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....