First‐Time Demonstration of a Two‐Step Mechanism for Azo Dye Removal by Bacillus proteolyticus Using Molecular Docking and FT‐IR

ABSTRACT

Azo dyes represent one of the most persistent classes of industrial pollutants due to their structural stability and resistance to biodegradation. In this study, the azo dye removal mechanism of Bacillus proteolyticus was investigated using an integrated approach combining microbiological assays, FT‐IR spectroscopy, molecular docking, and molecular dynamics simulations. The isolate ( B. proteolyticus OA7), obtained from textile wastewater, was identified by 16S rRNA gene analysis. Decolorization experiments demonstrated a markedly higher removal efficiency for Acid Red 88 (69.74%) compared to Acid Blue 193 (41.01%), indicating substrate‐dependent biodegradation behavior. Solid medium assays revealed distinct removal patterns, where Acid Red 88 underwent both biosorption and biodegradation, while Acid Blue 193 was primarily removed via biosorption. FT‐IR analysis showed significant alterations in hydroxyl, lipid, protein (Amide I–II), and extracellular polymeric substance (EPS) functional groups following dye exposure, suggesting the involvement of both surface interactions and intracellular responses. The emergence of aromatic vibration bands (900–600 cm ⁻¹ ) in dye‐treated samples supports the occurrence of azo bond transformation. Molecular docking analyses demonstrated strong binding affinities of azo dyes toward key redox enzymes, including azoreductase, OYE‐like flavoprotein reductase, FMN‐dependent NADPH‐quinone reductase, and laccase, indicating their potential role in enzymatic degradation. Notably, Acid Red 88 exhibited favorable binding without hydrogen bond formation, highlighting the contribution of hydrophobic interactions. According to molecular simulations, ligand binding induced moderate conformational changes and local flexibility, but did not affect the global structural stability of the enzyme. Overall, the findings support a two‐stage azo dye removal mechanism involving initial biosorption followed by enzymatic redox‐mediated degradation. This study provides new mechanistic insights into the role of B. proteolyticus in azo dye bioremediation and highlights the influence of dye structure on bacterial response and removal efficiency.