The Function of Cytoplasmic Flavin Reductases in the Reduction of Azo Dyes by Bacteria

Abstract

A flavin reductase, which is naturally part of the ribonucleotide reductase complex of Escherichia coli, acted in cell extracts of recombinant E. coli strains under aerobic and anaerobic conditions as an "azo reductase." The transfer of the recombinant plasmid, which resulted in the constitutive expression of high levels of activity of the flavin reductase, increased the reduction rate for different industrially relevant sulfonated azo dyes in vitro almost 100-fold. The flavin reductase gene (fre) was transferred to Sphingomonas sp. strain BN6, a bacterial strain able to degrade naphthalenesulfonates under aerobic conditions. The flavin reductase was also synthesized in significant amounts in the Sphingomonas strain. The reduction rates for the sulfonated azo compound amaranth were compared for whole cells and cell extracts from both recombinant strains, E. coli, and wild-type Sphingomonas sp. strain BN6. The whole cells showed less than 2% of the specific activities found with cell extracts. These results suggested that the cytoplasmic anaerobic "azo reductases," which have been described repeatedly in in vitro systems, are presumably flavin reductases and that in vivo they have insignificant importance in the reduction of sulfonated azo compounds.Various bacterial strains reduce azo dyes under anaerobic conditions. The most generally accepted hypothesis for this phenomenon is that many bacterial strains possess rather unspecific cytoplasmic enzymes, which act as "azo reductases" and under anaerobic conditions transfer electrons via soluble flavins to the azo dyes (35). We have recently suggested a different mechanism for the unspecific anaerobic reduction of azo dyes by Sphingomonas sp. strain BN6 and other bacteria. In this system, the reduction of the azo dyes is catalyzed extracellularly by the action of mediator compounds, which are either formed during the metabolism of certain substrates by the bacteria themselves or which may be added externally (e.g., compounds such as anthraquinonesulfonates). These mediators enable the transfer of redox equivalents from the cell membrane of the bacteria to azo dyes. In addition, it was demonstrated that Sphingomonas sp. strain BN6 also possesses cytoplasmic azo reductase activities, which could be assayed in vitro with cell extracts under anaerobic conditions (14,15,17). It was shown that the reduction rate of whole cells of Sphingomonas sp. strain BN6 for amaranth after growth with glucose was 0.3 mol min Ϫ1 g of soluble cell protein Ϫ1 and could be increased by the addition of anthraquinone-2-sulfonate to 3 mol min Ϫ1 g of soluble cell protein Ϫ1 . In contrast, in vitro with cell extracts or solubilized membrane preparations, specific activities of 20 to 40 mol min Ϫ1 g of soluble cell protein Ϫ1 were determined (15, 17). A possible explanation for the significantly lower reaction rates of the whole-cell preparations compared to those in the in vitro systems was that in vivo the cytoplasmic azo reductase activity does not function, because the highly polar sulfonat...

Bacterial diversity and composition in major fresh produce growing soils affected by physiochemical properties and geographic locations

“…Most of these enzymes are shown to be ineffective in vivo (Blumel et al, 2002;Russ et al, 2000). Limited transport of dyes across the cell membrane and high reactivity of many of the ortho aminohydroxy aromatics that are formed after the reductive cleavage of the azo dyes restrict the reaction of azoreductases (Grundmann, 1979;Kudlich et al, 1999).…”

“…Limited transport of dyes across the cell membrane and high reactivity of many of the ortho aminohydroxy aromatics that are formed after the reductive cleavage of the azo dyes restrict the reaction of azoreductases (Grundmann, 1979;Kudlich et al, 1999). Russ et al (2000) suggested that the cytoplasmic anaerobic azoreductases are flavin reductases and could function for extracellular reduction of azo dyes via an electron mediator. These mediators allow transfer of redox equivalents from the cell membrane of bacteria to azo dyes.…”

A New Alkali-Thermostable Azoreductase from Bacillus sp. Strain SF

“…Cell extracts generally showed much higher rates of anaerobic reduction of azo dyes than did preparations of resting cells (23,36). This has generally been explained by the low permeability of the cell membranes for the highly polar sulfonated azo compounds (32).…”

“…The ability of cytosolic flavine reductases to act in vitro as azoreductases was recently demonstrated by experiments using a recombinant flavine reductase in different genetic backgrounds (32). Cell extracts generally showed much higher rates of anaerobic reduction of azo dyes than did preparations of resting cells (23,36).…”

Catalyzing denitrification of Paracoccus versutus by immobilized 1,5-dichloroanthraquinone

“…That was why an explanation for the accelerating effect on the denitrification could be sought in terms of an alteration of the enzyme properties and of cofactor regeneration. This also explained the similar role of NADH and redox mediator for the accelerating effect during the decolorization (Russ et al 2000;Keck et al 1997). In our research group, the catalyzing mechanism of redox mediator on the denitrification is also studying by using cell extracts and purified enzymes, which would allow a better understanding on the nature of the process.…”