Enzymatic synthesis optimization of isoamyl butyrate

Article abstract of DOI:10.1590/S0103-50532011001100018

Lipozyme TL IM was used to catalyse the esterification of isoamyl alcohol and butyric acid. A fractional factorial design was employed to evaluate the effects of temperature (30, 40, 50 °C), alcohol:acid molar ratio (1:1, 2:1, 3:1), enzyme concentration (0.003, 0.0115, 0.020 g mL^-1), butyric acid concentration (0.1, 0.3, 0.5 mol L^-1) and shaking rate (50, 115, 180 rpm) on the ester yield. With these results, the levels were redefined to a 2³ factorial design. The maximum yield of ester was obtained at 30 °C, 180 rpm, alcohol:acid molar ratio of 1:1, enzyme concentration of 0.021 g mL^-1 and butyric acid concentration of 0.5 mol L^-1. Under the optimal conditions, 92% esterification was attained with an ester concentration of 0.9 mol L^-1. Isoamyl alcohol from fusel oil was used under the same conditions and resulted in 93% esterification and an ester concentration of 1.0 mol L^-1.

Full text of DOI:10.1590/S0103-50532011001100018

J. Braz. Chem. Soc., Vol. 22, No. 11, 2148-2156, 2011.
Printed in Brazil - ©2011 Sociedade Brasileira de Química
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Article
A
Enzymatic Synthesis Optimization of Isoamyl Butyrate
Andréia Anschau,* Vitor C. Aragão, Barbara D. A. Porciuncula, Susana J. Kalil,
Carlos A. V. Burkert and Janaína F. M. Burkert
Escola de Química e Alimentos, Universidade Federal do Rio Grande, CP 474,
96201-900 Rio Grande-RS, Brazil
Lipozyme TL IM foi usada para catalisar a esterificação de álcool isoamílico e ácido butírico.
Um planejamento fatorial fracionário foi utilizado para avaliar os efeitos da temperatura (30,
40, 50 ºC), razão molar álcool:ácido (1:1, 2:1, 3:1), concentração de enzima (0,003, 0,0115,
0,020 g mL-1), concentração de ácido butírico (0,1, 0,3, 0,5 mol L-1) e agitação (50, 115, 180 rpm)
na produção de éster. Com esses resultados, os níveis foram redefinidos para um planejamento
fatorial 2³. A máxima produção de éster foi obtida a 30 ºC, 180 rpm, razão molar álcool:ácido de
1:1, concentração de enzima 0,021 g mL-1 e concentração de ácido butírico de 0,5 mol L-1. Sob
essas condições ótimas foi obtido 92% de esterificação e concentração de éster de 0,9 mol L-1.
Álcool isoamílico obtido a partir de óleo fúsel foi utilizado nas mesmas condições resultando em
93% de esterificação e concentração de éster de 1,0 mol L-1.
Lipozyme TL IM was used to catalyse the esterification of isoamyl alcohol and butyric acid.
A fractional factorial design was employed to evaluate the effects of temperature (30, 40, 50 °C),
alcohol:acid molar ratio (1:1, 2:1, 3:1), enzyme concentration (0.003, 0.0115, 0.020 g mL-1),
butyric acid concentration (0.1, 0.3, 0.5 mol L-1) and shaking rate (50, 115, 180 rpm) on the ester
yield. With these results, the levels were redefined to a 23 factorial design. The maximum yield
of ester was obtained at 30 ºC, 180 rpm, alcohol:acid molar ratio of 1:1, enzyme concentration
of 0.021 g mL-1 and butyric acid concentration of 0.5 mol L-1. Under the optimal conditions,
92% esterification was attained with an ester concentration of 0.9 mol L-1. Isoamyl alcohol from
fusel oil was used under the same conditions and resulted in 93% esterification and an ester
concentration of 1.0 mol L-1.
