Mass spectrometric mapping of the enzymes involved in the phenol degradation of an indigenous soil pseudomonad.

The enzymes involved in the degradation of phenol by a new soil bacterium referred as Pseudomonas sp. strain phDV1 were characterized. The key enzyme catalyzing the second step in the phenol degradation meta-cleavage pathway, catechol 2,3-dioxygenase (C23O), was isolated using sucrose density centrifugation and anion exchange chromatography. The purified C23O 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 and energy source. Sucrose density centrifugation was used to collect and concentrate the cell fraction exhibiting C23O 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. We found a different expression of 19 proteins depending on the growth substrate (phenol or glucose) and 10 were identified as enzymes involved in the phenol degradation.