2.4.2. Esterase Activity Assay

The esterase activity was determined by measuring the amount of p-nitrophenol (p-NP) released from p-nitrophenyl butyrate (p-NPB) [31]. Briefly, 250 μL of suitably diluted sample was added to 730 μL of 50 mM potassium phosphate buffer (pH 7.5), and the mixture was pre-incubated at 37 °C for 5 min. Then, 20 μL of 50 mM p-NPB (dissolved in absolute ethanol) was added, and the reaction mixture was incubated at 37 °C for exactly 10 min. At the end of the incubation, the reaction tubes were placed on ice and the absorbance of p-NP was measured immediately at 410 nm. A blank control was prepared in the same manner except that the enzyme was added in the reaction mixture immediately before reading the absorbance.

The amount of p-NP released from the substrate was determined using the Beer-Lambert equation, knowing that the molar extinction coefficient of p-NP was determined as 10,400 M⁻¹⋅cm⁻¹ [32]. One unit of esterase activity was defined as the amount of enzyme required to release 1 μmol of p-NP per minute under the assay conditions. All tests were performed in triplicate.