Isolation and partial characterization of extracellular NADPH-dependent phenol hydroxylase oxidizing phenol to catechol in Comamonas testosteroni.

OBJECTIVE: Comamonas testosteroni Pb50 is a microorganism that possesses high tolerance for phenol and shows strong phenol degrading activity. This bacterial strain is capable of utilizing phenol as the sole carbon and energy source. Although examples are known in which the C. testosteroni utilizes phenol for growth or metabolism, much less information are known on the nature of the phenol-oxidizing enzymes in this microorganism. Therefore, the occurrence and cellular location of phenol hydroxylase (EC 1.14.13.7), the enzyme participating in the first step of phenol degradation, catalyzing its hydroxylation to catechol in a bacterial Comamonas testosteroni Pb50 strain grown in the presence of phenol as a sole carbon and energy source are the aims of this study. METHODS: Combination of fractionation with polyethylene glycol 6000 and gel permeation chromatography on columns of Sepharose 4B and Sephacryl S-300 was used for isolation of phenol hydroxylase detectable in the medium in which C. testosteroni was cultivated. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel chromatography on Sephacryl S-300 were used to evaluate the molecular mass of the enzyme. The enzyme activity was followed by HPLC (phenol consumption and/or catechol formation). RESULTS: Whereas low activity of phenol hydroxylase was detected in cytosol isolated from C. testosteroni, more than 16-fold higher activity of this enzyme was found in the medium in which C. testosteroni was cultivated. The presence of phenol hydroxylase extracellular activity suggests that this microorganism may secrete the enzyme into the extracellular medium. Using the procedure consisting of fractionation with polyethylene glycol 6000 and gel permeation chromatography on columns of Sepharose 4B and Sephacryl S-300, the enzyme was isolated from the medium to homogeneity. The formation of catechol mediated by purified phenol hydroxylase is strictly dependent on the presence of NADPH, which indicates that this enzyme is the NADPH-dependent phenol hydroxylase. The enzyme is a homotetramer having a molecular mass of 240 000, consisting of four subunits having a molecular mass of 60 000. The optimum pH of the enzyme for the phenol oxidation is pH 7.6. CONCLUSION: The results are the first report showing isolation and partial characterization of extracellular NADPH-dependent phenol hydroxylase of a bacterial C. testosteroni Pb50 strain capable of oxidizing phenol to catechol. The data demonstrate the progress in resolving the enzymes responsible for the first step of phenol degradation by bacteria.

Isolation and partial characterization of catechol 1,2-dioxygenase of phenol degrading yeast Candida tropicalis.

OBJECTIVES: Candida tropicalis yeast is a microorganism that possesses high tolerance for phenol and shows strong phenol degrading activity. This yeast is capable of utilizing phenol as the sole carbon and energy source. While the enzyme participating on the first step of phenol biodegradation, NADPH-dependent phenol hydroxylase, has already been characterized, information on the enzyme participating in the second step of its degradation, catechol 1,2-dioxygenase, is scarce. The development of the procedure suitable for catechol 1,2-dioxygenase isolation and partial characterization of this enzyme are the aims of this study. METHODS: Combination of chromatography on DEAE-Sepharose and gel-permeation chromatography on Sephadex G-100 was used for isolation of cytosolic catechol 1,2-dioxygenase from C. tropicalis yeast. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel chromatography on Sephadex G-100 were used to evaluate the molecular mass of the enzyme. The enzyme activity was followed by HPLC (catechol consumption and/or cis,cis-muconic acid formation). RESULTS: Using the isolation procedure consisting of chromatography and re-chromatography on a column of DEAE-Sepharose and gel filtration on Sephadex G-100, catechol 1,2-dioxygenase was purified from C. tropicalis cytosol to homogeneity. Catechol 1,2-dioxygenase was found to be a homodimer with a subunit molecular mass of 30000 +/- 5000. The enzyme oxidized catechol producing cis,cis-muconic acid. The optimal temperature and pH were 30 degrees C and 7.7, respectively. CONCLUSIONS: The data are the first report showing the isolation of eukaryotic catechol 1,2-dioxygenase from C. tropicalis to homogeneity and its partial characterization.

Isolation of cytoplasmic NADPH-dependent phenol hydroxylase and catechol-1,2-dioxygenase from Candida tropicalis yeast.

The efficiencies of NADPH-dependent phenol hydroxylase (EC 1.14.13.7) and catechol 1,2-dioxygenase (EC.1.13.11.1) in biodegradation of phenol in the cytosolic fraction isolated from yeast Candida tropicalis were investigated. Enzymatic activities of both NADPH-dependent phenol hydroxylase and catechol 1,2-dioxygenase were detected in the cytosolic fraction of C. tropicalis grown on medium containing phenol. Using the procedure consisting of chromatography on DEAE-Sepharose, fractionation by polyethylene glycol 6000 and gel permeation chromatography on Sepharose 4B the enzyme responsible for phenol hydroxylation in cytosol, NADPH-dependent phenol hydroxylase, was isolated from the cytosolic fraction of C. tropicalis close to homogeneity. However, fractionation with polyethylene glycol 6000 lead to a decrease in catechol 1,2-dioxygenase activity. Therefore, another procedure was tested to purify this enzyme. Gel permeation chromatography of proteins of the eluate obtained by chromatography on a DEAE-Sepharose column was utilized to separate phenol hydroxylase and catechol 1,2-dioxygenase. Among gel permeation chromatography on columns of Sephadex G-100, Sephacryl S-300 and Sepharose 4B tested for their efficiencies to isolate phenol hydroxylase and catechol 1,2-dioxygenase, that on Sephacryl S-300 was found to be suitable for such a procedure. Nevertheless, even this chromatographic method did not lead to obtain catechol 1,2-dioxygenase in sufficient amounts and purity for its further characterization. The data demonstrate the progress in resolving the enzymes responsible for the first two steps of phenol degradation by the C. tropicalis strain.