AN INTERNAL CELLULOSE-BINDING DOMAIN MEDIATES ADSORPTION OF AN ENGINEERED BIFUNCTIONAL XYLANASE CELLULASE

A chimeric xylanase/endoglucanase (XynCenA) with an internal cellulose -binding domain was constructed by fusing the Bacillus subtilis xyn ge ne fragment to the 5'-end of the Cellulomonas fimi cenA. A polyhistidi ne-encoding sequence was also fused to the 5'-end of the xyn gene. The gene fusion was overexpressed in Escherichia coli and the fusion poly peptide purified from the cell extracts using the polyhistidine tail. The hybrid protein behaved like the parental endoglucanase or xylanase when assayed on a number of soluble and insoluble cellulosic substrat es or xylans. The presence of two distinct active sites and the intern al cellulose-binding domain did not significantly affect the hydrolysi s of any of these substrates. However, the fusion protein exhibited a strong affinity for both microcrystalline cellulose (Avicel) and regen erated chitin. Like the parental endoglucanase, bound XynCenA could no t be eluted from these polysaccharides with either low or high salt bu ffer or distilled water. More stringent conditions, such as 1% SDS or 8 M guanidinium hydrochloride, fully desorbed the protein. The fusion protein did not adsorb significantly to insoluble xylan.