Enzyme and Microbial Technology 25 (1999) 194 200

Continuous enzymatic transesterification of high oleic sunflower oil in a

packed bed reactor: influence of the glycerol production
Valerie Dossata, Didier Combesb, Alain Martyb,*
a

La Toulousaine de Cereales, 26, avenue Didier Daurat, BP 4106, 31030 Toulouse Cedex 09, France
Institut National des Sciences Appliquees, Laboratoire Biotechnologie-Bioprocedes, UMR 5504, L.A. INRA,
Complexe Scientifique de Rangueil, 31 077 Toulouse Cedex 04, France
Received 4 November 1998; received in revised form 25 January 1999; accepted 10 February 1999

Abstract
The transesterification of high oleic sunflower oil with butanol by the immobilized Lipozyme in n-hexane was carried out in a
continuous packed bed reactor, oleic acid, butyl ester, and glycerol being formed as the main products. It was found that glycerol, insoluble
in n-hexane, remained in the reactor adsorbed onto the enzymatic support, leading to a drastic decrease in enzymatic activity. The
phenomenon involved in this loss of activity was attributed to the formation of an hydrophilic hindrance around the enzyme resulting in
diffusion limitations of the hydrophobic substrate from the organic phase to the enzyme. To recover enzymatic activity, several solutions
are proposed. The addition of silica gel into the enzymatic bed to adsorb the produced glycerol did not enable this loss of activity to be
avoided. In order to enhance the solubility of glycerol in the reaction medium as soon as it was produced, n-hexane amended acetone was
used as solvent, but high conversion of sunflower oil was not restored. Finally, by intermittent rinsing of the catalyst bed with a solution
of tertiary alcohol amended with water to obtain the optimal thermodynamic water activity of 0.54, glycerol was eliminated from the reactor,
and high conversion was maintained. This semi-continuous process allowed not only the synthesis of oleic acid butyl ester but also the two products
to be recovered separately. 1999 Elsevier Science Inc. All rights reserved.
Keywords: Transesterification; Continuous reactor; Organic solvent; Glycerol; Water activity; Lipase; Sunflower oil

1. Introduction
Esters of long chained carboxylic acids find a variety of
industrial applications (surfactants, lubricants . . . ) [1].
Vegetable oils are an interesting source of fatty acids and
have a number of potential advantages compared to their
mineral counterparts. They present lower toxicity, high biodegradability, and are renewable. From a European and
economic point of view, it would be advantageous to enhance the value of vegetable oils, from the surplus of agricultural products, in a non-alimentary field.
Among the fatty acids, oleic acid is particularly stable
towards thermo-oxidation, due to the presence of only one
unsaturated bond in its structure. While classical sunflower
oil contains around 40% of oleic acid, the new hybrid

* Corresponding author. Tel.: 33-5-6155-9439; fax: 33-5-6155-9403.

E-mail address: marty@insa-tlse.fr (A. Marty)

variety named high oleic sunflower oil presents more than

80% of this fatty acid in its composition [2].
Esterification and transesterification reactions are commonly practiced in industry using acids as catalysts at high
temperature (100C300C) and pressure. This chemical
way often offers poor reaction selectivity leading to undesirable side reactions and low yields. In recent years, the use
of triacylglycerol lipases (E.C. 3.1.1.3) as biocatalysts for
synthesis reactions has been developed as an alternative
route to the conventional chemical process [37]. The main
reason is a preference for better quality and the possibility
of achieving more efficient processes with higher selectivity
and fewer environmental problems.
Although high synthesis activity and good stability are
generally observed in hydrophobic solvents like n-hexane
[8], hydrophilic compounds used as substrate or obtained as
product are immiscible in this kind of reaction medium.
These problems of solubilization result in the adsorption of
polar molecules onto enzymatic hydrophilic support leading
to very low esterification rate [8,9,12].

