Degradation of acetochlor by consortium of two bacterial strains and cloning of a novel amidase gene involved in acetochlor-degrading pathway

Two bacterial strains Sphingobium quisquiliarum DC-2 and Sphingobium baderi DE-13 were isolated from activated sludge. Acetochlor was transformed by S. quisquiliarum DC-2 to a transitory intermediate 2-chloro-N-(2-methyl-6-ethylphenyl)acetamide (CMEPA), which was further transformed to 2-methyl-6-ethylaniline (MEA), and MEA could not be degraded by strain DC-2. S. baderi DE-13, incapable of degrading acetochlor, showed capability of degrading MEA to an intermediate 2-methyl-6-ethylaminphenol (MEAOH). MEAOH was further transformed to 2-methyl-6-ethylbenzoquinoneimine (MEBQI), which was mineralized by strain DE-13. A gene, cmeH, encoding an amidase that catalyzed the amide bond cleavage of CMEPA was cloned from strain DC-2. CmeH was expressed in Escherichia coli BL21 and homogenously purified using Ni-nitrilotriacetic acid affinity. CmeH efficiently hydrolyzed CMEPA and other important herbicide, such as propanil, fenoxaprop-p-ethyl and clodinafop-propargyl. (C) 2013 Elsevier Ltd. All rights reserved.

从活性污泥中分离出两株细菌，即鞘氨醇单胞菌DC - 2（Sphingobium quisquiliarum DC - 2）和鞘氨醇单胞菌DE - 13（Sphingobium baderi DE - 13）。乙草胺被鞘氨醇单胞菌DC - 2转化为一种短暂的中间产物2 - 氯 - N -（2 - 甲基 - 6 - 乙基苯基）乙酰胺（CMEPA），该中间产物进一步转化为2 - 甲基 - 6 - 乙基苯胺（MEA），而菌株DC - 2无法降解MEA。鞘氨醇单胞菌DE - 13不能降解乙草胺，但能够将MEA降解为一种中间产物2 - 甲基 - 6 - 乙基氨基酚（MEAOH）。MEAOH进一步转化为2 - 甲基 - 6 - 乙基苯醌亚胺（MEBQI），并被菌株DE - 13矿化。从菌株DC - 2中克隆出一个基因cmeH，它编码一种能够催化CMEPA酰胺键裂解的酰胺酶。CmeH在大肠杆菌BL21中表达，并使用镍 - 次氮基三乙酸亲和层析进行均一纯化。CmeH能够高效水解CMEPA以及其他重要的除草剂，如敌稗、精恶唑禾草灵和炔草酯。（C）2013爱思唯尔有限公司。保留所有权利。