title: Purification and characterization of 2-halocarboxylic acid dehalogenase II from Pseudomonas spec. CBS 3.
abstract: 2-Halocarboxylic acid dehalogenase II from Pseudomonas spec. CBS 3 (EC 3.8.1.2), which had been cloned in E. coli Hb 101 was purified to electrophoretic homogeneity from crude extracts of E. coli Hb 101 clone 1164. Ammonium sulfate fractionation and three subsequent chromatographic purification steps yielded a pure enzyme in a 230-fold enrichment. The relative molecular masses as determined by gelfiltration on Superose 12 and SDS-polyacrylamide gel electrophoresis were 64,000 Da for the holoenzyme and 29,000 Da for the subunit. The isoelectric point, determined by isoelectric focusing, was at pH 6.2. Substrate specificity towards chlorinated and brominated substrates was limited to short chain monosubstituted 2-halocarboxylic acids. Fluorocompounds were not converted. The reaction proceeded best at a pH above 9.5 and at a reaction temperature of 40-45 degrees C.
authors.affiliation: Fraunhofer-Institut für Grenzflächen- und Bioverfahrenstechnik, Stuttgart.
authors.forename: F M
authors.initials: FM
authors.lastname: Mörsberger
authors.forename: R
authors.initials: R
authors.lastname: Müller
authors.forename: M K
authors.initials: MK
authors.lastname: Otto
authors.forename: F
authors.initials: F
authors.lastname: Lingens
authors.forename: K D
authors.initials: KD
authors.lastname: Kulbe
mesh_terms: D002845:Chromatography; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007526:Isoelectric Point; D008970:Molecular Weight; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D006867:Hydrolases; C034507:2-haloacid dehalogenase