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The gene encoding Aquifex pyrophilus (Apy) pyrophosphatase was cloned and sequenced. The deduced amino acid sequence of Apy pyrophosphatase showed a 94.2% homology to Aquifex aeolicus (Aae) pyrophosphatase. The gene exhibits a difference in the codon usage at the third position from Aae pyrophosphatase. The gene was expressed under the control of a tac promoter in E. coli. The recombinant Apy pyrophosphatase was purified 18.7-fold with a 52.8% yield and a specific activity of 26.2 U mg−1 protein. The native enzyme has a homotetramer of 177 amino acids. The enzyme shows optimal activity in pH 7.5. The optimum temperature was approximately 70°C. A divalent cation was absolutely required for the enzyme activity; Mg2+ was the most effective.