<abstract xml:lang="en" xmlns="http://www.w3.org/1999/xhtml"><p>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 <italic>Candida tropicalis</italic> were investigated. Enzymatic activities of both NADPH-dependent phenol hydroxylase and catechol 1,2-dioxygenase were detected in the cytosolic fraction of <italic>C. tropicalis</italic> 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 <italic>C. tropicalis</italic> 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 <italic>C. tropicalis</italic> strain.</p></abstract>

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.

Isolation of cytoplasmic NADPH-dependent phenol hydroxylase and catechol-1,2-dioxygenase from <italic>Candida tropicalis</italic> yeast

Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic

Department of Fermentation Chemistry and Bioengineering, Institute of Chemical Technology, Prague, Czech Republic

Interdisciplinary Toxicology

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

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

Publié en ligne: 01 nov. 2010

Pages: 225 - 230

DOI: https://doi.org/10.2478/v10102-010-0046-7

Mots clés
environmental pollutants, phenol, biodegradation, yeast, NADPH-dependent phenol hydroxylase, catechol-1,2-dioxygenase

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