title: Cloning, purification, and characterization of inorganic pyrophosphatase from the hyperthermophilic archaea Pyrococcus horikoshii.
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.
authors.affiliation: College of Life Science and Technology, Zhanjiang Normal University, Zhanjiang City, Guangdong Province, 524048, PR China. Electronic address: ludongmei2012@hotmail.com.
authors.forename: Dongmei
authors.initials: D
authors.lastname: Lu
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130023, PR China.
authors.forename: Guiqiu
authors.initials: G
authors.lastname: Xie
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130023, PR China.
authors.forename: Renjun
authors.initials: R
authors.lastname: Gao
mesh_terms: D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D043564:Inorganic Pyrophosphatase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D043902:Pyrococcus horikoshii / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D043564:Inorganic Pyrophosphatase
keywords: Archaea·Pyrococcus horikoshii; Inorganic pyrophosphatase; Ni-chelating chromatography; Thermostability