Bacillus subtilis produces alpha-l-arabinofuranosidases (EC 126.96.36.199; AFs) capable of releasing arabinosyl oligomers and l-arabinose from plant cell walls. Here, we show by insertion-deletion mutational analysis that genes abfA and xsa(asd), herein renamed abf2, encode AFs responsible for the majority of the intracellular AF activity in B. subtilis. Both enzyme activities were shown to be cytosolic and functional studies indicated that arabino-oligomers are natural substrates for the AFs. The products of the two genes were overproduced in Escherichia coli, purified and characterized. The molecular mass of the purified AbfA and Abf2 was about 58 kDa and 57 kDa, respectively. However, native PAGE gradient gel analysis and cross-linking assays detected higher-order structures (>250 kDa), suggesting a multimeric organization of both enzymes. Kinetic experiments at 37 degrees C, with p-nitrophenyl-alpha-l-arabinofuranoside as substrate, gave an apparent K(m) of 0.498 mM and 0.421 mM, and V(max) of 317 U mg(-1) and 311 U mg(-1) for AbfA and Abf2, respectively. The two enzymes displayed maximum activity at 50 degrees C and 60 degrees C, respectively, and both proteins were most active at pH 8.0. AbfA and Abf2 both belong to family 51 of the glycoside hydrolases but have different substrate specificity. AbfA acts preferentially on (1-->5) linkages of linear alpha-1,5-l-arabinan and alpha-1,5-linked arabino-oligomers, and is much less effective on branched sugar beet arabinan and arabinoxylan and arabinogalactan. In contrast, Abf2 is most active on (1-->2) and (1-->3) linkages of branched arabinan and arabinoxylan, suggesting a concerted contribution of these enzymes to optimal utilization of arabinose-containing polysaccharides by B. subtilis.