0141-0229/99/$ see front matter 1999 Elsevier Science Inc. All rights reserved.
PII: S 0 1 4 1 - 0 2 2 9 ( 9 9 ) 0 0 0 2 6 - 5

V. Dossat et al. / Enzyme and Microbial Technology 25 (1999) 194 200

In the case of reactions producing hydrophilic molecules,

several solutions have been proposed. The simplest one is to
carry out the reaction under reduced pressure to ensure their
continuous elimination. However, this solution is only possible if the hydrophilic compound is easily evaporated,
which is not the case, for instance, if glycerol is produced.
In this case, Stevenson et al. [10] overcame this problem by
removing the glycerol as it was formed using silica gel and
other adsorbents to extract glycerol from the batch reaction
medium.
The problem is even more acute when considering continuous operation of a reactor. The continuous fixed-bed
reactors have been reported as being the most suitable kind
of reactors to develop new industrial applications for ester
synthesis by reversion of hydrolysis [12]. Marty et al. [13]
have studied the esterification of oleic acid with ethanol and
transesterification of triolein with ethanol in n-hexane in a
continuous packed bed reactor catalyzed by immobilized
Lipozyme and have shown a drastic loss of enzymatic
activity with time. This negative effect was explained by the
adsorption of the polar product, water, or glycerol onto the
support. The creation of this hydrophilic layer should result
in a limitation in hydrophobic substrate diffusion from medium to enzyme or in a modification of the optimal thermodynamic water activity of the catalyst. Colombie et al.
[14] have proposed controlling thermodynamic water activity in the fixed-bed reactor during continuous esterification
of oleic acid with ethanol in order to maintain a high and
constant conversion. This control is realized by adjusting
solvent polarity to the requisite evacuation of the produced
water.
In this work, our final objective is to propose an efficient
process to produce butyl oleate using direct transesterification of high oleic sunflower oil with butanol. This product
has a good potential as a lubricant, and this reaction is a
good model for the study of monoester production. With a
view to developing an industrial process, one of the aims
was to develop a continuous reaction method. An elucidation of the phenomena associated with the adsorption of
glycerol onto the enzymatic support might allow appropriate solutions to be put forward.

2. Materials and methods

2.1. Materials
Commercial immobilized lipase from Rhizomucor miehei, namely Lipozyme, was a gift from Novo Industri S/A
(Denmark). The enzyme is immobilized on macroporous
anionic resin beads (Duolite A568) (approximately 14%
protein are adsorbed onto the resin g/g). High oleic sunflower oil supplied by the Toulousaine de Cereales (Toulouse, France) contained 83% oleic acid, 10% linoleic acid,
5% stearic acid, and 2% palmitic acid; the main triglyceride
present in the oil is triolein (80%). Glycerol, oleic acid butyl

195

Fig. 1. Semi-continuous reactor for the production/fractionation of ester

and glycerol.

ester, oleic acid ethyl ester, monoolein, diolein and triolein

were purchased from Sigma Chemical Co. (St. Louis, MO,
USA). All other chemicals and solvents (butanol, 2-methylbutan-2-ol, methanol, n-hexane) were of high purity and
were purchased from Prolabo (France).
2.2. Experimental methods
Glycerol and water were adsorbed onto enzymatic support as described by Castillo et al. [15]: 100 mg of enzymatic support and various amounts of glycerol or water
were carefully mixed until a homogeneous suspension was
obtained.
In order to study the variation in enzymatic activity
versus amounts of glycerol adsorbed onto the support, an
esterification reaction without production of glycerol was
studied. Synthesis was performed in a discontinuous way in
n-hexane in a glass tube containing oleic acid (200 mM),
ethanol (300 mM), and the immobilized enzyme (100 mg).
The reaction mixture was incubated at 40C stirred magnetically.
For the continuous process in n-hexane, the reactor was
a fixed-bed type. It consisted of a thermostated (40C)
column (9 mm diameter) containing the immobilized enzyme (bed length: 4 cm/g). The reaction medium containing
high oleic sunflower oil (20 mM) and butanol (100 mM) in
n-hexane was pumped using a Gilson pump percolating the
packed bed of enzyme. At the outlet of the reaction vessel,
samples were collected and analyzed.
For the semi-continuous process in n-hexane, the previously described fixed-bed reactor is percolated alternatively
by the substrate solution and the rinsing solution (Fig. 1).
The 3-way electric valves placed at the outflow of the
reactor enable the two products of the reaction, ester and

