2.5. Enzymatic Activity Assay

The esterase activity was determined by monitoring the hydrolysis of p‐nitrophenyl esters using a spectrophotometric method that involved measuring the optical density (OD) at 410 nm. The standard reaction mixture, which contained 100 μL of various substrates (p‐nitrophenyl acetate (pNP‐A), p‐nitrophenyl butyrate (pNP‐B), p‐nitrophenyl caprate (pNP‐C), p‐nitrophenyl octanoate (pNP‐O), p‐nitrophenyl dodecanoate (pNP‐D), p‐nitrophenyl palmitate (pNP‐P), and p‐nitrophenyl myristate (pNP‐M)), 2,870 μL of reaction buffer (50 mmol/L Tris‐HCl pH 9.0) and 30 μL of diluted protein (concentration of 8 μg/ml), was incubated at 50°C for 5 min. One unit of enzyme activity was defined as the amount of enzyme that released 1 μmol of p‐nitrophenol per minute under the above conditions. To eliminate the influence of substrate self‐decomposition, a solution with reaction buffer but lacking enzyme was used as a control. All reactions were performed in triplicate.