Synthesis of trimethylolpropane esters with immobilized lipase from Candida sp. 99-125

Article abstract of DOI:10.1016/j.molcatb.2011.07.017

The lubricants of the future have to be more environmentally adapted, have a higher level of performance. Synthesis esters (SEs) which can be used as raw materials for biodegradable lubricant base oils are increasing in popularity due to superior technical properties. Direct esterification of trimethylolpropane (TMP) with fatty acid in a solvent free system, by immobilized lipase from Candida sp. 99-125 was studied. Investigations of important factors were carried out involving temperature, time, enzyme amount, substrates molar ratio and water content. For 2 g caprylic acid, under the optimal conditions, with 0.4 g immobilized lipase, at substrates molar ratio 1:10 (TMP to acid), temperature 40 °C and water content controlled under 0.8% (w/w), the total conversion of fatty acid with TMP reached up to 96% and the formation of trisubstituted TMP esters reached 93%. Water content controlled during esterification process was found to be critical for high yield of direct esterification.

Full text of DOI:10.1016/j.molcatb.2011.07.017

Journal of Molecular Catalysis B: Enzymatic 74 (2012) 151–155
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Synthesis of trimethylolpropane esters with immobilized lipase from Candida sp.
99–125
Yifeng Tao, Biqiang Chen, Luo Liu, Tianwei Tan∗
Beijing Key Lab of Bioprocess, The Biorefinery Research and Engineering Center of the Ministry of Education, Beijing University of Chemical Technology, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The lubricants of the future have to be more environmentally adapted, have a higher level of perfor-
mance. Synthesis esters (SEs) which can be used as raw materials for biodegradable lubricant base oils
are increasing in popularity due to superior technical properties. Direct esterification of trimethylol-
propane (TMP) with fatty acid in a solvent free system, by immobilized lipase from Candida sp. 99–125
was studied. Investigations of important factors were carried out involving temperature, time, enzyme
amount, substrates molar ratio and water content. For 2 g caprylic acid, under the optimal conditions,
with 0.4 g immobilized lipase, at substrates molar ratio 1:10 (TMP to acid), temperature 40 ◦C and water
content controlled under 0.8% (w/w), the total conversion of fatty acid with TMP reached up to 96% and
the formation of trisubstituted TMP esters reached 93%. Water content controlled during esterification
process was found to be critical for high yield of direct esterification.
Received 15 March 2011
Received in revised form 19 July 2011
Accepted 23 July 2011
Available online 12 August 2011
Keywords:
Biodegradable
Esterification
Immobilized lipase
Lubricant base oils
Polyolesters
Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.
1. Introduction
3.1.1.3) are enzymes which normally catalyze the hydrolysis of
glycerol esters at lipid/water interfaces. In organic solvent sys-
Since the late 1960s, scientists’ attention for lubricants has
been turned to biobased raw materials for its excellent physical
properties, including biodegradability, renewablility, high lubric-
ity and high flash points, and possibility to achieve tailor-made.
Meanwhile, petroleum derived lubricants may soon no longer be
available, industries have been searching for a cheap and renewable
source of lubricants [1]. Furthermore, the lubricants of the future
have to be more environmentally adapted [2–5]. The world annual
consumption of lubricants is 40 million tonnes, and is projected to
continue to rise 1.6% annually for at least the next 3 years [6]. The
pollution problem is so severe that approximately 50% of all lubri-
cants sold worldwide end up released in the environment [3,7]. Due
to poor oxidative stability of natural vegetable oil and there is not
enough arable land to support the widespread use of vegetable oil
based lubricants [1], synthesis esters (SEs) are an attractive alter-
native to conventional petrobased lubricants.
tems, lipases have also been shown to catalyze ester synthesis [11].
Unlike alkali-based reactions, the products of enzyme catalyzed
reaction can easily be collected and separated. The particular bene-
fits offered by enzymes are specificity, mild conditions and reduced
waste [12,13]. Studies on the synthesis of lubricant oils by chemi-
cal or enzymatic transesterification were reported [14]. Uosukainen
et al. [10] studied the reaction of alcoholysis of rapeseed oil methyl
ester (biodiesel) and TMP. In the chemical synthesis of triesters,
the reaction required high temperature (up to 120 ◦C) with 0.5%
alkaline. In enzymatic synthesis, rapeseed oil methyl ester was syn-
thesized chemically. Secondly, transesterification for the synthesis
of trimethylolpropane triester of rapeseed oil fatty acids was car-
ried out. Finally, 85% conversion to triester was achieved with 90%
total conversion at 60 h. Meanwhile, elimination of the by-product
methanol which led to lipase denaturation was found to be the
principal factors affecting the product yield. However, studies of
direct esterification have not been reported.
