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

Simple models for the continuous aerobic biodegradation of phenol in a packed bed reactor

This paper proposes the use of a preliminary, phenol removal step to reduce peak loads arriving at a conventional effluent plant. A packed bed reactor (PBR) using polyurethane foam, porous glass and also cocoa fibres as the inert support material was used. Experiments have been carried out where the flow-rates, plus inlet and outlet phenol concentrations were measured. A simple, plug-flow model is proposed to represent the results. Zero, first order, Monod and inhibited kinetics rate equations were evaluated. It was found that the Monod model gave the best fit to the experimental data and allowed linear graphs to be plotted. The Monod saturation constant, K, is approximately 50 g m-3, and ka is around 900 s-1.Este artigo propõe o uso de uma etapa preliminar de remoção de fenol para redução de picos de carga na entrada de sistemas convencionais de tratamento de efluentes. Um reator de leito fixo (RLF) foi usado, tendo como suportes inertes espuma de poliuretano, vidro poroso e também fibras de coco. Nos experimentos foram controladas a vazão e as concentrações de fenol de entrada e saída. Um simples modelo plug-flow é proposto para representar os resultados. Cinéticas de zero e primeira ordens, Monod e de inibição foram avaliadas. Foi verificado que o modelo de Monod foi o que melhor se ajustou aos dados experimentais, permitindo que gráficos lineares fossem traçados. A constante saturação de Monod, K, é de aproximadamente 50 g m-3 e ka em torno de 900 s-1