Screening And Enzymatic Properties Of Ligninolytic Enzyme Producing Fungi

China's crop straw resources are abundant,due to the presence of lignocellulose in the straw,its utilization and degradation are difficult,the microorganisms can rely on the lignin-degrading enzyme system to completely degrade the lignin in the straw.The purpose of this thesis is to screen out highly efficient lignin-degrading strains,study and analyze the degradation characteristics of straws,solid-state fermentation conditions,laccase purification and enzyme properties.The result is as follows:1.39 strains of fungi isolated and purified,through primary screening and rescreening,two fungi with strong laccase and manganese peroxidase activity were obtained:EM03?Pholiota nameko?and EM07?Pleurotus eryngii?,they were used as the target strain for further experiments.2.For solid fermentation of EM03 and EM07,the production of laccase and manganese peroxidase are periodic,after 12 days of culture,the enzyme activity peaked,the maximum laccase and manganese peroxidase activities of EM03 are 37.58 Ug^-11 and 20.78 Ug^-1,respectively;The maximum activity of the two enzymes in EM07 are 36.16 Ug^-1and 8.97Ug^-1,respectively.For solid fermentation of EM03,60 days later,the loss rate of solid matrix is 49.43%,the degradation rate of hemicellulose is 57.75%,the degradation rate of cellulose is 46.03%,and the degradation rate of lignin is 74.45%.For solid fermentation of EM07,60days later,the loss rate of the solid matrix is 17.35%,the hemicellulose degradation rate is12.95%,the cellulose degradation rate is 9.70%,and the lignin degradation rate is 51.69%.Combined enzyme production and degradation experiments,it was found that EM03 had higher enzyme-producing ability and ability to degrade lignocellulose,it can be used as the target strain for further experiments.3.The factors affecting the solid-state fermentation of EM03 were analyzed and optimized through partial factor experiments,center combination experiments,and response surface analysis experiments.The results showed that three factors were the key factors affecting solid-state fermentation:CuSO₄ concentration,initial water content in culture medium,and culture temperature.Through optimization,the optimal fermentation conditions for the degradation of enzymes and lignocellulose produced by the strains under solid conditions were obtained.The optimization results can provide a theoretical basis for the setting of key factors in the solid-state fermentation process,and the cultivation temperature parameters in the solid state fermentation process,increasing the enzyme production and degradation efficiency,shortening the fermentation time,and lowering the fermentation cost.4.A laccase was isolated from solid state fermentation extract of EM03?pholiota nameko?by applying successively precipitation,ionexchange and gel filtration chromatography,designated Lac-B.The SDS-PAGE gave a single band indicating that Lac-B appears as a monomeric protein with a molecular mass of 50.2 k Da.Domain spectroscopic analysis revealed that Lac-B contained a typical copper II binding domain,combination of N-terminal amino acid sequences and spectroscopic features,speculated that the enzyme may be a yellow laccase.Lac-B showed maximal activity at 70?and high thermostability,it retained more than 100%of its original activity after 90 min incubation at 4-50?.In the acidic environment?pH 3.0-5.0?,the substrate has high reactivity and stability.Lac-B have a certain degree of tolerance to heavy metals,low concentrations of K⁺can be used as weak activators to increase laccase activity.NaN₃ and DTT have a strong inhibitory effect on enzyme activity,In the presence of the chelating agent EDTA,Lac-B activity is slightly inhibited.The K_m and V_maxax for Lac-B toward ABTS as a substrate were 0.32?M and 0.53mM/min,respectively,and for Lac-B toward DMP,they were respectively 0.43?M and 0.48mM/min.Inaddition,Lac-Bmanifestseffectivedecolorizingactivitytowards triphenylmethane,heterocyclic,and azo.The results show that Lac-B has better theoretical research value and application prospect in the treatment of acidic waste liquid and dye decolorization.