Lipase-catalyzed synthesis of ethyl hexanoate in microemulsion system

Article abstract of DOI:10.14233/ajchem.2013.15126

This paper studied lipase-catalyzed synthesis of ethyl hexanoate in dodecylbenzenesulfonic acid/isooctane/water microemulsion system. The effect of several parameters, such as w0 ([H₂O]/[surfactant]) value, reaction time, reaction temperature, oil phase solvent, buffer solution pH value of microemulsion system on the esterification have been investigated. The results showed that the best experimental conditions for catalytic synthesis ethyl hexanoate were as follows: w0 = 4, reaction time 4 h, reaction temperature 40 °C, buffer solution pH 7. Under these conditions, the conversion of ethyl hexanoate can reach 98.5 %. Lipase-catalyzed synthesis of ethyl hexanoate in dodecylbenzenesulfonic acid inverse microemulsion system has triple mechanism, namely acid catalyzes, microemulsion catalyzes and enzyme catalyzes.

Full text of DOI:10.14233/ajchem.2013.15126

Asian Journal of Chemistry; Vol. 25, No. 17 (2013), 9675-9678
http://dx.doi.org/10.14233/ajchem.2013.15126
Lipase-Catalyzed Synthesis of Ethyl Hexanoate in Microemulsion System
*
ZHONGQIN TAN, XIAOXIANG HAN , XIAOLI HU, HUAN DU and XIUXIU BAO
Department of Applied Chemistry, College of Food Science & Biotechnology Engineering, Zhejiang Gongshang University, Hangzhou 310035,
P.R. China
*Corresponding author: Tel: +86 571 88071024-7581; E-mail: hxx74@126.com; han-xx74@163.com
(Received: 10 January 2013;
Accepted: 11 October 2013)
AJC-14252
This paper studied lipase-catalyzed synthesis of ethyl hexanoate in dodecylbenzenesulfonic acid/isooctane/water microemulsion system.
The effect of several parameters, such as w0 ([H2O]/[surfactant]) value, reaction time, reaction temperature, oil phase solvent, buffer
solution pH value of microemulsion system on the esterification have been investigated. The results showed that the best experimental
conditions for catalytic synthesis ethyl hexanoate were as follows: w0 = 4, reaction time 4 h, reaction temperature 40 ºC, buffer solution pH
7. Under these conditions, the conversion of ethyl hexanoate can reach 98.5 %. Lipase-catalyzed synthesis of ethyl hexanoate in
dodecylbenzenesulfonic acid inverse microemulsion system has triple mechanism, namely acid catalyzes, microemulsion catalyzes and
enzyme catalyzes.
Key Words: Esterification, Dodecylbenzenesulfonic acid, Microemulsion, Lipase.
The thermodynamically stable, isotropic, optically trans-
parent, highly decentralized microemulsion system that consists
of surfactant, surface active agent, oil and water in certain
proportion, as a micro reactor, has been widely used in many
fields8-14. Not only it can expand the reaction contact area to
improve the reaction rate and the conversion, but also the water
droplets of inverse microemulsion system can simulate the
natural environment of enzyme, make the enzyme maintains
catalytic activity and stability in the water environment. There-
fore, inverse microemulsion system is suitable for lipase
catalytic reaction. At present, lipase catalyzing synthesis of
short chain fatty acid ester in microemulsion has also been
reported in the literature15-17. In this paper, we prepared the
dodecylbenzenesulfonic acid (DBSA)/isooctane/water
microemulsion system and investigated several single factors
in this system on the esterification caproic acid and ethanol.
The results showed that lipase-catalyzed synthesis of ethyl
hexanoate in DBSA inverse microemulsion system with acid
catalysis, microemulsion catalysis and enzyme catalysis triple
mechanism.
INTRODUCTION
The method of producing ester catalyzed by lipase, in the
quality and the smell, is better than traditional method of synthe-
sizing products which have natural quality1-3. In the future,
the application of lipase is an important way to catalytically
synthesize ester. Ethyl hexanoate is colourless or transparently
light yellow liquid and has strong fruit and wine aroma, widely
used in food, beverage, tobacco and wine industry and also
used for cosmetics, soap, shampoo, as everyday life product
spices raw material. Ethyl hexanoate not only has a broad range
of applications, but also with the development of the related
industries, especially the increasing production of wine renders
the increasing demands of ethyl hexanoate. The output of ethyl
hexanoate only used in the luzhou-flavor liquor reached 3000
tons per year all over the country and the production value
achieved hundreds of millions4. At present, ethyl hexanoate is
mainly prepared by hexanoic acid and ethanol using sulfuric
acid as catalyst in industry. But this method has many side
effects, such as serious equipment corrosion, complex acid
liquid post-treatment, difficult catalyst regeneration and
doesn’t belong to the green process. Using these catalysts such
as solid acid, metal salt, alkaline lipase5-7 could solve traditional
catalysts’ shortcomings to some extent, but there were still some
defects such as the low relative activity, easily accumulating
carbon on the surface, low acid density and unequal acid
intensity distribution, which limited their applications.
EXPERIMENTAL
Dodecylbenzenesulfonic acid (DBSA, ≈ 97 % purity) was
purchased from Tokyo Kasei Kogyo Co. Ltd. (Japan). Candida
rugosa lipase (Type VII) was obtained from Sigma Co. Ltd.
Isooctane, cyclohexane, hexane, caproic acid, butyric acid,
isoamyl acid, ethanol, n-butanol and isoamyl alcohol were all

