Molecular characterization of a novel bacterial aryl acylamidase belonging to the amidase signature enzyme family

Abstract

In seeking aryl acylamidase (EC 3.5.1.13) acting on an amide bond in p-acetaminophenol (Tylenol (TM)), we identified a novel gene encoding 496 residues of a protein. The gene revealed a conserved amidase signature region with a canonical catalytic triad. The gene was expressed in E. coli and characterized for its biochemical properties. The optimum pH and temperature for the activity on p-acetaminophenol were 10 and 37A degrees C, respectively. The half-life of enzyme activity at 37A degrees C was 192 h and 90% of its activity remained after 3 h incubation at 40A degrees C. Divalent metals was found to inhibit the activity of enzyme. The K (m) values for various aryl acylamides such as 4-nitroacetanilide, p-acetaminophenol, phenacetin, 4-chloroacetanilide and acetanilide were 0.10, 0.32, 0.83, 1.9 and 19 mM, respectively. The reverse reaction activity (amide synthesis) was also examined using various chain lengths (C-1 similar to C-4 and C-10) of carboxylic donors and aniline as substrates. These kinetic parameters and substrate specificity in forward and reverse reaction indicated that the aryl acylamidase in this study has a preference for aryl substrate having polar functional groups and hydrophobic carboxylic donors.