3.3 Optimization of hydrolysis condition for the lipase from
Candida sp.
Firstly, pH were studied in the optimal range proposed by the
manufacturer (5 ≤ pH ≤9). we studied the effect of different pH of the
3-MCPD on the yield. Hydrolysis was performed by adding 3 mL of 30%
(w/v) sodium bromide aqueous solution (pH 5.0, pH 6.0, pH 7.0, pH 8.0,
pH 9.0) containing 150 U/mL of lipase. As shown in
Fig.4, among the tested pH, the
yield of
3-MCPD was the highest when pH was
5.0, achieving acceptable results at all three levels.It has been shown
that the change of pH value will cause the change of enzyme
conformation, leading to the change of enzyme catalytic
activity[14]. The results show that the optimal pH
of lipase from Candida sp. is 5. What is more, the
lipase from Candida sp. is
inactivated under alkaline conditions.
Secondly, to determine optimal lipase concentration in the hydrolysis
buffer, we analyzed 3-MCPD esters standard solution at 0.1 mg/mL by
using the hydrolysis buffer containing a concentration of lipase in the
range between 90 and 210 U/mL were studied in triplicate spanning, and
by adding 3 mL of 30% (w/v) sodium bromide aqueous solution (pH 5.0)
after completion of the hydrolysis step of 30 min. Good yield for 3-MCPD
(95%) were obtained when the lipase concentration was 150 U/mL at all
concentrations tested (Fig.5). From the result, it was decided to use
150 U/mL of lipase from Candida sp. in the hydrolysis buffer.
Thirdly, the effects of enzymatic
hydrolysis time were studied by varying from 10 min to 60 min. As shown
in Fig.6, the yield for 3-MCPD
increased and then decreased with the increase of enzymatic hydrolysis
time. It was suggested that optimum time for enzymatic hydrolysis is 30
minutes. And with the increase of time, the lipase activity decreased.
Lastly, in order to find appropriate temperature for enzymatic
hydrolysis of 3-MCPD esters, different temperatures such as 15℃, 20℃,
25℃, 30℃ and 35℃ were studied in triplicate spanning. As shown in Fig.7,
as the temperature rises, the yield
for 3-MCPD increased first, then decreased, When the hydrolysis
temperature reached 25℃, the lipase activity reached the highest value,
After that, the higher the temperature, the smaller the yield for
3-MCPD. It can be seen that the
lipase is sensitive to temperature,
and the activity of the lipase can be increased by increasing the
hydrolysis temperature appropriately. On the other hand, the high
hydrolysis temperature will reduce the lipase activity and reduce the
service life of the lipase.