Enzymatic synthesis of monoglycerides by esterification reaction using Penicillium camembertii lipase immobilized on epoxy SiO2-PVA composite

Article abstract of DOI:10.1016/j.molcatb.2009.12.009

Glycerol-fatty acid esterification has been conducted with lipase from Penicillium camembertii lipase immobilized on epoxy SiO₂-PVA in solvent-free media, with the major product being 1-monoglyceride, a useful food emulsifier. For a given set of initial conditions, the influence of reaction was measured in terms of product formation and selectivity using different fatty acids as acyl donors. Results were found to be relatively dependent of the chain length of the fatty acids, showing high specificity for both myristic and palmytic acids attaining final mixture that fulfills the requirements established by the World Health Organization to be used as food emulsifiers.

Full text of DOI:10.1016/j.molcatb.2009.12.009

Journal of Molecular Catalysis B: Enzymatic 65 (2010) 87–90
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Enzymatic synthesis of monoglycerides by esterification reaction using
Penicillium camembertii lipase immobilized on epoxy SiO2-PVA composite
Larissa Freitasa, Ariela V. Paulaa, Julio C. dos Santosa, Gisella M. Zaninb, Heizir F. de Castroa,∗
a Engineering School of Lorena, University of São Paulo, PO Box 116-12602-810, Lorena, São Paulo, Brazil
b State University of Maringa, Department of Chemical Engineering, Av. Colombo 5790, E-46, 87020-900 Maringa, PR, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 16 December 2009
Glycerol-fatty acid esterification has been conducted with lipase from Penicillium camembertii lipase
immobilized on epoxy SiO2-PVA in solvent-free media, with the major product being 1-monoglyceride,
a useful food emulsifier. For a given set of initial conditions, the influence of reaction was measured in
terms of product formation and selectivity using different fatty acids as acyl donors. Results were found
to be relatively dependent of the chain length of the fatty acids, showing high specificity for both myristic
and palmytic acids attaining final mixture that fulfills the requirements established by the World Health
Organization to be used as food emulsifiers.
Keywords:
Monoglycerides
Glycerol
Lipase
Esterification
Epoxy SiO2-PVA
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
this process produces environmentally undesirable by-products
[8]. The product is a mixture containing 35–60% monoacylglyc-
The use of mono- and diacylglycerols as non-ionic emulsifiers
in the food and pharmaceutical industries and as synthetic chemi-
cal intermediates has been a growing research area in recent years
[1,2]. Furthermore, they have a generally recognized as safe status,
which contributes to their larger application. Mono- and diglyc-
erides are consumed at an annual level of 85,000,000 kg in the
United States, corresponding roughly to 70% of the total emulsifiers
used in the food industry [3]. Besides bulk applications in the food
and dairy industries, some other applications for special monoacyl-
glycerols have been described [4,1,5]. Recently, the antimicrobial
activities of particular types of monoglycerides such as monolau-
rin, monomyristin, monolinolein, and monolinolenin have been
reported [6]. It has also been proposed that fatty acids and mono-
glycerides (lauric acid, monolaurin) can be used by the humam
or animal to destroy lipid-coated viruses, such as HIV, herpes,
cytomegalovirus, influenza, various pathogenic bacteria, including
Listeria monocytogenes and Helicobacter pylori, and protozoa such
as Giardia lamblia [7].
erols, 35–50% diacylglycerols, 1–20% triacylglycerols, 1–10% free
fatty acids, and their alkali metal salts [3].
Synthesis of MG by means of an enzymatic process is an envi-
ronmentally friendly approach. Highly stable lipases in organic
solvent offers the possibility of employing various approaches to
the enzyme-catalyzed synthesis of MG, such as selective hydroly-
sis or alcoholysis using 1,3-regiospecific lipases [9], esterification of
glycerol with fatty acids [10] and glycerolysis of fats or oils [11]. The
advantages of enzymatic synthesis are higher yields and mild reac-
tion conditions, resulting in products of higher quality and lower
energy consumption [4,12].
