Mode of action, kinetic properties and physicochemical characterization of two different domains of a bifunctional (1-÷4)-fI-D-xylanase from Ruminococcus flavefaciens expressed separately in Escherichia coli

Two catalytic domains, A and C, of xylanase A (XYLA) from Ruminococcusflavefaciens were expressed separately as truncated gene products from lacZ fusions in Escherichia coli. The fusion products, referred to respectively as XYLA-Al and XYLA-C2, were purified to homogeneity by anion-exchange chromatography and chromatofocusing. XYLA-A1 was isoelectric at pH 5.0 and had a molecular mass of 30 kDa, whereas XYLA-C2 had a pl of 5.4 and a molecular mass of 44 kDa. The catalytic activity shown by both domains was optimal at 50 °C, but XYLA-Al was more sensitive than XYLA-C2 to temperatures higher than the optimum. XYLA-Al showed a higher sensitivity to pH than XYLA-C2. The enzyme activity of both domains was completely inactivated in the presence of copper or silver ions