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«“√ “√ √“™∫— ≥±‘ µ¬ ∂“π ªï ∑’Ë Û ©∫— ∫∑’Ë Ò ¡.§.-¡’ .§. ÚıÙ¯ 65 Somsak Damronglerd, et al. substrates may act as electron donors for reduction of azo dyes. This would likely explain the observation that azo dye reduction occurs more readily as a co-metabolic event when addi- tional readily degradable substrates (glucose) are provided (Fig. 2). There were at least two possible ways for glucose to enhance reduction of sulfonated azo dyes. It could act as a donor of reducing equivalents [e.g., via NADH or FADH 2 ], or its addition could result in more actively respiring cells, thus rapidly removing the oxygen in culture medium and enabling corresponding enzymes to transfer reducing equivalent to azo dyes (46). In many intestinal bacterial isolates, a flavin compound (riboflavin, flavin adenine dinucleotide, of flavin mononucleotide (FMN)) is required for azoreductases activity (19, 22, 45, 46). The most generally accepted hypothesis for this phenomenon is that many bacterial cells possess a rather unspecific cytoplasmic favin- dependent reducatses (flavin reduc- tases) which transfers electron under anaerobic conditions via (soluble) flavins to the azo dyes (46, 50). In the present study, a rather rapid decolorization of all azo dyes was observed when incubated them with cytoplasmic fraction of strain A5 in oxygen-free buffer with NADH as a source of reduction equivalents (Table 2). However, the reaction rate increased dramatically in the presence of flavin adenine dinucleotide (FAD). A possible explanation for this phenomenon is that FAD is reduced enzymatically by NADH and reduced FAD (FADH 2 ) can then sponta- neously reduce the three sulfonated azo dyes to the corresponding amines (19, 22, 45, 46). In contrast, it was shown that the addition of FAD did not lead to enhancement of the reduction rates of sulfonated azo dyes by whole cells of strainA5 (Table 2). Thus, this has generally been explained by the low permeability of the cell membranes for the highly polar sulfonated azo compounds (56). Moreover, the bacterial membrane are also hardly permeable for flavin- containing cofactors and restrict the transfer of reducing equivalents by flavins from cytoplasm of intact cells to extracellular sulfonated azo dyes (56). In addition, it was clearly demonstrated in our study that the almost activity of flavin reductase, which hypothesized to function under adequate conditions as flavin- dependent azo reductase, was present in the cytoplasmic fraction (Table 3). Therefore, it appears reasonable that, with intact cells, intracellular enzymes like flavin reductases are of little importance for reduction of extracellular sulfonated azo com- pounds by strain A5. These results supported the hypothesis of Russ and coworkers that the reduction of sulfonated azo dyes by reduced flavins formed by cytosolic flavin- dependent azo reductases is mainly observed in vitro and in vivo is of insignificant importance (29, 46, 50). Thus, in the intact cells, other enzyme systems, which does not require transport of the azo dyes though the cell membrane, are presumably responsible for the unspecific reduction of various sulfonated azo dyes by Paenibacillus sp. strain A5. Several quinoid redox mediator compounds, for example; anthaquinone-2-sulfonate (AQS), 2- hydroxy-1,4-naphthoquinone (law sone), 4-amino-1,2-naphthoquinone and AQDS, have been shown to enhance degradation of sulfonated azo dyes by acting as electron shuttles that facilitate reduction of the azo dye (26, 27, 29, 30, 42, 43). In this article we report that external added AQDS not only stimulate quinone- dependent azo reductase activity in detergent-soluble membrane fractions but also enhance anaerobic reduction of intact strain A5 cells. In addition, it could be demonstrated in the cell-fractioning experiments that the NADH: quinone oxidoreductase activity was almost restrictively present in the membrane fraction (Table 3). These results suggest the existence of an NADH-dependent quinone reductase in Paenibacillus sp. strainA5 membranes that catalyze the reduction of endogenous quinones (e.g., menaquinone), may responsible for the reduction of exogenous qui- nones (e.g.,AQDS), which then transfer reduction equivalents to sulfonated azo dyes outside the cells. The anaerobic decolorization occurred only in the presence of cells, indicting that the cells reduced the AQDS to the corresponding hydro- quinone (AH 2 QDS). This reaction may be catalyzed anaerobically by NADH-quinone oxidoreductase (NDH) of the respiratory chain, which appeared to have a low substrate specificity of the quinone-binding