title: Purification and characterization of a dehalogenase from Pseudomonas stutzeri DEH130 isolated from the marine sponge Hymeniacidon perlevis.
abstract: 2-haloacid dehalogenases are enzymes that are capable of degrading 2-haloacid compounds. These enzymes are produced by bacteria, but so far they have only been purified and characterized from terrestrial bacteria. The present study describes the purification and characterization of 2-haloacid dehalogenase from the marine bacterium Pseudomonas stutzeri DEH130. P. Stutzeri DEH130 contained two kinds of 2-haloacid dehalogenase (designated as Dehalogenase I and Dehalogenase II) as detected in the crude cell extract after ammonium sulfate fractionation. Both enzymes appeared to exhibit stereo-specificity with respect to substrate. Dehalogenase I was a 109.9-kDa enzyme that preferentially utilized D-2-chloropropropionate and had optimum activity at pH 7.5. Dehalogenase II, which preferentially utilized L-2-chloropropionate, was further purified by ion-exchange chromatography and gel filtration. Purified Dehalogenase II appeared to be a dimeric enzyme with a subunit of 26.0-kDa. It had maximum activity at pH 10.0 and a temperature of 40 °C. Its activity was not inhibited by DTT and EDTA, but strongly inhibited by Cu²⁺, Zn²⁺, and Co²⁺. The K(m) and V(max) for L-2-chloropropionate were 0.3 mM and 23.8 μmol/min/mg, respectively. Its substrate specificity was limited to short chain mono-substituted 2-halocarboxylic acids, with no activity detected toward fluoropropionate and monoiodoacetate. This is the first report on the purification and characterization of 2-haloacid dehalogenase from a marine bacterium.
authors.affiliation: Marine Bioproducts Engineering Group, Dalian Institution of chemical physics, Chinese Academy of Sciences, Zhongshan Road 457, Dalian, 116023, People's Republic of China.
authors.forename: Jinyou
authors.initials: J
authors.lastname: Zhang
authors.forename: Xupeng
authors.initials: X
authors.lastname: Cao
authors.forename: Yanjuan
authors.initials: Y
authors.lastname: Xin
authors.forename: Song
authors.initials: S
authors.lastname: Xue
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D059001:Aquatic Organisms / Q000201:enzymology / Q000302:isolation & purification; D005591:Chemical Fractionation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004791:Enzyme Inhibitors / Q000032:analysis; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011161:Porifera / Q000382:microbiology*; D044223:Pseudomonas stutzeri / Q000201:enzymology* / Q000302:isolation & purification; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D006867:Hydrolases; C034507:2-haloacid dehalogenase
references.citation: Eur J Biochem. 1971 Jul 15;21(1):99-109
references.pmid: 5568680
references.citation: Biol Chem Hoppe Seyler. 1991 Oct;372(10):915-22
references.pmid: 1772590
references.citation: Biotechnol Bioeng. 1984 Jul;26(7):805-6
references.pmid: 18553452
references.citation: Anal Biochem. 1976 May 7;72:248-54
references.pmid: 942051
references.citation: J Bacteriol. 1991 Dec;173(24):7925-33
references.pmid: 1744048
references.citation: J Bacteriol. 1991 Feb;173(4):1530-5
references.pmid: 1995594
references.citation: Mar Drugs. 2010 Jun 23;8(6):1920-34
references.pmid: 20631875
references.citation: Mar Drugs. 2010 Apr 19;8(4):1323-72
references.pmid: 20479981
references.citation: J Bacteriol. 2012 Oct;194(19):5477-8
references.pmid: 22965097
references.citation: J Bacteriol. 2012 Mar;194(5):1277-8
references.pmid: 22328767
references.citation: Appl Environ Microbiol. 1994 Sep;60(9):3375-80
references.pmid: 7944368
references.citation: J Gen Microbiol. 1990 May;136(5):881-6
references.pmid: 2380688
references.citation: J Gen Microbiol. 1982 Aug;128(8):1755-62
references.pmid: 7142958
references.citation: J Gen Microbiol. 1992 Apr;138(4):675-83
references.pmid: 1588303
references.citation: J Basic Microbiol. 2011 Jun;51(3):318-24
references.pmid: 21298680
references.citation: J Biol Chem. 1996 Aug 23;271(34):20322-30
references.pmid: 8702766
references.citation: Appl Environ Microbiol. 1994 Jul;60(7):2389-93
references.pmid: 8074519
references.citation: J Biol Chem. 1968 Jan 25;243(2):428-34
references.pmid: 5635785
references.citation: Appl Biochem Biotechnol. 2009 Nov;159(2):382-93
references.pmid: 19266316
references.citation: Eur J Biochem. 1996 Feb 1;235(3):744-9
references.pmid: 8654424
references.citation: Curr Opin Biotechnol. 1999 Aug;10(4):365-9
references.pmid: 10449315
references.citation: Biol Chem Hoppe Seyler. 1993 Jul;374(7):489-96
references.pmid: 8216900
references.citation: Biochem J. 1993 May 15;292 ( Pt 1):69-74
references.pmid: 7684900
references.citation: Microbiol Rev. 1994 Dec;58(4):641-85
references.pmid: 7854251
references.citation: J Bacteriol. 1982 May;150(2):522-7
references.pmid: 7068529
references.citation: Hoppe Seylers Z Physiol Chem. 1983 May;364(5):529-35
references.pmid: 6873881
references.citation: J Bacteriol. 1992 Apr;174(8):2612-9
references.pmid: 1556080
references.citation: J Bacteriol. 1999 Apr;181(8):2535-47
references.pmid: 10198020