H. Zhong et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 114–117
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2.2. Reaction procedures
conversion. This could be explained by the increase in amount of
the substrate bonded to the enzyme with the increase in enzyme
amount. When the lipase concentration was higher than 5%, the
increase of conversion was not remarkable as not all the enzyme
particles were exposed to the substrates and the excess of enzyme
present in the reaction mixture was not actively involved in the
reaction, consistent with other reports [9]. The conversion of ester-
ification completed in 4 h and 2 h, at the lipase concentration of 5%
(w/w) and 10% (w/w) respectively. Considering the price of lipase,
it was concluded that the 5% (w/w of substrates) was the optimal
concentration for the esterification. Operating at this condition a
total esterification percentage of about 55% after 24 h of bioconver-
sion was attained. Therefore, all further studies in this work were
performed at this enzyme concentration.
2.2.1. General procedures of esterification
All the esterification experiments were carried out in a 50 mL
round bottom flask. The reaction procedure was described as fol-
lows: To a mixture of 0.05 mol oleic acid and corresponding alcohol,
catalyst was added at the given temperature. Normally, the reac-
tants mixture kept stirring for 24 h until the reaction completed.
The samples were taken out every 1 h in the first 12 h, and the acid
value was determined according to the standard GB1668-81. In the
end, the chromaticity of the oleates was recorded on PFX-i Series
from Tintometer Ltd.
2.2.2. Analytical methods
2.2.2.1. Determination of acid number. The ester content was quan-
tified by calculating the residual fatty acid amount in the reaction
mixture. Using a volumetric method (standard GB1668-81), a
0.2–0.3 g sample of the reaction mixture was diluted in 20 mL
of 0.1% (w/w) phenolphthalein solution in absolute ethanol and
titrated with standardized potassium hydroxide solution in water.
The acid number (AN) was calculated from the equation:
3.2. Effect of temperature
It is well-known, that temperature is an important parameter
The effect of temperature was studied by varying the temperature
in the range between 10 ◦C and 60 ◦C, with the lipase concentration
of 5% (w/w) and acid/alcohol ratio of 1:3, at the rate of 400 rpm stir-
rer speed. The results are illustrated in Fig. 2. In contradiction to the
generally reported literatures [7,10,11], the lipase from Candida sp.
99-125 had high catalytic activity at 20 ◦C instead of 40 ◦C. How-
ever, the results were in good agreement with the Tan’s report [12]
in which the optimal temperature for the immobilized lipase from
Candida sp. 99-125 ranged from 15 ◦C to 25 ◦C. All the experiments
here were carried out three times to show the reproducibility. The
result suggested that the lipase from Candida sp. 99-125 had high
catalytic activity at room temperature, which means it would have
a wider application in industry. Additionally, the esterification at
10 ◦C was also conducted, which resulted in a low conversion due to
the coagulation of oleic acid. Therefore, the further reactions were
operated at 20 ◦C.
56.1 × V × N
AN =
W
V, volume of NaOH, (mL); N, molarity concentration of titrant
(mol/L); W, weight of the sample in grams.
2.2.2.2. Calculation of esterification rate.
ANorg. − ANeq
esterification rate (%) =
× 100%
ANorg.
ANorg., acid number at the starting point; ANeq, acid number at the
checking point.
3. Results and discussion
3.1. Effect of lipase concentration
3.3. Effect of the molar ratio between oleic acid and alcohols
The investigation of the lipase concentration influence on the
esterification rate was performed by varying the concentration of
lipase in the range between 0.5% and 10% (w/w of substrates) in
the reaction bulk of 0.05 mol oleic acid and 0.15 mol ethanol at
30 ◦C, with magnetic stirring rate of 400 rpm. As shown in Fig. 1,
the lipase concentration affected the initiate rate as well as the final
Acid/alcohol molar ratio is one of the most important param-
eters in enzymatic esterification. Since the reaction is reversible,
an increase in the alcohol concentration should result in higher
ester yields and shift the chemical equilibrium toward the ester
Fig. 2. Effect of temperature on the esterification. Oleic acid, 0.05 mol/L; ethanol
alcohol, 0.15 mol/L; lipase, 5%; speed, 400 rpm. (ꢀ) 20 ◦C; (᭹) 30 ◦C; (ꢁ) 40 ◦C; (ꢂ)
50 ◦C.
Fig. 1. Effect of lipase concentration on esterification rate. (ꢀ) 0.5% (w/w); (᭹) 1%
(w/w); (ꢁ) 3% (w/w); (ꢂ) 5% (w/w); (ꢃ) 10% (w/w).