Keywords: favor ester, Lipozyme TL-IM, organic media, response surface methodology
(RSM), fusel oil
Introduction
enzymes (when immobilized) can be reused, therefore,
minimizing the reaction residues. The most used reaction
media in enzymatic syntheses still are organic solvents
such as hexane or heptane, which can be recovered
for reuse.2
Enzymatic catalysis in organic media allows to obtain
pure products, due to enzyme specificity. In addition, the
catalysis is carried out under mild reaction conditions,
which minimize side reactions when compared to the
chemical process. Moreover, enzymatic syntheses can
be performed using less toxic or non-toxic solvents as
compared to chemical syntheses.3 Interest in the use of
organic solvents is mainly due to enhancement of the
solubility of hydrophobic substrates, the elimination of
side reactions caused by water, ease of product recovery
and protection from microbial contamination.4
Short chain fatty acid and alcohol esters are important
components of natural aromas used in the food industry.
Currently, most of the favour and fragrance components are
obtained by traditional methods, which include chemical
synthesis or extraction from natural sources. The concept
of a natural ester made by enzymatic synthesis with lipase
and natural substrate components is an attractive alternative
to such routes.1
Biotechnological processes are more expensive
than chemical ones but they have clear environmental
advantages, since the use of inorganic acids (employed
as catalysts in chemical syntheses) is avoided, and the
*e-mail: andreiaanschau@hotmail.com

Vol. 22, No. 11, 2011
Anschau et al.
2149
Lipases (triacylglycerol acylhydrolases; EC 3.1.1.3) are
one of the most important classes of hydrolytic enzymes,
catalyzing both the hydrolysis and synthesis of esters.
Microbial lipases are widely diversified in their properties
and substrate specificity, which make them attractive
tools for industrial applications.5 Lipases display catalytic
activity towards a large variety of alcohols and acids in
reactions involving ester synthesis.6
and provided by Novozymes (Bagsvaerd, Denmark). It
is produced by genetically modified Aspergillus oryzae,
under submerged fermentation. The microbial donor of the
gene that expresses the lipase production is Thermomyces
lanuginosus.
Chemicals
Response surface methodology (RSM) is an efficient
statistical technique for the optimization of multiple
variables, in order to predict the best performance
conditions with a minimum of experiments, and the
optimization of the lipase-catalyzed production of various
esters has been investigated by RSM .7
In countries where ethanol is produced as a fuel on a large
scale, such as Brazil, the use of the by-products has become
an important issue to make the production of ethanol less
polluting and more profitable.8 The fusel oil obtained from
the distillation of fermented agricultural products is currently
used as a raw material for the production of amyl and butyl
alcohols. Fusel oil had been used as foam coating or as fuel for
energy source.Also, esters obtained from alcohols of fusel oil
can be used industrially as solvents and favoring, medicinal
and plasticizer agents.7 However, a great part of the fusel oil
produced is generally discarded.9 Its average composition is
51% i-amyl-alcohol, 15% i-butanol, 13% n-propanol, 10%
ethanol, 11% miscellaneous alcohols and water.10
Here, we wish to report a study employing isoamyl
alcohol from fusel oil instead of pure isoamyl alcohol. The
present work focused on the reaction parameters that affect
the use of Lipozyme TL IM in the esterification of isoamyl
alcohol and butyric acid with n-hexane as the organic
solvent. The main objectives of this work were to develop
an approach that would enable a better understanding
of the relationships between the variables (temperature,
concentrations of enzyme and butyric acid, alcohol: acid
molar ratio and the rate of shaking) and the responses
(percent esterification and ester concentration), to obtain the
optimum conditions for the synthesis of isoamyl butyrate
using a central composite rotatable design (CCRD) and
RSM analysis, and to compare the results obtained under
the optimum conditions, using commercial isoamyl alcohol
and isoamyl alcohol obtained from fusel oil, in order to use
this by-product in future studies.
All chemicals were of analytical grade. The fusel oil
(Sugarcane Technology Centre- CTC, Piracicaba-SP,
Brazil) was distilled in order to obtain the isoamyl alcohol,
which was used in the synthesis. Fractions above the
boiling point of 120 ºC (rich in isoamyl alcohol, its boiling
temperature is 128 ºC) were collected and distilled again.
Isoamyl alcohol obtained by distillation at purity of 97.6%
(v/v) was used in esterification.
