Study On The Recombinant Manganse Peroxidase From Ganoderma Lucidum And Its Application

Manganese peroxidase（MnP）is one of the most common lignin degradation enzymes.With lack of substract specificity,MnP is capable of oxidizing range of aromatic compounds,and is widely used in lignin degradation and dye decolorization.Therefore,the exploration of the characterization and application of MnP will lay a foundation for producing this enzyme in large-scale and environmental protection.Firstly,the yeast transformants（Pichia pastoris SMD1168H）carrying with the pAO815::GlMnP were induced to express for the production of recombinant Ganderma lucidum manganese peroxidase（rGlMnP）,in which the protein concertration was determined by Brandford method.Meanwhile,the enzymatic properties of rGlMnP were also explored.The results showed that the yield of total protein in the expressed products was3.3 mg/mL.The optimal temperature and pH of the rGlMnP-substract reaction are at 30℃and pH5.0,reapectively.rGlMnP remainded its stable activity in the range of pH 3.0-6.0 at the temperature of below 50℃.The metal ion,such as Ca²⁺,Mn²⁺,Na⁺,K⁺and Fe²⁺,could promote the enzymic activity of rGlMnP,while Fe³⁺could inhibit the enzymic activity.Secondly,the cultured conditions of yeast transformants,such as pH,temperature,concentration of methanol and heme for the influence of enzymic activity were explored by a response surface methodology（RSM）.The results showed that the enzymic activity of rGlMnP reached to 764.3U/L under the condition of optimal conditions（original pH 5.7,31℃,1.4%methanl and 1.0 mM heme）.Thirdly,four dyes,Drimaren Blue CL-BR,Drimaren Yellow X-8GN,Drimaren Red K-4Bl and Disperse Navy Blue HGL,were used for enzymatic decoloration treatment.The results showed that the decolorization rates of rGLMnP for the four dyes all reached to 70%in 90min,which indicated that rGlMnP has a high decolorizing ability.Finally,the crude enzymic solution containing rGlMnP was employed for breaking the Ganoderma lucidum spores（GLS）.The morphological change of the GLS was observed and evaluated by a microscope,the content of polysaccharides and triterpenoids in the treated/untreated GLS products was determined by spectrophotometric method.The results demonstrated that the effect of the sporoderm-broken GLS treated by MnP is better than untreated.The content of polysaccharides and triterpenoids in the sporoderm-broken of GLS products treated by rGlMnP increased by14.68%and 14.22%respectively,comparing with the sporoderm-broken of GLS products untreated by rGlMnP.The results demonstrated that rGlMnP has a significant effect on breaking GLS.