Purification And Characterization Of Laccase From Physisporinus Sp And Decolourization Of Azo Dyes

Laccase (EC1.10.3.2, benzenediol: oxygen oxidoreductase) are multicopper oxidases thatcan catalyze the oxidation of several, phenolics. Laccase selectively catalyze the one-electron oxidation of a phenolic substrate to a phenoxy radical, which can react further innon-enzymatic radical reactions. Moreover, laccase has special catalytic properties andbroad substrate range thus can be used as biocatalysts for diverse biotechnologicalapplications.The effects of different carbon, nitrogen sources and inducers on laccase of Physisporinussp. were studied. The extracellular laccase was purified. Laccase properties and itsdecolorization of azo dyes were analyzed.The laccase production by Physisporinus sp., under liquid fermentation was investigated.Optimization studies were done for different inducers such as veratryl alcohol, Zinc,copper under liquid fermentation. The results revealed that best inducers were veratrylalcohol.The effects of various carbon and nitrogen sources on the production of laccase byPhysisporinus sp. were investigated. Glucose and tryptone were found to be the optimalcarbon and nitrogen sources, respectively. The laccase was successfully purified by usingammonium sulphate (80%saturation). The specific activity was116.4U/mg, the yield was80.8%with purification1.5-fold. The hydrophobic interaction chromatography wascarried out by Phenyl Sepharose6Fast Flow. The obtained specific activity was781.9U/mg, the yield was42.1%with purification10.1-fold. On the other hand, ionexchange chromatography was carried by DEAE Sephadex. In this case the specificactivity was amounted by1515.1U/mg, the yield was20.4%, and19.6-fold of purification.The denatured electrophoretic analysis of the purified enzymatic demonstrated thepresence of one major protein bands, with a molecular weight of62kDa. Thecharacterizations of the purified enzymatic indicated an optimum pH for laccase activity atpH3for2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS)used as substrate, with an optimum reaction of temperature at60oC. The laccase activityremained stable at40°C for1h then residual activity was obtained at50°C. The half-life of the laccase at60°C was less than60min, indicating excellent temperature stability.The pH stability of the purified enzyme was noticed at pH6.0. The purified laccaseshowed an affinity trend in the order of ABTS> DMP, with a Kmvalue of1.27×10-3,2.82×10-3M and a Vmaxvalue of16.87and5.24μM/(L·min), respectively. In addition, theactivity of enzyme is slightly enhanced by metal ions such as Cu2+, Mn2+, Mg2+, Na2+, K+,Zn2+and Ca2+. On the other hand, the heavy metals such as Ni2+, Co2+and Hg2+arereducing the activities of the laccase enzyme even at low concentration. Arguably, theobvious effect of metal ion is inhibited by Fe2+. The results showed that the organicsolvent methanol reduced the laccase activity comparing with control,5%methanolcaused inhibition of laccase activity. The decolourization of different azo dyes by laccasewithout mediator and with mediator was analyzed. Without mediator, the decolorizationrates of Sunset Yellow, Direct red5B, Chlorazol Black38and Direct Black22at180minwere13%,2%,7%and12%, respectively. Syringaldehyde as the mediator couldobviously enhance the decolorization rates of four azo dyes. The effects of initial dyeconcentration with different syringaldehyde concentration were investigated. The optimaldecolourization of four azo dyes was different. The optimal decolorization rates were98%at0.2mM syringaldehyde for Sunset Yellow,97%at0.1mM syringaldehyde for Directred5B,53%at0.1mM syringaldehyde for Chlorazol Black38and48%at0.2mMsyringaldehyde for Direct Black22. The decolorization rates decreased with the increaseof concentration of four azo dyes.In conclusion, the strain Physisporinus sp. is an efficient producer of laccase. The purelaccase can slightly decolour of four without mediator, syringaldehyde as mediator canevidently enhance the decolorization ability of azo dyes by pure laccase.