9676 Tan et al.
Asian J. Chem.
0.00
from Shanghai chemical reagent factory (China).All the other
chemicals used were analytical grade and were used without
further purification.
1.00
0.25
0.75
Preparation of lipase microemulsion and the biggest
reaction point: DBSA surfactants acts as a component, oil
phase acts as the second component. First, make the mixture
of surfactants and oil, the oil content in this mixture is called
starting oil content. Then use a liquid gun to titrate dissolved
lipase in water to the mixture in the magnetic blender, to
dissolve all surfactants at the first phase change point, where
solution of this system is homogeneous, generating micro-
emulsion and presenting clear transparent state. After that,
continue adding water, sample transforming from transparent
to turbid, this time at the second phase change point, among a
clear solution called microemulsion.
0.50
0.50
Multiphase area
0.75
0.25
Microemulsion area
1.00
0.00
0.00
1.00
0.25
0.50
Isooctane
0.75
Fig. 1. Three-component phase diagram of DBSA/isooctane/water
microemulsion system
Change with the starting oil content of the surfactant oil
mixture and repeat the above titration procedure, get different
starting oil content samples at the phase change point. Then
calculate the mass ratio of three components surfactant, oil,
water at phase change point, by using Origin software to sketch
three-phase figure and determine the formation regional of
microemulsion system18.
without drop water in any ratio, it didn’t have the first phase
transition point. All experiment were studied in this micro-
emulsion area.
The water content in microemulsion system expressed by
w0, defined as the amount of water divided the amount of
DBSA and had an important influence to esterification. We
have studied the effect of w0 on the esterification conversion
(Fig. 2). Fig. 2 showed that the conversion of hexanoic acid
increased with increasing the amount of water. When w0 = 4,
98.5 % of hexanoic acid was converted into ethyl hexanoate
in 4 h. However, when the w0 exceeded 4, the conversion of
hexanoic acid and yield of ethyl hexanoate decreased slightly.
Take several special points at microemulsion area, work
out every component mass according to the mass ratio, prepare
the microemulsion system. In the same reaction time and under
the same temperature conditions, use lipase catalyzed synthesis
of ethyl hexanoate in this system, respectively. According to
the conversion, get the biggest reaction point of this micro-
emulsion area, this point aims to explore the single factor
influence point of ethyl hexanoate synthesis reaction.
Lipase-catalyzed synthesis of ethyl hexanoate in micro-
emulsion system:Add microemulsion system formed by three
components into 50 mL round bottom flask, stir continuously
in the magnetic blender for 0.5 h to form a clear, transparent
microemulsion system. Then esterification was carried out in
the above microemulsion system by addition of 3 mmol caproic
acid and 6 mmol ethanol.After some time, samples were with-
drawn from the reaction medium and analyzed subsequently
by GC.
100
98
96
94
Gas chromatography analysis:After being analyzed by
gas chromatography, take 200 µL reaction mixtures and a
certain amount of butyl acetate as internal standard into a test
tube, to be dissolved with 5 mL ethanol, attaining a processed
samples. Qualitative study by retention time and quantitative
study by internal standard method, calculate the conversion
per cent of the hexanoic acid.
92
1
2
3
4
5
6
7
w
w0
0
Fig. 2. Results of esterification under different water content w0
Gas chromatograph was equipped with a FID detector
using an Agilent technologies HP-5 column (30 m × 0.32 mm
× 0.25 mm). Injection port temperature is 240 ºC, detector
temperature is 260 ºC, carrier gas is nitrogen. The temperature
program used began at 50 ºC for 2 min, then increased to 260 ºC
at a gradient of 20 ºC/min, maintained for 20 min.
The reason of forming this phenomenon as follows:When
w0 was low, the water content of microemulsion system was
low, almost all water in system was combined with surfactant
to form microemulsion and kept enzyme activity. The amount
of water was small which inhibited the enzyme activity. The
conversion of hexanoic acid was low. With the rising of w0,
the water content increased, the extra water increased the
activity of enzyme. Also the produced water in reaction
system could easily enter the water mocrodomain and enlarge
the water droplet. In microemulsion system, the organic
reactions occurred at interface. The larger interface area of
microemulsion provided more opportunity of enzyme and
RESULTS AND DISCUSSION
Effects of microemulsion w0 value: The three-phase
diagram of DBSA/isooctane/water was shown in Fig. 1. Fig. 1
showed that the microemulsion area was small. We also found
that DBSA and isooctane could form transparent solution

