Cloning, purification, and characterization of inorganic pyrophosphatase from the hyperthermophilic archaea Pyrococcus horikoshii

• Purification and characterization of PPiase from Pyrococcus horikoshii.
• Pho PPiase has optimum activity at alkaline pH and high temperature.
• Pho PPiase is an excellent study model of thermostability and folding dynamics.

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