ABSTRACT
Enzyme immobilization has gained considerable attention due to the incredible properties exhibited by the enzymes in immobilized condition. Therefore, in the present study, a comparative analysis of free and immobilized laccase is reported using various substrates. The ideal substrate for immobilization was found to be copper alginate with an immobilization yield of 91.078%. The optimum pH was 4 and 5, respectively, for free and immobilized enzymes while the optimum temperature was found to be 50 and 60?C, respectively. The kinetic parameters Vmax and Km were found from the Line weaver-Burk plot and were 48.076 U/mL and 0.480 mM for free enzyme while 55.55 U/mL and 1.277 mM for the immobilized enzyme, respectively. The catalytic efficiency Kcat was found as 100.01s?1 for free enzyme and 43.5s?1 for its immobilized counterpart. Out of the various metal ions used, Co2+ was found to enhance the activity of an immobilized enzymes. The storage stability of the enzyme was studied and was found that only 32.44% of initial activity was retained by free enzyme whereas, 70.21% of activity was retained by immobilized enzyme upon storage for four weeks at 4?C. The thermal stability studies shows that the immobilized enzyme retained 32.60% of its initial activity and the free enzyme retained 1.14% of initial activity on exposure to 60?C for 3 h. Finally, the reusability of immobilized laccase beads was evaluated by decolorization of methyl orange for five repeated cycles and a percentage decolorization of 32.04% could be retained at the fifth cycle. This study therefore, suggests copper alginate-immobilized beads to be an effective option for various applications