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.