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Y. Zhao et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 157–162
Although immobilized lipase in many cases can hyperactivate
200 ml petroleum ether:ethyl acetate:acetic acid (70:30:0.7, v/v);
(IV) 100 ml petroleum ether:ethyl acetate:acetic acid (50:50:1,
v/v); (V) 100 ml ethyl acetate; (VI) 150 ml methanol. Eluents were
collected by an automatic fraction collector, and then the com-
ponents of each tube were analyzed by TLC-FID. The eluents
containing the same component combined, and the solvent evap-
orated, giving the final products.
lipases and also can improve it stability, modulate it specificity
and allow reutilization, almost no esterification of glycerol and
oleic acid catalyzed by immobilized Candida sp. 99-125 lipase was
noted in our previous study, this maybe due to the high viscos-
ity of the excessive glycerol in the system, the glycerol forms a
layer around the immobilized lipase, making it not disperse in the
system as well as lipase power, moreover, beta-cyclodextrin as an
assistant was mixed with lipase in this paper, it was more exer-
cisable to use the lipase power. So lipase powder from Candida sp.
99-125 was employed to catalyze the esterification of glycerol and
oleic acid to synthesize MAG and DAG in solvent-free system in
this study. And the effects of glycerol/oleic acid molar ratio, initial
water content, catalyst load, reaction temperature, agitator speed
and beta-cyclodextrin/lipase mass ratio were investigated, then
product was purified by silica column separation.
2.4. Analysis by TLC-FID
Samples were analyzed by thin layer chromatography coupled
with a flame ionization detector (Iatroscan MK-6s, Iatron Laborato-
ries, Japan). Aliquots were dissolved in 0.5 ml of n-hexane, and 1 l
of diluted sample was spotted onto silica-coated chromarod quartz
rods by a semiautomatic sample spotter. Samples were developed
with the developing system of methylbenzene/chloroform/acetic
acid mixture (70:30:2, v/v). The rods were dried for 3–5 min at
60 ◦C in an oven prior to analysis. Data handling was performed on
a computer equipped with SES I-Chromstar1 6.0 software. The area
percentages of TAG, DAG (1,3- and 1,2-isomers separately), MAG,
and free fatty acids (FFA) were used for the calculation of prod-
uct concentration. All the reactions in this work were conducted in
duplicates. The means of duplicated determinations were used for
result evaluation.
2. Materials and methods
2.1. Materials
cyclodextrin (analytical grade), silica gel (300–400 mesh) were
purchased from Beijing Chemicals Factory, Beijing, China. Lipase
powder from Candida sp. 99-125 namely LS-20 was purchased from
Beijing CTA New Century Biotechnology Co., Ltd, Beijing, China.
Candida sp. 99-125 was screened by our lab [19–21], and 99-125 is
a deposit number of a cell bank. All other reagents were obtained
commercially and were of analytical grade.
3. Results and discussion
3.1. Optimization of reaction variables
2.2. Esterification reaction
3.1.1. Effect of initial water content
It is well known that water content is one of the key factors
that affect the activity of an enzyme in a non-aqueous medium.
molar ratio 4:1, catalyst load (relative to the weight of total sub-
strates) 10%, beta-cyclodextrin/lipase mass ratio 1.5:1, reaction
temperature 40 ◦C, agitator speed 190 r/min, the effect of water
was investigated by varying initial addition content of water in the
range of 0–14% (w/w) of the substrate mass (Fig. 1). Almost no reac-
tion was observed at 0% initial water content. The production of
MAG and DAG increased when the initial water content increased.
As indicated, the esterification at 10% initial water content is fastest
with high reaction degree, in which MAG concentration amounts
up to 35.2% after 8 h, DAG concentration amounts up to 29.5% after
6 h, and the concentration of MAG plus DAG achieves maximum
of 61% around 8 h. However, 14% initial water reduced the con-
centration of MAG and DAG on the contrary. The result validated
the water is necessary to maintain enzyme activity, once the water
content reached the proper catalytic amount, the rate of the ester-
ification reaction decreased along with a further increase in water
content, which seemed to be due to inhibition of the esterification
because one of the reaction products of the esterification between
glycerol and oleic acid is water, thus prompted the esterification to
the reverse direction. The results were similar to the synthesis of
ethyl oleate catalyzed by 15 biocatalysts (native and immobilized
lipases) in the solvent-free system reported by Foresti [26]. He also
centages of 10% led to much higher esterification rate than systems
the need of very small amounts of water (0.2–0.3%) to successfully
employ lipases in esterification reactions in organic/solvent-free
media [27,28], the Candida sp. 99-125 employed in this study need
much more water to maintain activity, this result was in consistent
with former studies [21,29]. So the initial water content was fixed
at 10% of the substrate mass in the following reactions.
round-bottomed flask. The substrates were pre-mixed by agitation
with an impeller at the desired temperature. Cyclodextrin (CD) has
been reported to improve the functional and stability properties of
enzymes [22–25]. So beta-cyclodextrin was mixed with lipase as
an assistant in the process, powdered enzyme–cyclodextrin conju-
gates were added gradually to the reaction mixture with agitation,
water was added at last. The reaction was stopped until the con-
centration of products had no raise over time.
Initial water content, glycerol/oleic acid molar ratio, catalyst
load (enzyme–cyclodextrin conjugates), reaction temperature, agi-
tator speed and beta-cyclodextrin/lipase mass ratio were changed
to study their effects on the esterification, only one parameter was
varied at a time in the optimization studies. Aliquots of 10 l were
periodically withdrawn, and analyzed by thin layer chromatogra-
phy coupled with a flame ionization detector (TLC-FID). All the
experiments were replicated at least three times and the results
presented were the mean values for the replicated data.
2.3. Separation of product by silica column
After the reaction, acylglycerols and free fatty acid were
extracted with n-hexane. The resultant solution was evaporated
to remove the solvent under reduced pressure, preparing for the
separation on silica column.
A silica column, 300 mm in height and 30 mm in diameter,
was used to as the separation device. 5 g reactant was applied
to silica column in a loading solvent of petroleum ether. A
minimal volume (3 ml) was used to load the sample, and the
loading solvent was then pulled through under gravity. Reac-
tants containing TAG, FFA, DAG and MAG were then sequentially
eluted from the column using the solvents of follows: (I) 200 ml
petroleum ether:ethyl acetate:acetic acid (90:10:1, v/v); (II) 200 ml
petroleum ether:ethyl acetate:acetic acid (80:20:1, v/v); (III)