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We have determined the nucleotide sequence of the gene encoding thermostable L-2-halo acid dehalogenase (L-DEX) from the 2-chloroacrylate-utilizable bacterium Pseudomonas sp. strain YL. The open reading frame consists of 696 nucleotides corresponding to 232 amino acid residues. The protein molecular weight was estimated to be 26,179, which was in good agreement with the subunit molecular weight of the enzyme. The gene was efficiently expressed in the recombinant Escherichia coli cells: the amount of L-DEX corresponds to about 49% of the total soluble proteins. The predicted amino acid sequence showed a high level of similarity to those of L-DEXs from other bacterial strains and haloacetate dehalogenase H-2 from Moraxella sp. strain B (38 to 57% identity) but a very low level of similarity to those of haloacetate dehalogenase H-1 from Morarella sp. strain B (10%) and haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (12%). By searching the protein amino acid sequence database, we found two E. coli hypothetical proteins similar to the Pseudomonas sp. strain YL L-DEX (21 to 22%). Various halogenated organic compounds have been synthesized and are now used as pesticides, herbicides, solvents, and so on in a large scale. Several halogen compounds such as polychlorinated biphenyls (PCBs), 1,1,1-trichloro-2-bis(chlorophenyl)ethane (DDT), and 1,1,1-trichloroethane have accumulated in the environment and cause environmental pollution. Various enzymes involved in the degradation of halogenated compounds have been found, purified, and characterized. They are categorized into reductive, oxygenative, and hydrolytic dehalogenases (15, 31, 32). 2-Halo acid dehalogenases (DEXs) catalyze the hydrolytic dehalogenation of 2-halo acids and are further classified into four groups based on their stereospecificities. L-DEX acts on L-2-halo acids to give D-2-hydroxy acids (16, 18). D-DEX catalyzes the conversion of D-2-halo acids into L-2-hydroxy acids (27). There are two different types of DL-DEXs, which act on both Land D-2-halo acids: one catalyzes the dehalogenation of Land D-2-halo acids with retention of the C2-configuration of the substrates, and the other catalyzes

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The gene encoding thermostable L-2-halo acid dehalogenase of Pseudomonas sp. YL was isolated, and its overexpression system was constructed. Gene library was prepared from Sau3AI fragments of total DNA from Ps. sp. YL, pUC118 as a vector and Escherichia coli JM109 as a host. The recombinant cells resistant to bromoacetate, a germicide, were isolated and shown to produce L-2-halo acid dehalogenase. Subsequently, subcloning was carried out with pKK223-3 as a vector, and the length of DNA inserted was reduced to 1.1 kbp. One of the subclones showed very high activity, and the amount of the dehalogenase produced corresponded to about 30% of the soluble protein. From 5 g (wet weight) of cells, 105 mg of dehalogenase was efficiently purified by heat treatment and DEAE-Toyopearl chromatography. This overexpression system provides a large amount of the thermostable enzyme to enable us to study the properties, structure and application of the enzyme.