Analysis On Degradation And Enzymatic Properties Of Lignocellulose By Common Edible Fungi

Lignocellulosic biomass is one of the most abundant resources containing C and H elements on earth,which can be converted into clean renewable biofuels,providing continuous energy for the sustainable development of human society,and reducing dependence on fossil resources and environmental pollution.Lignocellulosic biomass has a complex structure.Hemicelluloses and lignin are connected by different types of hydrogen bonds and chemical bonds to form a protective shell similar to adhesive,which increases the difficulty of cellulose development and utilization.Further utilization of lignocellulosic biomass requires pretreatment.Edible fungi take lignocellulosic biomass as the main raw material and secrete a large number of extracellular enzymes to degrade lignocellulose into small molecules that can be absorbed and utilized during the growth process.Through the destruction of lignocellulosic structure by the growth of edible fungi,the pretreatment of lignocellulosic biomass under mild,low-cost and environmentally friendly conditions is a green and ideal new method in line with the sustainable development of society.Guizhou is one of the main production areas of Kiwifruit in China,kiwifruit harvest will produce a large number of abandoned branches of lignocellulosic biomass every year,and are currently treated by traditional incineration,resulting in environmental pollution and resource waste.Therefore,in this study,kiwifruit branches were used as raw materials to grow 10 kinds of common edible fungi.By comparing the growth of edible fungi mycelia,the ability to secrete lignocellulose-degrading enzymes and the change of lignocellulose-degrading content in the substrate,the excellent fungi that can effectively degrade lignin were screened out.Then,the liquid culture conditions of the strain were optimized and laccase protein was isolated and purified.Finally,the enzymatic kinetic properties of purified laccase were analyzed to provide a theoretical basis for the pretreatment of lignocellulosic biomass and the practical application of Laccase.The research includes the following three aspects:（1）The growth of mycelia,the ability to secrete degrading enzymes of lignocellulose and the changes of the main components of lignocellulose in kiwifruit branches were studied when 10 edible fungi such as Lentinus edodes,Pleurotus ostreatus and Pleurotus geesteranus were cultivated in kiwifruit branches.It was found that kiwifruit branches could be used as the cultivation substrates of 10 edible fungi,but there were differences in growth adaptability.There were differences in lignocellulose-degrading enzyme production among different edible fungi.The ones with the strongest ability to secrete laccase,manganese peroxidase,cellulase and hemicellulase were Pleurotus geesteranus,Pleurotus ostreatus and Ganoderma amboinense.Different edible fungi growth had certain selectivity for the degradation of lignin,cellulose and hemicellulose in kiwifruit branches.Pleurotus geesteranus was the strongest lignin-degrading strain,whose degradation rate reached 35.6%after 20 days of cultivation,while its degradation of cellulose and hemicellulose was poorer.Pleurotus ostreatus was the strongest cellulose-degrading strain with the rate reaching 48.3%after 25 days of cultivation,and Ganoderma amboinense was the strongest hemicellulose-degrading strain with the rate reaching 68.3%after 30days of cultivation.（2）The laccase production conditions of Pleurotus geesteranus was optimized by Response surface methodology,and the laccase protein was isolated and purified.It was found that Cu²⁺,Mn²⁺,gallic acid and guaiacol promoted laccase production in liquid culture,while Fe²⁺,Ca²⁺,Al³⁺and tannic acid inhibited.The optimal culture conditions were Cu²⁺concentration 0.50 mmol/L,Mn²⁺concentration 1.08 mmol/L,gallic acid concentration 1.04 mmol/L,under which the laccase activity was up to 7223.56 U/L,which was 4.76 times higher than that of the control group.By salting out,dialysis and ion chromatography,laccase protein with molecular weight of 69 KD was obtained,whose purification ratio was 11.91 and enzyme activity recovery was 28.94%.（3）The effects of temperature,p H and metal ions on the laccase activity of Pleurotus geesteranus were investigated.The optimal reaction temperature of laccase was 50℃,the optimal reaction p H was 4-5,and it had excellent stability at 20-40℃and p H 5-7.When K⁺concentration was lower than 0.625 mmol/L,it promoted laccase activity,while Ca²⁺,Cu²⁺,Mg²⁺,Fe²⁺,Fe³⁺,Zn²⁺,Al³⁺,Pb²⁺,Ni⁺,Co²⁺,Li⁺,Cd²⁺,Mn²⁺and Hg²⁺inhibited laccase activity,and Fe²⁺had the strongest inhibitory effect.In addition,the laccase had extensive substrate specificity with the highest affinity for ABTS,and lower K_m(4.64×10^-6mol/L)and V_max(5.57×10^-4mol/（L·min）).In conclusion,edible fungi can degrade lignin,which can be used for the pretreatment of lignocellulosic biomass.And Pleurotus geesteranus had the best pretreatment effect,which can effectively remove lignin and improve the yield of cellulose hydrolyzed sugar.At the same time,it also had a strong ability to produce laccase that had excellent physical and chemical properties such as enzymatic kinetics and substrate specificity,which can be used in commercial production.However,in practical applications,it is necessary to control temperature,p H and metal ions to avoid enzymes activity being affected.