PURIFICATION CHARACTERIZATION CLONING AND EXPRESSION OF LACCASE FROM PLEUROTUS FLORIDA NCIM 1243 POTENTIAL APPLICATION IN TREATING TEXTILE AND PAPER EFFLUENT

Abstract

Laccases belong to the quotmulticopper oxidasequot family of proteins, can newlineoxidize o-diphenols and p-diphenols in the presence of molecular oxygen. newlineLaccases have been well characterized in white rot fungi where they appear to play newlinea role in lignin degradation, morphogenesis, and stress defense. This thesis is an newlineinvestigation into the production, purification, characterization, cloning and newlineexpression of laccase from Pleurotus florida NCIM 1243, further the laccase newlineenzyme was used in the bioremediation processes such as synthetic dye newlinedecolorization and in vitro treatment of textile and paper industrial effluents. newlineWhite rot fungi Pleurotus florida NCIM 1243 produces laccase as the newlinepredominant ligninolytic enzyme during the dye decolorization process. newlineStatistically-based Plackett Burman design followed by Response surface newlinemethodology was applied for the optimization of laccase production in submerged newlinefermentation. The optimal concentration of variables for maximum (4.8 U/ml) newlinelaccase production was glucose (15.21 g/1), asparagine (6.40 g/1), CuSO4 (91.78 newline[tM) and incubation period (178.55 h), respectively. newlinePleurotus florida NCIM 1243 produces two extracellular laccase newlineisoenzymes (L1 and L2) under optimized culture conditions. Laccase (L1 newlineisoenzyme) which is dominantly involved in the dye decolorization process was newlinepurified and characterized. The purified laccase corresponding to the L I isoenzyme newlinein SDS PAGE was monomeric with an apparent molecular mass of 54 lcDa. newlinePurified Li isoenzyme was assessed for the decolorization of synthetic dyes and in newlinevitro treatment of textile and paper industrial effluents. Li isoenzyme effectively newlinedecolorized triarylmethane (malachite green) and anthraquinone (RBBR) dyes. newlineDemethylation was the main reaction of malachite green decolorization by this newlineAbstract newlinefungal laccase. In the case of RBBR dye decolorization, oxidation of amino group newlinefollowed by the formation of anthraquinone was the major mechanism. However, newlinethe presence of N-hydroxybenzotriazole (HBT) greatly enhanced