Identification and characterization of azoreductase enzyme AzoR2 from Bacillus velezensis for biodegradation of azo dyes

Azoreductase presents an interesting group of enzymes due to its widespread occurrence in all living forms, high sequence heterogeneity and debatable physiological role. Some bacteria have been reported to contain multiple azoreductase enzymes with differing properties, raising questions about their phylogenetic, functional and structural relationships. A YhdA azoreductase was characterized from Bacillus velezensis strain AB earlier. The present investigation reports another azoreductase AzoR2 from this culture. The amino acid sequence, physico-chemical and kinetic properties of AzoR2 were very different from YhdA, indicating that these enzymes were phylogenetically distant. AzoR2 was a flavoprotein containing FMN with subunit molecular weight of 25 kDa. It preferentially utilized NADH as electron donor with Vmax and Km of 400 U/mg and 40.2 μM respectively for methyl red decolourization. It also decolourized methyl orange and congo red effectively. The highest activity was observed at pH 7. The optimum temperature for enzyme activity was 20 °C with 84.8% activity at 60 °C. The 3-D enzyme structure was modeled, where the FMN binding residues were found to be conserved and the enzyme was predicted to be homodimeric. It is the first report on AzoR2-type enzyme from B. velezensis according to our extensive literature review.