Vol. 25, No. 17 (2013)
Lipase-Catalyzed Synthesis of Ethyl Hexanoate in Microemulsion System 9677
100
substrate collision frequency and more completely of the
reaction. Esterification was a dehydration reaction, when w0
was high, so water content was too much in the reaction system,
which made the microemulsion transform into emulsifiable
or unstable system and the phase interface area decreased. This
would lead to the reaction equilibrium towards reverse oppo-
site direction, which facilitated ester hydrolysis and reduced
the conversion of acid.
95
90
85
80
Effects of reaction temperature: Generally, high tempe-
rature could contribute to the enhancement of reaction rate as
well as conversion efficiency. Similar results were also observed
in our enzyme system. The effect of reaction temperature on
the esterification of hexanoic acid with ethanol was shown in
Fig. 3. In the systematic condition of w0 = 4 and molar ratio of
acid to ethanol 1:2, 87.5 % yield of ethyl hexanoate was
obtained at 30 ºC for 4 h. The yield of ethyl hexanoate increased
with a rise in temperature and reached a maximum of 98.5 %
at 40 ºC. Nevertheless, further increase of temperature resulted
in the decrease of yield of ethyl hexanoate. It was owing to
every enzyme had its flexible and optimal reaction temperature
and more or less temperature would constrain the speed of
enzyme catalytic reaction and the stability of enzyme. The
enzyme catalytic reaction was faster while the stability of enzyme
becomed worse with higher temperature. The optimum tempe-
rature was that the enzyme kept high activity and stability.
6.0
6.5
7.0
pH
7.5
8.0
pH
Fig. 4. Results of esterification under different buffer pH
Effects of different solvent: Under the condition of w0 =
4, molar ratio of acid to alcohol 1:2 at 40 ºC, we studied the
effects of different solvent (isooctane, cyclohexane, hexane)
on the esterification of hexanoic acid with ethanol. The results
were listed in Fig. 5. Fig. 5 showed that the different solvent
with DBSA can form the microemulsion and catalyze the
esterification reaction. The conversion of hexanoic acid in
isooctane was the highest, cyclohexane next and hexane was
the last. This might be because of the reactants had different
solubility in these solvent. The higher solubility was, the faster
reaction rate was. So, choose a suitable solvent to improve the
esterification reaction rate and equilibrium conversions are
important.
100
96
92
88
84
100
95
90
85
isooctane
80
30
35
40
45
50
cyclohexane
Temperature (°C)
hexane
75
Fig. 3. Results of esterification under different reaction temperature
70
Effects of buffer solution pH value: There were many
side chain groups of alkaline and acid amino acids in lipase
molecules, these groups had different solubility in acidic or
alkaline solution. The solubility had an important effect on
enzyme combine with substrate and the next reaction, could
also change the space conformation of the enzyme and
enzyme activity. That was, changing the buffer solution pH
value of microemulsion system would alter the conformation
of enzymes, which transformed the enzyme catalytic effect.
Fig. 4 showed the yield of ethyl hexanoate operating at desired
pH value and w0 = 4 at 40 ºC. It could be found 98.5 % of
ethyl hexanoate was achieved at pH of 7 and the yield of ethyl
hexanoate became feeble while the more or less pH was
adjusted. Thus, considering active environment and activity,
the optimum buffer solution pH value should be around 7.
1
2
3
4
Time (h)
Fig. 5. Results of esterification under different solvent
Esterification of different acid and alcohol: The DBSA
system was also applied to esterication with other alcohols
and acids as substrate and the results were displayed in Fig. 6.
As shown in Fig. 6, when a mixture of ethanol with butyric
acid (isovaleric acid and hexanoic acid) and a mixture of
hexanoic acid with ethanol (butanol and isoamyl alcohol) were
subjected to esterification in DBSA microemulsion system at
313 K for 4 h, these five equlibrium conversions were 98.2,
94.1, 98.5, 92.5 and 96.0 %, respectively.
These results indicated that the conversion of substrates
decreased at the equilibrium position with the alkyl chain and

