Characterisation of azoreductase produced by Brevibacillus panacihumi during the decolourisation of reactive black 5

Azoreductase plays an important role in the decolourisation of azo dyes by cleaving the azo bonds in azo dye structure. In view of this, Brevibacillus panacihumi, azo dye-degrading bacteria, was used for decolourisation of Reactive Black 5 (RB5) dye. Decolourisation of RB5 was carried out by growing the bacteria culture in RB5 dye solution (100 mg/L) at pH 9, supplemented with glucose 0.4 % (v/v) and yeast extract 1.2 % (v/v) and incubated at 37 °C under sequential anaerobic-aerobic condition. Azoreductase was produced during which the enzyme with the highest activity obtained during the end of log phase. Since the azoreductase activity related to the decolourisation of RB5 in anaerobic condition, the cells were harvested during this condition. Then, to determine whether the enzyme produced is found intracellular or extracellular, the cells was collected via centrifugation and the cell pellet was disrupted using sonication technique, and Lowry assay was used to determine the protein concentration. Azoreductase was found to be produced intracellularly as the cell free extract has the highest specific activity of 0.334 U/mg compared to the culture supernatant (extracellular), resting cell and cell debris which has significantly lower 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, RB5 dye concentration of 100 mg/L and NADH concentration of 0.2 mM. In addition, the optimum pH and Ionic liquids [emim][EtSO4] concentration was pH 7 and 70 %, respectively. Phosphate buffer, pH 7 showed a higher enzyme activity compared to the Ionic liquids as a stabiliser in azoreductase assay. Decolourisation of RB5 by azoreductase under the optimum assay conditions occured up to 93 % at 8th hour of incubation was successfully achieved