Esterase activity assay

Esterase activity assay was routinely used to assess relative activity of CA. 100 μL of 30 mM p-nitrophenyl acetate (p-NPA; Sigma-Aldrich) dissolved in acetonitrile was added to a cuvette containing 800 μL of buffer (20 mM Tris-sulfate; pH 7.5) and 100 μL of enzyme solution. The reaction was performed at 25 °C inside the spectrophotometer and the absorbance change at 405 nm was monitored for 3 min. The uncatalyzed rate of p-NPA hydrolysis was also measured by adding Tris-sulfate buffer instead of enzyme solution and subtracted from the observed data to obtain the enzyme-catalyzed rates. The enzyme activity was calculated using the extinction coefficient of p-nitrophenol (p-NP; 16,500 M⁻¹ cm⁻¹) at 405 nm. One unit (U) of esterase activity was defined as the activity that produces 1 μmol of p-NP per min.