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
Methomyl,an extremely toxic pesticide,is widely used in agriculture.A strain named mdw-1 capable of degrading methomyl rapidly was successfully isolated from activated sludge in this study.It could utilize methomyl as...Methomyl,an extremely toxic pesticide,is widely used in agriculture.A strain named mdw-1 capable of degrading methomyl rapidly was successfully isolated from activated sludge in this study.It could utilize methomyl as the sole carbon or nitrogen source.The optimal temperature and medium pH for its growth and methomyl biodegradation were 30-C and 7.0,respectively.It was identified as a Paracoccus sp.according to its morphological features,physiological and biochemical characteristics,and phylogenetic analysis based on the sequence of 16S rDNA.Gas chromatography-mass spectrometry (GC-MS)analysis showed that methomyl could be completely transformed to S-methyl-N-hydroxythioacetamidate in 10 h of incubation with the isolate mdw-1.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Project supported by the National Natural Science Foundation of China(No.30600016)the National High Technology Research and Development Program of China(863 Program)(No.2007AA10Z405)+2 种基金the National Program for Sci-Tech Basic Platform Construction of China(No.2005DKA21201-2)the Program for Science and Technology Development ofJiangsu Province,China(No.BE2008669)the Program for Integration of Production,Education,and Research of Guangdong Province and Ministry of Education,China(No.2006090204007)
文摘Methomyl,an extremely toxic pesticide,is widely used in agriculture.A strain named mdw-1 capable of degrading methomyl rapidly was successfully isolated from activated sludge in this study.It could utilize methomyl as the sole carbon or nitrogen source.The optimal temperature and medium pH for its growth and methomyl biodegradation were 30-C and 7.0,respectively.It was identified as a Paracoccus sp.according to its morphological features,physiological and biochemical characteristics,and phylogenetic analysis based on the sequence of 16S rDNA.Gas chromatography-mass spectrometry (GC-MS)analysis showed that methomyl could be completely transformed to S-methyl-N-hydroxythioacetamidate in 10 h of incubation with the isolate mdw-1.