Ester synthesis
In previous study, Aragão et al.,11 tested different
solvents, such as acetone, chloroform, toluene, hexane
and heptanes, which have logP values of -0.23, 2, 2.5, 3.5
and 4.0, respectively. The organic solvents, n-hexane and
n-heptane, with similar chemical nature presented similar
results, reaching a conversion of 80.1 and 84.5% at 48 h,
respectively. Based on RDC No. 2, from ANVISA,12 the
use of solvents is allowed in the preparation of favouring
in prescribed quantities, among which acetone, n-hexane
and toluene are allowed in small concentrations. However,
n-hexane can be used for food applications,13 and was
selected for further studies to optimize the reaction
parameters on isoamyl butyrate synthesis.
Synthesis of the ester was carried out in a 100 mL
stoppered flask, with a working volume of 40 mL of
n-hexane, containing glass beads and the required
concentrations of isoamyl alcohol and butyric acid
according to the experimental designs. An appropriate
enzyme concentration was added to the freshly prepared
reaction mixture, which was incubated in an orbital shaking
incubator (Tecnal TE-420) at 180 rpm at the specified
temperatures.
Determination of the percent esterification
Aliquots of the reaction mixture were withdrawn at
definite time intervals (3 h, 6 h, 18 h, 24 h and 48 h) and the
extent of esterification monitored by a titration procedure
to estimate the decrease in total acid content of the reaction
mixture. Titration was carried out using 0.02 mol L-1
standard potassium hydroxide with phenolphthalein as the
indicator14,15 and 3 mL of ethanol used as the quenching
Experimental
Enzyme
The biocatalyst employed was the commercial microbial
lipase Lipozyme TL IM (53.1 Ug-1), immobilized on silica

2150
Enzymatic Synthesis Optimization of Isoamyl Butyrate
J. Braz. Chem. Soc.
agent to determine the residual acid content.2 The percent
esterification was calculated from the concentration of
acid consumed in the reaction mixture, determined from
the values obtained in the titration for both the blank and
the test sample,16 using equation 1:
satisfying desired specifications, and which values (within
the range) of the factors will yield a maximum for a specific
response.7 A quadratic response surface model was fitted to
equation 3.
Y = β0 + Σ βixi + Σ βiix2ii + Σ βijxixij + ε
(3)
C0 – C
C0
(1)
molar % =
×100
where Y is the dependent variable (response variable)
to be modelled, xi and xj are the independent variables
(factors), β0, βi, βii and βij are the regression coefficients
of the model and ε is the error of the model (residual
term). Where possible, the model was simplified by
dropping terms which were not statistically significant
(p > 0.05) according to the analysis of variance.20 The
value for R2 indicates the percent of the variability in the
optimization parameter that is explained by the model.
Three-dimensional surface plots were drawn to illustrate
the main and interactive effects of the independent
variables on the dependent ones.6
where: C0 = concentration of free fatty acid residues at 0 h;
C = concentration of free fatty acid residues after time t.
Determination of ester concentration
Ester concentration was defined as the ratio of ester
amount (mmol) to total amount of substrates (grams
of isoamyl alcohol plus grams butyric acid), [mmolester
grammixture],17 and converted to mol L-1.
/
Experimental design and optimization by RSM
Statistical analysis
The infuence of various reaction parameters such
as temperature, enzyme concentration, butyric acid
concentration, alcohol:acid molar ratio and shaking rate in
the synthesis using Lipozyme TL IM, was evaluated using a
2v5-1 fractional factorial design,18 with central points, giving
a total of 20 trials, to study the percent esterification and
ester concentration (Table 1).
All the data were analysed with the aid of Statistica 7.0
(Statsoft, Inc., Tulsa, OK, USA).
Results and Discussion
2v5-1 Fractional factorial design
According to the results of the first factorial design,
a second experimental design was used to identify the
optimum levels of the parameters for the synthesis of
isoamyl butyrate (Table 2).A 23 central composite rotatable
design (CCRD) with a star configuration (2 axial points)
and 4 central points, giving a total of 18 trials, was used
to obtain a second order model. The distance of the axial
points was 1.68 as calculated from equation 2.19
Effect of the reaction parameters on the percent esterification
Monitoring of the enzymatic synthesis was carried
out for 48 h. However, after 18 h there was no significant
increase for most of the trials, and therefore this time was
chosen to analyse the effect of the reaction parameters
on the responses. The maximum percent esterification
after 18 h of reaction was 94.0% (Table 1) for trial 14 at
50 ºC, with 0.1 mol L-1 butyric acid, a concentration of
0.020 g mL-1 of Lipozyme TL IM, an alcohol:acid molar
ratio of 3:1 and a shaking rate of 180 rpm.
