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 Par...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.展开更多
The contamination of soils by polycyclic aromatic hydrocarbons （PAHs） is a widespread environmental problem and the remediation of PAHs from these areas has been a major concern. The effectiveness of many in situ bi...The contamination of soils by polycyclic aromatic hydrocarbons （PAHs） is a widespread environmental problem and the remediation of PAHs from these areas has been a major concern. The effectiveness of many in situ bioremediation systems may be constrained by low contaminant bioavailability due to limited aqueous solubility or a large magnitude of sorption. The objective of this research was to evaluate the effect of methyl-β-cyclodextrin （MCD） on bioaugmentation by Paracoccus sp. strain HPD-2 of an aged PAH-contaminated soil. When 10% （W/W） MCD amendment was combined with bioaugmentation by the PAH-degrading bacterium Paracoccus sp. strain HPD-2, the percentage degradation of total PAHs was significantly enhanced up to 34.8%. Higher counts of culturable PAH- degrading bacteria and higher soil dehydrogenase and soil polyphenol oxidase activities were observed in 10% （W/W） MCD-assisted bioaugmentation soil. This MCD-assisted bioaugmentation strategy showed significant increases （p 〈 0.05） in the average well color development （AWCD） obtained by the BIOLOG Eco plate assay, Shannon-Weaver index （H） and Simpson index （λ） compared with the controls, implying that this strategy at least partially restored the microbiological functioning of the PAH-contaminated soil. The results suggest that MCD-aided bioaugmentation by Paracoccus sp. strain HPD-2 may be a promising practical bioremediation strategy for aged PAH-contaminated soils.展开更多
基金supported by National Basic Research Program of China(973 program,Grant No.2009CB724700)the Hundred Talent Program of the Chinese Academy of Sciences(A1097)National Natural Science Foundation of China(No.31100092)
文摘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.
基金supported by the Natural Science Foundation of Jiangsu Province (No. BK2009016)the Chinese National Environmental Protection Special Funds for Scientific Research on Public Causes (No. 2010467016)the National High Technology Research and Development Program (863) of China (No. 2007AA061101)
文摘The contamination of soils by polycyclic aromatic hydrocarbons （PAHs） is a widespread environmental problem and the remediation of PAHs from these areas has been a major concern. The effectiveness of many in situ bioremediation systems may be constrained by low contaminant bioavailability due to limited aqueous solubility or a large magnitude of sorption. The objective of this research was to evaluate the effect of methyl-β-cyclodextrin （MCD） on bioaugmentation by Paracoccus sp. strain HPD-2 of an aged PAH-contaminated soil. When 10% （W/W） MCD amendment was combined with bioaugmentation by the PAH-degrading bacterium Paracoccus sp. strain HPD-2, the percentage degradation of total PAHs was significantly enhanced up to 34.8%. Higher counts of culturable PAH- degrading bacteria and higher soil dehydrogenase and soil polyphenol oxidase activities were observed in 10% （W/W） MCD-assisted bioaugmentation soil. This MCD-assisted bioaugmentation strategy showed significant increases （p 〈 0.05） in the average well color development （AWCD） obtained by the BIOLOG Eco plate assay, Shannon-Weaver index （H） and Simpson index （λ） compared with the controls, implying that this strategy at least partially restored the microbiological functioning of the PAH-contaminated soil. The results suggest that MCD-aided bioaugmentation by Paracoccus sp. strain HPD-2 may be a promising practical bioremediation strategy for aged PAH-contaminated soils.
