2.7. Substrate Specificity Measured Using p-Nitrophenyl Esters

Lipolytic activity tests were performed by incubating the enzyme with a 1 mM substrate (stock solution 50 mM in acetonitrile) at 40 °C in 50 mM Tris-HCl buffer (pH 8.0) for 5 min. Eight p-nitrophenyl esters (pNP) with different carbon chain lengths including acetate (C2), propionate (C3), butyrate (C4), valerate (C5), caproate (C6), caprylate (C8), caprate (C10), and laurate (C12) were used to determine the specificity of Est6. For convenience, reaction mixtures (final volume, 150 μL) were prepared in eight strip PCR tubes. The reaction mixtures were transferred immediately to a microplate, and the liberated p-nitrophenol was quantified by measuring the absorbance at 405 nm using a microplate spectrophotometer (PowerWave XS2, Bio-Tek, Winooski, VT, USA). The data were then corrected for nonenzymatic degradation of the ester substrate using an enzyme-free blank of the same reaction mixture treated in the same manner. All samples and blanks were analyzed in triplicate, and activities were quantified by comparison with p-nitrophenol standards in the same buffer. One unit of esterase is defined as the amount of enzyme releasing 1 μmol of free p-nitrophenol per min. The highest enzyme activity on a substrate (pNP acetate) was defined as 100%. Seven different concentrations of pNP acetate (C2) ranging from 0.08 to 1.0 mM were used for the kinetic study, and the K_m, V_max, and k_cat of Est6 were calculated by the Lineweaver–Burk plot.