Molecular cloning and characterizing of Bacillus subtilis cellulase collected from central-northern Iran forests

Abstract

In the large-scale commercial bioconversion of complex lignocellulosic materials to biofuels, a cost-effective purification and production of cellulase is a huge concern. In this regard, genetically engineered strains have shown great merits for producing active cellulase. As an attempt to improve cellulase production; herein, bacterial strains with cellulolytic activity were collected from forest soils located across Mazandaran Province, in central-northern Iran. To verify cellulolytic activity, carboxymethyl cellulose (CMC) in aka cellulose gum medium was used. Afterward, in order to characterize the bacteria with most cellulolytic activity, 16S rDNA of bacterial colonies were amplified and then phylogenetic analysis was conducted. Cellulase gene was isolated from the most active bacterial strain, Bacillus subtilis B2 and then it was cloned into the bacterial expression vector pET-26b using HindIII and BamHI restriction endonuclease sites. The generated plasmid containing cellulase and expression promoter system was transformed into the BL21 (DE3) competent Escherichia coli. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed to verify presence of the protein. The sequencing of cloned gene revealed 77% identity with endoglucanase of Bacillus subtilis subsp. Moreover, the molecular weight of cloned cellulase was 48 k Da as revealed by SDS-PAGE. Significance statementHerein, a newly isolated bacterial strain with high potential of cellulase production is introduced that benefits commercial bioconversion of complex lignocellulosic materials to biofuels.