Studies on the lipase-catalyzed esterification of alkyl oleates in solvent-free systems

Article abstract of DOI:10.1016/j.molcatb.2013.01.013

The alkyl oleates were prepared by esterification of oleic acid with alkyl alcohols catalyzed by the lipase from Candida sp. 99-125 in solvent-free system. The influence of several factors, including enzyme concentration, temperature, molar ratio between oleic acid and alkyl alcohols, the structure of alcohols and water content, was also investigated. The results indicated that the reactions catalyzed by Candida sp. lipase at 20 °C, in the presence of 5% (w/w) lipase, on the molar ratio of 1:1 between oleic acid and alcohols, afforded products in high yield and showed high selectivity to primary alcohols. The enzymatic synthesis gave purer products, compared with the conventional chemical synthesis. The lipase from Candida sp. 99-125 was identified to be an effective catalyst in the esterification of alcohol and oleic acid at low temperature.

Full text of DOI:10.1016/j.molcatb.2013.01.013

Journal of Molecular Catalysis B: Enzymatic 90 (2013) 114–117
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Studies on the lipase-catalyzed esterification of alkyl oleates in
solvent-free systems
Hui Zhonga, Zheng Fangb, Baohua Zoub, Xin Lia, Pingkai Ouyanga,c, Kai Guoa,∗
a College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, PR China
b School of Pharmaceutical Sciences, Nanjing University of Technology, Nanjing 211816, PR China
c National Engineering Research Center for Biotechnology, Nanjing University of Technology, Nanjing 211816, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The alkyl oleates were prepared by esterification of oleic acid with alkyl alcohols catalyzed by the lipase
from Candida sp. 99-125 in solvent-free system. The influence of several factors, including enzyme con-
centration, temperature, molar ratio between oleic acid and alkyl alcohols, the structure of alcohols and
water content, was also investigated. The results indicated that the reactions catalyzed by Candida sp.
lipase at 20 ◦C, in the presence of 5% (w/w) lipase, on the molar ratio of 1:1 between oleic acid and alco-
hols, afforded products in high yield and showed high selectivity to primary alcohols. The enzymatic
synthesis gave purer products, compared with the conventional chemical synthesis. The lipase from Can-
dida sp. 99-125 was identified to be an effective catalyst in the esterification of alcohol and oleic acid at
low temperature.
Received 11 October 2012
Received in revised form 10 January 2013
Accepted 14 January 2013
Available online 24 January 2013
Keywords:
Enzymatic esterification
Candida sp. lipase
Oleates
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
maltose, leucrose, maltotriose and n-dodecyl maltosides in 2000
[6]. In 2006, Tan et al. also synthesized 2-ethylhexyl palmitate via
Recently, fatty acid esters, which have surfactant and com-
bustible properties, have received widespread attention from
scientists due to their wide applications in cosmetics, pharma-
ceutics as well as food and chemical industries [1]. Especially,
biodiesels, which is defined as the mono-alkyl esters of long chain
fatty acids, have received an increasing attention from researchers
with the global shortage of fossil fuels, the increasing price of crude
oil and environmental pollution concerns. Nowadays, the majority
of biodiesel is produced by base-catalyzed trans-esterification of
edible oils with methanol, which has some shortage such as the
complicated procedure, more methanol content, hard to reclaim,
and environmental pollution [2]. Therefore, the lipase-catalyzed
synthesis has become a promising method to synthesize biodiesels
owe to the advantages of mild conditions, simplified downstream
processing, high region- and stereo-selectivity, low energy con-
sumption, and environmental friend over the chemical catalysis
[3].
an immobilized lipase membrane reactor [7]. The most recently
reported work of the enzymatic synthesis is the syntheses of
fructose, sucrose and lactose esters from the corresponding sugars
using Candida antarctica type B lipase immobilized in two different
supports, namely acrylic resin and chitosan by Goncalves’ group
this year [8]. Due to the limitation of enzyme catalyzed reactions
including high cost of enzyme, low yield, a long reaction time, the
demand for investigation on the details of the enzymatic synthesis
was still pressing. Herein, we report the esterification using one
cheap lipase from Candida sp. 99-125 as an efficient catalyst at 1:1
reactant ratio at low temperature. In addition, the full investigation
and optimization of the esterification are performed.
2. Experimental
2.1. Materials and reagents
The lipase-catalyzed syntheses of oleates were reported by
several groups. In 1996, the lipase-catalyzed syntheses of fruc-
tose oleates and sucrose fatty acid esters have been reported by
Ghoul’s group and Vulfson’s group, respectively [4,5]. Ferrer et al.
described the region-selective synthesis of fatty acid esters of
The lipase from Candida sp. 99-125 was presented by Beijing
University of Chemical Technology. Oleic acid was purchased from
Shanghai Lingfeng Chemical Reagent Co., Ltd. All reagents, includ-
ing methanol, ethanol, i-propanol, n-butanol, and n-octanol, were
of analytical grade and purchased from Shanghai Lingfeng Chemical
Reagent Co., Ltd.
Chromaticity was recorded on the PFX-i Series SpectroColorime-
ter from Tintometer Ltd., Amesbury, SP4 7SZ, UK.
∗
Corresponding author. Tel.: +86 25 58139926.
E-mail address: xinxin2019@hotmail.com (K. Guo).
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.01.013

