The Research On Manganese Peroxidase Of Poria Cocos And The Application

Manganese perxidases(MnP) that exist in various basidiomycetes is one of the most common lignin degredation enzymes. The basidiomycetes Poria cocos is one of the medicinal material famous and precious. Nobody studied the lignin degredation enzymes produced by P. cocos.In this dissertation,we focused on MnP produced by P. cocos. First,ascertained optimum condition of MnP production; seconde, isolated and purified MnP by modern bio-chromatography. meanwhile researched the characteristics of the enzyme. Third studied the ability of decolorization of two typical dyestuff and black liquor by MnP.The basic culture medium is nitrogen-limited because the MnP production ability of most fungi was higher when they grown in nitrogen-limited cultivation. The optimal conditiaon of MnP production were obtained as follows:carbon concentraton 20g/L, nitrogen concentraton 0.2g/L, other element concentration 150ml/L (MnSO4·2H2O 0.2g,CaCl2·2H2O 0.1g,NaCl 1g,ZnSO4·7H2O 0.1g,FeSO4·7H2O 0.1 g,CuSO4·5H2O0.1 g,CoCl20.1 g,KAl(SO4)2·12H2O0.01 g,HBO30.01 g,Na2MoO4·2H2 O 0.01g),K+ concentration 1.5mmol/L,temperature 35℃,pH 4.5,volum 80ml/250ml and rotate speed 160r/min.Adding 1g/L tween 80.In the end cultivation cycle is 10d,the highest activity of MnP 1249.7U/L.Manganese peroxidase from P. cocos has been purified by some steps:over concentration,35% and 85% ammonium sulfate deposition, The column chromatograph was Q-SepharoseTM Fast Flow. Finally two MnP isozymes(MnP I and MnPⅡ)were purified from the liquid cultivation of P. cocos,with the yield were 6.21% and 7.33%.Both enzymes had approximate molecular weight as determined by SDS-PAGE,46.66kDa for MnPⅠand 43.61kDa for MnPⅡ. The characteristics of MnP as follows:the optium temperature 40℃, stabled from 20℃to 40℃and optimum pH 5.0, stabled from pH4.0-5.0. DMP was one kind of substrate,the Km values of MnP for DMP was 21.27umol/L,VMAX 0.9975umol/min.It has reported that MnP play an obvious role in decolourization of dyestuffs of antithesis nitrogen, three benzene methane, quinone amine and black liquor. This experiment focused on the optimal conditiaon of MnP decolored crystal violet, acid Orange and black liquor. The result indicated two aspects.Firest, crystal violet and acid Orange decoloration condition was tmperature 40℃,pH5.0, H2O2 concentration 0.15mmol/L.Crystal violet decoloration is 53% that is higher than acid orange.Second, black liquor decoloration condition was dilute 50 times tmperature 40℃,pH 3.0.