Construction Of Protein Expression System In Chaetomium Ther Mophilus And The Properties Of Polysaccharide Monooxygenase

Filamentous fungi are widely used in the production of industrial enzymes because of their strong protein secretion and processing capabilities.With the development of molecular biology and genetic engineering,more and more people are focusing on the use of filament ous fungi to produce recombinant proteins.However,a single strain cannot meet the needs of various industrial protein production,so more filamentous fungi have been added to the ranks of protein expression host bacteria.Chaetomium thermophilum is a saprophytic fungus,which has a wide distribution range,can normally grow and reproduce in the range of 45 ° C ~ 50 ° C,and has the ability to produce a variety of heat-resistant cellulose hydrolases.,Is a potential industrial cellulose production strain.Compared with mesophilic fungi,the cellulase produced by thermophilic fungi has high activity and thermal stability,and is more abundant.Polysaccharide monooxygenases(PMOs)are a class of cellulose-degrading enzymes newly discovered in recent years.They have attracted much attention because of their unique mechanism of oxidative degradation of cellulose.This study relied on the good genomics and proteomics research resources of Chaetomium thermophilus,and carried out research from the transformation of the vector and the induced expression of proteins.The AA9(Auxiliary Activity 9,AA9)family The PMO enzyme gene PMO1 was used as the target.The enzyme was successfully expressed using the thermophilic protein expression system for the first time.The oxidative activity and enzymatic hydrolysis products of the enzyme were studied,and the thermophilic protein Expression system.The proteome sequencing analysis of the extracellular protein of C.thermophilus found that two proteins with high expression levels under CCM culture conditions were screened,among which CTHT-0002240(glucoamylase gla)had the highest expression level,which can be Maltose was significantly induced;CTHT-0031000(cellobiose hydrolase cbh)was the second most expressed,and it was significantly induced by lactose.The 1000-1500 bp sequences upstream of its gene were amplified as inducible promoters that regulate PMO1 expression,respectively,forming the Pgla-PMO1-Tgpd and Pcbh2-PMO1-Tgpd expression cassettes,and constructing the Ppmo1-PMO1-Tgpd and the constitutive promoter Pactin's Pactin-PMO1-Tgpd expression cassette for endogenous expression of C.thermophilus.Four expression cassettes and pNK54 plasmid were used to PEG-mediated transformation of C.thermophilus,and PMO1-positive transformed strains were obtained.Subsequently,the inducible promoter was fermented with 2% maltose,lactose,and microcrystalline cellulose,the constitutive promoter was fermented with CCM,and the fermentation supernatant was detected by Western Blot and SDS-PAGE 7 days after fermentation.The results showed that the inducible recombinant proteins Pgla-PMO1,Pcbh2-PMO1 and Ppmo1-PMO1 were successfully expressed and secreted,while the constitutive recombinant protein Pactin-PMO1was not expressed.Then the protein was purified with His-tag magnetic beads.SDS-PAGE showed that the protein size was 26KD.The results showed that the enzyme was correctly expressed,which was consistent with the theoretical molecular weight.Among them,the secretion level of Pgla-PMO1-Tgpd fusion protein induced by maltose was 0.54 mg/L,the secretion level of Pcbh2-PMO1-Tgpd fusion protein induced by lactose was 0.32 mg/L,and Pactin-PMO1 induced by microcrystalline cellulose-Tgpd secretion level was 0.12mg/L.The results showed that maltose-induced Pgla had the highest start-up efficiency in the thermophilic protein expression system.The purified PMO1 enzyme was tested with phosphate-expanded cellulose as the substrate to determine its enzymatic activity and composition of degradation products.TLC results showed that PMO1 had the highest enzymatic activity at pH 5.0,produced the most enzymatic products,and had good tolerance between pH 3 and 7;the reducing sugar concentration was highest when the temperature was 50 ° C This temperature is consistent with the optimal temperature for the growth of thermophilic capsid fungus,which is its optimal reaction temperature.When the temperature is increased to 60 ° C and 70 ° C,a small amount of reducing sugar is also produced,indicating the use of thermophilic capsid protein The PMO1 enzyme expressed by the expression system also has some heat resistance.With the increase of reaction time,the concentration of the product also increased,and the composition of the product was cellobiose to cellopentaose,and only under the condition of vitamin C as a reducing agent,PMO1 can exert the activity of oxidizing and lysing cellulose,which proved that Polysaccharide monooxygenases are a type of oxidase.