title: PURIFICATION and characterization of ddt-dehydrohalogenase from Pseudomonas putida T5.
abstract: DDT-dehydrohalogenase is involved in the catalytic degradation of p,p'-DDT by eliminating either HCl or Cl(-) to form DDD/DDE. Isolation, purification and characterization of DDT-dehydrohalogenase from a bacterial source is reported in this manuscript. Ten bacterial cultures belonging to DDT degrading microbial consortium were screened for the DDT-dehydrohalogenase activity. Among these, the clarified cell homogenate of Pseudomonas putida T5 showed higher DDT-dehydrohalogenase activity and enzyme was purified to apparent homogeneity with 73% overall recovery. The relative molecular mass of the enzyme estimated by the SDS PAGE method was ∼32 kDa. Native PAGE revealed the presence of a single band. The purity of the enzyme was confirmed by HPLC and capillary electrophoresis. The enzyme was stable for 4-5 h at pH 7.0 at the temperature optima of 37 °C. The K( m ) and V( max ), values for DDT-dehydrohalogenase were 3.7 µM and 6.8 µM min(-1), respectively. The enzyme was a glycoprotein with mannose forming the backbone. AIG-formed the N-terminus chain. Serine and tryptophan appeared to be involved at the active site. The enzyme appeared to be a metalloprotein containing Zn, Mg, and Ca ions. Monovalent and divalent cations (1 mM) inhibited the enzyme strongly. The primary sequence of HPLC purified enzyme was deduced by LC-MS-MALDI-ESI.
authors.affiliation: Department of Fermentation Technology and Bioengineering , Central Food Technological Research Institute, Council of Scientific and Industrial Research, Mysore, India.
authors.forename: Latha
authors.initials: L
authors.lastname: Rangachary
authors.forename: Ramasarma Pallavur
authors.initials: RP
authors.lastname: Rajagopalan
authors.forename: Thakur Munna
authors.initials: TM
authors.lastname: Singh
authors.forename: Manonmani Haravey
authors.initials: MH
authors.lastname: Krishnan
mesh_terms: D000595:Amino Acid Sequence; D004791:Enzyme Inhibitors; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008190:Lyases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016958:Pseudomonas putida / Q000201:enzymology* / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D008190:Lyases; C019990:DDT-dehydrochlorinase