The Construction Of Cellulase Gene In Bacillus Subtilis And Characterization Of Recombinant Enzyme

Cellulases, a multicomponent enzyme system? could degrade cellulose into oligosaccharide and cellose, hydrolying glucose enventually. Cellulases play the very important role in the treat process of animal feeds and environment. Moderate amount of cellulases in animal feeds could hydrolyse cellobiose into monosaccharide glucoses finally, which are beneficial for the absorption and transformation of animals. By hydrolyzing the cellobioses to glucose in crop straws and feces, cellulases could abate the burden of environment pressure and simultaneously produce considerable number of fertilizers.In order to obtain the cellulose genes with high efficient enzyme activity, The An-BglI、Sf-BglI、Tv-EgⅦ、Tv-EgⅦ-S genes had been cloned into Bacillus subtilis.The main results as follows:1. basing on An-BglI、Sf-BglI、Tv-EgⅦ、Tv-EgⅦ-S and shuttle plasmid of pMA5, Sf-BglI-pMA5、An-BglI-pMA5、Tv-EgⅦ-pMA5and Tv-EgⅦ-S-pMA5had been constructed.2. An-BglI-pMA5、Tv-EgⅦ-pMA5、Tv-EgⅦ-S-pMA5are transformed into Bsl68by electroporation. Three engineered bacteria had been obtained, Which were Bsl68-An-BglI-pMA5, Bsl68-Tv-EgⅦ-pMA5, Bs168-Tv-EgⅦ-S-pMA5.3. The recombinant of Bs168-An-BglI-pMA5and Bsl68-Tv-EgⅦ-pMA5had well expressed by the Congo red method and SDS-PAGE method; Protein molecular weight are90.3kDa and26.8kDa, but the recombinant of Bs168-Tv-EgⅦ-S-pMA5has no or less efficient enzyme activity of cellulase in Bacillus subtilis.4. We used the whatman No.l filter paper to determine the filter paper activity (FPA) of cellulose enzyme Engineering bacteria.The results showed that the enzyme activity in the extracellular supernatant of Bs168-Tv-EgⅦ-pMA5was0.273U/mL, Bsl68-An-BglI-pMA5was0.049U/mL. The CMC-Na enzymatic activity of Bsl68-Tv-EgⅦ-pMA5was0.131U/mL. The enzyme activi ty of Bs168in the filter paper test was0.002U/mL, but the enzyme activity of CMC-Na and pNPG could barely be detected. 5. The optimum culturecondition of engineering bacteria had been demonstrated. To Bs168-Tv-EgVII-pMA5, The range of pH was from5to7and the most suitable pH was5.6, while the range of proper temperature from35to70℃and the most suitable was60℃, the enzymatic activity of endonuclease could maintain at least65%of the highest enzyme activity; To Bs168-An-BglI-pMA5, the range of pH was from5to7and the most suitable pH was5, while the range of the proper temperature was from50to80℃and the most suitable was70℃, the enzymatic activity could maintain at least70%of the highest enzyme activity.Therefore, both two constructed bacterial strainscould express relative high enzyme activity, which provide credible theoretical evidences for the construction of multifunctional engineering bacteria of Bacillus subtilis.