Purification And Property Of Laccase From White Rot Fungus Pleurotus Ostreatus

Laccase is a kind of polyphenol oxidase,which mainly comes from microorganisms,plants and animals.Laccase secreting microorganisms are mainly fungi.Laccase is a kind of green catalyst,its catalytic substrate is very wide,and the only product of most catalytic reactions is water,harmless to the environment,the excellent performance of laccase makes it widely used in various fields.Laccase yield and laccase activity of bacterial strains growing under natural conditions are generally low,so it is particularly important to screen the strains with high laccase production.It is of great significance to obtain the laccase with excellent performance and develop its application.In this paper,a high-yielding strain of Pleurotus ostreatus is used as the strain to isolate and purify a laccase from its fermentation broth.The enzymatic properties of purified laccase are studied to provide some theoretical and technical support for the further application of laccase in industrial mass production.The decolorization of seven dyes with different structure types is carried out by crude enzyme solution,and the decolorization effect of laccase on different dyes is studied.Take Pleurotus ostreatus C1 mutagenic strain F-13 as raw materials,shake flask culture in liquid medium,the temperature of 30?,the speed of 220r/min,cultivation of laccase activity after 9d maximum 148U/L.After ammonium sulfate precipitation,DEAE-Sepharose anion exchange column chromatography and Sephasex G-100 gel chromatography,the crude enzyme solution is purified by electrophoresis.The specific activity of laccase is 96.4U/mg,the purification ratio is 27.5 times,and the recovery rate is 6.5%.Some enzymatic properties of the purified laccase are studied.The results shows that the molecular weight of laccase is 60.5kDa.The optimal pH and temperature is 5.0 and 50?,when the pH is 4.0-6.0,laccase activity is higher,when the pH is 3.0,4.0 and 5.0 has good stability,temperature of 40?and 50?laccase stability is very good;The degree of suitability for substrates is from high to low as follows:ABTS,DMP,guaiacol.Under the conditions of optimal substrates ABTS,K_m of this laccase is 0.194mmol/L,and V_max is9.25?molL^-1min^-1;Cu²⁺can significantly promote laccase activity,while Al³⁺,Ba²⁺,Zn²⁺,Mg²⁺and K⁺can promote laccase activity at a certain concentration.Hg²⁺,Pb²⁺and Cd⁺can inhibit laccase activity,while Fe³⁺,Mn²⁺and Ca²⁺have no significant effect on laccase activity.The decolorization of seven dyes with different structure types is carried out by using crude enzyme solution.The results shows that the decolorization rate of alialiin red,an anthraquinone dye,is 95.3%at the optimum pH and temperature of 100mg/L dye at 2h.The decolorization rates of the benzene-methane dyes basic fuchsin and malachite green reach90.1%and 93.6%respectively after 2h.The decolorization rates of aniline dyes neutral red and heterocyclic dyes methylene blue reach 81.5%and 85.9%respectively after 2h.The decolorization rates of methyl orange and congo red are only 20.8%and 13.8%,respectively.The results shows that laccase has significant decolorization effect on anthraquinone and benzene-methane dyes,good decolorization effect on aniline and heterocyclic dyes,and poor decolorization effect on azo dyes.