656

DOI 10.1002/ejlt.200300871

Mohamed M. Soumanoua,b,
Uwe T. Bornscheuera

Lipase-catalyzed alcoholysis of vegetable oils

Department of Technical
Chemistry and Biotechnology, Institute of Chemistry
and Biochemistry, Greifswald University, Greifswald,
Germany
Polytechnic College University, Research Laboratory
for Applied Chemistry and
Biology (LARECBA),
Abomey-Calavi University,
Cotonou, Benin

Eur. J. Lipid Sci. Technol. 105 (2003) 656660

Fatty acid alkyl esters were produced from various vegetable oils by transesterification
with different alcohols using immobilized lipases. Using n-hexane as organic solvent,
all immobilized lipases tested were found to be active during methanolysis. Highest
conversion (97%) was observed with Thermomyces lanuginosa lipase after 24 h. In
contrast, this lipase was almost inactive in a solvent-free reaction medium using
methanol or 2-propanol as alcohol substrates. This could be overcome by a three-step
addition of methanol, which works efficiently for a range of vegetable oils (e.g. cottonseed, peanut, sunflower, palm olein, coconut and palm kernel) using immobilized
lipases from Pseudomonas fluorescens (AK lipase) and Rhizomucor miehei (RM
lipase). Repeated batch reactions showed that Rhizomucor miehei lipase was very
stable over 120 h. AK and RM lipases also showed acceptable conversion levels for
cottonseed oil with ethanol, 1-propanol, 1-butanol and isobutanol (50-65% conversion
after 24 h) in solvent-free conditions. Methyl and isopropyl fatty acid esters obtained by
enzymatic alcoholysis of natural vegetable oils can find application in biodiesel fuels
and cosmetics industry, respectively.
Keywords: Immobilized lipase, alcoholysis, vegetable oils, fatty acid alkyl esters,
stability, organic solvent.

Research Paper

1 Introduction
Oils and fats are essential constituents of all plant and animal life. They are produced world-wide at a level of approximately 67 Mt per year, more than 90% of which is
used in edible products [1-2]. In addition to this nutritional
function, more than 2 Mt is used as starting material for
synthesis of valuable intermediates in oleochemistry
which are used as lubricants, plasticizers, biodiesel, in
cosmetics etc. [3-4]. To achieve the synthesis of such
products, high energy consuming processes such as hydrolysis, glycerolysis and alcoholysis are used. Alcoholysis is widely employed to convert lipids directly to ester
without prior isolation of free fatty acids.
For methanolysis, the fat or oil is dissolved in an excess of
methanol in a continuous process using an alkaline catalyst at 240 C and 10 MPa. A procedure for converting
crude vegetable oils (up to 30% free fatty acid) to methyl
esters on a pilot-plant scale (500 kg/h or 3000 t/year) has
been described [5]. However, the resulting products are
often impure and require re-distillation to remove degradation by-products, resulting from the severe reaction
conditions. In addition, highly unsaturated oils cannot be
used without prior hydrogenation.

Correspondence: Uwe T. Bornscheuer, Department of Technical Chemistry and Biotechnology, Institute of Chemistry and Biochemistry, Greifswald University, Soldmannstrae 16, D-17487
Greifswald, Germany. Phone: +49-3834-86-4367, Fax: +493834-86-4346; e-mail: uwe.bornscheuer@uni-greifswald.de,
web site: http://www.chemie.uni-greifswald.de/ ~biotech

2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Alternatively, a biocatalytic process can be used to replace the conventional chemical process, thus saving energy and minimizing thermal degradation. Consequently,
the application of lipases in the oleochemical industry has
become very attractive [6]. In spite of their potential industrial application, however, alcoholysis of vegetable oils
using lipases has not been extensively investigated.

Among various studies on enzymatic alcoholysis of vegetable oils, two main difficulties have been reported. First,
the insolubility of free glycerol in oil or organic solvent can
inhibit the reaction by limiting substrate and product diffusion. To solve this problem silica gel and other adsorbents
have been used, which allow glycerol extraction from the
alcoholysis reaction mixture, leading to near quantitative
conversion of various fats or oils to fatty alkyl esters [7]. A
second problem is inactivation of the enzymes by shortchain alcohols such as methanol [8-9]. Using a MichaelisMenten type equation for substrate inhibition, variation of
the initial reaction rate with methanol has been analyzed
[10]. Based on this equation, a procedure for the stepwise
addition of methanol has been shown to protect sensitive
lipases such as Candida antarctica lipase against inactivation [9].

