Abstract
The occurrence of NADPH-dependent phenol hydroxylase (EC 1.14.13.7) and its efficiency in hydroxylation of phenol in the cytosolic fraction isolated from yeast Candida tropicalis grown in the absence or presence of phenol were investigated. While low levels of NADPH-dependent phenol hydroxylase activity were detected in the cytosolic fraction of C. tropicalis grown on glucose as the carbon source, 20-fold higher activities of this enzyme were found in cytosolic fractions of C. tropicalis grown on media containing phenol. The major metabolite formed from phenol by its NADPH-dependent hydroxylation in cytosol of C. tropicalis was characterized by UV/vis absorbance and mass spectroscopy as well as by the chromatographic properties on HPLC. The characteristics are identical to those of catechol. Using a 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 to homogeneity. The enzyme is a bright-yellow protein with an absorption spectrum typical of flavoproteins. NADPH-dependent phenol hydroxylase shows a molecular mass of 240,000, consisting of four identical subunits with a molecular mass of 60,000. The optimum pH range of the enzyme for the phenol oxidation lies in the region of 7.4–7.6. This is the first report showing isolation and partial characterization of cytosolic NADPH-dependent phenol hydroxylase of yeast C. tropicalis capable of oxidation of phenol to catechol. The data demonstrate the progress in resolving the enzymes responsible for the first step of phenol degradation by the C. tropicalis strain.
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