Purification and characterization of a recombinant β-D-xylosidase from Thermobifida fusca TM51.

The subject of our investigations was a recombinant β-D-xylosidase (TfBXyl43) from Thermobifida fusca TM51 which was expressed in E. coli BL21DE3 and was purified to apparent homogeneity. The SDS-PAGE investigations demonstrated that the molecular weight of the monomer unit is 62.5 kDa but the native-PAGE studies indicated that the mass of the enzyme is 240-250 kDa which proves the presence of a characteristic homo oligomer quaternary structure in solution phase. Optimal parameters of the enzyme activity were at pH 6.0 and 50 °C. The enzyme showed little stability under pH 4.5 and above 60 °C. The substrate specificity investigations indicated that the TfBXyl43 is an exo-glycosidase, hydrolyzing only xylobiose and -triose from the nonreducing end. Besides the enzyme shows very high specificity on the glycon part of the substrate, since it can only hydrolyze β-D-xylopyranoside derivatives. The importance of hydrophobic interactions in the binding of the substrates are supported that the enzyme can hydrolize about four times more efficiently the artificial p-nitrophenyl-β-D-xylopyranoside substrate compared to the natural one, xylobiose. Furthermore we could detect transxylosidase activity both in the case of xylobiose and p-nitrophenyl-β-D-xylopyranoside donors which is the first example among the inverting β-D-xylosidases from T. fusca.