Molecular breeding of a toxic chemicals-degrading-bacterium

1.Soluble methane monooxygenase(SMMO)gene was cloned from a methane-utilizing bacterium，Methylocystis sp.M(Strain M)，and sequenced.(1)sMMO structural gene(About6kb)and its promotor region were sequenced，and those were confirmed to be sMMO genes by comparing them with complemental DNA sequences derived from N-terminal amino acid sequences of purified mMO genes by comparing them with complemental sequences derived from·Metal-binding site in a active center of sMMO，high G/C content in the third letter of the used codons，and prothsetic groups binding site were confirmed in that DNA sequence.2.Plasmid pWM3coding sMMO genes was introduced into E.coli.Trichloroethylene degradation activity was not detected in E.coli harboring pWM3，and no difference in lysates was observed between E.coli harboring pWM3 and host by SDS-PAGE.Hydroxylase，one of the components of sMMO，consisted with alpha2beta2gamma2subunits and requires ferric ion in the active center，indicating that the precise assembly of those subunits and a metal would required to expylhydroivity。

1. Soluble methane monooxygenase (sMMO) gene was cloned from a methane-utilizing bacterium, Methylocystis sp.M (strain M), and sequenced.(1) sMMO structural gene (about 6kb) and its promotor region were sequenced, and those were confirmed to be sMMO genes by comparing them with complemental DNA sequences derived from N-terminal amino acid sequences of purified sMMO in strain M.(2) Some characteristics were cleared as follows ;・ sMMO from strain M was related closely with those of methylosinus trichosporium OB3b and Methylococcus capsulatus Bath in DNA sequences and putative amino acid sequences.・ Metal-binding site in a active center of sMMO,high G/C content in the third letter of the used codons, and prothsetic groups binding site were confirmed in that DNA sequence.2. Plasmid pWM3 coding sMMO genes was introduced into E.coli. Trichloroethylene degradation activity was not detected in E.coli harboring pWM3, and no difference in lysates was observed between E.coli harboring pWM3 and host by SDS-PAGE.Hydroxylase, one of the components of sMMO,consisted with alpha 2 beta 2 gamma 2 subunits and requires ferric ion in the active center, indicating that the precise assembly of those subunits and a metal would be required to express hydroxylase activity.