Hybrid biocatalytic system of Kocuria rhizophila DyP peroxidase–nanobioconjugate for enhanced synthetic textile dye decolorization and parallel comparison in real textile effluent

Textile dye effluents remain difficult to remediate because single-dye assays rarely reproduce the chemical complexity of industrial wastewater. In this work, a dye-decolorizing peroxidase (DyP) from Kocuria rhizophila was heterologously expressed in E. coli , purified, and covalently immobilized onto Syzygium aromaticum -derived cobalt nanoparticles (SaCoNPs) using EDC/NHS chemistry. The biocatalyst was evaluated for its ability to transition the decolorization of synthetic dyes to the substantial reduction of parent dye concentrations of dyes in real textile effluents. The SaCoNPs exhibited phytochemical capping, crystalline spinel Co₃O₄ character, a hydrodynamic size distribution of 13–30 nm, and a zeta potential of −27.0 ± 3.9 mV. Covalent immobilization via EDC/NHS chemistry yielded an 82.03 ± 1.59% immobilization efficiency, confirmed by FTIR spectroscopy showing characteristic amide I and II bands followed by XPS and TEM. The DyP–SaCoNP conjugate demonstrated superior decolorization of Reactive Blue 19 (94.65 ± 0.22%), Reactive Black 5 (95.63 ± 0.21%), and Malachite Green (96.74 ± 0.58%) at 100 µg/mL regimen concentration over 24 h, significantly outperforming free DyP and SaCoNPs alone. Crucially, the system was validated on real textile effluent, where HPLC-DAD quantification confirmed 93.58%, 94.12%, and 95.32% removal of the respective dyes, showing strong correlation with synthetic dye performance. The conjugate retained 38–41% of initial activity after six reuse cycles, demonstrating operational stability. TOC analysis further substantiated the substantial organic matter mineralization, with the conjugate achieving 48.3% reduction in total organic carbon from real textile effluent. This study demonstrates proof-of-concept for evaluating laboratory-scale decolorization systems under real-world conditions, bridging the critical gap between synthetic dye studies and practical industrial wastewater treatment.