Experimental design optimization and decolorization of an azo dye by cross-linked peroxidase aggregates

Synthetic textile azo-dyes are important water contaminants in industrial textile wastes and peroxidases have potential for textile-dye degradation. The optimization of enzyme activity as crosslinked enzyme aggregases (CLEAs) without precipitant is important before use in any enzymatic process. In the proposed research, the immobilization of horseradish peroxidase in the form of crosslinked enzyme aggregates (HRP-CLEAs) without precipitant addition 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 cross-linker ratio and albumin amount were found as important parameters while 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. It was estimated that hydrogen peroxide decreased the enzyme aggregate activities at higher amounts than 0.012 mM. Under optimal conditions, The HRP-CLEAs completely oxidised the textile azo dye, Reactive Blue 160 within 90 h and it was found that decolorization time of azo dye was shortened at higher temperatures.