When the production of a high degree pure monoglyceride is
desired, an interesting route is the direct lipase-catalyzed esteri-
fication. Thus, the objective of the present work was to study the
synthesis of monoacylglycerols in a medium solely composed of
substrates by direct lipase-catalyzed esterification of glycerol with
fatty acids, without any solvent or surfactant. Such a system avoids
the problems of separation, toxicity, and flammability of organic
solvents, thereby lowering the cost of the final product and allowing
product recovered without further complex purification or evapo-
ration steps. Moreover, a solvent-free system was chosen because
the target products have a potential use in foods [13].
The conventional chemical method to produce monoglyc-
erides (MG) involves the glycerolysis of fats and oils at higher
temperatures (220–260 ◦C) and elevated pressure under nitro-
gen atmosphere while employing inorganic alkaline catalysts. The
major drawbacks of this process include high-energy consumption,
low yield, and poor product quality [8]. Molecular distillation is usu-
ally needed for the production of high purity MG [1,4]. Moreover,
Lipase-catalyzed synthesis of these partial acylglycerols by the
direct esterification of glycerol with fatty acids in different reac-
tion media have been studied and the media investigated include
the use of aqueous-organic two-phase systems [14], microemul-
sions [14,15] and anhydrous organic solvents [15] as well as the use
of solvent-free systems [3,16]. The selection of a lipase is another
factor affecting the success of this method.
∗
Corresponding author. Tel.: +55 12 3159 5063; fax: +55 12 3153 3224.
E-mail address: heizir@dequi.eel.usp.br (H.F. de Castro).
1381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2009.12.009

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L. Freitas et al. / Journal of Molecular Catalysis B: Enzymatic 65 (2010) 87–90
Previous work demonstrated that lipases from Candida antarc-
amount (100 ␮l/g of support). Lipase-support system was main-
tained in contact for 16 h at 4 ◦C under static conditions. The
immobilized lipase derivative was filtered (nylon membrane 62HD
from Scheiz Seidengazefabrik AG, Thal Schweiz, Switzerland) and
thoroughly rinsed with hexane. The esterification activity of result-
ing immobilized derivative was in range from 35 to 40 U/g following
methodology previously described [24].
tica and Penicillium camembertii (Lipase G) immobilized on
SiO2-PVA composite were found to be potential catalysts to pro-
duce monolaurin by direct esterification of glycerol with lauric acid
[17]. Reaction conditions were optimized by factorial design and
results indicated that P. camembertii gave the best performance, in
terms of lauric acid incorporation into glycerol giving about twice
the concentration of diglyceride and low amount of trilaurin [17].
In this paper, we report further studies dealing with the direct
acylation of glycerol with different fatty acids using P. camembertii
lipase immobilized on SiO2-PVA composite. This lipase has been
shown consistent high production of monoglyceride in the direct
acylation of glycerol with fatty acids having different chain lengths.
Although several reports have been found using this lipase prepa-
ration [18,19,20], data relating its performance on an immobilized
state is scarce in the literature. However, the use of immobilized
lipases is often recommended for lipase-catalyzed reactions, since
it is provided non-aqueous conditions necessary for synthetic reac-
tions [21]. Inaddition, immobilized lipasecanbereusedmany times
without significant loss of activity and may also exhibit substantial
thermo stability, which is another advantage for industrial appli-
cation. A promising approach to lipase immobilization makes use
of epoxy SiO2-PVA composite [22,23]. This type of support can be
easily recognized by the lipases, at molecular level, as solid sur-
faces. Thus, lipases can be immobilized on such supports leading to
open immobilized structures. In accordance with this methodology,
microbial lipase from P. camembertii was successfully immobilized
on epoxy SiO2-PVA rendering active and stable samples [24]. In
this work, the ability of this immobilized preparation to catalyze
the direct esterification of glycerol with short, medium and long
fatty acids was investigated.
