Molecule Evolution And Characterization Of Thermostable Cellulases From Thermophilic Fungi

In the context of energy shortages and the gradual depletion of the resource, countries are actively looking for effective development and utilization of biomass. Cellulose is the most abundant and renewable biomass on the earth. But people did not make full use of them. A lot of cellulose was discarded as rubbish. The unsuited treatment causes many environmental problems and huge economic losses every year, and it also violate the concept of sustainable development.Because of the low enzyme activity, thermostability, and pH stability, cellulase could not reach the standards of industrial production, so it is hard to meet the demands. Cellulase from the thermophilic fungi have been reported to be stable and highly active at high temperature. Thermoascus aurantiacus var. Levisporus is a widespread thermophilic fungus. Several thermostable enzymes have been isolated from the thermophilic fungi. Cellulase of thermophilic fungi have great research and industry value.In this study, an endo-β-glucanase encoding gene, egl (GenBank accession number: AY847014) has been isolated from T.aurantiacus var.levisporus and expressed in Pichia pastoris. The high expression efficiency strains were named GpN25, which were gotten through screening. GpN25can express recombinant endo-β-glucanase with excellent thermostability. The molecular of the GpN25was33kDa, the optimum temperature and pH of the recombinant enzyme activity were55℃and5.0respectively. The recombinant enzyme remained50%of its original activity after30min at90℃and the activity of the recombinant enzyme can remain stable in pH between3.0and5.0.In order to enhance the activity, we use the error-prone PCR to construct the mutant library. By using the method of High-throughput screening, finally we obtained two mutants. We have named them NT0252and NT0253. The Enzyme activity of NT0252was increased by2.19times.The other one was increased by1.88times. The result of alignment of the NT0252’s and the wild type amino acid sequences showed that the wild type’s52amino acid F was replaced with E, and the wild type’s91amino acid D changed to N, and wild type’s170amino acid W mutate to H. The result of alignment of the NT0253’s and the wild type amino acid sequences showed that the wild type’s14amino acid A was replaced with G, and the wild type’s198amino acid H changed to M, and wild type’s224amino acid S mutate to C.Both the mutants NT0252and NT0253were purified by fractional ammonium sulphate precipitation, ion exchange chromatography on DEAE-Sepharose. The preliminary mechanism was studied by comparing the two purified endo-β-glucanases.The results show that:the optimum temperature of NT0252changes from50℃to60℃. The other one changes from50℃to60℃. It shows that NT0252and NT0253can adapt to higher temperature. The optimum pH of NT0252changes from4to5. NT0253changes from4to6. It shows that both NT0252and NT0253have high Enzyme activity in a wide range of pH.