H. Zhong et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 114–117
115
2.2. Reaction procedures
conversion. This could be explained by the increase in amount of
the substrate bonded to the enzyme with the increase in enzyme
amount. When the lipase concentration was higher than 5%, the
increase of conversion was not remarkable as not all the enzyme
particles were exposed to the substrates and the excess of enzyme
present in the reaction mixture was not actively involved in the
reaction, consistent with other reports [9]. The conversion of ester-
ification completed in 4 h and 2 h, at the lipase concentration of 5%
(w/w) and 10% (w/w) respectively. Considering the price of lipase,
it was concluded that the 5% (w/w of substrates) was the optimal
concentration for the esterification. Operating at this condition a
total esterification percentage of about 55% after 24 h of bioconver-
sion was attained. Therefore, all further studies in this work were
performed at this enzyme concentration.
2.2.1. General procedures of esterification
All the esterification experiments were carried out in a 50 mL
round bottom flask. The reaction procedure was described as fol-
lows: To a mixture of 0.05 mol oleic acid and corresponding alcohol,
catalyst was added at the given temperature. Normally, the reac-
tants mixture kept stirring for 24 h until the reaction completed.
The samples were taken out every 1 h in the first 12 h, and the acid
value was determined according to the standard GB1668-81. In the
end, the chromaticity of the oleates was recorded on PFX-i Series
from Tintometer Ltd.
2.2.2. Analytical methods
2.2.2.1. Determination of acid number. The ester content was quan-
tified by calculating the residual fatty acid amount in the reaction
mixture. Using a volumetric method (standard GB1668-81), a
0.2–0.3 g sample of the reaction mixture was diluted in 20 mL
of 0.1% (w/w) phenolphthalein solution in absolute ethanol and
titrated with standardized potassium hydroxide solution in water.
The acid number (AN) was calculated from the equation:
3.2. Effect of temperature
It is well-known, that temperature is an important parameter
affecting reaction rate, enzyme activity and chemical equilibrium.
The effect of temperature was studied by varying the temperature
in the range between 10 ◦C and 60 ◦C, with the lipase concentration
of 5% (w/w) and acid/alcohol ratio of 1:3, at the rate of 400 rpm stir-
rer speed. The results are illustrated in Fig. 2. In contradiction to the
generally reported literatures [7,10,11], the lipase from Candida sp.
99-125 had high catalytic activity at 20 ◦C instead of 40 ◦C. How-
ever, the results were in good agreement with the Tan’s report [12]
in which the optimal temperature for the immobilized lipase from
Candida sp. 99-125 ranged from 15 ◦C to 25 ◦C. All the experiments
here were carried out three times to show the reproducibility. The
result suggested that the lipase from Candida sp. 99-125 had high
catalytic activity at room temperature, which means it would have
a wider application in industry. Additionally, the esterification at
10 ◦C was also conducted, which resulted in a low conversion due to
the coagulation of oleic acid. Therefore, the further reactions were
operated at 20 ◦C.
56.1 × V × N
AN =
W
V, volume of NaOH, (mL); N, molarity concentration of titrant
(mol/L); W, weight of the sample in grams.
2.2.2.2. Calculation of esterification rate.
ANorg. − ANeq
esterification rate (%) =
× 100%
ANorg.
ANorg., acid number at the starting point; ANeq, acid number at the
checking point.
3. Results and discussion
3.1. Effect of lipase concentration
3.3. Effect of the molar ratio between oleic acid and alcohols
The investigation of the lipase concentration influence on the
esterification rate was performed by varying the concentration of
lipase in the range between 0.5% and 10% (w/w of substrates) in
the reaction bulk of 0.05 mol oleic acid and 0.15 mol ethanol at
30 ◦C, with magnetic stirring rate of 400 rpm. As shown in Fig. 1,
the lipase concentration affected the initiate rate as well as the final
Acid/alcohol molar ratio is one of the most important param-
eters in enzymatic esterification. Since the reaction is reversible,
an increase in the alcohol concentration should result in higher
ester yields and shift the chemical equilibrium toward the ester
Fig. 2. Effect of temperature on the esterification. Oleic acid, 0.05 mol/L; ethanol
alcohol, 0.15 mol/L; lipase, 5%; speed, 400 rpm. (ꢀ) 20 ◦C; (᭹) 30 ◦C; (ꢁ) 40 ◦C; (ꢂ)
50 ◦C.
Fig. 1. Effect of lipase concentration on esterification rate. (ꢀ) 0.5% (w/w); (᭹) 1%
(w/w); (ꢁ) 3% (w/w); (ꢂ) 5% (w/w); (ꢃ) 10% (w/w).