α = (2n)1/4
(2)
where α is the distance of the axial points and n is the
number of independent variables.
The variables studied in the CCRD were temperature
(30-50 ºC), substrate concentration (0.1-0.9 mol L-1) and
enzyme concentration (0.005-0.025 g mL-1).
RSM is used in the empirical study of relationships
between one or more measured responses and a number
of input variables, and the objective is to optimize the
response(s). It offers solutions to critical questions such
as how a particular response is affected by a given set
of input variables over some specified region of interest,
what settings of factors will give a product simultaneously
In a previous study, Aragão et al.,11 investigated the
isoamyl butyrate production using free and immobilized
lipases by esterification of butyric acid with isoamyl
alcohol in a solvent-free system and in an organic media.
Among the studied enzymes, Lipozyme TL IM was found
to be the most active catalyst in n-hexane as a solvent.
The effects of different solvents and the amount of water
added on conversion rates were studied. A maximum
conversion yield of 80% in n-hexane at 48 h was obtained
under the following fixed conditions: 0.003 g mL-1 of
Lipozyme TL IM, 30 ºC, 180 rpm, alcohol:acid molar ratio
of 1:1 and 0.6 mol L-1 butyric acid.

Vol. 22, No. 11, 2011
Anschau et al.
2151
In trial 4 the responses were very low during the reaction
time, reaching 0% after 18 h. This may have occurred
because of reversibility (hydrolysis) when using such a low
enzyme concentration (0.003 g mL-1) or also due to the high
acid concentration (0.5 mol L-1) as well as in trials 3 and 11.
Macedo et al.,21 obtained similar results for the synthesis of
isoamyl butyrate when it was used an alcohol:acid molar
ratio of 4:1 and only 1% of lipase from Geotrichum sp. Using
these parameters the authors obtained about 7% conversion
in 72 h. However, using the same molar ratio and an enzyme
concentration of 10%, the conversion reached 50% in 72 h.
Intrial11, withlowenzymeconcentration(0.003gmL-1),
high acid concentration (0.5 mol L-1) and high alcohol:acid
molar ratio (3:1), the maximum esterification of 69.6% was
obtained after 6 h then decreased, reaching to 0% at the
end of the trial (18 h). It may be that around the critical
molar ratio, competitive alcohol binding reduces formation
of the acyl-enzyme complex, resulting in a decrease in
alcoholysis. This observation may also refect the ability
of excess alcohol to distort the essential water layer from
around the enzyme molecules.22
Figure 1a, where increasing the reaction temperature,
enzyme concentration and shaking rate from their lowest
levels to their highest levels had a positive effect on
synthesis, increasing, on average by 19%, 42% and 12%
at 18 h, respectively.
The opposite occurred with the isoamyl alcohol:butyric
acid molar ratio and the butyric acid concentration,
both of which had a negative effect, causing a decrease
of, on average, 8% and 38% at 18 h, respectively. The
negative effect of the butyric acid concentration indicates
the importance of working at low levels of acid and
alcohol.
Effect of the reaction parameters on the ester concentration
From the monitoring of the synthesis, the highest
isoamyl butyrate concentration of 1.4 mol L-1 was shown
to occur at 18 h in trial 8 (Table 1). The parameters
temperature, enzyme concentration and butyric acid
concentration exerted a positive effect on the synthesis of
isoamyl butyrate as they increased from their lower levels
to their highest levels (Figure 1b). The molar ratio had a
small negative effect and an increase in shaking rate showed
no significant infuence (p > 0.05).
