The gene encoding an alpha-L-arabinofuranosidase from Geobacillus caldoxylolyticus TK4, AbfATK4, was isolated, cloned, and sequenced. The deduced protein had a molecular mass of about 58 kDa, and analysis of its amino acid sequence revealed significant homology and conservation of different catalytic residues with alpha-L-arabinofuranosidases belonging to family 51 of the glycoside hydrolases. A histidine tag was introduced at the N-terminal end of AbfATK4, and the recombinant protein was expressed in Escherichia coli BL21, under control of isopropyl-beta-D-thiogalactopyranoside-inducible T7 promoter. The enzyme was purified by nickel affinity chromatography. The molecular mass of the native protein, as determined by gel filtration, was about 236 kDa, suggesting a homotetrameric structure. AbfATK4 was active at a broad pH range (pH 5.0-10.0) and at a broad temperature range (40-85 degrees C), and it had an optimum pH of 6.0 and an optimum temperature of 75-80 degrees C. The enzyme was more thermostable than previously described arabinofuranosidases and did not lose any activity after 48 h incubation at 70 degrees C. The protein exhibited a high level of activity with p-nitrophenyl-alpha-L-arabinofuranoside, with apparent K(m) and V(max) values of 0.17 mM and 5 88.2 U/mg, respectively AbfATK4 also exhibited a low level of activity with p-nitrophenyl-beta-D-xylopyranoside, with apparent K(m) and V(max) values of 1.57 mM and 151.5 U/mg, respectively. AbfATK4 released L-arabinose only from arabinan and arabinooligosaccharides. No endoarabinanase activity was detected. These findings suggest that AbfATK4 is an exo-acting enzyme.