Gene Cloning And Expression Of Endoglucanase From Trichoderma Viride

Cellulase system consists essentially of three types of enzymes:(1) exo-1,4-β-D-glucanases,also called cellobiohydrolases(CBH),(2) endo-1,4-,β-D-glucanases(EG),and(3)1,4-β-D-glucosidases (BG).Different cellulolytic enzymes cooperate in decomposing cellulose.Most of filamentous fungi produces cellulase,especially the Trichoderma spp which produces endoglucanases and cellobiohydrolases in high level,and Aspergillus which produces 1,4-β-D-glucosidases in high level.The cellulase formation can be induced by cellulose,lactose, cellobiose,sophorose,L-Sorbose et al.L-Sorbose has previously been assumed to stimulate cellulase formation in an indirect manner.In this work,we induced the cellulase formation with L-Sorobse in Trichoderma viride AS3.3711.after induced for 9-12 huors,when the mRNA of egl1 and egl3 transcription culminates, we collected the mycelia through filtration and washed them twice with 0.9%NaCl,then the mycelia were used to extract total RNA,and the cDNA was obtained by reverse transcription.The cDNA clones encoding endoglucanaseⅠand endoglucanaseⅢwhoes signal peptide were deleted were isolated by PCR protocol. then constructed recombined plasmids pET-egl1 and pSE-egl3 to be expressed in E.coli.the results of analysing with SDS-PAGE were that Protein bands of 46kDa and 42kDa were present as expressed products of pET-egl1 and pSE-egl3 transformations,the enzyme activity of EGⅢculminates 0.04U/mL.The egl1 with signal peptide was isolated from cDNA by PCR protocol,and then it was cloned into a yeast vector to construct a cellulases-producing Saccharomyces cerevisiae engineering strains with stable heredity.Recombinant yeast strains can secrete EGI by the guide of the signal peptide itself.So the transcription and expression of EGI can be screened by activity plate assays with Congo Red method respectively.The endoglucanase activity was assayed as CMCase activity with CMC-Na as a substrate.The results showed that The optimal reaction conditions of the enzyme were 47℃and pH5.2,the enzyme activity of EGI culminates 0.08U/mL when induced for 70h.