2.3. Monoglycerides synthesis
Esterification reactions between glycerol and fatty acids (lauric,
myristic, palmytic, steriac and oleic) at a fixed molar ratio (molar
ratio 8:1 between glycerol:fatty acid) was performed in solvent-
free system. These reactions were carried out in a closed spherical
glass reactor containing 40 g of medium and 5% (w/w) of lipase G
immobilized on SiO2-PVA corresponding to 5 U/g (activity units) of
substrate. The reaction mixture was incubated in a thermo stated
bath at temperature range from 45 to 65 ◦C, according to the melt-
ing point of each fatty acid. The reaction medium was magnetically
stirred at 200 rpm and samples taken at regular intervals were
treated for extraction of the water and glycerol following method-
ology previous described [16].
2.4. Gas chromatography analysis
Mono-, di- and triacylglycerols were analyzed by gas chromato-
graph using a Varian 3800 model equipped with flame-ionization
detector and with a (10 m × 0.25 mm × 0.12 ␮m CP Sil 5CB) cap-
illary column (Varian Inc., Corporate Headquarters, Palo Alto, CA,
USA). The chromatograms were processed using a Varian data inte-
grator version 4.51 computational program. Nitrogen was used as
the carrier gas with a flow rate of 2 ml min−1. The detector and
injector temperatures were 300 ◦C. The column temperature was
set to 80 ◦C for 1 min and was then programmed at 20 ◦C min−1
to 320 ◦C which was maintained constant for 2 min. Other con-
ditions were split ratio of 1:20 and attenuation equal to 1 [16].
Reaction medium was previously treated for extraction of the water
and glycerol and organic phase was dissolved in hexane/ethyl
acetate (proportion of 1:1) which contained tetradecane as internal
standard, and a direct injection was carried out into the gas chro-
matograph. For the determination of calibration curves, solutions
of pure glyceride (Sigma–Aldrich) were used. Concentrations were
expressed as molar fractions calculated from the peak area using
calibration curves. The selectivity values obtained in the reaction
systems were determined by Eq. (1) [3].
2. Experimental
2.1. Materials
Lipase G (P. camembertii) from Amano Pharmaceutical (Japan)
was used. Tetraethoxysilane was acquired from Aldrich Chemi-
cal Co. (Milwaukee, WI, USA). Epichlorohydrin, hydrochloric acid
(minimum 36%), ethanol (minimum 99%), polyvinyl alcohol (MW
72,000) and polyethylene glycol (molecular weight-1500) were
supplied by Reagen (Brazil). Substrates for esterification reactions
(glycerol and fatty acids: lauric, myristic, palmytic, stearic and oleic)
were purchased from Merck and were dehydrated with 0.32 cm
molecular sieves (aluminum sodium silicate, type 13 X-BHD Chem-
icals, Toronto, Canada), previously activated in an oven at 350 ◦C
for 6 h. Other used reagents were hexane (Quimex; Brazil), ethyl
acetate (Reagen; Brazil), tetradecane (minimum 99%, Fluka, Ger-
many).
%MG
%MG + %DG + %TG
%selectivity =
(1)
where %MG = is the amount of monoglycerides formed in the reac-
tion, %DG = is the amount of diglycerides formed in the reaction and
%TG is the amount of triglycerides formed in the reaction.
2.2. Support synthesis and lipase immobilization
A silica-polyvinyl alcohol (SiO2-PVA) hybrid composite was
prepared by the hydrolysis and polycondensation of tetraethoxysi-
lane according to methodology previously described [22] rending
particles having the following properties: average pore diame-
ter (22.91 Å); surface area BET (461.00 m2/g) and porous volume
(0.275 cm3/g) [23]. The activation of SiO2-PVA particles was car-
ried out with epichlorohydrin 2.5% (w/v) at pH 7.0 for 1 h at room
temperature, followed by exhaustive washings with distilled water.
