Functional and structural characterization of family 6 carbohydrate-binding module (CtCBM6A) of Clostridium thermocellum α-L-arabinofuranosidase

The gene encoding the family 6 carbohydrate-binding module ( Ct CBM6A) from Clostridium thermocellum , cloned in pET-21a(+) expression vector, was overexpressed using Escherichia coli BL-21(DE3) cells and purified by immobilized metal-ion affinity chromatography. SDS-PAGE analysis of the recombinant Ct CBM6A showed molecular size of approximately 15 kDa. Ligand-binding analysis of Ct CBM6A with rye arabinoxylan and oat spelt xylan by affinity gel electrophoresis showed low affinity for these ligands ( K a of 40 and 26 liter/g, respectively), and analysis by fluorescence spectroscopy ( K a of 33 and 15 liter/g, respectively) corroborated lower binding affinity with the above soluble ligands. However, Ct CBM6A displayed significantly higher ligand-binding affinity with insoluble wheat arabinoxylan with equilibrium association constant K a of 230 M −1 and binding capacity ( N 0 ) of 11 μmole/g. The protein melting curve of Ct CBM6A displayed a peak shift from 53 to 58°C in the presence of Ca 2+ , indicating that Ca 2+ imparts thermal stability to the Ct CBM6A structure. Homology modeling of Ct CBM6A revealed a characteristic β-sandwich core structure. The Ramachandran plot of Ct CBM6A showed 89% of the residues in the most favorable region, 10% in additionally favored region, and 1% in generously allowed region, indicating that Ct CBM6A has a stable conformation.