116
H. Zhong et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 114–117
Fig. 4. Effect of alcohol structure on the esterification.
Fig. 3. Effect of the ratio of acid/alcohol on the esterification. Lipase, 5%; tempera-
ture, 20 ◦C; speed, 400 rpm.
the increasing length of carbon chain, as shown in Fig. 4. As we
know, short chain alcohols, such as methanol and ethanol are toxic
to the lipase. However, the synthesis of ethyl oleate catalyzed by the
lipase from Candida sp. 99-125 was reported by Tan’s group [12].
It is deduced that ethanol mere inhibits the lipase from Candida sp.
99-125 but no deactivate, so does methanol. The lipase from Can-
dida sp. 99-125 was higher sensitive to methanol compared with
ethanol. This could explain why the esterification between oleic
acid and methanol afford a low conversion while that between oleic
acid and ethanol gave a high conversion.
synthesis. However, high substrates concentration may slow down
the reaction rates due to inhibition to the enzyme.
To investigate the effect of molar ratio between oleic acid and
alcohols, the reactions were run by varying the amount of ethanol,
which was supposed to be an inhibitor to the lipase from Can-
dida sp. 99-125, whilst keeping a constant amount of oleic acid
of 0.05 mol, with magnetic stirring rate of 400 rpm at 20 ◦C, in
the presence of 5% (w/w) lipase. The results are demonstrated
in Fig. 3. The esterification of an equimolar solution of oleic acid
and ethanol reached to the chemical equilibrium fast and afforded
the highest conversion of 94%, compared with 66% conversion
of the esterification at the reported optimum ratio of 1:2 [13].
It could be explained as following description. As we know, an
increase in amount of alcohol will shift the chemical equilibrium
to the product point as well as increase the reaction rate. These
manifest themselves as an increased conversion of esterification
over a specified reaction time. Meanwhile, an increased amount
of ethanol will decrease the reaction rate due to the inhibition
on the enzyme. It manifests itself as the decreased conversion of
esterification for a specified period of reaction. At the low ethanol
concentration, inhibition of alcohol to the enzyme predominate the
reaction. While the ethanol concentration increased, the contribu-
tion of alcohol to the reaction equilibrium dominated the reaction.
The esterification at 1:1 the acid/alcohol ratio gave the highest
conversion, because the enzyme had the highest activity without
inhibition from excess alcohol. As the acid/alcohol ratio decreased
till to 1:4, the conversion decreased due to the inhibition. When
ratio of acid/alcohol decreased to 1:5, the conversion was similar
with that of 1:1 acid/alcohol. However, it is obvious that reac-
tion with 1:1 acid/alcohol ratio has huge advantages for industrial
cost saving.
3.5. The effect of water content
Water plays an important role in the enzyme-catalyzed ester-
ification because of the following reasons: Firstly, proper protein
hydration state contributes to the enzyme structural integrity,
active site polarity and protein stability [14,15]. Secondly, water
produced in the esterification affects the reaction equilibrium
position since it is one of the two products in the reaction. The com-
parison of free powdered-lipase-catalyzed reaction in the optimal
condition above with that in the presence of molecular sieve was
performed, with the results as shown in Fig. 5. It indicated that the
molecular sieves removed the water produced in the reaction and
contributed the esterification.
3.4. The effect of alkyl chain of alcohols
The influence of alcohol structure on the esterification was stud-
ied via the esterification of oleic acid and alkyl alcohols, including
methanol, ethanol, propanol, i-propanol, n-butanol, and n-octanol,
at the ratio of 1:1, with 5% lipase at 20 ◦C. The results are demon-
strated in Fig. 4. It was found that straight long-chain contributed
to the reaction well. As shown in Fig. 4, the esterification gave much
lower conversion starting from i-propanol and t-butanol compared
with the others. It is deduced that the primary alcohols, which have
less hindrance to the nucleophilic hydroxyl group, benefited the
esterification. Also, the activity of the enzyme was elevated with
Fig. 5. Effect of water content on esterification between oleic acid and n-butanol
with 1:1 acid–alcohol molar ratio, 5% enzyme, 20 ◦C.

