The gene encoding inorganic pyrophosphatase (PP(i)ase) from the hyperthermophilic archaea Pyrococcus horikoshii (Pho PP(i)ase) was cloned in the Escherichia coil strain BL21/pET15b, and the recombinant PP(i)ase was purified by Ni-chelating chromatography in only an one-step procedure. The PP(i)ase showed optimal activity at 88 degrees C and pH of 10.3. Kinetic analysis revealed Km, kcat, Vm of 14.27 mu M, 3436 s(-1), and 34.35 mu mol/min/mg protein, respectively. Pho PP(i)ase was stable against denaturant chemicals as well as heat. It retained 19.61% of the original activity after incubation at 100 degrees C for 12 h and 25.96% of the original activity in the presence of 8 M urea after incubation at 50 degrees C for 120 h. Pho PP(i)ase showed high specificity for inorganic pyrophosphate but low reactivity to sodium tripolyphosphate and sodium tetrapolyphosphate. ADP and ATP could not serve as substrates. (C) 2014 Elsevier Inc. All rights reserved.