The entire relationship between the reaction factors
and the response can be understood better by examining
Table 1. 2v5-1 Fractional factorial design with the real values for the variables and the percent esterification and ester concentration after 18 h
Variables
E / (g mL-1)
0.003
Responses
Trial
T / °C
30
50
30
50
30
50
30
50
30
50
30
50
30
50
30
50
40
40
40
40
S / (mol L-1)
0.1
Alcohol:acid molar ratio
V / rpm
180
50
PE
EC
0.2
0.3
0.0
0.0
0.2
0.2
1.0
1.4
0.1
0.2
0.0
0.4
0.3
0.3
0.4
1.1
0.2
0.2
0.2
0.2
1
1:1
1:1
1:1
1:1
1:1
1:1
1:1
1:1
3:1
3:1
3:1
3:1
3:1
3:1
3:1
3:1
2:1
2:1
2:1
2:1
85.7
84.8
3.2
2
0.1
0.003
3
0.5
0.003
50
4
0.5
0.003
180
50
0.0
5
0.1
0.020
78.1
83.2
89.5
91.8
30.1
87.9
0.0
6
0.1
0.020
180
180
50
7
0.5
0.020
8
0.5
0.020
9
0.1
0.003
50
10
11
12
13
14
15
16
17
18
19
20
0.1
0.003
180
180
50
0.5
0.003
0.5
0.003
26.4
93.4
94.0
28.3
92.3
31.8
29.5
30.7
32.6
0.1
0.020
180
50
0.1
0.020
0.5
0.020
50
0.5
0.020
180
115
115
115
115
0.3
0.0115
0.0115
0.0115
0.0115
0.3
0.3
0.3
PE = percent esterification (%); EC = ester concentration (mol L-1).

2152
Enzymatic Synthesis Optimization of Isoamyl Butyrate
J. Braz. Chem. Soc.
Figure 1. Effects of the reaction parameters on (a) percent esterification and (b) ester concentration in the synthesis of isoamyl butyrate. T: temperature;
BA: butyric acid concentration; E: enzyme concentration; MR: alcohol: acid molar ratio; SR: shaking rate.
Similar results were obtained by Güvenç et al.,17 who
studied the effect of enzyme concentration. An increase in
the enzyme concentration from 2.5 to 10% (m/m) resulted
in an increase from 0.7 to 3.2 mol L-1 in the synthesis of
isoamyl acetate when the lipase Novozymes 435 was used
in a solvent-free system, showing that the effect of enzyme
concentration is important in the synthesis.
The variables temperature, enzyme concentration and
butyric acid concentration were selected and studied using
a CCRD (Table 2). Despite the positive effect of increasing
the temperature level, this parameter was maintained at
the same level, due to the risk of enzyme denaturation.
The enzyme and butyric acid concentration levels were
expanded due to the positive effects shown.
The isoamyl alcohol:butyric acid molar ratio was fixed
at 1:1 (level -1) in the CCRD, since the change from 1:1 to
3:1 showed a negative effect, reducing esterification to 8%.
Although an increase in the shaking rate resulted in a greater
percent esterification, its variation was not statistically
significant for the ester concentration, and therefore it was
set at 180 rpm (level +1).
After 18 h of reaction time there was no significant
increase in the percent esterification and ester concentration
in most of the trials, and, therefore, this time was chosen to
optimize the synthesis. Table 2 shows the variables in real
values, percent esterification, ester concentration and values
predicted by the model, plus their standard deviations.
The ANOVA (Table 3) indicated that the model
was significant and adequate to represent the current
relationship between the responses and the significant
variables, showing a very small p-value (0.05) and a
satisfactory coefficient of determination (p-values below
0.05 indicate that the terms of the model are significant).22
The pure error was very low, indicating good
reproducibility of the data obtained. The Fisher F-test
also demonstrated very high significance for the
regression model, since the F-value computed (30.98)
was much higher than the tabulated F-value (3.39) for the
percent esterification at the 5% level. The coefficient of
determination (R2) was 0.972, which indicates that only
2.8% of the overall variation was not explained by the
model. Thus the model adequately represented the real
relationships between the selected parameters, and similar
results were obtained for ester concentration. From the
analysis of variance (Table 3), the coefficient of correlation
obtained for the percent esterification and the result of the
F-test (9.1 times higher than Ftab) were good indicators of
a representative model of the current relationship amongst
the selected reaction parameters (equation 4).
Model fitting
23 Central composite rotatable design (CCRD)
Response surface methodology consists of an empirical
modelization technique, which can be used to evaluate the
relationship between experimental and observed results.