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V. Dossat et al. / Enzyme and Microbial Technology 25 (1999) 194 200

glycerol, to be recovered separately. The pumps were controlled by a timer which fixed a synthesis cycle of 2 h and
a rinsing cycle of 15 min. The inversion of the valves takes
into account the residence time of the reactor.
The optimal thermodynamic water activity of the enzyme is 0.54 and was obtained by putting the enzyme before
use in a closed chamber at room temperature containing an
aqueous saturated Mg(NO3)2 solution.
2.3. Analytical methods
Samples of the reaction mixture were filtered off, and the
n-hexane was thoroughly evaporated from the reaction samples in a thermostated bath at 90C. The samples were
resuspended in an equal volume of the HPLC mobile phase
and analyzed by HPLC with a Hewlett-Packard integrated
HPLC system HP 1050 (Hewlett Packard, Palo-Alto, CA,
USA) with a C18 reverse phase analytical column (Hewlett
Packard) Spherisorb ODS-2 (5 m, 250 4.6 mm) thermostated at 50C. Concentrations of monoolein, oleic acid,
oleic acid butyl ester, and oleic acid ethyl ester were detected with a HP 1047A refractive index detector (Hewlett
Packard) after calibration in the range of 0 to 50 mM for
each component. Elution was carried out isocratically with
a methanol/acetic acid (99.7/0.3, v/v) mobile phase and a
flow rate of 1 ml/min.
Diolein and triolein concentrations were measured by
HPLC using the same HP 1050 system and the same C18
column thermostated at 45C. The detector was a UV/VIS
HP 1050 at a wavelength of 210 nm. The eluent was acetone:acetonitrile (50:50). Elution of products was realized at
a flow of 0.5 ml/min for 7 min, which was then increased
over a period of 3 min to 3 ml/min and after 6 min was
decreased to 0.5 ml/min over 4 min. The conversion was
calculated as follows:
X

100*[Butyloleate]t
3*[Triolein]t 2*[Diolein]t
[Monoolein]t [Butyloleate]t

Where [Triolein]t: concentration of triolein at time t. [Diolein]t: concentration of diolein at time t. [Monoolein]t: concentration of monoolein at time t. [Butyloleate]t: concentration of butyl oleate at time t.
Analysis of the amount of glycerol adsorbed onto the
support was realized by rinsing immobilized Lipozyme
with aliquots of water in order to ensure that all the glycerol
is desorbed. Then, the glycerol concentrations in washing
volumes were measured by HPLC analysis with the previously described HPLC system and an analytical column
(Applied Biosystemes, Foster City, CA, USA) Polypore H
(10 m, 220 4.6 mm) thermostated at 30C. Glycerol was
detected by the previously described refractive index detector after calibration in the range of 0 to 25 g/l. Elution was
carried out isocratically with a H2SO4 5 mM mobile phase
at a flow rate of 0.3 ml/min.

Fig. 2. Conversion of high oleic sunflower oil with time during transesterification reaction with butanol in n-hexane in a continuous packed bed
reactor of immobilized Lipozyme. Reaction conditions: substrate concentrations: 20 mM triglyceride and 100 mM butanol in n-hexane; immobilized Lipozyme: 250 mg; flow rate: 0.35 ml/min; temperature: 40C.

2.4. Determination of the water content

Water content was determined by the Karl-Fisher titration method. The Karl-Fisher device consisted of a burette
automatically controlled by a titrator (Metrohm, Germany).
The titrant (Hydranal, composite 2 or 5) was a pyridine-free
solution from Merck. The solvent used was methanol.
2.5. Determination of the water activity
Thermodynamic water activity was determined by the
NOVASINA device (Humidat-IC-II, Rogo-Sampaic, Wissous, France) calibrated with saturated salt solutions (LiCl:
aw 0.11; Mg(NO3)2, 6H2O: aw 0.53; K2Cr2O7: aw
0.98). The sample is introduced in the air-tight sensor. As
soon as the sample has produced in the measuring chamber
a moisture equilibrium, the value can be read.