In this paper, we report the enzymatic alcoholysis of various vegetable oils in n-hexane and a solvent-free system.
In view of industrial processing, stability of immobilized lipases has also been investigated.
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Eur. J. Lipid Sci. Technol. 105 (2003) 656660

2 Material and methods

2.1 Chemicals and lipases
All chemicals and solvents used for alcoholysis and
analysis of reaction products were of reagent grade and
purchased from common commercial suppliers. The carrier material polypropylene EP 100 (Akzo Nobel Faser
AG, Obernburg, Germany) was used for lipase immobilization. Vegetable oils were obtained from IBCG industry
(Cotonou, Benin), except sunflower oil which was purchased from ATI industry (Cotonou, Benin). Lipase from
Pseudomonas fluorescens (AK) was a gift from Amano
Pharmaceutical Co. Ltd. (Nagoya, Japan). Lipases from
Rhizomucor miehei (Lipozyme RM, IM) immobilized on
an anion exchange resin, and Thermomyces lanuginosa
(Lipozyme TL, IM) immobilized on silica gel were gifts
from Novozymes (Bagsvaerd, Denmark).

2.2 Lipase immobilization

Lipase from Pseudomonas fluorescens was immobilized
by an adsorption method. Before immobilization, the
polypropylene carrier EP100 (0.5 g) was soaked with 2 ml
absolute ethanol. Lipase (0.5 g) was dissolved in 20 ml
phosphate buffer (pH 7.0, 0.1 M). This solution was added
to the wet support and stirred slowly overnight at room
temperature. The immobilized lipase preparation was collected by filtration, washed twice with 20 ml phosphate
buffer followed by distilled water (20 ml) and dried
overnight under vacuum.

2.3 Alcoholysis reaction

2.3.1 Reactions in organic solvent
Reactions were performed as batch process in 2 ml
n-hexane in glass tubes containing vegetable oil
(0.2 mmol), methanol (0.6 mmol) and 10% (w/w vegetable oil) immobilized lipase. The reaction mixture was
incubated in a water bath at 40 C and agitated with a
magnetic stirrer at 200 rpm. For time course studies, 5 l
aliquots of reaction medium were taken at various time intervals and diluted in n-heptane for gas chromatography
(GC)-analysis.

2.3.2 Reactions in a solvent free system

Reactions were carried out in eppendorf tubes (2 ml) containing a mixture of 0.2 mmol vegetable oil and alcohol
(0.6 mmol) using 10% (w/w vegetable oil) immobilized lipase. The reaction mixture was incubated at 40 C and
shaken in an eppendorf thermomixer. The stability of
commercially immobilized lipases in batch alcoholysis reactions with stepwise addition of methanol was investi 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Alcoholysis of vegetable oils

657

gated in the reaction media. After each batch reaction, immobilized lipase was recovered by filtration, washed three
times with chilled acetone and dried at room temperature.
Dried immobilized lipase was used in the next batch reaction with new substrates. Residual activity was expressed
in terms of conversion achieved after each 24 h reaction
cycle.

2.4 GC-analysis
Samples from organic solvent medium and solvent-free
systems were analyzed by injecting 1 l of reaction mixture and an internal standard (methyl pentadecanoate),
into a gas chromatograph equipped with a polar column
(25 0.25 mm id., Macherey & Nagel, Dren, Germany).
Analysis was carried out with a temperature program running from 150 C to 250 C at 5 C/min. Injection and detection temperature were fixed at 250 C using hydrogen
as carrier gas. The conversion level was calculated from
the amount of fatty acid alkyl ester formed.

3 Results and discussion

3.1 Alcoholysis in organic solvent
Unsaturated vegetable oils such as cottonseed oil and
palm olein were used for methanolysis, in n-hexane as organic solvent, with three immobilized lipases (two commercially immobilized lipases RM and TL, and lipase from
AK immobilized on EP100). All lipases tested were active
in this reaction medium (Fig. 1). As can be seen,
methanolysis using both vegetable oils, progressed faster
with immobilized lipase from TL. The highest conversion
in this reaction medium after 24 h was observed with TL lipase using palm olein (97%). For cottonseed oil, however, AK lipase showed the best performance (82% conversion). Thus, in n-hexane, palm olein appeared to be the
best substrate for lipases TL and RM, whereas AK lipase
was most effective in converting cottonseed oil to methyl
esters. This may be due to the different specificities displayed by lipases towards certain fatty acids and their position along the glycerol backbone in vegetable oils. In another interesterification reaction reported in the literature,
TL and RM showed experimentally similar activity profiles
[11]. Despite these high activities displayed by lipases in
organic media, some important disadvantages of organic
solvents remain, namely higher costs, toxicity and flammability, which can limit enzymatic processes in industry.
Therefore, a solvent-free system [12] which offers greater
safety, increased reactant concentrations and high volumetric productivity was investigated.
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658

Soumanou and Bornscheuer

Eur. J. Lipid Sci. Technol. 105 (2003) 656660

Fig. 1. Time course of methanolysis reaction of cottonseed oil

(open symbols) or palm olein (full
symbols) in n-hexane using immobilized lipases. Symbols: , lipase AK; , lipase RM; , lipase
TL. See Materials and methods
for details.

