pET-21a(+)

β-Xylosidase is an important industrial enzyme that has a synergistic effect with xylanase and can hydrolyze hemicellulose and xylan to the greatest extent. Beta-xylosidase plays an important role in breaking down xylo-oligosaccharides (the building blocks of xylan) into xylose. In this study, researchers cloned a β-xylosidase from Bacillus licheniformis and successfully expressed it in Escherichia coli for saccharification of plant biomass. By using this method, plant biomass, which mainly constitutes cellulose, can be converted into reducing sugars, which can then be easily converted into bioethanol through a simple fermentation process. The production of bioethanol will help overcome energy requirements due to fossil fuel consumption and will also help protect the environment by reducing greenhouse gas emissions.

Here, researchers cloned the β-xylosidase (xynB) gene from Bacillus licheniformis and expressed the cloned gene in Escherichia coli strain BL21(DE3) using the expression vector pET-21a(+). Finally, the saccharification potential of the cloned β-xylosidase gene on plant biomass was also determined.

The expression of the recombinant xynB gene was analyzed by SDS-PAGE (Figure 1). Different types of controls: cell lysate of wild-type E. coli BL21 (DE3), cell lysate of E. coli BL21 (DE3) containing only pET-21a(+) vector without any insert and cell lysate of recombinant E. coli BL21 (DE3) containing non-induced pET-21a(+) with gene of insert were run in parallel in order to investigate the expression of cloned xynB gene (Fig. 1). An obvious band of 52 kDa was observed in the cell lysate of IPTG-induced recombinant E. coli BL21 (DE3), while no band was observed in all controls, indicating that the cloned β-xylosidase gene was successfully expressed.

Figure 1. SDS-PAGE analysis of the expression of cloned β-xylosidase gene. Lane 1 to 5 represents: protein marker, extract of wild E. coli, pET 21a(+) vector only, non-induced vector plus β-xylosidase gene and induced vector plus β-xylosidase gene. (Aftab, Muhammad N., et al., 2017)