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

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Molecules and Cells

Abstract

In seeking aryl acylamidase (EC 3.5.1.13) acting on an amide bond in p-acetaminophenol (Tylenol™), 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 37°C, respectively. The half-life of enzyme activity at 37°C was 192 h and 90% of its activity remained after 3 h incubation at 40°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 (C1∼C4 and C10) 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.

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Authors and Affiliations

  1. Division of Biotechnology, Korea University, Seoul, 136-713, Korea

    Hyeok-Jin Ko, Eun Woo Lee, Won-Gi Bang, Cheol-Koo Lee & In-Geol Choi

  2. School of Life Sciences and Biotechnology, Korea University, Seoul, 136-713, Korea

    Kyoung Heon Kim

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  1. Hyeok-Jin Ko
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  2. Eun Woo Lee
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  3. Won-Gi Bang
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  4. Cheol-Koo Lee
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  6. In-Geol Choi
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Correspondence to In-Geol Choi.

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Ko, HJ., Lee, E.W., Bang, WG. et al. Molecular characterization of a novel bacterial aryl acylamidase belonging to the amidase signature enzyme family. Mol Cells 29, 485–492 (2010). https://doi.org/10.1007/s10059-010-0060-9

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  • DOI: https://doi.org/10.1007/s10059-010-0060-9

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