Optimization of trichloroethylene degradation using soluble methane monooxygenase of Methylosinus trichosporium OB3b expressed in recombinant bacteria. Biotechnol. Bioeng

By complementing cell-free extracts of fseudomonas putida Fl/pSMM020 with purified soluble methane mo-nooxygenase (sMMO) components of Methylosinus tri-chosporium OB3b, the low cloned-gene sMMO activity in the recombinant strain was found to be due to incomplete activity of the hydroxylase component. To address this incomplete activity, additional sMMO-expressing strains were formed by transferring mmo-containing pSMM020 and pSMM050 into various bacterial species including pseudomonads and a-2 subdivision strains such as meth-anotrophs, methylotrophs, Agrobacterium tumefaciens A114, and Rhizobium meliloti 102F34 (11 new strains screened); sMMO activity was detected in the last two strains. To increase plasmid segregational stability, the hok/sok locus originally from Escherichia coli plasmid R1 was inserted downstream of the mmo locus of pSMMO2O (resulting in pSMM040) and found to enhance plasmid stability in f. putida F1 and R. meliloti 102F34 (first report of hok/sokin Rhizobium). To further increase sMMO activ-ity, a modified Whittenbury minimal medium was se-lected from various minimal and complex media based on trichloroethylene (TCE) degradation and growth rates and was improved by removing the sMMO-inhibiting metal ions [Cu(ll), Ni(ll), and Zn(ll)l and chloramphenicol from the medium and by supplementing with an iron source (3.6 pM of ferrous ammonium sulfate). Using chemostat-grown f. putida Fl/pSMM040, it was found that sMMO activity was higher for cells grown at higher dilution rates. These optimization efforts resulted in a twofold increase in the extent of TCE degradation and more consistent sMMO activity. 0 1996 John Wiley &amp