H. Zhong et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 114–117
117
Table 1
Comparison between enzymatic esterification and chemical esterification.
Method
Chemical method
Enzymatic method
Catalyst
Conversion of acid, %
Chroma
p-TsOH
87
87.7
MeSO3H
88
>505
NaHSO4·H2O
54
120.7
Tf-OH
94
133.5
Powdered lipase
96
67.6
Novozyme 435
81
70.2
3.6. Comparison of the chemical synthesis and enzymatic
synthesis of ethyl oleate
enzymatic synthesis indicated that the enzymatic catalysis
afforded purer oleates. Further investigations on the immobilized-
lipase-catalyzed synthesis of oleates will be performed to gain
deeper insight into esterification catalyzed by lipase from Candida
sp. 99-125.
As known, the enzymatic catalysis has advantages of mild con-
dition, high selectivity and high conversion. In order to compare the
acid-catalyzed synthesis of ethyl oleate with the lipase-catalyzed
synthesis in the optimal condition above, the chromaticity of the
alkyl oleate produced were determined. The results were shown in
Table 1, the chromaticity of ethyl oleate by lipase-catalyzed syn-
thesis was much lower than that by chemical synthesis, which
means that some impurities were introduced in downstream of
chemical synthesis. And the esterification catalyzed by Novozyme
435 was also performed, employing a 5% lipase loading, with 1:1
acid/alcohol ratio at 20 ◦C, which is equal to the optimal condition
of lipase from Candida sp. 99-125. As shown in Table 1, esterifica-
tion catalyzed by the lipase from Candida sp. 99-125 afforded high
conversion and purer oleates.
Acknowledgements
This work was supported by a grant from the Major State
Basic Research Development Program of China (1073 Program)
(Nos. 2012CB725204 and 2011CB710803) and the Program for
Changjiang Scholars and Innovative Research Team in University
(No. IRT1066).
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4. Conclusions
Alkyl oleates were successfully produced via enzymatic synthe-
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