Abstract
Azo dyes are commonly used in many commercial industries. Some of the azo dyes can produce carcinogenic compounds after being metabolized by azoreductase. Several human intestinal microbiota possess azoreductase activity which plays an important role in the toxicity and mutagenicity of these azo dye compounds. The acpD gene product (AzoEf1) responsible for the azoreductase activity of Enterococcus faecium, an intestinal bacterium, was heterologously expressed, purified and characterized. The protein sequence shares 67% identity with the azoreductase from Enterococcus faecalis, AzoA. Although AzoEf1 possesses many commonalities with AzoA, there are differences in coenzyme preference, residues associated with FMN binding, substrate specificity, and specific activity. AzoEf1 utilized both NADH and NADPH for the reduction of azo dyes, and it contains a leucyl residue at position 104 and threonyl residue at position 19 which differ from AzoA at the active site. Its specific activity was 5095 M/min/mg and its catalytic efficiency for Methyl red reduction was lower than AzoA.
Highlights
An important function associated with intestinal bacteria is their ability to metabolize xenobiotics, such as azo dyes (Chung, KT. et al, 1978; Brown, JP. 1981; Cerniglia, CE. et al, 1982; Manning, BW., Cerniglia, CE. & Federle, TW., 1985)
Identification of the E. faecium acpD gene The AzoA protein sequence from E. faecalis and the AzoR from Escherichia coli were initially identified as an acyl carrier protein phosphodiesterase (AcpD)
Expression and purification of the acpD gene from E. faecium Specific primers designed to amplify the acpD gene, renamed as azoEf1, successfully amplified the predicted 730 bp fragment from the genomic DNA of E. faecium The amplicon was directly cloned into the TOPO-TA vector and the sequencing results showed that the insert contained the complete open reading frame (ORF) for the gene
Summary
An important function associated with intestinal bacteria is their ability to metabolize xenobiotics, such as azo dyes (Chung, KT. et al, 1978; Brown, JP. 1981; Cerniglia, CE. et al, 1982; Manning, BW., Cerniglia, CE. & Federle, TW., 1985). An important function associated with intestinal bacteria is their ability to metabolize xenobiotics, such as azo dyes Et al, 1982; Manning, BW., Cerniglia, CE. The introduction of some of these dyes into the human body results in the reduction of their azo bonds and subsequent biotransformation to carcinogenic metabolites (Cerniglia et al, 1986; Bragger et al, 1997; Brown et al, 1983, Cerniglia, CE., 1982). Several intestinal bacteria are known to have azoreductase activity from culture studies (Chung K-T. et al, 1992b), thereby the isolation and characterization of azoreductase genes from these intestinal bacteria will provide information to their biochemical function within the cells and perhaps even allude to their evolutionary origin.
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