Trichloroethylene and chloroform degradation by a recombinant pseudomonad expressing soluble methane monooxygenase from Methylosinus trichosporium OB3b.
Soluble methane monooxygenase (sMMO) from Methylosinus trichosporium OB3b can degrade many halogenated aliphatic compounds that are found in contaminated soil and groundwater. This enzyme oxidizes the most frequently detected pollutant, trichloroethylene (TCE), at least 50 times faster than other enzymes. However, slow growth of the strain, strong competition between TCE and methane for sMMO, and repression of the smmo locus by low concentrations of copper ions limit the use of this bacterium. To overcome these obstacles, the 5.5-kb smmo locus of M. trichosporium OB3b was cloned into a wide-host-range vector (to form pSMMO20), and this plasmid was electroporated into five Pseudomonas strains. The best TCE degradation results were obtained with Pseudomonas putida F1/pSMMO20. The plasmid was maintained stably, and all five of the sMMO proteins (alpha, beta, and gamma hydroxylase proteins, reductase, and component B) were observed clearly by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting. TCE degradation rates were quantified for P. putida F1/pSMMO20 with a gas chromatograph (Vmax = 5 nmol per min per mg of protein), and the recombinant strain mineralized 55% of the TCE (10 microM) as indicated by measuring chloride ion concentrations with a chloride ion-specific electrode. The maximum TCE degradation rate obtained with the recombinant strain was lower than that of M. trichosporium OB3b but greater than other TCE-degrading recombinants and most well-studied pseudomonads. In addition, this recombinant strain mineralizes chloroform (a specific substrate for sMMO), grows much faster than M. trichosporium OB3b, and degrades TCE without competitive inhibition from the growth substrate.
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Department of Chemical and Biochemical Engineering, University of California, Irvine 92717-2575.
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D001483:Base Sequence; D001673:Biodegradation, Environmental; D002725:Chloroform / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008772:Methylococcaceae / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism*; D012989:Soil Pollutants / Q000378:metabolism; D012995:Solubility; D014241:Trichloroethylene / Q000378:metabolism*; D014874:Water Pollutants, Chemical / Q000378:metabolism
D004269:DNA, Bacterial; D012989:Soil Pollutants; D014874:Water Pollutants, Chemical; D014241:Trichloroethylene; D002725:Chloroform; D010105:Oxygenases; C021555:methane monooxygenase