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

Latha, R. and Ramasarma, P. R. and Thakur, M. S. and Manonmani, H. K. (2012) Purification and Characterization of DDT-Dehydrohalogenase from Pseudomonas Putida T5. Preparative Biochemistry and Biotechnology, 42. pp. 60-76. ISSN 1082-6068

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Abstract

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.

Item Type:	Article
Uncontrolled Keywords:	catalytical degradation, characterization, DDT-dehydrohalogenase, Pseudomonas putida T5, purification
Subjects:	500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 26 Pesticide Chemistry 500 Natural Sciences and Mathematics > 07 Life Sciences > 04 Microbiology > 02 Bacteriology
Divisions:	Fermentation Technology and Bioengineering Flour Milling Bakery and Confectionary Technology
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	27 Jan 2012 05:08
Last Modified:	03 Oct 2018 05:52
URI:	http://ir.cftri.res.in/id/eprint/10589

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