3. Results
3.1. Study of the transesterification reaction in n-hexane
From a study carried out in a batch reactor of the transesterification of high oleic sunflower oil with butanol, the
operating conditions necessary to obtain 95% conversion in
a plug-flow reactor (flow rate and enzyme quantity) were
determined. This value of the conversion is in fact obtained
at the beginning of the experiment, but later the conversion
decreases with time and becomes lower than 10% after 14 h
of reaction (Fig. 2). Surprisingly, no mono and diglyceride
were detected at the outflow of the reactor, the only product
being butyl oleate.
At the end of the experiment, the enzymatic support was
recovered and rinsed with water in order to desorb the

V. Dossat et al. / Enzyme and Microbial Technology 25 (1999) 194 200

197

thermodynamic water activity from its optimum value and

consequently to the decrease in catalytic activity. Indeed,
the role of water is crucial. Chulalaksananukul et al. [16]
have studied the kinetics of oleic acid esterification by
immobilized Lipozyme in n-hexane and have demonstrated the crucial influence of the water content of the solid
phase on the enzyme activity. They found an optimal enzyme activity when water content represents 10% (by
weight of water to weight of initial dry support). This water
content corresponds to an optimal thermodynamic water
activity of 0.54 [17].

Fig. 3. Conversion of high oleic sunflower oil with time during transesterification reaction with butanol in n-hexane in a continuous packed bed
reactor with increasing quantities of enzymatic support. Reaction conditions: substrate concentrations: 20 mM triglyceride and 100 mM butanol in
n-hexane; immobilized Lipozyme: (F) 500 mg, () 1 g, () 2 g; flow
rate: 0.5 ml/min; temperature: 40C.

glycerol, which was then measured: 180 mg of glycerol was

adsorbed onto the enzymatic support which represented
73% (by weight of glycerol to the weight of initial dry
enzymatic support). It was calculated that during the reaction 172 mg of glycerol must be formed, no mono and
diglyceride being produced, which means that all the glycerol produced by the reaction stays adsorbed onto the support.
An increase in the amount of enzymatic support, for the
same operating conditions (flow rate, substrate concentrations), leads to the achievement of a time-lag before the
conversion value starts to decrease (Fig. 3). Higher quantities of glycerol are necessary to deactivate the enzyme,
glycerol thus formed being distributed onto a higher quantity of enzymatic support. Indeed, doubling the enzyme
amount results in approximately a doubling of the steady
state duration and approximately a halving of the conversion gradient with time: with 500 mg of Lipozyme, the
activity started to decrease after 5 h of reaction, and the
slope of the curve is about 18 (%/h) while with 1 g and 2 g
of Lipozyme, the fall appears after 8 h and 20 h respectively, the slope of the curves being respectively approximately 8.6 and 5 (%/h).
To solve this problem of a non-steady state, it is necessary to understand the actual phenomenon involved. In fact,
two hypotheses can be proposed:
1. The accumulation of adsorbed glycerol molecules generates a hydrophilic barrier around the immobilized catalyst
leading to diffusion limitations of the hydrophobic substrate, the triglyceride, from the medium to the enzyme.
This hypothesis has already been proposed in the case of
reactions producing water to explain the decrease in activity
during continuous esterification reactions [12].
2. The adsorbed glycerol decreases the availability of the
water adsorbed onto the support leading to a decrease in the

3.2. Investigation of the phenomenon associated with the

loss of enzyme activity in the presence of glycerol
adsorbed onto the support
As previously described (Fig. 2), the continuous transesterification reaction was carried out for 10 h, at the end of
which the conversion was about 20%. The enzymatic support with adsorbed glycerol was then recovered and placed
at 4C in a closed chamber containing an aqueous saturated
Mg(NO3)2 solution to ensure the recovery of an optimal
thermodynamic water activity (aw) of 0.54. The continuous
transesterification was then carried out with this new catalytic preparation, containing always the same quantity of
glycerol but with an aw of 0.54. The conversion obtained is
around 15% (data not shown). No improvement was observed after equilibration of the catalyst to its optimal thermodynamic water activity.
As first sight, glycerol seems responsible by itself for the
loss of activity by creating a hydrophilic hindrance. In order
to confirm these results, one series of enzymatic preparations was prepared with various amounts of adsorbed glycerol, a second series with various amounts of adsorbed
glycerol with aw 0.54 corresponding to a solution of
glycerol containing 20% of water (w/w) and finally, a series
with various amounts of water. The amounts of adsorbed
water were equal to those of the second series.
Activities of these catalytic preparations were tested in a
batch esterification reaction without production of glycerol:
200 mM of oleic acid and 300 mM of ethanol were added to
25 ml of n-hexane in the presence of the enzymatic preparation. Initial reaction velocities were measured and were
compared to a standard reaction, carried out with commercial Lipozyme.
Fig. 4 shows clearly that for each polar compound adsorbed onto the support, there was a decrease in enzymatic
activity, and the greater the amount adsorbed, the more
marked the effect. Glycerol amended with water has a
slightly higher negative effect than glycerol alone.
The formation of an hydrophilic layer of adsorbed glycerol seems to be the more valid explanation for the decrease
in enzyme activity. This hydrophilic hindrance is also observed in the case of adsorption of water onto the support
and is then further increased when glycerol amended with
water is adsorbed.

