrich, R. Fernandez- 31 D. Bezbradica, D. Mijin, S. Siler-Marinkovic and Z. Knezevic,
Lafuente, M. A. Z. Ayub and R. C. Rodrigues, Ultrason.
Sonochem., 2013, 20, 1155–1160.
9 H. Fazlena, S. Norsuraya and S. N. Nadiah, Ultrasonic assisted
enzymatic reaction: An overview on ultrasonic mechanism and
stability-activity of the enzyme, Langkawi, 2013.
0 V. B. Veljkovic, J. M. Avramovic and O. S. Stamenkovic,
Renewable Sustainable Energy Rev., 2012, 16, 1193–1209.
1 B. Kwiatkowska, J. Bennett, J. Akunna, G. M. Walker and
D. H. Bremner, Biotechnol. Adv., 2011, 29, 768–780.
2 M. M. Zheng, L. Wang, F. H. Huang, L. Dong, P. M. Guo,
J. Mol. Catal. B: Enzym., 2007, 45, 97–101.
32 A. V. de Paula, G. F. M. Nunes, J. D. L. Silva, H. F. De Castro
and J. C. Dos Santos, Appl. Biochem. Biotechnol., 2010, 160,
1146–1156.
33 S. W. Chang, J. F. Shaw and C. J. Shieh, Food Technol.
Biotechnol., 2003, 41, 237–242.
34 H. C. Chen, Y. K. Twu, C. M. J. Chang, Y. C. Liu and
C. J. Shieh, Ind. Crops Prod., 2010, 32, 522–526.
35 G. A. Macedo, G. M. Pastore and M. I. Rodrigues, Process
Biochem., 2004, 39, 687–692.
1
2
2
2
Q. C. Deng, W. L. Li and C. Zheng, Ultrason. Sonochem., 36 J. C. Santos and H. F. De Castro, World J. Microbiol.
2012, 19, 1015–1020.
Biotechnol., 2006, 22, 1007–1011.
2
2
3 J. V. Sinisterra, Ultrasonics, 1992, 30, 180–185.
4 L. Batistella, L. A. Lerin, P. Brugnerotto, A. J. Danielli,
37 H. Horchani, A. Bouaziz, Y. Gargouri and A. Sayari, J. Mol.
Catal. B: Enzym., 2012, 75, 35–42.
C. M. Trentin, A. Popiolski, H. Treichel, J. V. Oliveira and 38 L. Gubicza, A. Kabiri-Badr, E. Keoves and K. Bela-Bako,
D. De Oliveira, Ultrason. Sonochem., 2012, 19, 452–458. J. Biotechnol., 2000, 84, 193–196.
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