A thermophilic Bacillus sp. strain AN-7, isolated from a soil in India, produced an extracellular pullulanase upon growth on starch-peptone medium. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The optimum temperature and pH for activity was 90 degrees C and 6.0. With half-life time longer than one day at 80 degrees C the enzyme proves to be thermostable in the pH range 4.5-7.0. The pullulanase from Bacillus strain lost activity rapidly when incubated at temperature higher than 105 degrees C or at pH lower than 4.5. Pullulanase was completely inhibited by the Hg2+ ions. Ca2+, dithiothreitol, and Mn2+ stimulated the pullulanase activity. Kinetic experiments at 80 degrees C and pH 6.0 gave V-max and K-m values of 154 U mg(-1) and 1.3 mg ml(-1). The products of pullulan were maltotriose and maltose. This proved that the purified pullulanase (pullulan-6-glucanohydrolase, EC 3.2.1.41) from Bacillus sp. AN-7 is classified under pullulanase type I. to our knowledge, this Bacillus pullulanase is the most highly thermostable type I pullulanase known to date. (c) 2006 Elsevier Inc. All rights reserved.