Journal of Molecular Catalysis B: Enzymatic 84 (2012) 173–176
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Preparation of n-3 PUFAs ethyl esters by an efficient biocatalyzed solvent-free
process
∗
R. Morrone , N. D’Antona, D. Biondi, D. Lambusta, G. Nicolosi
CNR – Istituto di Chimica Biomolecolare, Via Paolo Gaifami 18, 95126 Catania, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 16 April 2012
alpha-Linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been sub-
jected to esterification with ethanol in presence of lipase B from Candida antarctica (Novozym 435 ),
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in solvent free condition. The use of alcohol donors triethyl orthoformate (TEOF) or diethyl carbonate
(DEC) instead of free ethanol, allowed working in irreversible esterification conditions and ALA-, EPA-
and DHA-ethyl esters were obtained in quantitative yields.
Keywords:
PUFA
Irreversible esterification
Lipase
©
2012 Elsevier B.V. All rights reserved.
Diethyl carbonate
Triethyl orthoformate
1
. Introduction
While an extensive series of data is available for the trans-
formation of PUFA into triglycerides, little is reported about the
preparation of PUFA ethyl ester by direct esterification. Recently
Today the design of new environmentally friendly and economi-
cally valuable syntheses is one of the main challenges for companies
operating in the chemistry sector. The “green approach” seeks to
respond to this requirement by developing procedures with high
or quantitative yields, safety of reagents and products, possibil-
ity to recycle the catalyst(s), and when feasible by performing the
reactions in water or solvent free conditions. Biocatalysis, exploit-
ing the action of natural catalysts such as enzymes that are able
to work in mild and eco-friendly conditions, can often accomplish
these “green” requirements. In chemical transformations of sensi-
tive compounds these conditions are mandatory. This is the case of
n-3 polyunsaturated fatty acids (n-3 PUFAs) esterification.
The interest towards n-3 polyunsaturated fatty acids dates back
to about thirty years ago when some studies suggested that the
elevated concentrations of n-3 PUFA in the diet of Greenland Eski-
mos were responsible for the low mortality for cardiovascular
disease in this population [1]. The beneficial effects of n-3 PUFAs
on human health are now well documented [2–4] and the main
macro-consequences have been their much higher consumption
and a consequent increase in the market demand.
Silva et al. [8] published an H PW12O40 catalyzed esterification
3
of different fatty acids, including unsaturated acids. Although the
process gives ethyl esters in satisfactory yield, the large excess of
alcohol and the use of tungsten-catalyst makes the process unsuit-
able for a green application. Considering that PUFA triglycerides
represent the natural substrates for lipases, these enzymes are opti-
mal candidates to act as mild catalysts in the preparation of their
corresponding ethyl esters. But so far, although this approach has
been reported for fatty acids [9], few investigation have been made
for the sensitive PUFAs and, to our knowledge, the only example
is the use of Phycomices nitens lipase in the esterification of EPA
with ethanol [10]. In the light of this, we decided to try extend-
ing our experience in the esterification of aryl acids to PUFAs, and
here we report the irreversible esterification, in presence of lipase
B from Candida antarctica, of three different n-3 PUFAs: ␣-linolenic
acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid
(DHA) (Fig. 1) in order to prepare the corresponding ethyl esters in
high yield.
Since they are easily oxidised, the free n-3 PUFAs are usually
preserved during storage in the form of triglycerides or ethyl esters
2. Materials and methods
[
5]. In general, having these acids in the form of esters also facilitates
2
.1. Materials
epoxy fatty acids [6,7].
ALA (9,12,15-octadecatrienoic acid), EPA (5,8,11,14,17-
eicosapentaenoic acid), DHA (4,7,10,13,16,19-docosahexaenoic
acid), stearic acid (SA), ethanol absolute and lipase B from C.
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∗ Corresponding author. Fax: +39 095 7338310.
antarctica (Novozym 435 ) were obtained from Sigma–Aldrich;
diethyl carbonate (DEC) and triethyl orthoformate (TEOF) were
1
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