Tomasek P H, Karns J S
Pesticide Degradation Laboratory, U.S. Department of Agriculture, Beltsville, Maryland 20705.
J Bacteriol. 1989 Jul;171(7):4038-44. doi: 10.1128/jb.171.7.4038-4044.1989.
A 14-kilobase-pair (kbp) EcoRI DNA fragment that encodes an enzyme capable of rapid hydrolysis of N-methylcarbamate insecticides (carbofuran hydrolase) was cloned from carbofuran-degrading Achromobacter sp. strain WM111. When used to probe Southern blots containing plasmid and total DNAs from WM111, this 14-kbp fragment hybridized strongly to a 14-kbp EcoRI fragment from the greater than 100-kbp plasmid harbored by this strain but weakly to EcoRI-digested total DNA from Achromobacter sp. strain WM111, indicating that the gene for N-methylcarbamate degradation (mcd) is plasmid encoded. Further subcloning localized the mcd gene on a 3-kbp ScaI-ClaI fragment. There was little or no expression of this gene in the alternative gram-negative hosts Pseudomonas putida, Alcaligenes eutrophus, Acinetobacter calcoaceticus, and Achromobacter pestifer. Western blotting (immunoblotting) of the protein products produced by low-level expression in P. putida confirmed that this 3-kbp fragment encodes the two 70+-kilodalton protein products seen in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified carbofuran hydrolase.
从能够降解克百威的无色杆菌属WM111菌株中克隆出一个14千碱基对（kbp）的EcoRI DNA片段，该片段编码一种能够快速水解N - 甲基氨基甲酸酯类杀虫剂的酶（克百威水解酶）。当用该14 kbp片段探测含有WM111菌株质粒和总DNA的Southern印迹时，它与该菌株携带的大于100 kbp质粒上的一个14 kbp EcoRI片段强烈杂交，但与无色杆菌属WM111菌株经EcoRI酶切后的总DNA杂交较弱，这表明N - 甲基氨基甲酸酯降解基因（mcd）是由质粒编码的。进一步亚克隆将mcd基因定位在一个3 kbp的ScaI - ClaI片段上。在恶臭假单胞菌、真养产碱菌、醋酸钙不动杆菌和有害无色杆菌等其他革兰氏阴性宿主中，该基因几乎没有表达。对恶臭假单胞菌中低水平表达产生的蛋白质产物进行的蛋白质印迹法（免疫印迹法）证实，这个3 kbp片段编码了在纯化的克百威水解酶的十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳中看到的两种70 + - 千道尔顿的蛋白质产物。
