Lipase-Catalyzed Synthesis of Structured Triacylglycerides from 1,3-Diacylglycerides

Article abstract of DOI:10.1007/s11746-004-0873-z

A new method for the lipase-catalyzed synthesis of structured TAG (ST) is described. First, sn1,3-dilaurin or -dicaprylin were enzymatically synthesized using different published methods. 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 performed in the presence of organic solvents or in solvent-free systems under reduced pressure. With this strategy, mixed ST containing the desired compounds 1,3-dicapryloyl-2-oleyl- glycerol or 1,3-dilauroyl-2-oleyl-glycerol (CyOCy or LaOLa) were obtained at 87 and 78 mol% yield, respectively, using immobilized lipases from Burkholderia cepacia (Amano PS-D) in n-hexane at 60°C. However, regiospecific analysis with porcine pancreatic lipase indicated 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 solvent system and moderate yield (>50%) in a solvent-free system under reduced pressure.

Full text of DOI:10.1007/s11746-004-0873-z

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)

152
S. WONGSAKUL ET AL.
Japan], Burkholderia cepacia (immobilized, Amano PS-D, FID analysis (Iatroscan; Iatron Laboratories, Tokyo, Japan)
formerly named Pseudomonas cepacia; Amano Pharmaceuti- (18). The FA compositions of acylglycerols were determined
cals Co.), Rhizomucor miehei (Lipozyme RM IM, Novozyme, by converting all FA into the corresponding FAME followed
Bagsvaerd, Denmark), lipase B from Candida antarctica by GC analysis as described previously (10). For ST analysis,
(
CAL-B; Novozyme), and porcine pancreatic lipase (Amano the reaction mixture was separated by TLC using a solvent sys-
Pharmaceuticals Co.). All chemicals and solvents used were tem of chloroform/methanol/ammonium (70:0.4:0.1 by vol)
reagent grade and purchased from common commercial sup- and visualized by iodine vapor. The band corresponding to
pliers, with the exception of EP 100 (particle size 200–400 TAG was scratched-off, followed by methylation for GC analy-
µm; Akzo, Obernburg, Germany).
sis.
HPLC separation of TAG. The composition of the TAG
Synthesis of 1,3-DAG by esterification of glycerol and FFA
or FA vinyl esters (FAVE). 1,3-DAG was synthesized from formed during the enzymatic esterification was determined by
glycerol (immobilized on silica gel) and FFA or FAVE in HPLC using a Nucleosil ODS column, (5 µm, 250 × 4.6 mm;
methyl-tert-butyl ether (MTBE) at room temperature cat- Agilent, Waldbronn, Germany) and an ELSD (Polymer Labs,
alyzed by Lipozyme RM IM as described (11).
Darmstadt, Germany) at a flow rate of 1.5 mL/min. Elution was
1
,3-DAG were also synthesized by esterification of glyc- performed using a gradient elution system of acetonitrile and
erol with FFA or FAVE in a solvent-free system or in an or- dichloromethane (70 to 55% acetonitrile over 10 min, followed
ganic solvent catalyzed by CAL-B according to a published by 55 to 70% acetonitrile over 8 min).
method (12) with minor modifications. Activated molecular
Regiospecific analysis of TAG. The distribution of the FA on
sieves were added or vacuum was applied in reactions with the TAG was determined by digestion of TAG with porcine
FFA to remove water produced during the reaction. The 1,3- pancreatic lipase followed by GC analysis (9). To a 5-mL
DAG were recovered and purified by crystallization in n- screw-capped tube, 20 mg of purified TAG, 10 mg pancreatic
hexane at –20°C. If high concentrations of 2-MAG were pre- lipase, and 3 mL Tris-HCl buffer (pH 8, 1 M) were added. After
sent, recrystallization in dry methanol at –20°C was per- shaking, 0.5 mL sodium deoxycholate solution (1 g/L) and 0.2
formed to obtain purer 1,3-DAG. Samples from the reaction mL calcium chloride solution (220 g/L) were added. After vig-
mixture were periodically withdrawn to determine the acyl- orous shaking, the tube was immediately placed in a water bath
glycerol composition by TLC-FID (see below).
(40°C) for 1 min. Then the tube was shaken for 2 min and
Alcoholysis of TAG for the synthesis of 1,3-DAG. This reac- cooled in running water before 1 mL hydrochloric acid (6 M)
tion was carried out using a modified method for 2-MAG syn- and 1 mL diethylether were added, followed by vigorous shak-
thesis (10). The substrate mixture was pre-equilibrated to a water ing for 1 min. After separation of the phases by centrifugation,
activity of a 0.11 for 48 h before use. The TAG/ethanol ratio the components of the resulting mixture of partial acylglycerols
w
was 1:1 to 1:2 (vol/vol). Samples from the reaction mixture were in the ether phase were separated by preparative TLC using ace-
periodically withdrawn and diluted with chloroform, followed tone/chloroform (96:4 by vol) as developing solvent and visu-
by the determination of acylglycerol composition by TLC-FID alized by iodine vapor. The 2-MAG band was scraped off,
(see below). After 24 h, the immobilized lipase was separated methylated, and analyzed by GC.
from the reaction mixture by centrifugation to stop the reaction.
Synthesis of ST from 1,3-DAG and FFA/FAVE. ST were
synthesized by esterification of 1,3-DAG and oleic acid (OA)
RESULTS AND DISCUSSION
or oleic acid vinyl ester (OAVE) in n-hexane at 60°C using Enzymatic synthesis of 1,3-DAG. The highest 1,3-DAG yields
PS-D as biocatalyst. 1,3-DAG (0.1 mmol) and OA or OAVE [93% for 1,3-dicaprylin (1,3-DCy) 73% for 1,3-dilaurin, (1,3-
(
0.2 mmol) were dissolved in 1 mL n-hexane in 2-mL screw- DLa)] were obtained after 4–6 h in the esterification between
capped vials. Activated molecular sieves were added, or vac- FAVE and glycerol at 0°C in a solvent-free system using CAL-
uum was applied when OA was used as acyl donor, for water B as biocatalyst. The molar ratio of 1,3-DAG and FAVE was
removal. The reaction was started by addition of PS-D (10% 1:2 (mol/mol). The yield thus obtained was higher than the one
w/w of 1,3-DAG). Samples from the reaction mixture were found in the esterification of glycerol adsorbed on silical gel
periodically withdrawn for the determination of acylglycerol (11) (75% 1,3-DCy; 65% 1,3-DLa). Direct esterification of FFA
composition by TLC-FID and HPLC.
and glycerol was slow and gave only moderate yields (60% 1,3-
Purification of produced TAG. Lipase was removed by DCy; 60% 1,3-DLa) with high amounts of MAG present. How-
centrifugation, and excess solvent was removed by a nitrogen ever, the yield could be raised with increasing temperature and
stream. The reaction mixture was dissolved in n-hexane and under reduced pressure (data not shown).
purified by silica gel column chromatography. Elution was
performed with a gradient starting from n-hexane/diethylether the introduction, the synthesis of an ST from sn1,3-DAG and a
99:1) and subsequent increase of diethylether concentration sn2-destined FA should be possible with a lipase having a well-
up to pure diethylether. The composition of each fraction was defined FA specificity. To demonstrate the practicability of this
determined separately by TLC-FID. new strategy, the nonregiospecific lipase PS-D was chosen, as
Enzymatic synthesis of ST from 1,3-DAG. As pointed out in
(
Analytical methods. Changes in acylglycerol composition it preferably acts on long-chain FA (LCFA) rather than MCFA
during the reaction were quantitatively determined by TLC- (Wongsakul, S., unpublished observations). Thus, using a 1,3-
JAOCS, Vol. 81, no. 2 (2004)

