Abstract
A hyperthermophilic alkaline phosphatase was purified from Thermotoga neapolitana by heat treatment at 100°C in the presence of Co 2+ followed by ion-exchange and affinity chromatographies. The enzyme was purified 2,880-fold with 44% yield. The purified enzyme showed a single protein band of M r 45,000 on SDS-PAGE and an apparent molecular weight of 87,000 estimated by gel filtration chromatography. This suggested a homogenous dimer structure. The optimal pH and temperature for enzyme activity were 9.9 and 85°C, respectively. Under optimal conditions, T. neapolitana alkaline phosphatase displayed 30% higher activity than calf intestine alkaline phosphatase did with p-nitrophenyl-phosphate as substrate. The hyperthermostable enzyme had a half-life of 238 min at 90°C and K m and V max values of 183 μ m and 1,352 U mg −1, respectively. Co 2+ enhanced the enzyme activity, thermostability, and ligand affinity during column chromatography. The alkaline phosphatase was twice as active with Co 2+ than with either Zn 2+ or Mn 2+ as the metal cofactor.
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