In the present work, the preparation, characterization and application of silica-coated magnetic nanoparticles immobilized papain and pectinase is reported. In this manner, the magnetite nanoparticles were synthesized by co-precipitation method. Next, silica-coated nanoparticles were prepared through the sol–gel reaction and then activated by 3-(chloropropyl)trimethoxysilane. The immobilized enzyme was characterized by FT-IR, XRD,SEM andVSM techniques. Response Surface Methodology was applied by for statistical designing of operating conditions in order to immobilize papain and pectinase on magnetic nanoparticles. Operation conditions included temperature of the immobilization solution, pH of the enzyme solution, enzyme concentration and immobilization time as numeric factors and immobilization amount of enzyme and activity of immobilized papain as responses. The immobilization of papain process was optimized at 27.3 o C, pH=7.1 and 3.3 mg papain for 10 h. The immobilization yield was 58.3% at optimum conditions. Compared with the native papain, the conjugated papain was found to exhibit enhanced enzyme activity, better tolerance to the variations of medium pH and temperature, and improved storage stability as well as good reusability. Furthermore, free and immobilized samples were used to compare their efficiency on clarification of pomegranate juice. Addition of both proteases individually, exerted major decreasing effects on turbidity development (haze formation) during cold storage. However, there was no significant difference between the effects of these proteases treatmenton juice turbidity. The optimal conditions for immobilization of pectinase were: 30 o C temperature, pH=5.5, 42.97 µl pectinase and 2 h. The immobilization yield was 53.18% at optimum conditions. Compared with the free pectinase, the immobilized pectinase was found to exhibit better tolerance to the variations of medium pH and temperature, and improved storage stability. Both free and immobilized samples reduced the viscosity of apple juice from 1.12 to 0.88 and 0.92 mm 2 s -1 , respectively, after 30 min at optimum temperature of each enzyme. Furthermore, the immobilized enzyme could be re-used six consecutive cycles and the efficiency loss in viscosity reduction was found to be only 8.16%. Keywords : Magnetic nanoparticles, Papain, Pectinase, Immobilization, Enzyme activity, Juice clarification ,
