Esterase activity of Sub1 on p-nitrophenyl esters

Esterase activity was assessed by spectrophotometrically measuring the absorbance of p-nitrophenol using the substrates p-nitrophenyl butyrate (C4), p-nitrophenyl octanoate (C8), p-nitrophenyl decanoate (C10), and p-nitrophenyl dodecanoate (C12) (Sigma-Aldrich). The molar extinction coefficient of p-nitrophenol in Tris-HCl (20‍ ‍mM, pH 7.5) at room temperature is 12,000‍ ‍M^–1‍ ‍cm^–1 at 420 nm. This enzymatic assay was performed as described previously (Komeil et al., 2013) with slight modifications. In a 1.5-‍mL plastic cuvette, 20 μL of 100×-diluted Sub1 (60‍ ‍ng) was added to 970 μL of Tris-HCl (20‍ ‍mM, pH 7.5), with or without Triton X-100 (0.5%), and 10 μL of a 20‍ ‍mM p-nitrophenyl ester substrate (0.2‍ ‍mM final concentration). The absorbance at 420 nm of this reaction mix was measured at room temperature every 10‍ ‍s for 1‍ ‍min. The increase in absorbance of each sample was read against a blank without purified protein. One unit (U) was the amount of enzyme liberating 1 μmol of p-nitrophenol min^–1 under the assay conditions. The V_max and K_m values of the enzyme Sub1 were assessed using the software GraphPad Prism7, according to the Michaelis-Menten equation, with different concentrations of the C4 substrate.