Stereochemical Course and Reaction Products of the Action of β‐Xylosidase from Thermoanaerobacterium saccharolyticum Strain B6A‐RI

β‐Xylosidases are grouped in families 39 and 43 of a general classification of glycosyl hydrolases based on amino acid sequence similarities [Henrissat, B. & Bairoch, A. (1993) Biochem. J. 293 , 781–788]. The β‐xylosidase from Butyrivibrio fibrisolvens , which belongs to family 43, has been shown to operate by a molecular mechanism which results in the inversion of the anomeric configuration [Braun, C., Meinke, A., Ziser, L. & Withers, S. G. (1994) Anal. Biochem. 212 , 259–262], Thermoanaerobacterium saccharolyticum B6A‐RI β‐xylosidase which belongs to family 39 was purified as a recombinant enzyme from Escherichia coli. The stereochemistry of the hydrolysis of p ‐nitrophenyl β‐d‐xylopyranoside was followed by 1 H NMR. The spectrum recorded after 2 h hydrolysis showed a large signal centred at 4.47 ppm ( J ≈ 10 Hz) assignable to H1 of free β‐xylose with a small amount of α‐xylose (5.05 ppm, J ≈ 3 Hz) attributable to mutarotation. This result indicates that T. saccharolyticum β‐xylosidase operates with overall retention of the anomeric configuration. This result, with the lack of sequence similarity between the two families of β‐xylosidases, suggests that these two families have major differences in their active‐site geometries. Consistent with its retaining mechanism, β‐xylosidase of T. saccharolyticum B6A‐RI also displayed transglycosylating activity: reverse‐phase HPLC showed approximately 30% conversion of p ‐nitrophenyl β‐D‐xylopyranoside into a number of higher nitrophenyl oligosaccharides after 5 min incubation with the enzyme. The structure of the most abundant oligosaccharides could be determined by total correlation spectroscopy NMR and showed that the enzyme can build β‐1,4, β‐1,3‐ and β‐1,2‐linked xylo‐oligosaccharides.