Experimental Design Optimization and Decolorization of an Azo Dye by Cross-Linked Peroxidase Aggregates

Abstract

The immobilization of horseradish peroxidase in the form of cross-linked enzyme aggregates (HRP–CLEAs) was optimized by three parameters full factorial experimental design at two levels and the degradation of an azo dye was tested. The optimal immobilization conditions were estimated as 0.06 mg enzyme/mL (0.96 U), 3 % (v/v, Glutaraldehyde)) cross-linker ratio, and 6 mg /mL Bovine Serume Albumine amount, respectively. The effects of variables were analysed by responce surface plots and the enzyme concentration effect was insignificant for the tested levels. The maximum CLEAs activity was 0.2188 U and the kinetic parameters (Km and Vmax) were 0.0314 mM and 0.1044 mM/min while the calculated values for soluble enzyme were 0.06 mM and 0.468 mM/min, respectively. Hydrogen peroxide has inhibitory effect on enzyme aggregate activities at higher amounts than 0.012 mM. Under optimal conditions, a textile azo dye, Reactive Blue 160 was completely oxidized within 90 h and higher temperatures also showed promising results in dye degradation.