STRUCTURED TAG BY LIPASE CATALYSIS
153
FIG. 1. Time course of the esterification between 1,3-DAG and oleic acid vinyl ester or oleic acid
in n-hexane catalyzed by lipase Amano PS-D at 60°C (small-scale synthesis). TAG from 1,3-DCy +
OAVE (■), TAG from 1,3-DCy + OA (●), DAG decrease from 1,3-DCy + OAVE (■), DAG de-
crease from 1,3-DCy + OA (●), TAG from 1,3-DLa + OAVE (▲), TAG from 1,3-DLa + OA (◆),
DAG decrease from 1,3-DLa + OAVE (▲), DAG decrease from 1,3-DCy + OA (◆); 1,3-DCy, 1-3-
dicaprylin; OAVE, oleic acid vinyl ester; OA, oleic acid; 1,3-DLa, 1,3-dilaurin.
DAG composed of MCFA and a LCFA as acyl donor, ST syn- hexane was very slow, and only 20% TAG was obtained after
thesis should be feasible. Indeed, esterification of 1,3-dicaprylin 24 h in the esterification starting from 1,3-dilaurin, and al-
or -dilaurin with OAVE in n-hexane resulted in high yields of most no reaction occurred using 1,3-dicaprylin (Fig. 1).
TAG (87 and 78%, respectively) at very short reaction times (3 Yields and reaction rates were considerably increased to 56%
and 8 h) (Fig. 1). GC analysis of these ST showed that the ratio (from 1,3-dicaprylin) and 53% (from 1,3-dilaurin) in the sol-
of MCFA to LCFA was about 2:1 (Table 1). Esterification of 1,3- vent-free system under reduced pressure (Fig. 3).
DCy with OAVE performed at gram-scale was finished after
A new enzymatic method for the synthesis of ST starting
only 2 h (Fig. 2). HPLC analysis of the product mixture revealed from 1,3-DAG and a FA (or its vinyl ester) was developed. The
that the desired ST CyOCy (59.4%) was most prominent fol- yields are comparable to the previously described two-step
lowed by CyOO (32.8%). Triolein was formed in only very method (7–9), which is based on TAG alcoholysis to yield 2-
small amounts (0.65%). It should be noted that this method does MAG followed by esterification at the sn1,3-positions. How-
not allow distinction between regioisomers. Thus, the TAG were ever, in this method, the yield of the desired ST (CyOCy or
purified and partially degraded using porcine pancreatic lipase, LaOLa) is lower due to the presence of other TAG species. In
followed by GC analysis of the 2-MAG fraction. The result addition, the regioisomers (i.e., CyCyO or LaLaO) were found
showed that CyOCy/CyOO contained 74.3% OA and 25.7% after analysis using PPL degradation. However, it cannot be ex-
caprylic acid at the sn2-position, whereas LaOLa/ LaOO con- cluded that partial acyl migration occurred during this analyti-
tained 88.9% OA and 11.1% lauric acid at the sn2-position.
cal method. Further improvement of the reaction conditions is
When free OA was used as acyl donor, water produced under study to avoid the formation of undesired ST and to apply
during the esterification was removed either by applying vac- alternative methods for regiospecific analysis.
uum (solvent-free system) or by adding molecular sieve (sol-
Still, several major advantages of this new method exist,
vent system), to avoid acyl migration and an unfavorable which include the easier synthesis of regioisomerically pure
equilibrium. Esterification between 1,3-DAG and OA in n- sn1,3-DAG. These are much more stable than the sn2-MAG,
obtained from alcoholysis of TAG, which can easily undergo
acyl migration to yield sn1(3)-MAG. We assume that it will
also be easier to produce ST bearing PUFA such as EPA or
DHA at the sn2-position, as they are difficult to obtain using
the two-step method owing to difficulties in the isolation of
their sn2-MAG. The alternative would be an acidolysis reac-
tion between, e.g., fish oil and another FA, but this results in
low yields, a mixture of a broad range of TAG species and
acyl migration can occur. In contrast, it has been demon-
strated here that lipase from B. cepacia (Amano PS-D) effi-
ciently introduced OA to the sn2-position of two 1,3-DAG
TABLE 1
FA Composition of Structured TAG Products
FA (%)a
Structured TAG
C8:0
C12:0
C18:1
CyOCyb
LaOLab
61.0
—
—
63.3
39.0
36.7
a
As determined by GC analysis
CyOCy, structured TAG obtained from esterification of 1,3-dicaprylin and
b
oleic acid vinylester; LaOLa, structured TAG obtained from esterification of 1,3-
dilaurin and oleic acid vinylester. Note: The data also include regioisomers
JAOCS, Vol. 81, no. 2 (2004)

