Expression Of Endoglucanase Gene From Bacillus Subtilis C-36 In Bacillus Megaterium

Cellulase is a group of complex phosphoesterasum which mainly comprised of endoglucanase, exoglucanase andβ-1,4-glucosidase. Cellulose enzymatic hydrolysis is considered to require the synergistic interaction of these enzymes. Recently, bacterial cellulase preparation had showing satisfactory application perforate and economic value. But the large scale industrialized application of cellulase was restricted by the low activity and high cost price. Improving the activity of cellulase by genetic engineering method is a efficient way to cut down the cost price.Based on the endoglucanase gene (Genbnk No. DQ782954 ) frome Bcillus subtilis C-36,a pair primer was designed to remove the codon sequence of signal peptide,Theendoglucanase gene fragment from the B. subtilis C-36 encoding matte enzyme was amplified by PCR with insertion of BglⅡand SphⅠsites at the 5' and 3' ends,respectively.The amplified PCR fragment was about 1.5Kb. The gene without the signal peptide was ligated with the expression plasmid pHIS1525.The recombinant plasmid was transformed into the protoplasts of Bacillus megaterium WH320 strains. The effective expression of the gene in the B.megaterium were proved by the way of Congo-red dyeing. SDS-PAGE showed that the molecular weight is about 55KDa.Farther more,the culture conditions of recombinant strain were studied.The results show that: the tulle volume is 50mL, the seed aga is 12h, the inoculation rate is 5%, the inducer concentration is 0.2%, and the induction time is 9 hours.Under the optimal culture condition, the supernatant enzyme activity of endoglucanase was up to 889U/mL which is about 11.22 times higher than that of the initial strain(79.2U/mL).The character of recombinant endoglucanase was studied in this experimentation.The results showed that: the optimum reaction pH and optimum reaction temperature is pH6.0 and 65℃,respectively. Enzyme stability experiment showed that: the residual activity was 70% and 80% after treatment at 40℃～75℃and at pH4.5～pH10.0, respectively.