PURIFICATION AND CHARACTERIZATION OF THERMOSTABLE AND NONTHERMOSTABLE2-HALOACID DEHALOGENASES WITH DIFFERENT STEREOSPECIFICITIES FROM PSEUDOMONAS SP STRAIN YL

Two novel hydrolytic dehalogenases, thermostable L-2-haloacid dehaloge nase (L-DEX) inducibly synthesized by 2-chloropropionate (2-CPA) and n onthermostable DL-2-haloacid dehalogenase (DL-DEX) induced by 2-chloro acrylate, were purified to homogeneity from Pseudomonas sp. strain YL. DL-DEX consisted of a monomer with a molecular weight of about 36,000 and catalyzed the dehalogenation of L and D isomers Of 2-CPA to produ ce D- and L-lactates, respectively. It acted on 2-haloalkanoic acids w ith a carbon chain length of 2 to 4. The maximum activity on DL-2-CPA was found at pH 10.5 and 45 degrees C. L-DEX, composed of two subunits with identical molecular weights of 27,000, catalyzes the dehalogenat ion of L-2-haloalkanoic acids to produce the corresponding D-2-hydroxy alkanoic acids. The enzyme acts not only on short-carbon-chain 2-haloa cids such as monochloroacetate and monoiodoacetate in aqueous solution but also on long-carbon-chain 2-haloacids such as 2-bromohexadecanoat e in n-heptane. L-DEX is thermostable: it retained its full activity u pon heating at 60 degrees C for 30 min. The pH and temperature optima for dehalogenation of L-2-CPA were 9.5 and 65 degrees C, respectively. L-DEX was strongly inhibited by modification of carboxyl groups with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and Woodward reagent K, but DL-DEX was not.