title: Isolation and characterization of a 4-nitrotoluene-oxidizing enzyme from activated sludge by a metagenomic approach.
abstract: To isolate a biocatalytic enzyme, metagenomic libraries were constructed in fosmids from samples of activated sludge used to treat coke plant wastewater. Six indigo-producing clones were isolated from approximately 40,000 metagenomic clones in the search for the oxygenase responsible. In vitro mutagenesis and whole-sequencing revealed one open reading frame to be responsible for the production of indigo in the fosmid clones. The deduced sequence of the gene product showed 60% identity with 2-naphthoate monooxygenase from Burkholderia sp. JT1500. Subclones carrying this open reading frame (icpA) retained indigo production, and indigo-producing enzymes expressed from subclones catalyzed the oxidization of 4-nitrotoluene to form 4-nitrobenzyl alcohol. These results suggested that the icp product is an enzyme involved in catalyzing 4-nitrotoluene's oxygenation.
authors.affiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1–1–1, Higashi, Tsukuba, Ibaraki 305–8566, Japan. n-kimura@aist.go.jp
authors.forename: Nobutada
authors.initials: N
authors.lastname: Kimura
authors.forename: Kayoko
authors.initials: K
authors.lastname: Sakai
authors.forename: Kazunori
authors.initials: K
authors.lastname: Nakamura
mesh_terms: D001483:Base Sequence; D001592:Benzyl Alcohols / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015698:Genomic Library; D007220:Industrial Waste; D007564:Japan; D056186:Metagenomics / Q000379:methods*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames / Q000235:genetics; D010084:Oxidation-Reduction; D017422:Sequence Analysis, DNA; D014050:Toluene / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D001592:Benzyl Alcohols; D007220:Industrial Waste; D014050:Toluene; C046885:4-nitrobenzyl alcohol; D004247:DNA; C017572:4-nitrotoluene; D006899:Mixed Function Oxygenases