PRODUCTION, PURIFICATION AND CHARACTERIZATION OF AN ALPHA-L-ARABINOFURANOSIDASE FROM BACTEROIDES XYLANOLYTICUS X5-1

Cell-free extracts of L-arabinose- and D-xylose-grown cells of the mes ophilic anaerobic bacterium Bacteroides xylanolyticus X5-1 contained h igh activities [2 units ((U)/mg] of an alpha-L-arabinofuranosidase (EC 3.2.1.55). The enzyme was also produced during growth on xylan, but n ot during growth on glucose or cellobiose. The enzyme was mainly extra cellularly attached to the cell when the organism was grown on xylan a nd was not released into the medium. The enzyme was purified 41-fold t o apparent homogeneity. The native enzyme had an apparent molecular ma ss of 364 kDa and was composed of six polypeptide subunits of 61 kDa. The enzyme displayed a pH optimum of 5.5-6.0, and a pH stability of 5. 5-9.0. The temperature optimum was 50 degrees C and the enzyme was sta ble up to 50 degrees C. Thiol groups were essential for activity, but the enzyme activity was not dependent on divalent cations. The Michael isconstant (K-m) and maximal reaction velocity (V-max for p-nitropheny l-alpha-L-arabinofuranoside were 0.5 mM and 155 U/mg protein, respecti vely. The enzyme was specific for the alpha-linked arabinoside in the furanoside configuration. The enzyme displayed activity with ara binos e-containing xylo-oligosaccharides with a polymerization degree of 2-5 , but not with the polymeric substrates oat-spelt xylan or arabinogala ctan. The enzyme belongs to the Streptomyces purpurascens-type of alph a-L-arabinofuranosidase.