Structure and Engineering of Clostridium thermocellum Cel5E, a Bifunctional Endoglucanase/Xylanase
Date Issued
2012
Date
2012
Author(s)
Yuan, Shuo-Fu
Abstract
Cellulose and hemicellulose are sources of biofuel production. Their degradation into monosaccharide requires action of cellulase (endoglucanase, exoglucanase, and β-glucosidase) and hemicellulase. Cel5E from Clostridium thermocellum belongs to family 5 of the glycoside hydrolases. It shows activity against both cellulose and hemicellulose (xylan), suggesting it is an unusual bifunctional β-1,4 endoglucanase/β-1,4 xylanase. The optimal conditions for endoglucanase and xylanase activities are 50°C, pH 5.0 and 60°C, pH 6.0, respectively.
By thin-layer chromatography (TLC) analysis and kinetic assays, CtCel5E displays a higher catalytic efficiency on hydrolysis of cellulose than on hydrolysis of xylan. In addition, some metal ions and chemical reagents such as Ca2+, Mg2+, Sr2+ as well as Dithiothreitol (DTT) can improve both endoglucanase and xylanase activities of CtCel5E. Glu209 and Glu314 were identified as the catalytic sites for its dual-activities. Besides, CtCel5E displays obvious synergistic effects with β-1,4 glucosidase, which can further degrade cellulolytic biomass into fermentable sugars. To my knowledge, there is no literature explained why family 5 endoglucanase can also hydrolyze heteroxylan. In order to investigate which amino residues are responsible for substrate specificity, CtCel5E was crystallized. Crystals of CtCel5E have been obtained using sitting drop method, and structure has been determined. To distinguish amino acids essential for catalysis, mutagenesis experiments were performed. By structures superimposition with TmCel5A (PDB: 3MMU), residues from Glu274 to Asp290 may be a shorter flexible loop and Tyr270 play crucial role in enzyme catalysis. Moreover, Arg128 is important for CtCel5E cellulase to function at pH 4.0.
Crystallization of ligand-bound CtCel5E is underway, and we believe it can provide detailed information for structure-based engineering, leading to potential biofuel industrial applications.
Subjects
cellulase
hemicellulase
biofuel
crystal structure
mutagenesis
Type
thesis
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