Azo Reductase Activity of IntactSaccharomyces cerevisiaeCells Is Dependent on the Fre1p Component of Plasma Membrane Ferric Reductase

Abstract

Unspecific bacterial reduction of azo dyes is a process widely studied in correlation with the biological treatment of colored wastewaters, but the enzyme system associated with this bacterial capability has never been positively identified. Several ascomycete yeast strains display similar decolorizing behaviors. The yeastmediated process requires an alternative carbon and energy source and is independent of previous exposure to the dyes. When substrate dyes are polar, their reduction is extracellular, strongly suggesting the involvement of an externally directed plasma membrane redox system. The present work demonstrates that, in Saccharomyces cerevisiae, the ferric reductase system participates in the extracellular reduction of azo dyes. The S. cerevisiae ⌬fre1 and ⌬fre1 ⌬fre2 mutant strains, but not the ⌬fre2 strain, showed much-reduced decolorizing capabilities. The FRE1 gene complemented the phenotype of S. cerevisiae ⌬fre1 cells, restoring the ability to grow in medium without externally added iron and to decolorize the dye, following a pattern similar to the one observed in the wild-type strain. These results suggest that under the conditions tested, Fre1p is a major component of the azo reductase activity.Research work on biodegradative processes for azo dyes usually exploits bacterial species, either isolated or in consortia (4, 36). Bacteria, under appropriate conditions (e.g., oxygen limitation and the presence of substrates utilized as carbon and energy sources), frequently reduce azo dyes, producing colorless amines. Nevertheless, many dyes are recalcitrant to conventional wastewater treatment processes with activated sludge (4). The overall impression in this research area is that many azo dyes can be reduced (and decolorized) by a considerable number of bacterial species, but as far as we know, the enzyme responsible for the unspecific primary reduction step has never been positively identified. What is currently postulated is that reductive decolorization of sulfonated azo dyes by living cells must occur extracellularly due to the impermeant nature of those compounds and that the primary reductant is a cytoplasmic electron donor, presumably NAD(P)H (36).Previous studies (30,31) have demonstrated that some nonconventional ascomycete yeasts are efficient azo dye decolorizers, acting, as many bacteria, by reducing the azo bond. Dye decolorization by yeasts is comparatively unspecific but is affected by the medium composition, by the yeast strain used, and by parameters such as pH and dissolved oxygen level. It also requires actively growing cells, being faster during the exponential growth phase, and displays an enzyme-like temperature profile, strongly suggesting its biotic nature. However, further information is required for successful application of yeasts in a wastewater treatment process. The present work was developed to demonstrate the participation of an externally directed plasma membrane redox system (PMRS) in azo dye reduction, linking an intracellular reductant to an extracellular electr...

Recent Advances in Azo Dye Degrading Enzyme Research

“…It restored the ability to grow in medium without externally added iron and to decolorize the dye, following a pattern similar to the one observed in the wild-type strain. This indicated that Fre1p (component of plasma membrane ferric reductase) is a major component of the azo reductase activity [25].…”

“…Fungal degradation of azo dyes mainly occurs from the lignin peroxidase activity under aerobic conditions [24]. In yeast, the ferric reductase system participates in the extracellular reduction of azo dyes [25].…”

Three-dimensional Structure of AzoR from Escherichia coli

“…Many kinds of bacterial azoreductases have been isolated and characterized for the progress of bioremediation (7)(8)(9)(10)(11)(12)(13)(14)(15). However, AzoR is different from other azoreductases reported thus far with respect to its requirements for cofactors, electron donors, and substrate specificity and its amino acid sequence.…”

Textile azo dye degradation by Candida rugosa INCQS 71011 isolated from a non-impacted area in Semi-Arid Region of Brazilian Northeast

“…The decolorization of a number of simple azo dyes derived from 2-naphtholaminoazobenzene and p-N,Ndimethylaminoazobenzene in concentrations of 10-50 mg l -1 were tested in liquid aerated batch cultures using a strain of the yeast Candida zeylanoides UM2, by which method a 90% adsorption was obtained from the best result after seven days of incubation (Martins et al, 1999). This same strain was used by Ramalho et al (2005) which, after changing some parameters, such as medium, for example, C. zeylanoides UM2 showed 100% efficiency of decolorization without traces of absorption after 60 h.…”

“…Within 16-48 h of incubition the color disappeared completely without residual color on the biomass in the case of only one of these dyes, the Reactive Black 5. The metabolic profile species-specific, such as enzymatic activity (Ramalho et al, 2005;Martorell et al, 2012), media culture (Ramalho et al, 2002;Kaushik and Malik, 2010) and the structure complexity of the dye (Fu and Viraraghavan, 2001;Bergsten-Torralba et al, 2009) can greatly influence the final response of fungal degradation. In the present work, C. rugosa INCQS 71011 was much more efficient degrading RR198, a single azo class dye, than it was towards RR 141, a double azo class dye, which took longer to be degraded and towards RB 241, another double azo class dye, which was not completely degraded under the conditions established in this study.…”