Characterization of a Highly Thermostable Alkaline Phosphatase from the EuryarchaeonPyrococcus abyssi

• DOI: 10.1128/aem.67.10.4504-4511.2001
• Publication Date: 2002-07-27T10:00:58Z
• Authors: Sébastien Zappa; Jean-Luc Rolland; Didier Flament; Yannick Gueguen; Joseph Boudrant; Jacques Dietrich
• Keywords: Dephosphorylation; Vanadate; Divalent; Molecular mass

ABSTRACT This work reports the first isolation and characterization of an alkaline phosphatase (AP) from a hyperthermophilic archaeon. An AP gene Pyrococcus abyssi , euryarchaeon isolated deep-sea hydrothermal vent, was cloned enzyme expressed in Escherichia coli . Analysis sequence showed conservation active site structural elements E. AP. The recombinant purified characterized. Monomeric homodimeric forms were detected, with monomer molecular mass 54 kDa. Apparent optimum pH temperature estimated at 11.0 70°C, respectively. Thus far, P. has been demonstrated to be most thermostable AP, half-lives 100 105°C 18 5 h, Enzyme activity found dependent on divalent cations: metal ion chelators inhibited activity, whereas addition exogenous Mg(II), Zn(II), Co(II) increased activity. by inorganic phosphate, but not molybdate vanadate. Strong inhibitory effects observed presence thiol-reducing agents, although cysteine residues incorporated within intra- or interchain disulfide bonds. In addition, dephosphorylate linear DNA fragments dephosphorylation efficiencies 93.8 84.1% regard cohesive blunt ends,