Characterization of a family I-liked alkaline-resistant inorganic pyrophosphatase from the hyperthermophilic archaeon Pyrococcus furiosus for rapid determination of sugar nucleotidyltransferase at high temperature

Abstract A gene-encoded inorganic pyrophosphatase (AE009950.1: AAL80381.1) from hyperthermophilic archaeon Pyrococcus furiosus (PfPPase) was cloned and expressed in Escherichia coli BL21 (DE3). PfPPase showed 95% identity with family I PPases from Pyrococcus horikoshii OT3. The recombinant PfPPase had a molar mass of 22 kDa and was activated by Mg 2+ . The optimum temperature and pH of PfPPase were 95 °C and 9.5, respectively. PfPPase showed the half-lives of heat inactivation about 6.85 × 10 3 , 2.76 × 10 3 and 0.85 × 10 3  min at 85, 95 and 100 °C in the presence of Mg 2+ , respectively; 3.94 × 10 3 , 1.90 × 10 3 and 0.49 × 10 3  min at 85, 95 and 100 °C in the absence of Mg 2+ , respectively. Inorganic pyrophosphate was the most specific substrate for PfPPase. The K m and the maximum velocity ( V max ) of the enzyme were 173 μM and 55.9 μmol min −1  mg −1 , respectively, at pH 9.5 and 95 °C. A simple and efficient colourimetric coupled enzyme assay to determine the activity of thermophilic glucose-1-phosphate uridylyltransferase from P. furiosus (PfG1PUTase) with PfPPase at high temperatures was also developed.