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V. Dossat et al. / Enzyme and Microbial Technology 25 (1999) 194 200

enzymatic support in order to adsorb preferentially the glycerol and thus to limit the loss in activity in reactors operating in a discontinuous way. This method has been tried in a
continuous plug flow reactor and have demonstrated that, in
this case, glycerol is partitioned between both solids (silica
gel and enzymatic support), and the fall in activity is just
delayed (data not shown).

Fig. 4. Relative initial velocity of esterification in presence of immobilized

Lipozyme pretreated with water and glycerol. Reaction conditions: substrate concentrations: 200 mM oleic acid and 300 mM ethanol in n-hexane;
immobilized Lipozyme: 100 mg; () glycerol adsorbed support; ()
glycerol with aw 0.54 adsorbed support; (F) water adsorbed support.

It could hypothesized that the thicker the hydrophilic

layer in the supported environment, the lower the activity of
the reaction involving high hydrophobic substrates.
In order to confirm this hypothesis, the reaction with
substrates less hydrophobic than oleic acid was studied. The
esterification reactions with lauric acid (C10) and valeric
acid (C5) catalyzed by Lipozyme were carried out. In each
case (oleic, lauric, and valeric acids), the initial rates obtained with commercial Lipozyme and with 100% adsorbed glycerol Lipozyme were compared.
As can be seen in Table 1, the less hydrophobic the
substrates, the less negative is the effect of glycerol adsorbed onto enzymatic support.
This result seems to confirm the creation of an adverse
hydrophilic layer by adsorption of glycerol.
3.3. Recovery of catalytic activity
Different methods were investigated to avoid this decrease in activity.
3.3.1 Use of silica gel to adsorb the produced glycerol
Stevenson et al. [10] have proposed an addition of another hydrophilic support like silica gel in addition to the
Table 1
Effect of glycerol adsorbed onto enzymatic support on initial reaction
rates for different substratesa

R
a

Oleic acid

Lauric acid

Valeric acid

25

18

Ratio of initial velocities (R) obtained with commercial Lipozyme and

Lipozyme with 100% of adsorbed glycerol.

3.3.2. Use of a polar co-solvent in n-hexane to increase

the medium polarity
Another solution proposed to maintain high conversion
at the outflow of a continuous reactor was the use of nhexane amended with a polar solvent in order to increase
glycerol solubility in the reaction medium and hence remove it continuously from the reactor. The use of 3 M
acetone amended n-hexane during continuous esterification
between oleic acid and ethanol has been successful [14].
The authors obtained a stable conversion of 85% for a
1-week period. They nevertheless observed a modification
of the enzyme kinetic parameters; in fact, initial velocities
were 3 times lower than in pure n-hexane, and no significant
change in the thermodynamic equilibrium position was detected.
The continuous transesterification of high oleic sunflower oil with butanol was carried out in the fixed bed
reactor using 3 M acetone amended n-hexane as solvent
taking into account the 3-times reduction in enzyme activity. The new conditions to obtain 95% conversion are a flow
rate of 0.5 ml/min and the use of 3.25 g of immobilized
catalyst. An initial conversion of high oleic sunflower oil of
90% was obtained, but as previously described in pure
n-hexane, a loss of activity with time was observed (data not
shown). After 80 h of reaction, the conversion was only
60%, which denotes that there was no improvement in the
stability of the reactor taking into account the new operating
conditions (higher quantity of enzymes and a lower flow
rate). The measurement of glycerol concentration on the
enzymatic support and in the outflow medium showed that
only 0.05 g/l of glycerol was evacuated by the medium, the
rest being adsorbed onto the enzymatic support (54%, w/w).
3.3.3. Intermittent washings of the enzymatic support.
Another solution proposed to recover the initial conversion is to solubilize and evacuate the glycerol out of the
reactor by intermittent washings of the Lipozyme with a
polar solvent. This solution is possible only if the adverse
adsorption of glycerol is a reversible phenomenon.
Butanol and 2-methylbutan-2-ol (2M2B) were used to
rinse the glycerol. These solvents were chosen in order to
avoid undesirable reaction, butanol is one of the substrates
of the reaction, and the Lipozyme does not catalyze the
esterification reaction with tertiary alcohols, like 2M2B.
These two polar solvents have to be equilibrated at the
optimal thermodynamic water activity of 0.54. The use of
dried butanol will lead to the drying of the enzymatic
support and to the decrease in enzyme activity [16]. Fig. 5

