Gene Cloning, Expression And Characterization Of Cellulases From Neosartorya Fischeri P1

Cellulases and hemicellulases are impartant to the transformation of the lignocellulose --the earth’s most widely distributed and abundant renewable resources. Because of its own characteristics, thermophilic cellulases and hemicellulases are better used in a variety of industries, such as feed, paper pulp, foods and biomass energy.In this paper, on the one hand, we excavated a series of thermophilic cellulases and hemicellulases from the secretion of thermophilic fungi N.fischeri P1, which were also successfully overexpressed by pichia expression system to be used efficiently for the industrial application; on the other hand, we modified the appropriate gene of thermophilic cellulases to improve the pH stability or thermal stability in order to meet the industrial demand better.Through specific primers, we obtained a series of different genes of thermophilic enzyme from the cDNA of thermophilic fungi N.fischeri P1, which were successfully and efficiently expressed in pichia system, including a GH5 family endoglucanase Egl5A, a GH7 family cellulase Cel7A, a GH12 family endoglucanase Eg112A and a GH12 family xyloglucanase Xyg12A. In addition, using GH5 family endoglucanase Eg15A as the material, we obtained four single point mutants Q177E, Q261E, Q288E and Q333E by molecular modification, which could significantly improve the pH stability and thermal stability of their wild type.This thesis not only provides several industrial candidate enzyme of different families of thermophilic cellulase and hemicellulase, but also proves some possible sites related to the pH stability and thermal stability of Eg15A by experiment, which provides a good research material and theoretical guidance for the further study of the mechanism of stability of cellulases from GH5 family.