Purification and Characterization of DDT-Dehydrohalogenase from Pseudomonas Putida T5.

DDT-dehydrohalogenase is involved in the catalytic degradation of p,p0-DDT by eliminating
either HCl or Cl� to form DDD=DDE. Isolation, purification and characterization of DDTdehydrohalogenase
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 Km and Vmax, values for DDT-dehydrohalogenase were
3.7 lM and 6.8 lM 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.