Expression Of Endoglucanase Gene From Bacillus Subtilis C-36 In Escherichia Coli

Cellulase is a group of complex phosphoesterasum which mainly comprised ofendoglucanase, exoglucanase andβ-1,4-glucosidase. Cellulose enzymatic hydrolysis isconsidered to require the synergistic interaction of these enzymes. Recently, bacterialcellulase preparation had showing satisfactory application performance and economicvalue. But the large scale industrialized application of cellulase was restricted by the lowactivity and high cost price. Improving the activity of cellulase by genetic engineeringmethod is a efficient way to cut down the cost price.According to the sequences of endoglucanase gene(GenBank AccessionNo.DQ782954) from Bacillus subtilis C-36, a pair of primers was designed to amplify thefull sequence. The KpnⅠsite was linked with the upstream primer, and the EcoRⅠsitewas linked the downstream primer which ended at stop codon. By using a high-fidelitypolyerase, an encoding region (1.5Kb) was amplified by PCR. After cut with KpnⅠ/EcoRⅠ, the fragment was ligated to E.coli expression vector pET-32a. The recombinantwas transformed into the E.coli BL21(DE3) strain. The positive colonies were screened onLB plate(100μg/mL amp added). The result of enzyme digestion of vector proved that therecombination vector(named pET-C36) was obtained. The tranformants on the selectiveplate(Amp, CMC-Na, IPTG added) were detected for CMCase by the Congo red method.The transformants were cultured in LB broth(100μg/mL amp added) and induced byImmol/L IPTG for 4～6h. The enzyme activity of the recombinant was 99.02 U/mL.SDS-PAGE showed that the molecular mass of recombinant protein was about 73KD.Farther more , the culture conditions of pET-C36 strain were studied. The resultsindicate that the recombinant E.coli was incubated at 43℃for 5h, and when inducing by0.1mmol/L IPTG or 0.1% lactose, its yield of endoglucanase can come to 141.22U/mLwhich is about 1.78 times higher than the activity of the initial strain. The optimumreaction temperature and pH for this enzyme activity were found to be 65℃and pH6.0,respectively. Moreover, the residual activity was over 70% after treatment at 30℃～75℃and pH4.5～pH10.0.