Cloning and expression of endo-1 , 4 – β-glucanase gene from Bacillus licheniformis ATCC 14580 into Escherichia coli BL 21 ( DE 3 )

An endo-1,4-β-glucanase gene from moderate thermophilic strain Bacillus licheniformis ATCC 14580 was cloned into Escherichia coli DH5α using pTZ57R/T as cloning vector. After confirmation of clone through polymerase chain reaction (PCR) and restriction analysis, endoglucanase gene (1.5 kb) was further expressed in E. coli BL21 (DE 3) using pET-22b (+) expression vector. The molecular weight of endoglucanase gene was found to be 52.2 kDa as determined by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). The addition of isopropyl-β-D-thio-galactoside (IPTG) as an inducer in the fermentation medium, caused an increase in expression of recombinant endoglucanase. The activity of enzyme in optimized condition was found to be 1.5 U/ml using carboxymethylcellulose as substrate. The optimum pH and the temperature of enzyme were also determined and found to be 6.0 and 60°C, respectively.