Continuous degradation of Direct Red 23 by calcium pectate-bound Ziziphus mauritiana peroxidase: identification of metabolites and degradation routes.

In the present study, oxido-reductive degradation of diazo dye, Direct Red 23, has been carried out by Ziziphus mauritiana peroxidases (specific activity 17.6 U mg-1). Peroxidases have been immobilized via simple adsorption and cross-linking by glutaraldehyde; adsorbed and cross-linked enzyme retained 94.28% and 91.23% of original activity, respectively. The stability of peroxidases was enhanced significantly upon immobilization; a marked widening in both pH and temperature activity profiles were observed. Adsorbed peroxidases exhibited similar pH and temperature optima as reported for the free enzyme. Thermal stability was significantly enhanced in case of cross-linked enzyme which showed 80.52% activity even after 2 h of incubation at 60 °C. Packed bed reactors containing adsorbed and cross-linked peroxidases were run over a period of 4 weeks; adsorbed peroxidases retained 52.86% activity whereas cross-linked peroxidases maintained over 77% dye decolorization ability at the end of the fourth week of its continuous operation. Gas chromatography coupled with mass spectrometry was used to analyze the degradation products; it showed the presence of four major metabolites. Degradation of dye starts with the 1-Hydroxybenzotriazole radical attack on the carbon atom of the phenolic ring bearing azo linkage, converting it into cation radical which underwent nucleophilic attack by a water molecule and results in cleavage of chromophore via symmetric and asymmetric cleavage pathways. Intermediates undergo spontaneous removal of nitrogen, deamination, and oxidation reactions to produce maleic acid as the final degradation product. Graphical abstract.