Decolourisation of Reactive Black 5 by Azoreductase Produced by Brevibacillus panacihumi ZBI

Azoreductases are often associated with decolourisation of non-degradable azo dyes via cleavage of azo bonds. In this study, Brevibacillus panacihumi ZBI, an azo dye-degrading bacterium which has not been reported before, was used for the decolourisation of Reactive Black 5 (RB5) dye. The highest activity of azoreductase was obtained during the end of log phase. Azoreductase produced intracellularly had the highest specific activity of 0.334 U/mg compared to the culture supernatant (extracellular), resting cell and cell debris with low enzyme activity of 0.034 U/mg, 0.010 U/mg and 0.200 U/mg respectively. The optimum assay conditions for the maximum azoreductase activity were at 37°C, pH 7, RB5 dye concentration of 100 mg/L and NADH concentration of 0.2 mM by using phosphate buffer as a medium for the enzyme reaction. Alternatively, the azoreductase assay was also carried out using ionic liquid, [emim][EtSO4] that may function to enhance the activity and stability of azoreductase. Results using phosphate buffer (pH 7) showed higher enzyme activity twice that of the ionic liquid besides enhancing the stability of enzyme. Under the optimum assay conditions up to 93 % of decolourisation was achieved after 8 h of incubation. In addition, growth of bacteria was also concurrently observed during the decolourisation of RB5.