In order to determine whether the quadratic model is
significant or not, it is necessary to carry out an regression
analysis and variance (ANOVA) using the usual Fisher
F-tests, in which the sum of squares in the regression is
subdivided into two parts that attributed to linear regression
and that attributed to the quadratic model.7
For the ester concentration, the correlation coefficient
obtained was 0.926 and the ratio of Fcalc/Ftab was 3.27,
indicating that the second order model was also statistically
significant and predictive (equation 5).
According to the t and p values from regression analysis,
all the variables have statistical significance on the percent

Vol. 22, No. 11, 2011
Anschau et al.
2153
Table 2. 23 Factorial designwith the real values for the variables and the percent esterification and ester concentration after 18 h plus the values predicted
by the model and the standard deviations
Response value for percent
esterification / (%)
Response value for ester
concentration / (mol L-1)
Variables
Trial
T / ºC
34
46
34
46
34
46
34
46
30
50
40
40
40
40
40
40
40
40
S / (mol L-1) E / (g mL-1)
PE
PV
SD
5.9
4.1
6.7
26.0
3.7
5.3
2.7
0.7
0.4
9.1
3.8
17.6
44.9
0.2
0.6
0.4
0.6
0.0
EC
0.5
0.5
0.3
0.6
0.5
0.5
1.0
1.5
1.1
0.9
0.2
0.4
0.2
0.7
0.7
0.7
0.7
0.7
PV
0.6
0.5
0.2
0.5
0.5
0.5
1.0
1.3
1.0
1.1
0.1
0.6
0.2
0.8
0.7
0.7
0.7
0.7
SD
17.6
8.8
1
0.26
0.26
0.74
0.74
0.26
0.26
0.74
0.74
0.5
0.009
0.009
0.009
0.009
0.021
0.021
0.021
0.021
0.015
0.015
0.015
0.015
0.005
0.025
0.015
0.015
0.015
0.015
90.2
88.3
16.6
35.8
90.4
91.8
88
84.9
84.7
15.5
26.5
93.8
87
2
3
8.2
4
17.2
4.6
5
6
10.4
2.1
7
85.7
90
8
90.7
93.7
89.4
86.6
29.1
19.2
88.9
89.1
88.9
89.1
88.6
9.9
9
94.1
97.5
89.9
34.2
27.8
88.7
88.5
88.5
88.5
88.5
8.2
10
11
12
13
14
15
16
17
18
0.5
19.1
18.7
27.8
6.8
0.1
0.9
0.5
0.5
13.0
1.1
0.5
0.5
0.9
0.5
1.1
0.5
0.6
PE = percent esterification (%); EC = ester concentration (mol L-1); PV = predicted value; SD = standard deviation (%).
Table 3. ANOVA for the responses of percent esterification and ester concentration
SS
MS
F-valuea
Source of variation
DF
PE
EC
PE
EC
PE
EC
Regression
Residual
Lack of fit
Pure error
Total
12802.65
367.35
367.19
0.17
1.705014
0.136434
0.136423
0.000011
1.841448
9
8
1422.52
45.92
0.189446
0.017054
30.98
11.11
5
3
13170.00
17
R² (percent esterification) = 0.972; R² (ester concentration) = 0.926; aF(0.95; 9; 8) = 3.39; PE = percent esterification; EC = ester concentration; SS, sum
of squares; DF, degrees of freedom; MS = mean square.
responses of esterification and ester concentration. The
application of RSM yielded the following regression model,
which is an empirical relationship between the response
variable and the test variables in real values, where T, S
and E are the temperature, butyric acid concentration and
enzyme concentration, respectively.
The sensitivity of the esterification conditions to enzyme
concentration, butyric acid concentration and temperature
can be studied from these response surfaces. The best way to
predict the relationships between the responses, parameters
and their interactions is to analyze the contour plots generated
from the predicted model.23 Each contour curve represented
an infinite number of combinations of two test variables,
while the other one remained at its respective zero-level.
