A strain US 149 having high pullulanase activity was isolated from Tunisian soil adjacent to hot spring. The cells of this strain were rod shaped, Gram(+), non-motile, and could grow only in acidic pH (4.5-5.0) between 37 and 55 degreesC. The strain is likely to be Bacillus naganoensis since the analysis of the 16S rRNA gene sequence showed highest similarity (96%) with that of B. naganoensis (accession no. AB021193). The enzyme was purified to homogeneity by ion exchange and size exclusion chromatography from cell free culture supernatant. The purified native form had an estimated molecular mass of 200 kDa and it is composed of two subunits each of 95 kDa determined by SDS-PAGE. The purified enzyme had an optimum pH of 5 and retained activity at pH lower than 5. It had half-life duration of 48 and 20 min at 80 and 90 degreesC, respectively. The enzyme could hydrolyze pullulan, but it was unable to hydrolyze amylose and maltoheptaose. It was demonstrated that the DP3 product of pullulan hydrolysis was isopanose since it was cleaved by alpha-glucosidase to form glucose and isomaltose. This proved that the PulUS149 is an isopullulanase (pullulan-4-glucanohydrolase, EC 3.2.1.57) classified under pullulanase type II. (C) 2003 Elsevier Inc. All rights reserved.