Lipase-Catalyzed Synthesis of Structured
Triacylglycerides from 1,3-Diacylglycerides
a,b
a
b,
Sirirung Wongsakul , Aran H-Kittikun , and Uwe T. Bornscheuer *
a
Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai 90112,
b
Thailand, and Institute of Chemistry and Biochemistry, Department of Technical Chemistry and Biotechnology,
University of Greifswald, D-17487 Greifswald, Germany
ABSTRACT: A new method for the lipase-catalyzed synthesis of rities can be achieved by a two-step process developed in our
structured TAG (ST) is described. First, sn1,3-dilaurin or -dicaprylin laboratories (7–9). In the first step, highly pure 2-MAG are ob-
were enzymatically synthesized using different published meth-
ods. Next, these were esterified at the sn2-position with oleic acid
or its vinyl ester using different lipases. Key to successful enzymatic
synthesis of ST was the choice of a lipase with appropriate FA
specificity, i.e., one that does not act on the FA already present in
the sn1,3-DAG, but that at the same time exhibits high selectivity
and activity toward the FA to be introduced. Reactions were per-
formed in the presence of organic solvents or in solvent-free sys-
tems under reduced pressure. With this strategy, mixed ST con-
taining the desired compounds 1,3-dicapryloyl-2-oleyl-glycerol or
tained by alcoholysis of a TAG with ethanol using a 1,3-re-
giospecific lipase followed by crystallization. The 2-MAG thus
obtained are subsequently esterified with a suitable FA in the
second step to obtain the desired ST. More recently, we also re-
ported the synthesis of ST containing PUFA from tuna oil in the
sn2-position (10). However, crystallization of 2-MAG contain-
ing unsaturated FA is rather difficult due to the very low m.p.
(i.e., –56°C for 2-docosahexaenoic acid glycerol ester).
An alternative would be the use of sn1,3-diacylglycerols
1
8
,3-dilauroyl-2-oleyl-glycerol (CyOCy or LaOLa) were obtained at (1,3-DAG) as starting materials, which are then esterified
7 and 78 mol% yield, respectively, using immobilized lipases with a sn2-specific lipase (Scheme 1). A range of enzymatic
from Burkholderia cepacia (Amano PS-D) in n-hexane at 60°C.
However, regiospecific analysis with porcine pancreatic lipase in-
dicated that in CyOCy, 25.7% caprylic acid and in LaOLa 11.1%
lauric acid were located at the sn2-position. Oleic acid vinyl ester
was a better acyl donor than oleic acid. Esterification of sn1,3-
DAG and free oleic acid gave very low yield (<20%) of ST in a sol-
vent system and moderate yield (>50%) in a solvent-free system
under reduced pressure.
methods for the synthesis of sn1,3-DAG in high yield and pu-
rity are described in the literature that use, e.g., glycerol ad-
sorbed on silica gel and FA vinyl esters as acyl donors (11) or
hydrolysis of TAG by sn1,3-specific lipases (12). 1,3-DAG
obtained by lipase catalysis (13) have been introduced re-
cently as alternative cooking and frying oils (14) and there-
fore are readily available on an industrial scale. Unfortu-
nately, sn2-specific lipases are not available from nature, al-
though some sn2-selectivity of a few lipases has been
Paper no. J10669 in JAOCS 81, 151-155 (February 2004).
KEY WORDS: Burkholderia cepacia lipase, Candida antarctica described (15,16). This was later attributed mostly to acyl mi-
lipase B, 1,3-DAG, immobilized lipases, structured TAG.
gration and not to a positional selectivity of the enzyme (17).
We envisaged that the enzymatic synthesis of ST also
should be feasible from 1,3-DAG by using a lipase with FA
The nutritional value of TAG and their physicochemical prop- chain-length or FA saturation specificity. Accordingly, this
erties not only are determined by the FA composition but also enzyme must not act on the FA already present in the 1,3-
depend on the positional distribution of the acyl groups DAG, but at the same time exhibit high selectivity and activ-
bonded to the glycerol. Structured TAG (ST) of the ABA-type ity toward the FA to be introduced at the sn2-position. The in-
containing medium-chain FA (MCFA; e.g., C ) in sn1,3-posi- vestigation of this strategy is described in this paper.
8
tions and a long-chain unsaturated FA (e.g., C –C ) in the
1
6
22
sn2-position are an effective energy source for patients with
malabsorption, e.g., pancreatic insufficiency. A range of
EXPERIMENTAL PROCEDURES
methods for the enzymatic synthesis of these compounds Lipases. Lipases (TAG hydrolases, EC 3.1.1.3) were from Rhi-
have been already described (1,2). This includes acidolysis of zopus oryzae [Amano D, D-EP100; immobilized on poly-
a TAG (3,4) or interesterification between two TAG (5,6). propylene (EP100) Amano Pharmaceuticals Co., Nagoya,
Unfortunately, yields of ABA-type TAG are low, and a vari-
ety of by-products are formed. These are difficult to separate
from the desired product. Significantly higher yields and pu-
*To whom correspondence should be addressed at Institute of Chemistry and
Biochemistry, Department of Technical Chemistry and Biotechnology, Uni-
versity of Greifswald, Soldmannstr. 16, D-17487 Greifswald, Germany.
R = –H (FFA); –CH = CH (FAVE)
3
2
E-mail: uwe.bornscheuer@uni-greifswald.de
SCHEME 1
Copyright © 2004 by AOCS Press
151
JAOCS, Vol. 81, no. 2 (2004)