A thermophilic strain, L14, isolated from Iranian hot springs, produces an extracellular pullulanase upon growth on optimized liquid medium. According to 16S rDNA analysis and biochemical tests, L14 seems to be a Geobacillus stearothermophilus strain. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion-exchange and gel filtration chromatographies with a yield of 30%. The purified enzyme showed a single band on SDS-PAGE with an estimated molecular mass of about 100 kDa for monomeric enzyme. Optimal conditions for the activity of the purified pullulanase were calculated as pH 5.5 and T-opt = 65 degrees C. A reasonable stability was also observed at 60-70 degrees C. The enzyme could hydrolyze pullulan, starch and malto-oligosaccharides and was unable to degrade alpha-cyclodextrin and panose. The apparent K-m and V-max for the enzyme activity on pullulan were 0.48 g/l and 44 mu mol min(-1) and for soluble starch were 5 g/l and 80 mu mol min(-1), respectively. The products of enzymatic reaction on pullulan were glucose, maltose and maltotriose, whereas conversion of starch produced glucose and maltose. This enzyme could also hydrolyze different types of malto-oligosaccharides like maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose. Consequently, the enzyme attacked both alpha-D-(1 -> 4) and alpha-D-(1 -> 6) glycosidic linkages of pullulan and branched polysaccharides like starch and produced glucose and maltose. Therefore. it is assumed that the newly purified annylopullulanase from Geobacillus sp. L14 is the first report on the presence of a specific debranching enzyme which can produce glucose from pullulan. (C) 2009 Elsevier Inc. All rights reserved.