V. Dossat et al. / Enzyme and Microbial Technology 25 (1999) 194 200

199

Fig. 6. Continuous () and semi-continuous (F) transesterification

reaction.
Fig. 5. Thermodynamic water activity versus water concentration, in butan1-ol () and in 2-methylbutan-2-ol (F).

presents the correlation between aw and water content for

both polar solvents; to obtain an aw 0.54, butanol has to
be amended with 70 g/l of water and 2-methylbutan-2-ol
with 42 g/l.
A semi-continuous process was therefore proposed
which enables not only the evacuation of the glycerol out of
the reactor and thus the maintenance of a high conversion
value but also the separate recovery of the two products, the
ester and the glycerol. This system is presented in Fig. 1.
Firstly, the transesterification reaction was carried out for
8 h; the conversion yield was less than 50%. Next, the
catalyst bed was rinsed with the butanol solution for 30 min.
The transesterification reaction was then restored by pumping the substrate solution through the catalyst bed; the initial
conversion of 95% was recovered (data not shown). The
washing step has enabled all the glycerol adsorbed onto the
enzymatic support to be eliminated; optimal hydration of
the catalyst has been conserved and initial conversion restored. The same results are obtained with 2M2B (data not
shown). Finally, the transesterification reaction was carried
out in a succession of 2-h synthesis steps in order to minimize the loss in conversion followed by 30 min washing
steps at a flow rate of 0.5 ml/min. The results obtained with
the butanol solution are presented in Fig. 6. With this
process, a conversion value of more than 80% at the end of
48 h of synthesis was retained, whereas conversion had been
almost 0 after 15 h.

Two hypothesis were proposed to explain this phenomenon. Firstly adsorption of glycerol may cause a decrease in
thermodynamic water activity of the enzyme whose optimum aw is 0.54. Various experiments carried out with
Lipozyme adsorbed glycerol at controlled aw of 0.54 did
not show any improvement in conversion. Alternatively, it
was assumed that glycerol forms a layer around the enzyme
which inhibits the diffusion of the hydrophobic substrate.
This hypothesis is supported by the fact that the more polar
the acid, the less marked is this negative effect.
Various solutions were proposed to try to maintain enzymatic activity.
Firstly, silica gel was added to the reactor bed in order to
preferentially adsorb the glycerol. No significant improvement was observed in the stability of the reactor. Secondly,
the reaction was carried out in n-hexane amended with
acetone as reaction medium, but unfortunately, glycerol was
not evacuated in any significant quantities. Finally, a semicontinuous process was proposed which consisted of cycles
of transesterification reaction and rinsing of the catalyst in
order to evacuate the adsorbed glycerol out of the reactor.
The rinsing solution was butanol amended water with the
thermodynamic water activity adjusted to 0.54. By rinsing
the enzymatic support, optimal hydration of the catalyst was
maintained, and the initial conversion was restored. A semicontinuous process for the synthesis of oleic acid butyl ester
was developed, which ensures not only that high conversion
was maintained but also that the products, ester and glycerol, were recovered separately without a further separation
step.

4. Conclusions
Continuous transesterification of high oleic sunflower oil
by immobilized Lipozyme was carried out in a continuous
way. It was demonstrated that all the glycerol produced
remains adsorbed onto the enzymatic support, leading to a
drastic decrease in enzyme activity.

Acknowledgments
The authors thank La Toulousaine de Cereales for the
financial support of this work. They are very grateful to
Yoann Francheteau and Dominique Pituello for their tech-

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V. Dossat et al. / Enzyme and Microbial Technology 25 (1999) 194 200

nical assistance and to Mel Sladdin for the English style

corrections.

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