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

Abstract

The gene encoding the family 6 carbohydrate-binding module (CtCBM6A) 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 CtCBM6A showed molecular size of approximately 15 kDa. Ligand-binding analysis of CtCBM6A with rye arabinoxylan and oat spelt xylan by affinity gel electrophoresis showed low affinity for these ligands (Ka of 40 and 26 liter/g, respectively), and analysis by fluorescence spectroscopy (Ka of 33 and 15 liter/g, respectively) corroborated lower binding affinity with the above soluble ligands. However, CtCBM6A displayed significantly higher ligand-binding affinity with insoluble wheat arabinoxylan with equilibrium association constant Ka of 230 M(-1) and binding capacity (N0) of 11 µmole/g. The protein melting curve of CtCBM6A displayed a peak shift from 53 to 58°C in the presence of Ca2+, indicating that Ca2+ imparts thermal stability to the CtCBM6A structure. Homology modeling of CtCBM6A revealed a characteristic β-sandwich core structure. The Ramachandran plot of CtCBM6A showed 89% of the residues in the most favorable region, 10% in additionally favored region, and 1% in generously allowed region, indicating that CtCBM6A has a stable conformation.