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

作　　者：ZHANG Jinyou XIN Yanjuan CAO Xupeng XUE Song ZHANG Wei 

机构地区：[1]Marine Bioproducts Engineering Group, Dalian Institute of Chemical Physics,CAS [2]University of Chinese Academy of Sciences [3]Flinders Centre for Marine Bioproducts Development (FCMB2), Medical Biotechnology, School of Medicine

出　　处：《Journal of Ocean University of China》2014年第1期91-96,共6页中国海洋大学学报（英文版）

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)

摘　　要：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.2-haloacid dehalogenases constitute a group of dehalogenases which are capable of dehalogenating the halogenatedorganic compounds. So far, the 2-haloacid dehalogenases have been found in many bacteria, but not in Paracoccus genus. In thepresent study, one enzyme 2-haloacid dehalogenase （designated as Deh99）, induced by DL-2-chloropropionate （DL-2-CPA）, waspurified from the marine bacterium Paracoccus sp. DEH99, isolated from marine sponge Hymeniacidon perlevis. The enzyme ofDeh99 was purified to homogeneity by ammonium sulfate precipitation, ion exchange chromatography （Q-Sepharose HP）, and Su-perdex 200 gel filtration chromatography. The molecular weight of Deh99 was estimated to be 25.0 kDa by sodium dodecyl sul-fate-polyacrylamide gel electrophoresis （SDS-PAGE）, and 50.0 kDa natively by gel filtration chromatography. The enzyme of Deh99stereospecifically dehalogenated L-2-CPA to produce D-lactate, with an apparent Michaelis-Menten constant （Kin） value of0（21 mmolL-1 for L-2-CPA. The optimal pH and temperature for Deh99 activity were 10.0 and 40°C, respectively. The enzyme ofDeh99 acted on short-carbon-chain 2-haloacids, with the highest activity towards monochloroacetate. The activity of Deh99 wasslightly affected by DTT and EDTA, but strongly inhibited by Cu2＋ and Zn2＋. The enzyme of Deh99 shows unique substrate specific-ity 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.

关 键 词：PARACOCCUS sp 2-Haloacid DEHALOGENASE PURIFICATION MARINE BACTERIUM MARINE SPONGE 

分 类 号：X172[环境科学与工程—环境科学]