An extracellular 1,3-1,4-beta-glucanase-producing strain S2 was isolated from booldice and identified as Bacillus methylotrophicus. Furthermore, a homogeneous extracellular 1,3-1,4-beta-glucanase GCS2 was purified by ammonium sulfate precipitation and cation-exchange chromatography. The gene for the 1,3-1,4-beta-glucanase was cloned, and the nucleotide sequence was determined. Characterization of the purified enzyme revealed the molecular mass of 26 kDa and the optimum activity at pH 7.5, 55 degrees C. The purified enzyme can highly hydrolyze carboxymethylcellulose including oat gum, barley beta-glucan, CMC and lichenan, while low activity on avicel, cellobiose, filter paper, p-nitrophenyl beta-D-cellobioside, and p-nitrophenyl beta-D-glucoside, but no activity against microcrystalline cellulose or salicin. The enzyme was stable at wide range of pHs 5-10 and still maintained above 60% activity at 70 degrees C. The enzyme activity was stimulated by Trixon X-100. The property of the enzyme GCS2 makes this enzyme a broad prospect in brewing and commercial detergent industry. To our knowledge, this is the first report of a 1,3-1,4-beta-glucanase from microbes associated with booldice. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.