Structural and Functional Characterization of Bacillus Azoreductase: A Computational Study for Sustainable Azo Dye Bioremediation

Abstract

Background: Azo dyes are major environmental pollutants due to their toxicity, persistence, and resistance
to conventional degradation methods. Azoreductases from Bacillus species play a crucial role in the reductive
cleavage of azo bonds, offering an eco-friendly solution for dye bioremediation. Computational approaches provide
valuable insights into enzyme structure and function for sustainable applications. Aim: To perform structural
and functional characterization of azoreductase from Bacillus velezensis using in silico approaches to evaluate
its potential in azo dye bioremediation. Methodology: The azoreductase sequence (Accession No. B3VPZ9)
was retrieved and subjected to homology modeling using MODELLER, based on Bacillus subtilis azobenzene
reductase (PDB ID: 1NNI). Structural validation was carried out using PROCHECK and Verify-3D. Molecular
docking with selected azo dyes was performed to assess binding interactions and affinity. Results: The sequence
showed high similarity (98.24%) with the template, enabling a reliable 3D model. Validation confirmed good
stereochemical quality, with 98.7% residues in favored regions and a Verify-3D score of 94.61%. A conserved
Rossmann fold and catalytic residues were identified. Docking studies revealed strong binding affinities, with
Congo red showing the highest binding energy (ΔG = −7.99 kcal/mol), supported by hydrogen bonding and
hydrophobic interactions. Conclusion: The findings demonstrate the structural stability and functional efficiency
of Bacillus velezensis azoreductase, highlighting its potential for sustainable azo dye bioremediation and eco-
friendly wastewater treatment.