3.2 Alcoholysis in solvent free media

Alcoholysis in solvent free medium using various alcohols
with cottonseed oil as substrates was investigated at
40 C. Results in Fig. 2 show alkyl ester production from
cottonseed oil after 24 h reaction time in a solvent free
system. Low conversion levels were observed with the
short chain alcohol methanol, especially with TL lipase.
This low performance in alkyl ester production from vegetable oils and methanol is in agreement with the work of
Mittelbach [8]. The low yield of methyl esters can be attributed to unfavorable viscosity conditions which affects
mixing of substrates with the lipase [13-14]. However, AK
and RM lipases displayed high catalytic activity in this re-

action medium with most alcohol substrates (Fig. 2). Lowest conversion was observed with TL lipase with all alcohol substrates, which is in striking contrast to its performance in organic solvent but in agreement with our previous work [15]. Using secondary alcohols as substrate for
ester production, conversion was found to be lower with
2-propanol than with isobutanol. In all cases, better conversion levels were obtained when cottonseed oil was
transesterified with 1-propanol and isobutanol, as demonstrated in Fig. 2.
To increase conversion levels with methanol as substrate,
a stepwise addition was used. The reaction was started at
a 1:1 molar equivalent methanol: cottonseed, oil followed

Fig. 2. Effect of alcohol type on

cottonseed oil alcoholysis, in solvent-free system using immobilized lipases. See Materials and
methods for details.

2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Eur. J. Lipid Sci. Technol. 105 (2003) 656660

Alcoholysis of vegetable oils

659

Fig. 3. Effect of stepwise

methanol addition on methanolysis of cottonseed oil in a solventfree system, using immobilized lipases. Symbols: , lipase AK; ,
lipase RM; , lipase TL. See Materials and methods for details.

by addition of 1 molar equivalent methanol after 4 and 8 h.

With this strategy, over 90% conversion was possible after
30 h using AK and RM lipases (Fig. 3). A lower conversion
level was observed with TL lipase, which was nevertheless
considerably higher than a one-step methanol addition with
this enzyme (10% conversion after 24 h, Fig. 2). This is in
agreement with results reported by Shimada et al. [9] who
demonstrated that stepwise addition of methanol is extremely effective in converting vegetable oil fatty acids to
their respective alkyl esters, by reducing lipase inactivation.
Particularly with water-miscible alcohol substrates such as
methanol, water required to maintain lipase structure can

be stripped leading to lower activity and eventual biocatalyst inactivation. Step-wise addition of methanol is thought
to reduce this effect, explaining the higher stabilities and
conversion levels observed.
The stability of immobilized lipases during consecutive
batch transesterification of cottonseed oil was studied under
these conditions. As shown in Fig. 4, both immobilized TL
and RM lipases exhibited high stability (above 75%) when
reused during the first five batch reactions (each 24 h). Additional reactions lead to further loss of enzyme activity,
which was particularly significant for TL lipase. This enzyme

Fig. 4. Stability of commercially

immobilized lipases, RM and TL
during eight consecutive batch
reactions. For conditions see Fig.
3 and Materials and methods.

2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Soumanou and Bornscheuer

Eur. J. Lipid Sci. Technol. 105 (2003) 656660

Tab. 1. Alcoholysis of vegetable oils by stepwise addition of methanol or 2-propanol using immobilized lipase. Alcoholysis reaction was carried out for 4 h in a mixture of 0.2 mmol vegetable oil/alcohol (1:1, mol/mol) using 10% immobilized lipase. After 4 h and 8 h, 0.2 mmol alcohol was added to the reaction mixture. RM: immobilized lipase from Rhizomucor miehei; AK: lipase from Pseudomonas fluorescens immobilized on polypropylene EP100. Conversion levels were determined after 24 h.
Conversion [%]
Vegetable oil

Cottonseed oil
Peanut oil
Sunflower oil
Palm olein
Coconut oil
Palmkernel oil

RM

AK

Methanol

2-Propanol

Methanol

2-Propanol

83
63
84
72
64
72

41
42
35
50
56
69

82
73
75
82
66
74

33
37
36
32
34
35

retained only 35% residual activity after 8 batch reactions.

This can be attributed to different factors such as, inactivation of the biocatalyst in the oil phase, type of carrier used
for immobilization or enzyme sensitivity to long-term
methanol exposure. In contrast, the RM lipase preparation
appears to be very suitable for alkyl esters production.
Finally, RM and immobilized AK lipase activities were
compared for alcoholysis of various vegetable oils using
stepwise addition of methanol or 2-propanol. Data in Tab.
1 demonstrates that both lipases can achieve conversion
levels between 63 and 84% after 24 h methanolysis.
Using the secondary alcohol 2-propanol, lower conversions were found with all vegetable oils. In these experiments RM lipase was found to be the most suitable enzyme with 69% conversion using palm kernel oil. These
conversion levels are nevertheless higher than those reported for one step esterification of 2-propanol with lauric
oils [14].

Acknowledgements
We gratefully acknowledge financial support for Dr. M. M.
Soumanou by the German Academic Exchange Service
(DAAD, Bonn, Germany). The gifts of lipases from
Novozymes, Bagsvaerd, Denmark and Amano Pharmaceuticals Inc., Nagoya, Japan are gratefully appreciated.

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[Received: July 8, 2003; accepted: July 29, 2003]

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