Genesys 10s bio uv vis spectrophotometer

This assay was performed by measuring the increase in absorbance at 410 nm, produced by the release of p-nitrophenol (pNP) that results from the hydrolysis of the p-nitrophenyl butyrate (pNPB) substrate. Initially, 100 μL of enzyme solution was added to a spectrophotometer cuvette containing 900 μL of substrate solution (0.5 mM pNPB in 25 mM sodium phosphate buffer; pH 7.0). Absorbance values were recorded at 410 nm with a Genesys 10S Bio UV-Vis spectrophotometer (Thermo-Fisher, Illkirch, France) at regular time intervals. Between each measurement, the cuvette was continuously agitated. Measurements were performed in triplicate. The enzymatic activity was deduced from the slope of the linearized curve of absorbance vs. time and the molar extinction coefficient of pNP. One unit of enzyme activity (U) was defined as 1 μmol of pNP liberated in 1 min by 1 mg of CALB at 25 °C (1 U/mg = 1 μmol pNP/min·mg). The average standard deviation of the calculated mean values of hydrolytic activity was estimated to be ±10%.