Molecular characterization of a novel bacterial aryl acylamidase belonging to the amidase signature enzyme family
Molecular characterization of a novel bacterial aryl acylamidase belonging to the amidase signature enzyme family
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DOI：
10.1007/s10059-010-0060-9
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发表时间：
2010-05-01
影响因子：
3.8
通讯作者：
Choi, In-Geol
中科院分区：
文献类型：
--
作者：
Ko, Hyeok-Jin;Lee, Eun Woo;Choi, In-Geol
在寻找作用于对乙酰氨基苯酚（Tylenol（TM））中的酰胺键的芳基酰基酰胺酶（EC www.example.com）时，我们鉴定了编码496个蛋白质残基的新基因。该基因揭示了一个保守的酰胺酶签名区与典型的催化三联体。该基因在E.大肠杆菌，并对其生化特性进行了表征。对乙酰氨基酚的最适pH和温度分别为10和37 ℃。在37 A ℃下酶活性的半衰期为192 h，在40 A ℃下孵育3 h后仍有90%的酶活性。二价金属离子对酶活性有抑制作用。各种芳基酰胺如4-硝基乙酰苯胺、对乙酰氨基苯酚、非那西丁、4-氯乙酰苯胺和乙酰苯胺的K（m）值分别为0.10、0.32、0.83、1.9和19 mM。还使用各种链长（C-1类似于C-4和C-10）的羧酸供体和苯胺作为底物检查逆反应活性（酰胺合成）。这些动力学参数和底物特异性的正向和反向反应表明，在本研究中的芳基酰基酰胺酶具有优先的芳基底物具有极性官能团和疏水性羧酸供体。
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.