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Detection and characterisation of catechol 2,3-dioxygenase in an indigenous soil Pseudomonad by MALDI-TOF MS using a column separation

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Abstract

The key enzyme catalyzing the second step in the phenol degradation meta-cleavage pathway (C23O) has been purified to homogeneity from a new bacterial strain, which belongs to genus Pseudomonas. The species was growing on phenol as carbon source. The C23O was detected and identified by absorption spectroscopy. The protein was isolated using sucrose density centrifugation and anion exchange chromatography. The purified protein showed a molecular mass of 32 kDa to sodium dodecyl sulfate polyacrylamid gel electrophoresis and an isoelectric point of 5 estimated by analytical isoelectrical focusing. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and peptide mapping was attempted for the identification of the isolated protein and proteins involved in the metabolic pathway.

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Abbreviations

C12O:

catechol 1,2-dioxygenase

C23O:

catechol 2,3-dioxygenase

MALDI-TOF:

matrix assisted laser desorption ionisation-time of flight

MS:

mass spectrometry

SDS PAGE:

sodium dodecyl sulfate polyacrylamid gel electrophoresis

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Authors and Affiliations

  1. Division of Biochemistry, Department of Chemistry, University of Crete, Knossos Avenue, Heraklion, P.O. Box 1470, GR-71409, Greece

    Eirini Tsirogianni, Michalis Aivaliotis & Georgios Tsiotis

  2. Institut für Pharmazeutische Chemie, Instrumentelle Analytische Chemie Johann Wolfgang Goethe Universität, Marie-Curie-Str. 9-11, 60439, Frankfurt am Main, Germany

    Michael Karas

Authors
  1. Eirini Tsirogianni
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  2. Michalis Aivaliotis
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  3. Michael Karas
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  4. Georgios Tsiotis
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Correspondence to Georgios Tsiotis.

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Tsirogianni, E., Aivaliotis, M., Karas, M. et al. Detection and characterisation of catechol 2,3-dioxygenase in an indigenous soil Pseudomonad by MALDI-TOF MS using a column separation. Biodegradation 16, 181–186 (2005). https://doi.org/10.1007/s10532-004-4885-9

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