Afterwards, the support was dried at 60 ◦C for 24 h. Epoxy SiO2-
PVA particles were soaked into hexane under stirring (100 rpm)
for 1 h at 25 ◦C. Then, excess of hexane was removed and lipase
was added at a ratio of 1:4 g of enzyme/g of support. PEG-1500
(5 mg/g) was added together with the enzyme solution at a fixed
3. Results and discussion
Lipases display varying degrees of selectivity towards the sub-
strates with which they interact. Steric hindrance (branching,
unsaturation and chain length) and electronic effects of the sub-
strates are the two major factors that determine selectivity. Since
it is difficult to generalize the effect of chain length on esterification
as this depends on individual lipase preparation and its specificity,
in this work the influence of fatty acids in terms of carbon chain size
and unsaturated degree on the performance of the esterification
reactions catalyzed by lipase from P. camembertii were investi-
gated using lauric, myristic, palmytic, steriac and oleic acids as acyl
donors. These acids are the major components in the oil extracted

L. Freitas et al. / Journal of Molecular Catalysis B: Enzymatic 65 (2010) 87–90
89
Fig. 1. Profile curve for fatty acid consumption in the esterification reaction of glyc-
erol with fatty acids catalyzed by lipase G immobilized on SiO2-PVA.
Fig. 3. Influence of fatty acid chain length on the diglycerides formation in the
esterification reaction of glycerol using lipase G immobilized on SiO2-PVA.
from babassu palm trees (Orbinya martiana) a potentially raw mate-
rial source for fatty acids found in abundance in tropical countries,
such as Brazil [25].
In all reaction systems, lipase G was immobilized on epoxy SiO2-
PVA and used as biocatalyst. These experiments were carried out
using glycerol in excess (molar ratio glycerol to fatty acid 8:1) and
5% (w/w) immobilized derivative.
Fig. 1 displays the curve profile for consumption for each tested
fatty acid as a function of time and the formation of the correspond-
ing monoglycerides and diglycerides are shown in Figs. 2 and 3.
Different profiles were obtained depending on the fatty acid
studied (Fig. 1). The rate at which the fatty acid was converted
decreased in the order of lauric C12, myristic C14, palmitic C16,
steric C18 and oleic C18:1, attaining in 6 h conversions of 93%, 60%,
50%, 60% and 46% of the corresponding acids. Extending the reac-
tion for an additional 6 h, did not favor the acid consumption. This
rank order can be attributed in part to differences in the solubilities
of these fatty acids at the reaction temperature used. However, the
rank order is also a consequence on the selectivity of this enzyme
for medium chain fatty acids rather than for long chain fatty acids.
In particular, this enzyme prefers saturated chain fatty acids lower
than C18.
In terms of monoglycerides formation the curve profile using
different fatty acids (Fig. 2) showed that the highest concentration
were obtained for the shortest fatty acid (C12), attaining almost
60 wt%, this performance decreased with increasing chain length.
Therefore, for myristic acid there was a decreased on formation of
monomyristin to 48 wt%. Further decreased on the monoglycerides
formation were also observed when palmytic and steriac acids were
used as acyl donors, attained, respectively, 46 and 44 wt% and even
lower monoglyceride formation was found using oleic acid (C18:1)
achieving only 32 wt% monoolein. Note also that the monoester
accumulation in the medium was as consequence of dispropor-
tionation and glycerolysis reactions of the diester formed earlier
as shown in Fig. 3. Nearly quantitative conversions to the corre-
sponding monoesters were found for myristic and palmytic acids.
However, both saturated (C18) and unsaturated C18:1 fatty acids
produce lower amounts of the corresponding diesters.
This could be related with the active site of the lipase G hav-
ing difficulty in interacting with longer chain fatty acids (steric
hindrance, unsaturation and chain length), decreasing in this way
its catalytic activity. This suggests that the acylation migration in
lipase G catalyzed synthesis is the rate-determining step and the
extent of the deacylation step will affect the overall reaction rate.
