Cloning of the High Alkaline Endoglucanase Gene from Bacillus pumilus AC-4 and Expressed in E.coli

The genomic DNA of Bacillus pumilus AC-4 strain, which were separated by our laboratory, was used as the DNA template. The β-1,4-endoglucanase gene fragment was amplified by PCR (Polymerase Chain Reaction). The PCR product was recovered and cloned into plasmid pMD-19T. The DNA sequence analysis showed that the length of the amplified fragment is 1898bp, which has 95% similarities to the β-1,4-endoglucanase gene sequence from other Bacillus strains. Sub-cloned the recombinant plasmid which containing β-1,4-endoglucanase and digested with BamHI and EcoRI. And then linked with the expression plasmid pGEX-4T-1 and transferred into the competent E. coli BL21 for expression. The result of protein electrophoresis showed that there has expressed protein whose molecular weight is about 70 kDa. Measured the enzyme activity of the expression protein of recombinant strains is 0.92 IU/mL, which is 1.52 times as the start strains.