A contribution for the identification of the azo reductase activity in intact yeast cells

Abstract

Azo dyes are synthetic organic colorants which are extensively used in textile, food and cosmetic industries. A large fraction of these dyes is released into the environment even after conventional wastewater treatments. This is a worldwide problem and particularly a problem in regions where textile industries release large quantities of coloured wastewater to water courses. In an attempt to develop a biological treatment for colour removal we isolated several ascomycete yeast strains from contaminated soil based on its capacity to decolourize soluble azo dyes. We studied the process in batch cultures and have demonstrated that the colour loss was due to a reductase activity, expressed constitutively, which could transform azo dyes into colourless amines [1]. In order to understand the process involved in this reduction we selected a Saccharomyces cerevisiae strain (BLC0276) with this capacity. This strain reduced model azo dyes in 8-16 h. We decided to further investigate which enzyme(s) could be involved in this reduction. It was found that in S. cerevisiae there is a membrane redox system with an externally directed reductase – the ferric reductase system, responsible for the extracellular reduction of ferric to ferrous iron previous to uptake, and involving the genes fre1 and fre2. Several know inhibitors of this system were investigated like excess of iron in the medium, CCCP, DPI, chloroquine and p-chloromercurybenzoic acid. Several of these compounds managed to inhibit the decolourizing activity. We also deleted the genes fre1 and fre2 in our strain and analysed the effect on decolourizing activity. Once again the process was retarded in fre1 and fre1fre2 strains. The effect of fre2 deletion was negligible. The deletion of fre1 retarded the decolourizing capability showing the involvement of this system. However the capacity was not completely removed indicating that cells have an alternative reducing system.

[1] Ramalho, P.A., Scholze, H., Cardoso, M.H., Ramalho, M.T., Oliveira-Campos, A.M. 2002 Improved conditions for the aerobic reductive decolourization of azo dyes by Candida zeylanoides. Enz Microbiol Technol 31: 848-854.