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 gene fragment to the 5'-end of the Cellulomonas fimi cenA. A polyhistidine-encoding sequence was also fused to the 5'-end of the xyn gene. The gene fusion was overexpressed in Escherichia coli and the fusion polypeptide 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 substrates or xylans. The presence of two distinct active sites and the internal cellulose-binding domain did not significantly affect the hydrolysis of any of these substrates. However, the fusion protein exhibited a strong affinity for both microcrystalline cellulose (Avicel) and regenerated chitin. Like the parental endoglucanase, bound XynCenA could not be eluted from these polysaccharides with either low or high salt buffer 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.
Tomme
P
P
Department of Microbiology, University of British Columbia, Vancouver, Canada.
Gilkes
N R
NR
Miller
R C
RC
Warren
A J
AJ
Kilburn
D G
DG
D000327:Adsorption; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics; D002482:Cellulose / Q000378:metabolism*; D002686:Chitin / Q000378:metabolism; D002846:Chromatography, Affinity; D004926:Escherichia coli; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D043325:Xylan Endo-1,3-beta-Xylosidase; D014990:Xylans / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D014990:Xylans; D002686:Chitin; D002482:Cellulose; D006026:Glycoside Hydrolases; D043325:Xylan Endo-1,3-beta-Xylosidase; D002480:Cellulase