Purification and Properties of Haloacetate Halidohydrolase Specified by Plasmid fromMoraxellasp. Strain B

Abstract
Haloacetate halidohydrolase II specified by a plasmid pUO1 was purified from haloacetate-assimilating Moraxella sp. B. The purification procedures included protamine treatment, ammonium sulfate fractionation, and column chromatographies with DEAE-cellulose, hydroxyapatite and Bio-gel P-150, resulting in a 200-fold purification. The purified enzyme was homogeneous by criteria of ultracentrifugation and disc electrophoresis. The molecular weight estimated by Sephadex G-I00 gel filtration was 43,000, and it was 26,000 by SDS-polyacrylamide gel electrophoresis. The sedimentation coefficient was 4.1 S, and the isoelectric point was pH 5.2. The amino acid composition was also estimated. The enzyme catalyzed the dehalogenation of monochloro-, monobromo- and monoiodoacetate, but not monofluoroacetate. 2,2-Dichloroacetate and 2-chloropropionate were slightly dehalogenated, but trichloroacetate and 3-chloropropionate were not. The enzyme was very sensitive to inhibition with thiol reagents.

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