The extremely thermophilic archaeon Thermococcus hydrothermalis, isolated from a deep-sea hydrothermal vent in the East Pacific Rise at 21 degrees N, produced an extracellular pullulanase. This enzyme was purified 97-fold to homogeneity from cell-Gee culture supernatant. The purified pullulanase was composed of a single polypeptide chain having an estimated molecular mass of 110 kDa (gel filtration) or 128 kDa (sodium dodecyl sulfate/polyacryl amide gel electrophoresis). The enzyme showed optimum activity at pH 5.5 and 95 degrees C. The thermostability and the thermoactivity were considerably increased in the presence of C2+. The enzyme was activated by 2-mercaptoethanol and dithiothreitol, whereas N-bromosuccinimide and alpha-cyclodextrin were inhibitors. This enzyme was able to hydrolyze, in addition to the alpha-1,6-glucosidic linkages in pullulan, alpha-1,4-glucosidic linkages in amylose and soluble starch, and call therefore be classified as a type II pullulanase or an amylopullulanase. The purified enzyme displayed Michaelis constant (K-m) values of 0.95 mg/ml for pullulan and 3.55 mg/ml for soluble starch without calcium and, in the presence of Ca2+, 0.25 mg/ml for pullulan and 1.45 mg/ml for soluble starch.