Abstract
We used the recombinant CotA‐laccase from the bacterium Bacillus subtilis to investigate the biotransformation of the commercial anthraquinonic dye Acid Blue 62. Kinetics of dye biotransformation at pH 6 follow a Michaelis–Menten model. NMR and several MS techniques allowed the identification of intermediates and final products of the enzymatic biotransformation. The main final product obtained, 1‐[(4‐amino‐9,10‐dioxo‐3‐sulfo‐9,10‐dihydroanthracen‐1‐yl)diazenyl]‐4‐cyclohexylamino‐9,10‐dioxo‐9,10‐dihydroanthracene‐2‐sulfonic acid, is formed through the creation of an azo link and has been previously identified as an intermediate compound in the biodegradation of Acid Blue 62 by crude fungal preparations. The identification of 1,4‐diamino‐9,10‐dioxo‐3‐sulfo‐9,10‐dihydroanthracene‐2‐sulfonic acid and of cyclohexanone, in reaction mixtures with CotA‐laccase and also its presence in reactions performed with the LAC3 laccase from the fungus Trametes sp. C30, suggest the occurrence of coupling reactions between the intermediate products of dye oxidation. Based on these results, we propose a mechanistic pathway for the biotransformation of Acid Blue 62 by laccases. A bioassay based on the inhibitory effects of the dye and its enzymatic products on the growth of Saccharomyces cerevisiae shows the importance of laccases in reducing dye toxicity.