X. Wang et al. / Process Biochemistry 50 (2015) 388–394
389
Table 1
because these compounds are not commercially available, and the
preparation of purified diacid 1,3-DAGs presents a challenge [11].
highly purified diacid 1,3-DAGs.
Experimental design for optimization of enzymatic transesterification between 1-
Level
X1
X2 (wt%)
X3 (mL)
X4 (◦C)
X5 (h)
To date, the method for the synthesis of diacid 1,3-DAGs has
received little attention. In general, diacid 1,3-DAGs have been
synthesized either by one-step enzymatic esterification [14,15]
esterification of glycerol with mixed fatty acids only produces
approximately 15% diacid 1,3-DAGs due to the generation of
monoacylglycerols (MAGs), monoacid 1,2-DAGs, diacid 1,2-DAGs,
monoacid 1,3-DAGs and triacylglycerols (TAGs) and the difficult
isolation of diacid 1,3-DAGs [14,15]. Therefore, the one-step enzy-
matic esterification is not feasible. In contrast, highly purified diacid
1,3-DAGs can be obtained by chemical synthesis. Generally, the
chemical method for the synthesis of diacid 1,3-DAGs requires
using toxic solvents, such as dichloromethane and triethylamine,
and toxic catalysts, such as 4-dimethylaminopyridine (DMAP) and
N,N-dicyclohexylcarbodiimide (DCC). Therefore, the synthesis of
purified diacid 1,3-DAG by the chemical method is not environ-
mentally sound.
1
2
3
4
5
6
Lipozyme RM IM
Novozym 435
4
6
8
10
12
0.5
1.0
1.5
2.0
30
35
40
45
50
10
1.5
2/0
2.5
3.0
4.0
a
X1 = the type of lipase; X2 = lipase load; X3 = solvent amount; X4 = reaction tem-
perature; X5 = reaction time.
conducted in 100 mL of 95% ethanol at room temperature for 24 h
with 2 g Amberlyst-15 resin as catalyst. At the end of the reaction,
the crude product was purified by recrystallization in hexane at
−30 ◦C as described previously [19].
2.3. Enzymatic synthesis of 1-oleoyl-3-palmitoylglycerol
To avoid the disadvantages of the previous methods, we have
established a two-step enzymatic method for the synthesis of
diacid 1,3-DAGs that included the use of lipase. First, 1-monolein
was synthesized by enzymatic esterification and the cleavage reac-
tion. Subsequently, enzymatic transesterification was conducted
between synthetic 1-monoolein and vinyl palmitate using a lipase
as catalyst to form 1-oleoyl-3-palmitoylglycerol. Compared to pre-
vious chemical synthesis, the two-step enzymatic reaction for the
synthesis of diacid DAG utilizes less toxic reagents. Compared to the
one-step enzymatic esterification, two-step enzymatic reaction is
more effective.
The design for the optimization experiments is presented in
Table 1. The effects of the type of lipase, lipase load, amount
of solvent, reaction temperature and duration on 1-oleoyl-3-
palmitoylglycerol content in the crude reaction mixture were
examined. When one factor was optimized, other factors were
maintained at fixed values. After a factor optimization was com-
pleted, the selected value of this factor was used for subsequent
factor optimizations. All reactions were performed in duplicate, and
data were expressed as means standard deviation (SD).
The enzymatic transesterification of 1-monoolein with vinyl
palmitate was conducted in hexane with agitation by reacting
2.1 mmol 1-monoolein with 2 mmol vinyl palmitate in 1 mL hex-
ane with agitation at 35 ◦C for 2 h. Lipase was used as a catalyst to
start the reaction. The effects of type of lipase (Novozym 435 and
Lipozyme RM IM), lipase load (4, 6, 8, 10 and 12%), amount of sol-
vent (0.5, 1.0, 1.5 and 2.0 mL), reaction temperature (30, 35, 40, 45
and 50 ◦C) and duration (1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 h) were
investigated. At the end of the reaction, the lipase was removed
by vacuum filtration, and the solvent was removed under reduced
pressure. The crude reaction product was diluted to 0.8 mg/mL with
hexane and subsequently quantified by HPLC, as described in the
following section.
2. Materials and methods
2.1. Materials
Palmitic acid vinyl ester (>96%) was purchased from Tokyo
Chemical Industry (Shanghai, China). 1-Oleoylglycerol (≥99%) and
diolein (85% 1,3-diolein and 15% 1,2-diolein) were obtained from
Sigma–Aldrich Chemical Co. Ltd. (Shanghai, China). Novozym 435
(lipase B from Candida antarctica, immobilized on a macropo-
rous acrylic resin) and Lipozyme RM IM (lipase from Rhizomucor
miehei, immobilized on an anionic exchange resin) were obtained
from Novozymes (Beijing, China). Novozym 435 and Lipozyme
RM IM are immobilized lipases and have declared activities of
10,000 PLU (propyl laurate unit)/g and 275 IUN (inter-esterification
units Novo)/g, respectively. All other reagents were of analytical
grade and were purchased from Sinopharm Chemical Reagent Co.
Ltd (Shanghai, China).
2.4. Purification of synthetic 1-oleoyl-3-palmitoylglycerol
After all factors were evaluated, the reaction was per-
formed again under the selected conditions. At the end of the
reaction, the resulting product was purified to separate 1-oleoyl-
mixture were vinyl palmitate and 1-monoolein. 1-Oleoyl-3-
palmitoylglycerol was separated from impurities based on their
differing solubilities in different solvents.
First, diacid 1,3-DAG was dissolved in hexane (1:10, w/v)
at 60 ◦C, and the mixture was maintained at 4 ◦C until com-
pletely crystallized. Thereafter, the crystal containing 1,3-DAG was
collected, and the liquid phase containing vinyl palmitate was
discarded. Second, semi-purified diacid 1,3-DAG was dissolved in
methanol (1:10, w/v) at 60 ◦C, and the mixture was placed at 4 ◦C
until completely crystallized. Subsequently, the crystal containing
diacid 1,3-DAG was collected, and the methanol phase containing
1-monoolein was discarded. Finally, the fully purified 1-oleoyl-3-
palmitoylglycerol was obtained after the removal of solvent under
reduced pressure and quantified by HPLC.
2.2. Enzymatic synthesis of 1-monoolein
1-Monoolein was synthesized by a two-step method based
on our previous method with some modifications [19,20].
Briefly, 1,2-acetonide-3-oleoylglycerol was first synthesized. 1,2-
Acetonide-3-oleoylglycerol was prepared in 25 mL hexane by
reacting 50 mmol oleic acid with 60 mmol 1,2-acetonide glycerol
at 65 ◦C for 12 h in the presence of 10% (w/w, relative to total
reactants) Novozym 435 as a catalyst. At the end of the reaction,
the crude product was used to synthesize 1-monoolein after the
removal of lipase and solvent by filtration and by evaporation under
reduced pressure, respectively.
The synthesis of 1-monoolein by the cleavage of unpuri-
fied 1,2-acetonide-3-oleoylglycerol (approximately 50 mmol) was