Thermostable amylopullulanases can catalyse the hydrolysis of both alpha-1,4 and alpha-1,6 glucosidic bonds and are of considerable interest in the starch saccharification industry. In this study, the gene Apu-Tk encoding an extracellular amylopullulanase was cloned from an extremely thermophilic anaerobic archaeon Thermococcus kodakarensis KOD1. Apu-Tk encodes an 1100-amino acid protein with a 27-residue signal peptide, which has a predicted mass of 125 kDa after signal peptide cleavage. Sequence alignments showed that Apu-Tk contains the five regions conserved in all GH57 family proteins. Full-length Apu-Tk was expressed in Escherichia coli and purified to homogeneity. The purified enzyme displayed both pullulanase and amylase activity. The optimal temperature for Apu-Tk to hydrolyse pullulan and soluble starch was >100 degrees C. Apu-Tk was also active at a broad range of pH (4-7), with an optimum pH of similar to 5.0-5.5. Apu-Tk also retained >30% of its original activity and partially folded globular structure in the presence of 8% SDS or 10% beta-mercaptoethanol. The high yield, broad pH range, and stability of Apu-Tk implicate it as a potential enzyme for industrial applications. (C) 2013 Elsevier Ltd. All rights reserved.