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

作者：Ko Hyeok Jin; Lee Eun Woo; Bang Won Gi; Lee Cheol Koo; Kim Kyoung Heon; Choi In Geol^*

来源：Molecules and Cells, 2010, 29(5): 485-492.

DOI：10.1007/s10059-010-0060-9

• amidase signature enzymes
• amide synthesis
• aryl acylamidase
• p-acetaminophenol

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.

• 出版日期2010-5