Functional Study Of Glycoside Hydrolase Family 61 Gene Form Penicillium Piceum On The Degradation Of Plant Lignocellulose

As the main component of plant biomass,lignocellulose can be degraded to liquid fuels and chemical products such as bioethanol.It's not only helps to protect the environment and reduce oil consumption,but also supports to solve energy problems and extent agricultural industry chain of our country.Lignocellulose is abundant in the world,at present,the production and application of lignocellulosic enzymes mainly depends on exocrine enzyme system of filamentous fungi.However,due to various factors such as chemical composition and complex structure,the highly production cost of the degradation enzyme and lowly hydrolysis activity is crucial restrictions of effective utilization of lignocellulose.Therefore,It is still the bottleneck that efficient production of lignocellulosic hydrolytic enzyme system,and the synergetic protein of traditional cellulase becomes a research focus gradually.Glycoside hydrolase 61 family is a kind of protease widespread in fiber hydrolysis fungi that can improve the hydrolase of cellulose enzyme system to the substrate significantly and is the only oxidase family relying on Cu2+.The research showed that essence of the hydrolysis of GH61 family to lignocellulose is oxidation.At present,the study of GH61 family has become one of research hotspots in the field of cellulose enzyme.Penicillium piceum was isolated from compost waste,sequenced and analyzed by comparative genomics analysis.The consequence shows that genome size of P.piceum was25.27 Mbp,with 8591 predictive coding gene.The results of the functional annotation of KOG and GO indicated that the secretion and metabolic function of secondary metabolites of P.piceum were more developed.Cellulase and hemicellulase were annotated in CAZyme showing that P.piceum had balanced cellulase system and a lot of hemicellulase coding genes,which was very good for producing cellulase.In this thesis,Agrobacterium-mediated transformation for P.piceum was established.The optimized ATMT method as follows: concentration of spore suspension was 106spores/m L and optical value of agrobacterium was 0.6 mixing with equal volume,coculture on AIM media with 200?mol/L AS,temperature was 22?,time was 48 h.Experiments showed that the transformants had largest number in this condition and great genetic stability.The only gene of P.piceum belonging to glycoside hydrolase family 61 was deleted by ATMT in order to study its biological function.Deletion strain of GH61 had well-nigh same growth rate with original strain of P.piceum,simultaneously,extracellular proteinconcentration,cellulase activity and biomass also decreased slightly.Besides,after deleting GH61 gene,hydrolysis rate had declined in 1% air-blast corn straw substrate with equal FPA activity.After 72 h,hydrolysis rate of deletion strain was reduced by 17.5% compared with original.GH61 gene was cloned and then expressed in Aspergillus niger,obtained 2 recombinants.By comparing the enzyme activity of filter paper and ?-glucosidase with original strain of A.niger,results shows that FPase and glucosidase activity of recombinants strain both better than original strain.Beyond that,in 1% air-blast corn straw substrate of hydrolysis system added to extracellular enzyme liquid with equal FPA activity,hydrolysis rate of recombinants were increased by 9.8% and 12.1% respectively compared with original strain after 72 h.