Purification and Characterization of 2-Haloacid Dehalogenase from Marine Bacterium Paracoccus sp. DEH99, Isolated from Marine Sponge Hymeniacidon perlevis

2-haloacid dehalogenases constitute a group of dehalogenases which are capable of dehalogenating the halogenated organic compounds. So far, the 2-haloacid dehalogenases have been found in many bacteria, but not in Paracoccus genus. In the present study, one enzyme 2-haloacid dehalogenase(designated as Deh99), induced by DL-2-chloropropionate(DL-2-CPA), was purified from the marine bacterium Paracoccus sp. DEH99, isolated from marine sponge Hymeniacidon perlevis. The enzyme of Deh99 was purified to homogeneity by ammonium sulfate precipitation, ion exchange chromatography(Q-Sepharose HP), and Superdex 200 gel filtration chromatography. The molecular weight of Deh99 was estimated to be 25.0 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), and 50.0 kDa natively by gel filtration chromatography. The enzyme of Deh99 stereospecifically dehalogenated L-2-CPA to produce D-lactate, with an apparent Michaelis-Menten constant(Km) value of 0.21 mmol L-1 for L-2-CPA. The optimal pH and temperature for Deh99 activity were 10.0 and 40℃, respectively. The enzyme of Deh99 acted on short-carbon-chain 2-haloacids, with the highest activity towards monochloroacetate. The activity of Deh99 was slightly affected by DTT and EDTA, but strongly inhibited by Cu2+ and Zn2+. The enzyme of Deh99 shows unique substrate specificity and inhibitor sensitivities compared to previously characterized 2-haloacid dehalogenases and is the reported one about purified 2-haloacid dehalogenase isolated from the bacteria of Paracoccus genus.

Lysobacter hymeniacidonis sp. nov., Isolated from a Crude Oil-Contaminated Marine Sponge

An aerobic, Gram-negative bacterium, strain 2-5~T, was isolated from a crude oil-contaminated marine sponge collected near Dalian Bay, China, and subjected to a polyphasic taxonomic investigation. Cells of strain 2-5~T were non-spore forming, non-motile, rods 0.2–0.3 μm wide and 1.1–1.2μm long. Strain 2-5~T grew well on nutrient agar, ~TSA, R2 A agar and LB agar. Colonies of strain 2-5~T on LB agar were circular, smooth with entire margins, non-transparent and pale yellow after 3 d of incubation at 30℃. Growth of strain 2-5~T occurred in LN medium with 0–6% Na Cl; no growth occurred in the presence of 8.0% NaCl. Strain 2-5~T grew at 15–42℃ and at pH 6.0–8.0. Comparative 16 S r RNA gene sequence analysis showed that strain 2-5~T clustered with the species of the genus Lysobacter. Its closet neighbors were the type strains of Lysobacter concretionis KC^TC 12205~T(97% similarity), Lysobacter arseniciresistens ZS79~T(96%), and Lysobacter defluii APB-9 ~T(96%). ~The value for DNA-DNA relatedness between strain 2-5~~T and L. concretionis KC^TC 12205 ~T was 23%. Branched fatty acids iso-C16: 0, iso-C15: 0, iso-C11: 0 3-OH, iso-C17: 1ω9c and iso-C11: 0 were found to be predominant. ~The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. Strain 2-5~T had a DNA G+C content of 63.8 mol%. On the basis of the phenotypic, chemotaxonomic, DNA-DNA hybridization and phylogenetic data, strain 2-5~T represents a novel species of the genus Lysobacter, for which the name Lysobacter hymeniacidonis sp. nov. is proposed. ~The type strain is 2-5~T(=CGMCC 1.12190 ~T = JCM 18137~T).