© 2005 Heron Publishing—Victoria, Canada
Characterization of the Family I inorganic pyrophosphatase from Pyrococcus horikoshii OT3
Sung-Jong Jeon (1, 2) and Kazuhiko Ishikawa (3, 4)
1. Department of Biotechnology and Bioengineering, Dong-Eui University, Busan, 614-714, Korea / 2. Department of Biomaterial Control, Dong-Eui University, Busan, 614-714, Korea / 3. Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST Kansai), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan / 4. Corresponding author ([email protected]) / Received January 8, 2005; accepted February 2, 2005; published online May 31, 2005
A gene encoding for a putative Family I inorganic pyrophosphatase (PPase, EC 184.108.40.206) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (Accession No. 1907) from P. horikoshii showed some identity with other Family I inorganic pyrophosphatases from archaea. The recombinant PPase from P. horikoshii (PhPPase) has a molecular mass of 24.5 kDa, determined by SDS-PAGE. This enzyme specifically catalyzed the hydrolysis of pyrophosphate and was sensitive to NaF. The optimum temperature and pH for PPase activity were 70 °C and 7.5, respectively. The half-life of heat inactivation was about 50 min at 105 °C. The heat stability of PhPPase was enhanced in the presence of Mg2+. A divalent cation was absolutely required for enzyme activity, Mg2+ being most effective; Zn2+, Co2+ and Mn2+ efficiently supported hydrolytic activity in a narrow range of concentrations (0.05–0.5 mM). The Km for pyrophosphate and Mg2+ were 113 and 303 µM, respectively; and maximum velocity, Vmax, was estimated at 930 U mg–1.
Keywords: archaea, hyperthermophile.
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