PURIFICATION and characterization of ddt-dehydrohalogenase from Pseudomonas putida T5.

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.