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better than hexanoic acid. DBSA, as a kind of surfactant, can
also form thermodynamic stability microemulsion system,
therefore, the combination of them not only possesses acid
catalysis property, but also the collision opportunities of the
reactant increase due to the large phase interface area. At the
same time, the water produced by esterification reactions can
be dissolved into the reverse micelle internal pool, which made
the esterification reaction balance move to product direction
and improved the esterification conversion. As a result, the
equlibrium conversion in this system was higher (91.7 %, 5 h)
than that in separate system. From Fig. 7, it is also shown that
the conversion of DBSA microemulsion system with lipase
was higher than without lipase system. Lipase system could
shorten the esterification reaction time of reaching the highest
conversion. This result also indicated that DBSA micro-
emulsion system with w0 = 4 could be helpful for lipase playing
its activity and enhanced the reaction conversion.
95
90
85
80
A
B
C
D
E
Fig. 6. Results of esterification under different acid with alcohol. A = ethyl
butyrate; B = ethyl isovalerate; C = butyl hexanoate; D = isoamyl
hexanoate; E = ethyl hexanoate
Based on the above experimental results, it can be con-
cluded that DBSA microemulsion system with lipase is the
most effective system for the esterification conversion under
mild conditions and have acid catalysis, microemulsion
catalysis and enzyme catalysis triple mechanism.
increased branch chain. This was because straight chain acid
with alcohol, their space resistance was small, easy to collision
and combine and conversion was high. But butyl hexanoate
was also a straight chain ester, the situation was special and
the specific reason was a subject of research. Based on the
above results, we found that the DBSA reverse microemulsion
system was preferential to esterification of the straight chain
acid and short alkyl alcohol.
Conclusion
We have described esterification reaction in DBSA/isooc-
tane microemulsion system with lipase. This reaction system
was found to be applicable to several esterification reactions
under mild conditions. 91.7 % yield of ethyl hexanoate was
reached in DBSA microemulsion system without lipase after
5 h on the esterification of ethanol and hexanoic acid. Lipase
in DBSA microemulsion system could improve the conversion
of hexanoic acid (98.1 %) and shorten reaction time (3 h). The
higher catalytic efficiency proved that DBSA microemulsion
with lipase should be a promising reaction system for esterifi-
cation reactions. The proton acid (DBSA) catalyzed, micro-
emulsion catalyzed and enzyme catalyzed ternary mechanism
was proposed in this microemulsion system.
Effects of different reaction system: Subsequently, we
studied the esterification in the different system and compared
them with that in DBSA microemulsion system. The results
were presented in Fig. 7. From Fig. 7, it is evident that the
conversion in DBSA microemulsion system is much higher
than that in other systems with the same substrates concen-
tration after reaction proceeding 5 h.
100
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