The gene encoding a glycoside hydrolase family 43 beta-xylosidase (GbtXyl43A) from the thermophilic bacterium Geobacillus thermoleovorans strain IT-08 was synthesized and cloned with a C-terminal His-tag into a pET29b expression vector. The recombinant gene product termed GbtXyl43A was expressed in Escherichia coli and purified to apparent homogeneity. Michaelis-Menten kinetic parameters were obtained for the artificial substrates p-nitrophenyl-beta-D-xylopyranose (4NPX) and p-nitrophenyl-alpha-L-arabinofuranose (4NPA), and it was found that the ratio k(cat)/K-m 4NPA/k(cat)/K-m 4NPX was similar to 7, indicting greater catalytic efficiency for 4NP hydrolysis from the arabinofuranose aglycon moiety. Substrate inhibition was observed for the substrates 4-methylumbelliferyl xylopyranoside (muX) and the arabinofuranoside cogener (muA), and the ratio k(cat)/K-m muA/k(cat)/K-m muX was similar to 5. The enzyme was competitively inhibited by monosaccharides, with an arabinose K-i of 6.8 +/- 0.62 mM and xylose K-i of 76 +/- 8.5 mM. The pH maxima was 5.0, and the enzyme was not thermally stable above 54 degrees C, with a t(1/2) of 35 min at 57.5 degrees C. GbtXyl43A showed a broad substrate specificity for hydrolysis of xylooligosaccharides up to the highest degree of polymerization tested (xylopentaose), and also released xylose from birch and beechwood arabinoxylan.