Cloning,Expression And Characterization Of Polysaccharide Monooxygenases Gene From Therrmoascus Aurantiacus

Lignocellulose is the most abundant renewable resource on the earth.At present,we produce about 600 million scrap wood and straw in our country every year,but people usually use the way of burning straw,that made the straw can't be made full use of,this approach not only wasted resources,increased environmental pollution,but also increased atmospheric carbon dioxide content of the greenhouse effect intensified.The lignocellulosic biomass can be used to remove the chemical treatment method of lignin,the cellulose and hemicellulose components loose,lignocellulose can be various cellulose hydrolase,monomer sugar,and then fermented into ethanol.In the plant cell wall,hemicellulose and lignin comes with covalent bond,contains cellulose microfibril core,surrounded by external matrix layer hemicellulose,the structure further hindered the hydrolysis of cellulase and hemicellulase decomposing enzyme.Therefore,how to use high efficiency and low cost of lignocellulose is a major problem that we present facing.Thermoascus aurantiacus is a class of fungi that can survive in high temperature environments and grows at 40 to 50°C.The enzymes produced by thermophilic fungi also have high temperature,thermal stability and strong characteristics,therefore,in the future of industrial production and production applications have great prospects.Polysaccharide monooxygenases(PMOs)is a class of copper oxidases,which can directly cleave cellulose molecules in an oxidative mechanism in the presence of reduced cofactor(such as vitamin C).In this study,thermophilic ascomycetes were used as materials to extract the fungal hyphae DNA,and two PMOs protein sequences are known,which named PMO4983 and PMO8913.After analyzing the two sequences,we found that the two PMO protein sequences contained the signal peptide,so both proteins were extracellular secretory proteins.PMO4983 and PMO8913,respectively,encode 228 and 333 amino acids(without signal peptide),and the protein molecular weight predicted 24.33 KDa and 34.67 KDa,respectively.A specific primer was designed and amplified with DNA as a template.The PMO gene sequence was obtained,and the pPICZ?A-PMO expression vector was constructed by gene recombination.The recombinant plasmid pPICZ?A-PMO was transferred into Pichia pastoris GS115 by electroporation,and the antimicrobial resistance of the recombinant plasmid was screened,after screening the positive transformants with high copy number,the recombinant protein were obtained by fermentation and methanol induced secretion of the recombinant protein.Collecting the fermentation broth when fermenting is carried out on the seventh day,the protein is separated by ammonium sulfate precipitation and then dialyzed against PBSA buffer for two days,then centrifuged to remove the impurities to obtain the crude enzyme.The crude enzyme was eluted with HisTrapTM FF nickel column chromatography,and the protein was isolated and purified at the time of peak collection.The purity and molecular weight of the protein were determined by SDS-PAGE.The results showed that the average molecular weight of PMO4983 and PMO8913 were 40.0KDa and 60.1KDa,respectively.After further purification and identification of the recombinant protein,enzymological properties and oxidative detection were further performed.It was found that the two polysaccharides monooxygenase did not have the ability to hydrolyze cellulose with phosphoric acid expanded cellulose as the reaction substrate,but under the condition that ascorbic acid as a reducing agent,PMO4983 can oxidize cellulose to fibrous oligosaccharide.However,the oxidative activity of PMO8913 was not significant,therefore,the nature of PMO8913 remains to be further identified.In the next experiment,we demonstrated that PMO4983 not only has the properties of oxidative cleavage,but also has the ability to improve the hydrolytic activity of cellulase hydrolase.In the environment of divalent copper ions and vitamin C,PMO4983 was used to treat the substrate for 48 h,and it was found that PMO4983 could improve the hydrolytic activity of cellulose.At present,many articles have been published about polysaccharide monooxygenase.Most scholars believe that the oxidation of polysaccharide monooxygenase mainly acts on the C1 position of cellulose chain.With the deepening of research,the C4 oxidation of polysaccharide monooxygenase has also been found,but whether the natural oxidation site of polysaccharide monooxygenase is C4 oxidation or C6 oxidation is still controversial in academia.