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Identifier uch.chemistry.msc//2004tsirogianni
Title Μελέτη του μεταβολισμού της φαινόλης από ένα νέο βακτήριο του γένους Pseudomonas
Creator Tsirogianni, Eirini
Abstract The subject of this study is the aerobic catabolism of aromatic compounds and specially the metabolic pathway of phenol degradation of the new soil bacterium Pseudomonas sp. Strain phDV1. The key enzyme catalyzing the second step in the phenol degradation meta-cleavage pathway, catechol-2,3-dioxygenase (C2,3O), was isolated using sucrose density centrifugation and anion exchange chromatography. The C2,3O was detected and identified by absorption spectroscopy and peptide mapping. Further, the Pseudomonas sp. strain phDV1 proteome was monitored under different growth substrate conditions, using glucose or phenol as sole carbon source. In the beginning, the cytosolic proteins were subjected to two-dimensional gel electrophoresis. The first dimension was isoelectric focusing (IEF) and the second dimension was SDS-PAGE electrophoresis. This method didn’t have high reproducibility because in the first dimension the pH gradient were formed by carried ampholites. To decrease the complexity of the sample was used ssucrose density centrifugation. In that way we succeeded to collect and concentrate the cell fraction exhibiting C2,3O activity and to reduce the complexity of the total protein mixture. 1-DE Tricine PAGE electrophoresis separation in combination with MALDI-TOF MS was attempted for the identification of the proteins involved in the metabolic pathway. From 19 proteins that are more abundant in phenol, 10 were identified as enzymes involved in the phenol degradation. Two-dimensional gel electrophoresis (Native-SDS PAGE) was used to study the protein interactions. The proteins were analysed by MALDI TOF MS.
Issue date 2004-04-01
Date available 2004-07-26
Collection   School/Department--School of Sciences and Engineering--Department of Chemistry--Post-graduate theses
  Type of Work--Post-graduate theses
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