A beta-xylosidase gene (xylA4) was identified in the genome sequence of thermoacidophilic Alicyclobacillus sp. A4. The deduced amino acid sequence was highly homologous with the beta-xylosidases of family 52 of the glycoside hydrolases (GH). The full-length gene consisted of 2097 bp and encoded 698 amino acids without a signal peptide. The gene product was successfully expressed in Escherichia coli with an activity of 564.9 U/mL. Recombinant XylA4 was purified by Ni2+-NTA affinity chromatography with a molecular mass of 78.5 kDa. The enzyme showed optimal activity at pH 6.0 and 65 degrees C, and remained stable over the pH range of 5.0-9.0. The thermostability of XylA4 is noteworthy, retaining almost all of the activity after 1 h incubation at 65 degrees C. Usingp-nitrophenyl-beta-D-xylopyranoside (pNPX) as the substrate, XylA4 had the highest specific activity (261.1 U/mg) and catalytic efficiency (601.5/mM/s) known so far for GH52 xylosidases. The enzyme displayed high tolerance to xylose, with a K-i value of approximately 88.7 mM. It also had synergy with xylanase XynBE18 from Paenibacillus sp. E18 in xylan degradation, releasing more xylose (up to 1.43 folds) than XynBE18 alone. Therefore, this thermostable xylose-tolerant beta-xylosidase may have a great application potential in many industrial fields. (C) 2014 Elsevier Ltd. All rights reserved.