Production, Purification, Characterization and Immobilization of Laccase from Phoma betae and its Application in Synthetic Dyes Decolorization

Abstract

LACCASE (EC 1.10.3.2, p-diphenol: dioxygen oxidoreductases) is an extracellular enzyme found mainly in bacteria, fungi and plants. It has the ability to decolorize the hazardous synthetic dyes. Our study aimed to produce, purify, characterize and immobilize laccase from the fungus Phoma betae and its application in decolorization of synthetic dyes. Laccase was produced using both submerged and solid-state fermentations. Some inducers (ferulic acid, vertyl alcohol and CuSO4) for laccase production were used singly and in mixture. Significant increase in laccase production with all tested compounds was detected. Submerged fermentation induced laccase production than solid-state fermentation. Fast protein liquid chromatography (FPLC) used for laccase purification and through Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) the molecular mass of the purified laccase was 37 kDa. Optimum temperature for lacasse activity was determined at 30°C using guaiacol assay. Enzyme was more stable at -4oC than at -20 and -80oC. Maximum laccase activity was found at pH 8. Effect of metal ions and inhibitors on laccase activity was studied. Ni2+ and K1+ induced laccase activity, while Na1+, Ag1+, Hg2+, Zn2+, Cu2+, EDTA and SDS decreased laccase activity with different ratios. Laccase was immobilized in alginate beads. Both free and immobilized laccase were used to decolorize five synthetic dyes (Eriochrome Cyanine R, Merantine Brilliant Yellow 8G, Rhodamine 123, Solvent Orange 20 and Solvent Yellow 47) at concentration 0.2 g dye/L. Immobilized laccase decolorized dyes with higher yields than free one in all tested dyes. Phoma betae laccase is an efficient biocatalyst for synthetic dye decolorization.