Immobilized Candida sp. 99–125 lipase has been established in
our laboratory and successfully applied in esterification synthesis
for fatty acid esters [15–17]. This study focused on synthesis of
trisubstituted TMP esters by lipase catalyzed direct esterification
(Fig. 1) in a solvent free system to avoid additional transesterifica-
tion step and release of toxic methanol. Caprylic acid is the common
name for the eight-carbon saturated fatty acid (also known as
octanoic acid), which can be found in coconut oil. Low temperature
performance of caprylic acid ester is better than pamilic acid (16C)
TMP ester or other long chain fatty acid TMP esters. Furthermore,
Traditional methods of producing SEs are alkali-based reactions.
A relatively new and promising development in the production
of biodegradable lubricant oils and additives is enzymatic ester-
ification with lipase as the catalyst. It has been suggested since
the late 1990s [8–10]. Lipases (triacylglycerol acylhydrolase, EC
∗
Corresponding author at: Chaoyang District North Third Ring, East Road No.
15th, Beijing University of Chemical Technology, Beijing, China.
E-mail addresses: ifengtaobuct@gmail.com (Y. Tao), chenbiqiang@gmail.com (B.
Chen), liuluo02@gmail.com (L. Liu), twtan@mail.buct.edu.cn (T. Tan).
1381-1177/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2011.07.017

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Y. Tao et al. / Journal of Molecular Catalysis B: Enzymatic 74 (2012) 151–155
Fig. 1. The reaction scheme of esterification of TMP with fatty acid.
caprylic acid served also as solvent in this reaction because its melt-
ing point is as low as 16.8 ◦C. Due to three-dimensional symmetric
structure of hydroxymethyl of TMP, these reactions produce unique
mono-, di-, and tri-substituted TMP esters, while TMP was esteri-
fied with single fatty acid. The goal of this work was by investigating
optimal reaction conditions involving effect of temperature, time,
enzyme amount, substrates molar ratio and water content by dif-
ferent pretreatments, in order to achieve the highest trisubstituted
TMP esters formation for that the hydroxyl group of mono-, di-
substituted TMP esters leads to pour point reversion of lubricant.
air in order to remove water in case of low environmental rela-
tive humidity in Beijing. 2.019 g caprylic acid was mixed with TMP
corresponding to different substrate molar ratios from 1:4 to 1:11
(TMP to fatty acid). About 25 min after addition of TMP to caprylic
acid at 40 ◦C, a clear, homogeneous solution was observed. Heat up
to 60 ◦C could accelerate the solution of TMP. Subsequently, differ-
ent amount of lipase were added. The reaction was carried out in an
orbital reciprocal shaker at 190 rpm at different temperatures from
30 ◦C to 50 ◦C with gradient of 5 ◦C and for different time periods
from 12 h to 72 h.
To investigate effect of water content, 25.263 g acid, 6.906 g
TMP, and 5 g immobilized lipase were added in a 100 ml airtight
shake at 40 ◦C. Enzyme preparations and the organic phase of
the reaction mixture were adjusted to different water content by
pre-equilibration with different pretreatments at 40 ◦C in separate
containers. Equilibration was performed overnight (at least 16 h).
Thus, different initial water content of system including 0.071%,
0.105%, 0.273%, 0.335%, and 0.488% (w/w) were obtained.
2. Materials and methods
2.1. Chemicals
Trimethylolpropane
propanedediol, TMP) with
caprylic acid with a purity of 98% were obtained from Fuchen
Chemical Co. Ltd. (Tianjin, China). The other solvents and salts
of analytical grade were obtained from Beijing Chemical Factory.
They were dried by molecular sieves before usage.
(2-ethyl-2-(hydroxymethyl)-1,
a
3-
melting range of 56–59 ◦C and
2.4. Analytical procedure
Samples were obtained at schedular time, and immediately
heated to ensure enzyme deactivation. The compounds includ-
ing fatty acid, mono-, di-, and trisubstituted esters in the reaction
mixture were quantified using a GC-2010 gas chromatography
(Shimadzu, Japan) equipped with a DB-1ht capillary column
(30 m × 0.25 mm × 0.1 ␮m; J&W Scientific, USA) and a flame ion-
izing detector (FID). The column temperature was held at 110 ◦C,
then heated to 132 ◦C at 12 ◦C/min, and to 180 ◦C at 30 ◦C/min and
finally to 300 ◦C at 20 ◦C/min and then maintained for 5 min. The
temperatures of the injector and detector were both set at 300 ◦C.
