Development and application of degradation system for polychlorinated dioxin

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We isolated a fluoroacetate-degrading bacterium from soil, and purified fluoroacetate dehalogenase, which catalyzes the hydrolytic dehalogenation of fluoroacetate, from this bacterium. The enzyme also acted on chloroacetate and bromoacetate, but did not catalyzed the hydrolysis of haloalkanoic acids whose carbon chain lengths are longer than three. We also carried out screening using a medium containing 2-chloropropionate as a carbon source, and isolated a bacterium degrading this substrate from lake water. The enzyme isolated from this bacterium, DL-2-haloacid dehalogenase, catalyzed the hydrolysis of both D-and L-2-chloropropionates. The enzyme acted on various haloacetates as well as 2-chloropropionamide. The gene encoding this enzyme was cloned, and its nucleotide sequence was determined. The gene product was estimated to be composed of 301 amino acid residues, and its molecular weight was calculated to be 34,049. We analyzed the reaction mechanism of DL-2-haloacid dehalogenase. When the single-turnover enzyme reaction was carried out using 2-chloropropionate as a substrate in the presence of HィイD22ィエD2ィイD118ィエD1O, ィイD118ィエD1O was found to be incorporated into the product, lactate. The enzyme was not labeled with ィイD118ィエD1O even after the multiple-turnover enzyme reaction in the presence of HィイD22ィエD2ィイD118ィエD1O. These results indicate that the water molecule activated by the enzyme directly attacks the substrate to displace the halogen atom.