Isolation and characterization of a 4-nitrotoluene-oxidizing enzyme from activated sludge by a metagenomic approach
| Autor: | Kayoko Sakai, Nobutada Kimura, Kazunori Nakamura |
|---|---|
| Rok vydání: | 2011 |
| Předmět: |
Oxygenase
Molecular Sequence Data Soil Science Mutagenesis (molecular biology technique) Industrial Waste Plant Science Biology Indigo Catalysis Microbiology Mixed Function Oxygenases Open Reading Frames Japan Escherichia coli Cloning Molecular Ecology Evolution Behavior and Systematics Benzyl Alcohols chemistry.chemical_classification Genomic Library Base Sequence General Medicine DNA Sequence Analysis DNA Monooxygenase Fosmid Open reading frame Mutagenesis Insertional Enzyme Activated sludge chemistry Biochemistry Metagenomics Oxidation-Reduction Toluene |
| Zdroj: | Microbes and environments. 25(2) |
| ISSN: | 1347-4405 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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