Detection and characterisation of catechol 2,3-dioxygenase in an indigenous soil pseudomonad by MALDI-TOF MS using a column separation.
The key enzyme catalyzing the second step in the phenol degradation meta-cleavage pathway (C230) 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 C230 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.
Tsirogianni
Eirini
E
Division of Biochemistry, Department of Chemistry, University of Crete, Knossos Avenue, P.O. Box 1470, GR-71409 Heraklion, Greece. tsiotis@chemistry.uoc.gr
Aivaliotis
Michalis
M
Karas
Michael
M
Tsiotis
Georgios
G
D000595:Amino Acid Sequence; D050557:Catechol 2,3-Dioxygenase; D002845:Chromatography; D049308:Dioxygenases / Q000032:analysis / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D019800:Phenol / Q000378:metabolism; D011548:Pseudomonadaceae / Q000201:enzymology* / Q000302:isolation & purification; D012988:Soil Microbiology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013057:Spectrum Analysis
D019800:Phenol; D049308:Dioxygenases; D050557:Catechol 2,3-Dioxygenase