The retention time of caprylic acid and mono-, di-, and trisubsti-
tuted TMP esters (Fig. 2) were 2.19 min and 5.43 min, 8.13 min, and
10.25 min, respectively. The fatty acid conversion was calculated
2.2. Lipase immobilization
Lipase (EC.3.1.1.3) was obtained from Candida sp. 99–125, its
characterisation and catalytic properties was described in previous
literatures [15]. Furthermore, the immobilized method has been
established in our lab, and its procedure was expatiated in our
previous literatures [16,17].
2.3. Enzymatic synthesis of TMP esters
The optimization of reaction conditions except studies of water
content took place in a 50 ml shake flask, and was exposed to the
Fig. 2. GC chromatograms of direct esterification enzymatic products. 0 – caprylic acid (2.19 min); 1 – monosubstituted TMP esters (5.43); 2 – disubstituted TMP esters
(8.13 min); 3 – trisubstituted TMP esters (10.25 min).

Y. Tao et al. / Journal of Molecular Catalysis B: Enzymatic 74 (2012) 151–155
153
as molar percentage of the maximum theoretic conversion (TMP
was completely converted to trisubstituted TMP esters), and the
formation of trisubstituted TMP esters was molar percentage of
trisubstituted TMP esters to all esters formed.
The compounds were identified by GC–MS. The mass spectrom-
eter (GC-QP2010, Shimadzu, Japan) was operated under electron
impact ionization conditions (electron energy 70 eV, source tem-
perature 200 ◦C, interface temperature 200 ◦C). Full scan mass
spectra were acquired ranging from 50 to and 600 amu, using a scan
rate of 2 scans/s from 2 min to 15 min. Trisubstituted TMP esters
m/z: 369, 297, 281, 191, 127, 98, and 57.
Karl-Fischer moisture titrator equipped with a double platinum
electrode from Bejing Xianqu Weifeng Technology Development
Co. Ltd. was applied to quantify the water content of sample. About
100 mg of sample was analyzed at room temperature with one-
component system using monopropellant Karl-Fischer reagent as
titrating solution and methanol-dry as the solvent.
Fig. 4. Effect of enzyme amount on esterification. -ꢀ- the total conversion, -ꢁ-
the formation of trisubstituted TMP esters, respectively. Reaction conditions: TMP
0.2684 g, fatty acid 2.0189 g, substrates molar ratio 1:7 (TMP to acid), at 40 ◦C and
190 rpm for 24 h.
3. Results and discussion
3.1. Effect of temperature
In enzymatic catalysis, reaction temperature plays one of the
most important roles. Higher temperature enhances mass transfer,
and the solubility of TMP, and then accelerates the release of water
from the system. However, too high temperature leads to enzyme
denaturation. The highest total conversion was observed at 40 ◦C
(Fig. 3), which was consistent with previous literature [15,18]. As
the temperature increased from 30 ◦C to 40 ◦C, the total conversion
increased 14%, and the formation of trisubstituted TMP esters rose
about 15%. However, the conversion did not show significant dif-
ference when the temperature further increased. The temperature
reported [10] was as high as 65–70 ◦C using Novozym 435 in order
to enhance the solubility of TMP. However, the higher conversion
was obtained in this study at lower temperature. This result sug-
gested that the impact of temperature on enzyme activity was more
important than others such as TMP solubility.
of trisubstituted were achieved after 24 h of reaction at enzyme
amount of 0.4 g (16% based on substrates weight). The lipase used
in this study was immobilized on textile, which had a large sur-
face of about 91 cm2/g. After immobilization the carrier surface
was hydrophobically modified, to avoid adsorption of water. At the
enzyme amount greater than 0.4 g, there is a reduction in the total
conversion but the formation of trisubstituted TMP esters is still
increasing. No side reaction was observed at the enzyme amount
greater than 0.4 g. It was assumed that the immobilized enzyme
showed surface effect due to the large and hydrophobic area. At
the enzyme amount greater than 0.4 g, the surface area of the solu-
tion was little smaller than that of the immobilized enzyme. The
mono- and disubstituted TMP esters may accumulate on the surface
due to their hydrophobicity, which shifted the reaction to trisubsti-
tuted TMP ester, and even the total conversion was slightly lower
than that at the enzyme amount 0.4 g. Upon increasing the enzyme
amount further, reaction conversion did not increase significantly
but even decrease slightly. This phenomenon may be explained that
the form of immobilized lipase led to the limit of mass transfer and
the lack of substrate to access the active site of enzyme.
