Enzyme assay

The esterase activity was determined by measuring the amount of p-nitrophenol released in the standard reaction mixture with the OD values at 405 nm monitored by Thermo Scientific Multiscan Spectrum. The standard reaction mixture contained 10 μl of enzyme, 2 μl of p-NP ester (20 mM) in ethanol and 188 μl of Tris–HCl buffer (50 mM, pH 8.5). The reaction mixture in which enzyme is replaced with PBS was considered as control. All experiments were carried out in triplicate and the data obtained were analyzed using GraphPad Prism 5.0 and Excel 2010 software. The calculated values were expressed as mean ± standard deviation (SD) with the statistical significance at p < 0.05 and standard deviation was calculated by standard deviation function (STDEV) in Excel. One unit of enzyme activity was defined as the amount of enzyme needed to release l μmol of p-nitrophenol per minute under the above reaction conditions. The experiments were performed in triplicate and average values were calculated with standard deviations.