Trichioroethylene and Chloroform Degradation by a Recombinant PseudomonadExpressing Soluble Methane Monooxygenase from Methylosinus Trichosporium OB3b

摘要

Soluble methane monooxygenase (sMMO) from Methylosinus trichosporium OB3b candegrade many halogenated-aliphatic compounds that are found in contaminated soil and groundwater. This enzyme oxidizes the most-frequently-sited 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 ion limit use of this natural 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 pSMMO2O), and this plasmid was electroporated into five Pseudomonas strains. The best TCE degradation results were obtained with P. putida F1/pSMMO20. The plasmid was maintained stably, and the sMMO proteins were observed clearly using SDS-PAGE and Western immunoblotting. The recombinant cells mineralize both TCE and chloroform (as indicated by measuring chloride ion concentrations with a chloride-ion-specific electrode), degrades TCE faster than other recombinants, grows much faster than M. trichosporium OB3b, and degrades TCE without competitive inhibition.