If one supposes that the acylation step is dependent on the affin-
ity between the fatty acids and enzyme and that deacylation of the
acyl-enzyme intermediate by alcohols is merely thermodynami-
cally controlled nucleophilic reaction, then it is expected that for an
enzymatic reaction for which the acylation is the rate-determining
step, the relative rate of synthesis of esters using fatty acids of dif-
ferent carbon chain length will be more dependent on the carbon
chain length of the fatty acids. Interpretation of these findings, with
respect to the relationship between the chain lengths of fatty acids,
seems to be consistent with the fact that the active site of lipase G
is composed of a cavity large enough to accommodate substrates
whose total carbon chain length occupies a space equivalent to 15
– CH – moieties. A similar spatial arrangement of the active site
has been also suggested for bile-salt stimulated human milk lipase
[26]. Thus, even though the longer chain fatty acids may intrude
Fig. 2. Influence of fatty acid chain length on the monoglycerides formation in the
esterification reaction of glycerol using lipase G immobilized on SiO2-PVA.

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L. Freitas et al. / Journal of Molecular Catalysis B: Enzymatic 65 (2010) 87–90
Table 1
trace amounts of triglyceride were detected. The final composition
observed after 6 h reaction in the monoglyceride synthesis fulfills
the requirements for emulsifier utilization in food, cosmetic, and
pharmaceutical industries. The results obtained by enzymatic syn-
thesis under mild experimental conditions were similar to those
achieved by chemical glycerolysis of fat and oils, without the
drawback of producing some secondary products such as acrolein,
polyethers, or polyesters of glycerol.
Selectivity for monoglyceride production in the esterification reactions of glycerol
with different fatty acids.
Fatty acid
MG (%)
DG (%)
TG (%)
Selectivitya (%)
Lauric acid (C12
)
59.45
47.92
45.86
42.16
32.92
26.67
10.56
6.39
13.37
7.56
8.38
1.48
3.51
4.20
3.40
62.91
79.92
83.80
70.58
75.02
Myristic acid (C14
Palmytic acid (C16
Stearic acid (C18
)
)
)
)
Oleic acid (C18:1
a
Values calculated for 6 h reaction.
Acknowledgments
into this active site, they will also block the pathway for approach
by the alcohols to form the products from the acyl-enzyme interme-
diate, thereby causing a decrease in the net rate of the monoesters
production.
The authors are grateful for the financial support provided by
FAPESP (Fundac¸ ão de Amparo à Pesquisa do Estado de São Paulo)
and CNPq (Conselho Nacional de Desenvolvimento Científico e Tec-
nológico), Brazil.
Regarding the selectivity of these reactions as displayed in
Table 1, the highest values were obtained when both myristic and
palmytic acids were used as acyl donors (about 80%). Langone et al.
[3], found similar values to 70% of selectivity for the monomyristin
formation in the esterification reaction catalyzed by lipozyme. The
results presented in Table 1 confirmed that although the lipase
from P. camembertii possesses a high affinity for lauric acid, higher
selectivity was shown for C14 and C16 fatty acids.
Selectivity values can be also related with the enzyme affinity
and reaction rates, as already previously discussed. Increasing the
fatty acid chain length led to a decrease on the reaction rate which
in this turn also decreased the diglycerides formation. However,
for C18:0 and C18:1 fatty acids which had much lower global reac-
tion rate, the monoglycerides formation from the glycerolysis of
the diglycerides earlier produced occurred at lesser extend, result-
ing in a decrease in the selectivity, compared with those attained
using myristic and palmytic acids as acyl donors.
According to the directives of the World Health Organization,
the requirements for these mixtures for utilization as food emulsi-
fiers are: (1) to have at least 70% of mono + diglycerides, (2) to have
a minimum of 30% of monoglyceride, and (3) to present contents
of both glycerol and triglyceride below 10% [27]. The enzymatic
synthesis of both monomyristin and monopalmytin as carried out
in this work, resulted in a final mixture that fulfills these require-
ments.
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