The azoreductase of yeast cells: a new feature of an old enzyme

Abstract

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]. To understand the process behind the azo decolourising capacity of some yeast strains isolated by our group [1,2] we selected a Saccharomyces cerevisiae strain (BLC0276) with this capacity. This strain was able to decolourize the media in 8 to 16 h. With evidences from a previous work and with the knowledge that the plasma membrane redox systems are known to be ubiquitous and multifunctional, we decided to explore the involvement of one of these systems on this feature of S.cerevisiae – the ferric reductase system. This system is responsible for the reduction of ferric to ferrous iron previous to its uptake by the yeast cell. First we compared both ferric and azo reducing activities along the growth phases. Both have an activity peak in the late exponential growth phase. These peaks correspond in growing cultures to the phase where the dye is more actively removed. Then several known inhibitors were added to the media, like carbonylcyanide m-chlorophenylhydrazone (CCCP), diphenylene iodonium (DPI), p-chloromercuribenzoate (pCMB) and chloroquine (CQ), and different levels of inhibition on both ferric and azo reductase activities were observed. The genes Fre1 and Fre2 were deleted and the effect on the decolourising activity was analysed in the mutant strains. 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 decolourising capability showing the involvement of this system in the azo decolorizing capacity of S.cerevisiae. 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. [2] Ramalho, P.A., Cardoso, M.H., Cavaco-Paulo, A., Ramalho, M.T. 2004 Characterisation of the azo reductase activity in a novel ascomycete yeast strain. Accepted for publication in Appl Environ Microbiol 70