A comparative study between an endoglucanase IV and its fused protein complex Cel5-CBM6.
The recombinant endoglucanase IV (Cel5; encoded by egIV) of Ruminococcus albus was compared with protein Cel5-CBM6 comprised of Cel5 fused at the C-terminus with the single-cellulose binding domain II (CBM6) of Clostridium stercorarium xylanase A, in order to improve its binding ability. Previous analyses using ball-milled cellulose had suggested that a cellulose binding domain of xylanase A could enhance cellulase activity, especially with insoluble substrates. Comparison of the catalytic activities of Cel5 and Cel5-CBM6 were determined using carboxymethylcellulose, Avicel, and filter paper as substrates. This study confirmed previous findings, and provided further evidence suggesting that Cel5-CBM6 exhibits enhanced activity with insoluble cellulose compared to native Cel5. However, its hydrolytic activity with soluble substrates such as carboxymethylcellulose was comparable to Cel5. For both cellulases, central linkages of cellulooligosaccharides (up to six glucose residues) were found to be the preferred points of cleavage. The rates of hydrolysis with both cellulases increased with cellulooligosaccharide chain length, and at least three consecutive glycosyl residues seemed to be necessary for hydrolysis to occur. Cel5-CBM6 showed a higher affinity for cellulose substrates than did Cel5, as demonstrated by transmission electron microscopy. Taken together, these results suggest that CBM6 increases the affinity of Cel5 for insoluble substrates, and this increased binding capacity seems to result in increased catalytic activity.
Bae
Hyeun-Jong
HJ
Département de Phytologie, Université Laval, G1K 7P4, Sainte-Foy, QC, Canada. baeyeunjong@hotmail.com
Turcotte
Ginette
G
Chamberland
Hélène
H
Karita
Shuichi
S
Vézina
Louis-Philippe
LP
D001665:Binding Sites; D002384:Catalysis; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D006046:Gold / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D045857:Ruminococcus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D011993:Recombinant Fusion Proteins; D006046:Gold; D002482:Cellulose; D002480:Cellulase