3.2. Effect of enzyme amount
The effect of enzyme amount on synthesis TMP ester was inves-
tigated from 0.1 g to 0.5 g (4–20% based on substrates weight). Fig. 4
shows that about 60% of total conversion and 30% of the formation
3.3. Effect of substrates molar ratio
To obtain the highest formation of trisubstituted TMP esters,
substrates molar ratio (TMP to acid) from 1:4 to 1:11 was inves-
tigated. Fig. 5 shows that there was no significant difference of
total conversion. The highest yield of trisubstituted TMP esters was
observed with molar ratio (TMP to acid) 1:10. However, when molar
ratio (TMP to acid) increased to 1:11, the formation of trisubstituted
TMP esters decreased slightly, possibly due to substrate inhibition
by excess fatty acid. The fatty acid amount was constant in this
study, and the TMP amount varied with substrates molar ratio.
Thus, with the lower substrates molar ratio, the relative higher TMP
concentration led to the generation of mono- and di-substituted
TMP esters (data not shown). Further experiments on the reuse of
fatty acid and mono- and di-esters are considered.
3.4. Effect of water content
Water content in the reaction medium is a key parame-
ter in nonaqueous enzymology both for the maintenance of
three-dimensional structural integrity and also for optimal cat-
alytic activity of the enzyme. Thus, water content of system was
Fig. 3. Effect of temperature on esterification. -ꢀ- the total conversion, -ꢁ- the for-
mation of trisubstituted TMP esters, respectively. Reaction conditions: TMP 0.2684 g,
fatty acid 2.0189 g, substrates molar ratio 1:7 (TMP to acid), enzyme amount 0.4 g,
at 190 rpm for 24 h.

154
Y. Tao et al. / Journal of Molecular Catalysis B: Enzymatic 74 (2012) 151–155
Fig. 5. Effect of substrates molar ratio (TMP to acid) on esterification. The left col-
umn represents the total conversion; the right column represents the formation
of trisubstituted TMP esters, respectively. Reaction conditions: fatty acid 2.0189 g,
enzyme amount 0.4 g, at 40 ◦C and 190 rpm for 24 h.
Fig. 7. The formation of trisubstituted TMP esters during esterification process.
Reaction conditions: TMP 6.906 g, fatty acid 25.263 g, enzyme amount 5 g, at 40 ◦
and 190 rpm. Initial water content, -ꢁ- 0.071%, -᭹- 0.105%, -ꢀ- 0.273%, -ꢂ- 0.335%,
-ꢃ- 0.488%.
C
analyzed to evaluate the effect of water in this work. Figs. 6 and 7
show the final total conversion with the initial water content of
0.105%, achieved 67.85%, and was almost 20% higher than the low-
est. However, the initial conversion rate showed no significant
difference, which is only 5% between the highest and the lowest
after 10 h. The final formation of trisubstituted TMP esters was only
about 15%. Worthy of noticing that, while the initial water content
was above 0.3% (Fig. 9), there was almost no trisubstituted TMP
esters generated in the first 4 h. As the water content reached over
0.8% (Fig. 9) after 10 h, the total conversion rate and the formation
rate to trisubstituted TMP esters slowed down due to the increased
water content. Possibly, the water tolerance limit of this immobi-
lized lipase to form trisubstituted TMP esters was 0.8% (w/w) which
needs to be confirmed by more sophisticated experiment design.
Meanwhile, esterification process (Fig. 8) was studied in a shake
flask exposed to air in order to control water content at a low
level. The bell-type curve of mono- and di-substituted TMP esters
showed that trisubstituted TMP esters were generated step by step.
