Expression Of Secretory Fusion Cellulases In Periplasmic Space Of Escherichia Coli

Cellulase is a group of enzymes that catalyzes cellulose,which includes exoglucanase(EC 3.2.1.176,EC 3.2.1.91),endoglucanase(EC 3.2.1.4)and ?-glucosidase(EC 3.2.1.21).Cellulases are widely used,remarkablely,the application in biofuel production is of great significance to solve the shortage of global fossil resources.In this research,genetic engineering bacteria were constructed,which expressed secretory fusion cellulases.The target amino acid sequences of Endoglucanase EG originating from Thermobifidafusca YX and ?-glucosidase BG originating from Bacillus subtilis were obtained from GenBank.According to E.coli preference codon,the target genes were designed after the restriction sites of BamH? and Xho?were added at the upstream and downstream,respectively.The amplified products by PCR were digested with BamH? and Xho?and then cloned into pET-39b(+)and pET-39b(+)-sumo.Consequently,three engineering bacteria of expressing secretory fusion cellulases were successfully constructed,which were denominated as E.coli DS-EG,DS-BG and D-BG,respectively.Induced by IPTG,the proteins with biological activity were successfully expressed in periplasmic space of E.coli.The results analyzed by SDS-PAGE showed that the molecular weight of DS-EG,DS-BG and D-BG were about 80 kDa,90 kDa and 76 kDa,respectively.The activity of endoglucanase was assayed using CMC-Na as substrate.The results showed that the activity of DS-EG was 0.41 IU/mL,the optimum pH and temperature were 9.0 and 50?,respectively.The activity of ?-Glucosidase was assayed using pNPG as substrate.The results showed that the activity of DS-BG and D-BG were 8.35 IU/mL and 3.29 IU/mL,respectively.The optimum temperature of DS-BG and D-BG were both 40 ? and the optimum pH were 6.0 and 7.0,respectively.Compared with the two recombinant ?-glucosidase enzymes,we found the activity of DS-BG was higher than that of D-BG.Further study showed that the DS-EG and DS-BG acted synergistically,which synergisticed strongly at the condition of pH 7.0 and 50?.