Wang Fei, Zhou Jie, Li Zhoukun, Dong Weiliang, Hou Ying, Huang Yan, Cui Zhongli
Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, Nanjing Agriculture University, Nanjing, People's Republic of China College of Bioscience and Bioengineering, Jiangxi Agriculture University, Nanchang, People's Republic of China.
Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, Nanjing Agriculture University, Nanjing, People's Republic of China.
Appl Environ Microbiol. 2015 Mar;81(6):2182-8. doi: 10.1128/AEM.03764-14. Epub 2015 Jan 16.
Acetochlor [2-chloro-N-(ethoxymethyl)-N-(2-ethyl-6-methylphenyl)-acetamide] is a widely applied herbicide with potential carcinogenic properties. N-Deethoxymethylation is the key step in acetochlor biodegradation. N-Deethoxymethylase is a multicomponent enzyme that catalyzes the conversion of acetochlor to 2'-methyl-6'-ethyl-2-chloroacetanilide (CMEPA). Fast detection of CMEPA by a two-enzyme (N-deethoxymethylase-amide hydrolase) system was established in this research. Based on the fast detection method, a three-component enzyme was purified from Rhodococcus sp. strain T3-1 using ammonium sulfate precipitation and hydrophobic interaction chromatography. The molecular masses of the components of the purified enzyme were estimated to be 45, 43, and 11 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Based on the results of peptide mass fingerprint analysis, acetochlor N-deethoxymethylase was identified as a cytochrome P450 system, composed of a cytochrome P450 oxygenase (43-kDa component; EthB), a ferredoxin (45 kDa; EthA), and a reductase (11 kDa; EthD), that is involved in the degradation of methyl tert-butyl ether. The gene cluster ethABCD was cloned by PCR amplification and expressed in Escherichia coli BL21(DE3). Resting cells of a recombinant E. coli strain showed deethoxymethylation activity against acetochlor. Subcloning of ethABCD showed that ethABD expressed in E. coli BL21(DE3) has the activity of acetochlor N-deethoxymethylase and is capable of converting acetochlor to CMEPA.
乙草胺[2-氯-N-(乙氧基甲基)-N-(2-乙基-6-甲基苯基)乙酰胺]是一种广泛应用且具有潜在致癌特性的除草剂。N-去乙氧基甲基化是乙草胺生物降解的关键步骤。N-去乙氧基甲基酶是一种多组分酶，可催化乙草胺转化为2'-甲基-6'-乙基-2-氯乙酰苯胺（CMEPA）。本研究建立了一种利用双酶（N-去乙氧基甲基酶-酰胺水解酶）系统快速检测CMEPA的方法。基于该快速检测方法，通过硫酸铵沉淀和疏水相互作用色谱从红球菌属菌株T3-1中纯化出一种三组分酶。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）测定，纯化酶各组分的分子量估计分别为45、43和11 kDa。基于肽质量指纹分析结果，乙草胺N-去乙氧基甲基酶被鉴定为一种细胞色素P450系统，由细胞色素P450加氧酶（43-kDa组分；EthB）、铁氧化还原蛋白（45 kDa；EthA）和还原酶（11 kDa；EthD）组成，该系统参与甲基叔丁基醚的降解。通过PCR扩增克隆了基因簇ethABCD，并在大肠杆菌BL21(DE3)中表达。重组大肠杆菌菌株的静息细胞显示出对乙草胺的去乙氧基甲基化活性。ethABCD的亚克隆表明，在大肠杆菌BL21(DE3)中表达的ethABD具有乙草胺N-去乙氧基甲基酶的活性，能够将乙草胺转化为CMEPA。
