Cloning of a Novel Arylamidase Gene from Paracoccus sp. Strain FLN-7 That Hydrolyzes Amide Pesticides
Cloning of a Novel Arylamidase Gene from Paracoccus sp. Strain FLN-7 That Hydrolyzes Amide Pesticides
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DOI：
10.1128/aem.00320-12
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发表时间：
2012-04
影响因子：
4.4
通讯作者：
Jun Zhang;Jin Yin;Bao-jian Hang;Shu Cai;Jian He;Shungui Zhou;Shun-Peng Li
中科院分区：
文献类型：
--
作者：
Jun Zhang;Jin Yin;Bao-jian Hang;Shu Cai;Jian He;Shungui Zhou;Shun-Peng Li
副球菌（Paracoccus sp.） FLN-7菌株通过酰胺键裂解水解酰胺类农药，如双氟苯脲、丙腈、氯苯胺和乐果。从该菌株中克隆出一种新的芳基酰胺酶ampA基因，该基因可催化酰胺类杀虫剂中酰胺键的裂解。ampA包含一个1395 bp的开放阅读框，编码一个465个氨基酸的蛋白。AmpA在大肠杆菌BL21中表达，采用ni -硝基三乙酸亲和层析纯化。AmpA是同型二聚体，等电点为5.4。AmpA在40°C和7.5 - 8.0的pH范围内表现出最大的酶活性，在5.5 - 10.0的pH范围和高达50°C的温度下非常稳定。AmpA能有效水解多种仲胺化合物，如丙烯、4-对乙酰氨基酚、苯胺、氯苯胺、乐果和乐果。最适宜的底物为丙醇，Km和k cat值分别为29.5 μM和49.2 s−1。苯甲酰脲类杀虫剂（二氟脲和六氟脲）也可水解，但效率较低。水解活性不需要辅因子。AmpA与已报道的芳基酰胺酶具有较低的同源性（小于23%），在系统发育树上与密切相关的芳基酰胺酶形成不同的谱系，并且与相关芳基酰胺酶具有不同的生化特性和催化动力学。本研究结果表明，AmpA是研究酰胺类农药水解机理、酰胺类除草剂抗性作物基因工程和酰胺类农药污染环境生物修复的良好候选者。
ABSTRACT The bacterial isolate Paracoccus sp. strain FLN-7 hydrolyzes amide pesticides such as diflubenzuron, propanil, chlorpropham, and dimethoate through amide bond cleavage. A gene, ampA, encoding a novel arylamidase that catalyzes the amide bond cleavage in the amide pesticides was cloned from the strain. ampA contains a 1,395-bp open reading frame that encodes a 465-amino-acid protein. AmpA was expressed in Escherichia coli BL21 and homogenously purified using Ni-nitrilotriacetic acid affinity chromatography. AmpA is a homodimer with an isoelectric point of 5.4. AmpA displays maximum enzymatic activity at 40°C and a pH of between 7.5 and 8.0, and it is very stable at pHs ranging from 5.5 to 10.0 and at temperatures up to 50°C. AmpA efficiently hydrolyzes a variety of secondary amine compounds such as propanil, 4-acetaminophenol, propham, chlorpropham, dimethoate, and omethoate. The most suitable substrate is propanil, with Km and k cat values of 29.5 μM and 49.2 s−1, respectively. The benzoylurea insecticides (diflubenzuron and hexaflumuron) are also hydrolyzed but at low efficiencies. No cofactor is needed for the hydrolysis activity. AmpA shares low identities with reported arylamidases (less than 23%), forms a distinct lineage from closely related arylamidases in the phylogenetic tree, and has different biochemical characteristics and catalytic kinetics with related arylamidases. The results in the present study suggest that AmpA is a good candidate for the study of the mechanism for amide pesticide hydrolysis, genetic engineering of amide herbicide-resistant crops, and bioremediation of amide pesticide-contaminated environments.