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

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

Abstract

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.

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

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cloning, Molecular
  • Enzyme Stability
  • Escherichia coli / genetics
  • Hot Temperature
  • Hydrogen-Ion Concentration
  • Inorganic Pyrophosphatase / biosynthesis*
  • Inorganic Pyrophosphatase / genetics
  • Inorganic Pyrophosphatase / isolation & purification
  • Pyrococcus horikoshii / enzymology*
  • Pyrococcus horikoshii / genetics
  • Substrate Specificity

Substances

  • Inorganic Pyrophosphatase