Background: Laccases are important enzymes that have numerous applications in different
Objective: The aim was to purify laccase from Aspergillus flavus PUF5, successfully immobilize it on
coconut fiber and characterize different physical and kinetic properties under both free and immobilize
Methods: Laccase from A. flavus PUF5 was purified using ammonium sulfate precipitation, followed
by DEAE column chromatography and gel filtration using Sephadex G100. The molecular weight was
determined through SDS-PAGE (12%). It was immobilized on pretreated coconut fiber through crosslinking
by glutaraldehyde (4% v/v). Physical and kinetic parameters like optimum temperature, pH,
thermostability, the effect of additives, activation energy, Km and Vmax for free and immobilized laccase
were also analyzed. Recycling stability of the immobilized laccase was further determined.
Results: The extracellular laccase (65 kDa) was purified up to homogeneity and was immobilized on
acid-pretreated coconut fiber by 4% (v/v) glutaraldehyde solution at 30°C, pH 5.0. Activation energy
(Ea) of free and immobilized laccase for oxidation of guaiacol was found to be 24.69 and 32.76 kJ mol-1
respectively. Immobilized laccase showed higher melting temperature (Tm) of (82.5°C) than free enzyme
(73°C). Km and Vmax for free and immobilized laccase were found to be 0.67 mM, 0.70 mM
and 280 U/mg, 336 U/mg respectively when guaiacol was used as substrate. Additionally, in immobilized
condition laccase retained ˃80% of its initial activity after use till six repeated cycles.
Conclusion: The purified laccase enzyme and the cheap immobilization seem to be a prospective process
for different biotechnological and industrial applications.