154
S. WONGSAKUL ET AL.
FIG. 2. Time course of the esterification between 1,3-DCy and OAVE in n-hexane catalyzed
by lipase Amano PS-D at 60°C (gram-scale synthesis). 1,3-DCy (▲), OAVE (■), CyOCy (▲),
CyOO (■) , triolein (●) , total TAG (×). CyOCy, 1,3-dicapryloyl-2-oleyl-glycerol; CyOO, 1-
capryloyl-2,3-dioleyl-glycerol; for other abbreviations see Figure 1.
FIG. 3. Structured triacylglycerol production by esterification of 1,3-DCy (■) or 1,3-DLa (●)
with OA in a solvent-free system catalyzed by Amano PS-D at 60°C under reduced pressure.
For abbreviations see Figure 1.
serving as model compounds and rarely acted on the MCFA mentioned in the Experimental Procedures section for the provision
of lipases. We are especially grateful to Dr. David Weiner, Dr. Tim
Hitchman, and Jonny Lyon (Diversa) for helpful discussions.
present at the sn1- or 3-positions.
Although enzymatic esterification of glycerol or 1,3-DAG
with a FAVE proceeded faster and gave higher yields of 1,3-
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[Received June 12, 2003; accepted December 24, 2003]
JAOCS, Vol. 81, no. 2 (2004)