Metagenomic analysis of a bioreactor biofilm treating raw textile effluent and biochemical characterization of a novel azoreductase gene

Abstract

Azo dyes are potent synthetic carcinogens that are widely used in the textile industries and their discharge into the surface water has detrimental consequences for human and ecosystem health. Oxidoreductases have been found to be involved in the metabolism of various xenobiotic compounds including azo dyes. Therefore, in this study shotgun metagenomic sequencing (SGMS) was used to bioprospect novel oxidoreductase genes in the biofilm of a fixed-film bioreactor treating raw textile effluent. According to the SGMS taxonomic and functional annotation, the bacteria found to be dominant in the bioreactor biofilm exhibited versatility and possessed genes that are capable of degrading azo dyes as well as other xenobiotics. A novel azoreductase gene (BM-AzoR) derived from the bioreactor biofilm metagenome was found to encode a previously uncharacterized FMN-dependent NADH azoreductase. In silico analysis of BM-AzoR gene encoded enzyme suggests that it is a thermostable and hydrophilic monomeric protein. The purified BM-AzoR is a flavin protein showing an optimal enzyme activity at pH 5 and 55 °C, respectively. The purified enzyme showed a specific azoreductase activity of 1.348 ± 0.12 U mg−1 in the presence of flavin mononucleotide (FMN) and preferred nicotinamide adenine dinucleotide (NADH) over nicotinamide adenine dinucleotide phosphate (NADPH) as an electron donor. A molecular docking analysis validated the in vitro results by identifying Methyl Red as the best substrate for the BM-AzoR. The purified enzyme retained its activity in a variety of metal ions and solvents, indicating its potential application in azo dye bioremediation.