Efficient degradation of azo dyes by an engineered myoglobin with a modified heme active site showing high peroxidase activity

Azo dyes, widely used in global textiles, persist in wastewater causing significant ecological and health risks, necessitating efficient degradation to protect ecosystems and public health. The development of artificial metalloenzymes has provided advanced biotechnological solutions to address this challenge. This study presents a highly efficient artificial metalloenzyme, F43Y/H64D myoglobin (F43Y/H64D Mb), engineered from sperm whale myoglobin with a modified heme active site by replacement of Phe43 with Tyr and His64 with Asp, respectively, for azo dye degradation. The engineered enzyme achieved degradation efficiencies of approximately 93%, 93%, and 90% for Acid Orange 7, Methyl Orange, and Acid Blue 92, respectively, within 10   seconds. Its overall catalytic efficiency was found to be approximately 3.6 times higher than that of horseradish peroxidase, a widely used natural enzyme. HPLC and ESI-MS analyses proposed degradation pathways, and bacterial toxicity tests confirmed reduced toxicity post-degradation. Furthermore, F43Y/H64D Mb exhibited excellent performance in natural aquatic environments, surpassing many recently reported results in efficiency, reaction speed, and operational simplicity. These findings highlight the feasibility of F43Y/H64D Mb for efficient azo dye decolorization and degradation in wastewater, offering a novel enzymatic tool for sustainable environmental remediation.