Molecule Evolution And Characterization Of Thermostable Cellulases From Thermophilic Fungi

In the context of energy shortages and the gradual depletion of the resource, countries inthe world all to seek effective development and utilization of biomass, cellulose is the mostabundant biomass on the earth，widely spreading as one of the cheapest renewal resources, butmost of it become cellulose waste. Almost all of the cellulose waste, such as crop residual,used paper and so on, are merely burned or deserted without treatment in China, which causesmany environmental problems and several billion yuan economic losses every year. Thecellulose waste is difficult to be used because it’s a tough molecule structure to break down,but it can be converted into small molecule cellulose, cellobiose, glucose which easily to befurther fermentation by cellulase. Take full advantage of the cellulase provide an importantway to solve the food and the environment problems, the energy crisis. Cellulase refers to aclass of enzymes produced chiefly by fungi, bacteria, and protozoans.Although the cellulase has been widely used as an important industrial enzyme, it shouldbe improved in enzyme activity, thermostability, pH stability to meet the industry need.Thermophiles are main sources of cellulase with high thermostability. Cellulase from thethermophilic fungi have been reported to be stable and highly active at high temperature, likemescophilic fungi, thermophilic fungi can produce multiple forms of the cellulasecomponents, mostly. Thermoascus aurantiacus var. levisporus is widespread thermophilicfungi. Several thermostable enzymes have been isolated from the thermophilic fungi. Currentstudy shows that the thermophilic fungi’s cellulase have multiple components and have highactivity and thermostability, which makes the thermophilic fungi’s cellulase have greatresearch and industry value.In this study, an endo-β-glucanase encoding gene, eg1（GenBank accession number:AY847014） and glycoside hydrolases encoding gene61f have been isolated fromT.aurantiacus var.levisporus and expressed in pichia pastoris. The high expression efficiencystrains named GpN24and GS-TA-61F, which were gotten through screening. GpN24canexpress recombinant endo-β-glucanase with excellent thermostability， GS-TA-61F canexpress recombinant glycoside hydrolases with excellent thermostability.The molecular of the GpN24was33kDa, the optimum temperature and pH of therecombinant enzyme activity were55℃and5.0respectively, the recombinant enzymeremained50％of its original activity after30min at90℃and the activity of the recombinant enzyme can remain stable in pH between3.0and5.0;The molecular of theGS-TA-61F was29kDa, the optimum temperature and pH of the recombinant enzyme activitywere65℃and5.0respectively, the recombinant enzyme remained50％of its originalactivity after40min at75℃. In order to study glycoside hydrolases61F on the role ofendo-β-glucanase N24，in this study, characteristics of pure protein of two strains, the resultsshow that,61F can promote the enzymatic activity of N24, the enzyme mixture showed3.89fold enzyme activity compare to N24. Differents of the metal icons shows promotion orsuppression to61F and61F+N24, effect of Mn2+,61F and61F+N24showed20fold and1.2fold enzyme activity.Direct evolution based on eg1’s error-prone PCR and high throughput screening forhigher activity in pichia pastoris was used to enhance activity and stability of theendo--β-glucanase from T.aurantiacus var. levisporus and the expression efficiency of itsencoding gene eg1. A mutant library of eg1was constructed by error-prone PCR. In thisresearch, multiple copies of screening and small amount ferment in1.5mL centrifuge tubeafter congo red substrate plate were used to screen for higher enzyme activity. Throughmutagenesis and screening, a mutant showed2.27fold enzyme activity and optimumtemperature changing from55℃to65℃, optimum pH changing from5.0to6.0. The resultof alignment of the mutant’s and the wild type amino acid sequences showed that the wildtype’s9amino acid S was replaced with P,and the wild type’s204amino acid D changed to V,and wild type’s295amino acid Q mutate to R. The mutant site204belongs to conserved sites.Both the mutant endo-β-glucanase and wild type endo-β-glucanase were purified by fractionalammonium sulphate precipitation, ion exchange chromatography on DEAE-Sepharose. Thepreliminary mechanism was studied by comparing the two purified endo-β-glucanases.