An extensive list of putative cellulosomal enzymes from C. thermocellum is now available in the public databanks, however, most of these remain unvalidated with regard to their activity and expression control mechanisms. This is particularly true of those enzymes putatively involved in hemicellulose deconstruction. Our research group has been working on mapping and characterization of glycoside hydrolases produced by C. thermocellum B8, that are critical for lignocellulosic biomass deconstruction. One of the identified genes expressed during growth on sugar cane bagasse and straw is axb8, which encodes a putative cellulosomal GH43_29 alpha-arabinofuranosidase (EC 3.2.1.55) that has not previously been characterized at the molecular or kinetic levels. The AxB8 predicted amino acid sequence presented GH43 and dockerin domains, as well as a family 6 carbohydrate-binding module (CBM6). Also, it is a close homologue of Firmicutes putatives alpha-arabinofuranosidases, including cellulosomal proteins. Multiple alignment analysis grouped AxB8 in a cluster with four uncharacterized putative GH43_29 subfamily enzymes, all containing dockerin type I domain and CBM6 modules. Purified heterologously expressed AxB8 showed activity against the synthetic substrates pNPX (p-nytrophenyl-beta-D-xylopyranoside) and pNPA (p-nytrophenyl-alpha-L-arabinofuranoside), as well as against the natural substrate wheat arabinoxylan (WAX), with maximal activity at 50 degrees C and pH between 5.0 and 6.0. The WAX degradation profile by AxB8 is different from those typically seen for alpha-arabinofuranosidases, presenting mainly xylose as a hydrolysis product, instead of arabinose. In addition, unlike other GH43_29 enzymes already characterized, AxB8 did not present activity against arabinan. Kinetic parameters using pNPA as a substrate were K-m of 23 +/- 3 mM and k(cat) of 104 +/- 7 s(-1). Despite its activity against pNPX, we did not observe AxB8 saturation with this substrate. AxB8 is the first member in its lade to be characterized regarding kinetic parameters, and together with its closest homologues could represent a large group of glycoside hydrolases with particular properties within the GH43_29 subfamily.