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Protein Expr Purif. 2014 Jul;99:94-8. doi: 10.1016/j.pep.2014.04.006. Epub 2014 Apr 20.

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

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College of Life Science and Technology, Zhanjiang Normal University, Zhanjiang City, Guangdong Province, 524048, PR China. Electronic address:
Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130023, PR China.


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 14.27μM, 3436s(-1), and 34.35μmol/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 12h and 25.96% of the original activity in the presence of 8M urea after incubation at 50°C for 120h. 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.


Archaea·Pyrococcus horikoshii; Inorganic pyrophosphatase; Ni-chelating chromatography; Thermostability

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