Please use this identifier to cite or link to this item: http://hdl.handle.net/10603/13612
Title: Bio-chemical and molecular characterization of DDT degrading dehalogenase from pseudomonas spp.
Researcher: Latha, R
Guide(s): Manonmani, H K
Keywords: Biotechnology
microorganisms
Energy Metabolism
dehydrohalogenase
Upload Date: 3-Dec-2013
University: University of Mysore
Completed Date: 2009
Abstract: 1, 1, 1- trichloro- 2,2- bis (p- chlorophenyl) ethane (DDT) is one of the most widely used organochlorine pesticides around the globe. It is one of the most toxic and environmentally persistent organic pollutant. Ten bacterial cultures belonging to DDT degrading microbial consortium were screened for the DDT- dehydrohalogenase activity. Among these, the cell free extract 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 ~32kDa. 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 µM and 6.8 and#956;M 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 (1mM) inhibited the enzyme strongly. The primary sequence of HPLC purified enzyme was deduced by LC-MS-MALDI-ESI. newlineUrea, sodium dodecyl sulfate (SDS) and guanidine hydrochloride (GdmCl) are often used as excellent denaturing or unfolding agents. The effect of these denaturing agents on the structural changes of DDT- enzyme of Psuedomonas putida T5 were studied. There was a progressive loss in catalytic activity of DDT- dehydrochlorinase with increasing concentrations of denaturants, namely urea, SDS and GdmCl. At 10 M urea, 5 % SDS and 1 M GdmCl, the extent of loss in enzyme activity was 98, 78 and 100% respectively. The emission spectrum of urea denatured enzyme did not show shift, but that of SDS and GdmCl treated enzyme showed very marginal shift.
Pagination: xxxiii, 317p.
URI: http://hdl.handle.net/10603/13612
Appears in Departments:Department of Biotechnology

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01_title.pdfAttached File116.39 kBAdobe PDFView/Open
02_declaration.pdf133.82 kBAdobe PDFView/Open
03_certificate.pdf84.54 kBAdobe PDFView/Open
04_acknowledgements.pdf45.4 kBAdobe PDFView/Open
05_contents.pdf86.25 kBAdobe PDFView/Open
06_list offigures.pdf98.99 kBAdobe PDFView/Open
07_list of tables.pdf44.73 kBAdobe PDFView/Open
08_abbreviations.pdf172.9 kBAdobe PDFView/Open
09_abstract.pdf90.23 kBAdobe PDFView/Open
10_synopsis.pdf219.4 kBAdobe PDFView/Open
11_introduction.pdf347.8 kBAdobe PDFView/Open
12_review of literature.pdf2.49 MBAdobe PDFView/Open
13_chapter 1.pdf2.82 MBAdobe PDFView/Open
14_chapter 2.pdf3.62 MBAdobe PDFView/Open
15_summary.pdf179.85 kBAdobe PDFView/Open


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