2.4. General assay for p-nitrophenyl ester hydrolysis

50 mM Tris·HCl pH 8.0 was added to a 96-well Flat Bottom Suspension Culture Plate (Cat#:25–104 Genesee Scientific) containing 30 µg of OleA C143S or C143A, 5% ethanol, and 200 µM p-nitrophenyl hexanoate in a 200 µL total volume. A SpectraMax Plus 384 Microplate Reader (Molecular Devices, San Jose, CA) was used, and p-nitrophenol absorbance was read at 410 nm. Absorbance was read at 410 nm every 5 min for at least 2 h at 37 °C. A standard curve was developed in a buffer containing 50 mM Tris·HCl at pH 8.0. The extinction coefficient for p-nitrophenol was determined to be 15,548 M⁻¹cm⁻¹, and the path length through the 200 µL liquids in microtiter wells was experimentally determined to be 0.58 cm. All reactions were run in parallel on the same microtiter plates with 3–5 replicates for each data point. Controls of each p-nitrophenyl alkanoate chain length containing no protein were used to correct for any non-enzymatic hydrolysis in buffer. For wells containing wild type OleA, only 4 µg of protein was added to the wells, and measurements were taken every minute for 30 min. Other mutant and inhibited assays were varied as described below.