From the analysis of the response surfaces and contour
plots for the percent of esterification it appears that an
increase in the butyric acid concentration decreases the
percent esterification at any point in the temperature range
studied, and the greatest percent esterification occurred
at 0.26 mol L-1 butyric acid. In general, increments in
Percent esterification (%) = 173.73 – 6.12T + 0.07T2 –
143.04S – 163.66S2 + 8.54E – 0.3E2 + 1.94TS –
0.05 TE + 10.64SE
(4)
Ester concentration (mol L-1) = 6.37 – 0.29T + 0.01T2 –
1.76S – 2.28S2 + 0.01E – 0.09E2 + 0.06TS + 0.01TE +
0.14SE
(5)

2154
Enzymatic Synthesis Optimization of Isoamyl Butyrate
J. Braz. Chem. Soc.
the butyric acid concentration lower the esterification
capacity of lipases. This effect has been reported for the
biosynthesis of isoamyl acetate isoamyl isovalerate and
for short-chain fatty acid ethyl esters in general.6 There
may be two reasons for the lower conversions reported
at higher butyric acid concentration: the accumulation of
water during the progress of the reaction, which favours
hydrolysis (the reverse reaction) or, more likely, inhibition
by the higher acid or alcohol concentrations. It has been
asserted that alcohols are terminal inhibitors of lipases and
acids, and may cause acidification of the micro-aqueous
interface, leading to enzyme inactivation.24,25
At a fixed temperature, esterification showed little
variation with increased enzyme concentrations, but with
0.021 g mL-1 enzyme, a temperature of 30 °C, and the
other conditions at the central point, total esterification
from the acid substrate could occur. Even an increase in
enzyme concentration had no significant effect at the higher
temperatures. Hence, the results show the beneficial effect
of using room temperature for the esterification reaction,
in order to achieve maximum conversions. Krishna et al.26
suggested that high temperatures reduced the operational
stability of the enzyme. Furthermore, the use of low
temperature is beneficial so that the power costs can be
reduced and the enzyme stability can be preserved during
prolonged operations.
Figure 2a shows the effects of the enzyme and butyric
acid concentrations on the percent esterification. It was
observed that when the butyric acid concentration was
high (+1.68 level), the reaction hardly occurred with a low
enzyme concentration, possibly due to enzyme denaturation
or substrate inhibition. It has been asserted that acids may
cause acidification of the micro-aqueous interface, leading
to enzyme inactivation.24,25 The highest values for percent
esterification were found between 0.15 and 0.6 mol L-1
with the enzyme concentration in the range from 0.012 to
0.024 g mL-1.
Enzyme concentration is known to be an important
variable in esterifications for the synthesis of various fatty
acid esters. The positive effect of enzyme concentration
on the synthesis of butyric ester is in agreement with
the reports of other authors dealing with the production
of favour esters (ethyl hexanoate, isoamyl isovalerate,
isoamyl acetate) using microbial lipases.24,27,28
Figure 2. Contour plots from (a) percent esterification (%) and (b) ester
concentration (mol L-1) obtained by equations 4 and 5, respectively, as a
function of enzymatic and butyric acid concentration.
Figure 2b shows the contour plot for the isoamyl butyrate
concentration as a function of enzyme concentration and
butyric acid concentration. The ester concentration was
higher at enzyme concentrations above 0.021 g mL-1 and
butyric acid concentrations between 0.7 and 0.9 mol L-1.
Considering the two evaluated responses and the
obtained contour plots, the optimized condition was shown
to be an enzyme concentration of 0.021 g mL-1 (level +1),
a butyric acid concentration of 0.5 mol L-1 (level 0),
temperature of 30 ºC (level –1.68), alcohol:acid molar ratio
of 1:1 and shaking rate of 180 rpm. Under these optimum
conditions the conversion rate was shown to be 92% and
the ester concentration 0.9 mol L-1.
Nogales and Contreras,6 used response surface
methodology to optimize the synthesis of ethyl butyrate in
an organic medium (n-heptane) using the immobilized lipase
Novozymes 435. They obtained values for esterification
above 70% with a butyric acid concentration of 0.04 mol L-1
and enzyme concentration of 7% (m/m) at 34 °C.
Atatemperatureof30ºC, thehighestesterconcentrations
were found with a butyric acid concentration ranging from
0.3 to 0.7 mol L-1 and an enzyme concentration between
0.015 and 0.025 g mL-1, respectively. At a temperature
of 50 ºC, the highest values for ester concentration were
found in the range from 0.5 to 0.9 mol L-1 with an enzyme
concentration from 0.015 to 0.025 g mL-1.
