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สารฮิ วมิ กช่วยเพิ่ มความสามารถการลดสี ... 212 The Journal of the Royal Institute of Thailand Vol. 37 No. 1 Jan.-Mar. 2012 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 ad- enine 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 reductases (flavin reductases) which transfers electron under anaerobic conditions via (soluble) fla- vins 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 spontaneously 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 strain A5 (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 compounds 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