The Release Expression Of β-Mannanase Of Trichoderma Reesei Rut-C30 In E.coli

In this study, the target DNA fragment of ManI gene was purified from the recombinant plasmid pGEM-ManΙ after digesting by restriction enzyme EcoRI Mung Bean Nuclease,HindⅢ, and then cloned into secretion expression vector pT002 containing Ω sequence, T7 promoter and signature sequence OmpT after digesting by restriction enzyme BamHΙ,Mung Bean Nuclease,HindⅢ. The ManI gene was fused in-frame after the signature sequence ompT in the resulting plasmid pT002-ManΙ. The positive clones were screened in E.coli BL21 transformed by the recombinant plasmid and then subjected to sequencing after identification by PCR. The sequence of cloned ManI gene are completely identical to corresponding cDNA sequences of Trichoderma Reesei Rut- C30 of β-Mannanase mature peptides. The secretion expression system was established with E.coli BL21 co-transformed by pT002-ManΙ and pUC18-kil according to the principle kil can enhance the permeabilization of cell membrane and promote the secretion of periplasmic protein. The fusion β-Mannanase with molecular weights of about 51 kD as indicated by SDS-PAGE analysis was expressed with high-efficency after inducing by IPTG. The activity of β-Mannanasein in the concentrated medium was determined with locost bean gum as substract by 3,5-dinitosalicylic acid method, and the result indicated that the purified product showed apparentβ-Mannanase activity and the recombinant β-Mannanase was correctly processed during the secretion.