The present results are in good agreement with their
investigation. Both investigations gave similar results,

Vol. 22, No. 11, 2011
Anschau et al.
2155
even though different types of substrate and lipase were
employed in the studies.
respect to the percent esterification and ester concentration
after18 h.
Thus, it was shown there was no significant difference
between the optimized conditions using commercial
isoamyl alcohol or using that extracted from fusel oil,
making the use of this by-product from the alcohol industry
a feasible option. If the levels of isoamyl alcohol produced
biotechnologically from fusel oil are high enough to make
it commercially available, this would be an alternative
way of obtaining natural isoamyl butyrate from cheap
agricultural residues.
Using the isoamyl alcohol from fusel oil in a solvent
free system with the commercial immobilized lipase
Novozym 435, Güvenç et al.7 enhanced the synthesis of
isoamyl acetate by analyzing a second order model using
response surface methodology, obtaining the following
optimal conditions: acid/alcohol mole ratio of 0.8 enzyme
concentration of 12% (m/m), 30 °C, 150 rpm, a reaction
system of 6 mL and 8 h of synthesis, obtaining a response
of 3.8 mol L-1 ofester concentration.
A comparative study between the optimum conditions for
use with commercial isoamyl alcohol and with isoamyl
alcohol from fusel oil
Figure 3a shows the comparison between the results
obtained under the optimized conditions using commercial
isoamyl alcohol and isoamyl alcohol obtained from the
distillation of fusel oil as the substrates. It can be seen in
Figure 3a that the percent esterification was 92% and 93%
after 18 h, respectively. Figure 3b shows the monitoring
of the ester concentration, where the use of the optimized
conditions and commercial isoamyl alcohol as the substrate
resulted in 0.9 mol L-1 after 18 h, and isoamyl alcohol
from fusel oil as the substrate, 1.0 mol L-1. The ANOVA
followed by the Tukey Test showed there was no significant
difference (p < 0.05) between the two isoamyl alcohols with
Bi et al.29 studied the lipase catalyzed production of
isoamyl acetate in a solvent of n-hexane. The isoamyl
alcohol obtained from fusel oil and acetic acid were
used as the reactants. The favourable reaction conditions
for maximum ester production (92%) were an enzyme
concentration of 9%, an acid:alcohol molar ratio of
1:2 mol L-1, temperature of 40 °C and reaction time of 6 h.
The environmental impact of biotechnological utilization
of food industry and agricultural wastes and by-products
is twofold: the utilization for manufacture of value-added
products on the one hand, and the environmentally safe,
waste-free biotechnological process using mild conditions
on the other.9 Owing to its rich alcohol composition and
availability in large quantities, fusel oil can be considered
an inexpensive source of starting material for the production
of important natural favor compounds.8
The enzymatic synthesis of isoamyl butyrate was carried
out using Lipozyme TL IM in an organic media. From an
evaluation of the effects of the fractional factorial design on
the synthesis of isoamyl butyrate, the variable that exerted the
greatest infuence was the enzyme concentration, followed
by the butyric acid concentration and the temperature.
Response surface methodology was used to optimize the
percent esterification and ester concentration. The optimized
condition considering the percent esterification and ester
concentration was the following: enzyme concentration
of 0.021 g L-1, butyric acid concentration of 0.5 mol L-1,
temperature of 30 ºC, alcohol:acid molar ratio of 1:1 and
shaking rate of 180 rpm. By comparing the use of the
optimized condition with commercial isoamyl alcohol as the
substrate and with that obtained from the distillation of fusel
Figure 3. Time course of the synthesis of isoamyl butyrate under the
optimized condition using commercial isoamyl alcohol (––) and
isoamyl alcohol obtained from fusel oil (––) with respect to the percent
esterification (a) and ester concentration (b). The values represent the
means of three independent experiments and the error bars indicate the
standard deviations.

2156
Enzymatic Synthesis Optimization of Isoamyl Butyrate
J. Braz. Chem. Soc.
oil, the percent esterification after 18 h was 92% and 93%,
respectively, and the ester concentration was 0.9 mol L-1
and 1.0 mol L-1. As a result, it can be said that the fusel oil
generated as a by-product during the production of alcohol
can be recycled by a green chemical process such as the one
used in this study.
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Published online: September 23, 2011