The process of direct esterification is similar to transesterification
using sodium methoxide as catalyst [19]. Before 6 h, due to the high
TMP concentration, the system mainly generated monosubstituted
TMP ester. However, the formation of di- and tri-substituted TMP
esters increased slightly. While the concentration of monosubsti-
tuted reached higher level, the production rate of disubstituted TMP
esters increased, and the highest yield was observed at 20 h. Mean-
while, the formation of trisubstituted TMP esters rose. Finally, the
total conversion and the formation of trisubstituted TMP esters
was 96% and 93%, respectively. The results of direct esterifica-
tion were comparable to transesterification [14], however, without
release of toxic methanol. Uosukainen et al. [10] screened several
lipases to catalyze transesterification of TMP with methanol esters,
including Lipozyme IM (sn-1, 3), and Novozym 435 (not specific)
etc. Lipozyme IM was able to catalyze completely substituted TMP
esters, which could be a result of spontaneous acylmigration [20]
catalyzed by the carrier resin or Lipozyme is not 1,3-specific in the
Fig. 8. Esterification process of TMP to acid. -ꢁ- the total conversion; -᭹- the forma-
tion of monosubstituted TMP ester, -ꢀ- the formation of disubstituted TMP esters,
-ꢂ- the formation of trisubstituted TMP esters, respectively. Reaction conditions:
TMP 0.2684 g, fatty acid 2.0189 g, enzyme amount 0.4 g, at 40 ◦C and 190 rpm for
24 h.
Fig. 6. The tendency of total conversion of fatty acid during esterification process.
Reaction conditions: TMP 6.906 g, fatty acid 25.263 g, enzyme amount 5 g, at 40 ◦
C
and 190 rpm. Initial water content, -ꢁ- 0.071%, -᭹- 0.105%, -ꢀ- 0.273%, -ꢂ- 0.335%,
-ꢃ- 0.488%.

Y. Tao et al. / Journal of Molecular Catalysis B: Enzymatic 74 (2012) 151–155
155
during esterification process. The optimum of enzyme amount was
16% (w/w) based on the weight of substrates. Substrates molar
ratio showed significant impact on formation of trisubstituted TMP
esters. Under the optimal reaction condition, the total conversion of
fatty acid with TMP reached up to 96% and the formation of trisub-
stituted TMP esters reached 93%. Water content controlled during
esterification process is found to be more important and effective
than at the beginning for direct esterification. Direct esterification
achieved comparable results to transesterification, however, with-
out the methanol toxicity. The purity of trisubstituted TMP esters
was 98% after one-step purification of molecular distillation. Fur-
ther experiments are on the way to study the interaction between
factors using response surface modeling and to control water under
a certain level during esterification process.
Acknowledgements
The authors express their thanks for the support from
the National High Technology Research and Development 863
Program of China (2009AA03Z232 and 2009AA02Z207), 973
Program (2009CB724703, 2011CB200905 and 2011CB710800),
the National Nature Science Foundation of China (20876011,
21106005), Beijing Municipal Science and Technology Commission
(Z09010300840901) and Beijing Municipal Science and Technology
Commission (2009GJA00016).
Fig. 9. The water content in the reactant during esterification process. Reaction con-
ditions: TMP 6.906 g, fatty acid 25.263 g, enzyme amount 5 g, at 40 ◦C and 190 rpm.
Initial water content, -ꢁ- 0.071%, -᭹- 0.105%, -ꢀ- 0.273%, -ꢂ- 0.335%, -ꢃ- 0.488%, -ꢀ-
the process water content of reaction in shake flask exposed to air.
conditions used. However, the same result was observed in this
work, which indicated equality of hydroxymethyl of TMP.
The initial water content of investigations in airtight shake flask
was in range of about 0.1–0.5% (w/w, Fig. 9). Without any pretreat-
ment, the initial water content was 0.335% due to the hygroscopic
capacity of TMP with three hydroxyl groups. Fig. 9 shows the ten-
dency of water content of system during esterification process.
With higher initial water content, water content of process kept in a
higher level. After about 10 h, water content with different pretreat-
ments all reached 0.6%. After then, the increasing rate slowed down,
and finally was in range of 1.4–1.8%. However, compared to airtight
shake flask reaction, the process water content of esterification in
a shake flask exposed to air kept in lower range of 0.03–0.06%.
Figs. 8 and 9 show that the best method to synthesize TMP esters
is working in an open air flask, where the water contents is kept
under 0.1%. Therefore, water content of system is a key parameter to
high yield of trisubstituted TMP esters. In addition, controlled water
content during esterification process at a certain level is required.
Meanwhile, controlled initial water content is not necessary for
direct esterification that is different to transesterification.
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The lipase from Candida sp. 99–125 immobilized on a textile
membrane was able to catalyze esterification of TMP with fatty
acid. In this study, the immobilized lipase had an optimal tempera-
ture at 40 ◦C. Water content should be controlled under 0.8% (w/w)