The family 6 carbohydrate-binding module (CtCBM6B) of Clostridium thermocellum alpha-L-arabinofuranosidase binds xylans and thermally stabilized by Ca2+ ions

Abstract

The gene encoding CtCBM6B of Clostridium thermocellum α-L-arabinofuranosidase (Ct43Araf) was cloned in pET-21a(+) vector, over-expressed using Escherichia coli BL-21(DE3) cells and purified by immobilized metal-ion affinity chromatography (IMAC). The recombinant CtCBM6B showed a molecular size close to 15 kDa by SDS-PAGE analysis, which was close to the expected size of 14.74 kDa. The ligand-binding affinity of CtCBM6B was assessed against ligands for which the catalytic enzyme, Ct43Araf showed maximum activity. The affinity-gel electrophoresis of CtCBM6B with rye arabinoxylan showed lower equilibrium association constant (Ka, 4.0% C− 1), whereas, it exhibited higher affinity (Ka, 19.6% C− 1) with oat spelt xylan. The ligand-binding analysis of CtCBM6B by fluorescence spectroscopy also revealed similar results with low Ka (3.26% C− 1) with rye arabinoxylan and higher affinity for oat spelt xylan (Ka, 17.9% C− 1) which was corroborated by greater blue-shift in case of oat spelt xylan binding. The CtCBM6B binding with insoluble wheat arabinoxylan by adsorption isotherm analysis showed significant binding affinity as reflected by the equilibrium association constant (Ka), 9.4 × 103 M− 1. The qualitative analysis by SDS-PAGE also corroborated the CtCBM6B binding with insoluble wheat arabinoxylan. The protein-melting curve of CtCBM6B displayed the peak shift from 53°C to 59°C in the presence of Ca2+ ions indicating that Ca2+ ions impart thermal stability to the CtCBM6B structure.