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

• A Pyrococcus furiosus inorganic pyrophosphatase gene has been cloned and expressed in E. coli.
• The optimum temperature and pH for PfPPase activity were 95 °C and pH 9.5.
• The half-life of PfPPase was about 6853, 2764 and 852 min at 85, 95 and 100 °C with Mg2+, respectively.
• The half-life of PfPPase was about 3937, 1901 and 491 min at 85, 95 and 100 °C without Mg2+, respectively.
• A coupled enzyme assay for the activity of P. furiosus glucose-1-phosphate uridylyltransferase at 80 °C was developed.

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 Mg2+. 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 × 103, 2.76 × 103 and 0.85 × 103 min at 85, 95 and 100 °C in the presence of Mg2+, respectively; 3.94 × 103, 1.90 × 103 and 0.49 × 103 min at 85, 95 and 100 °C in the absence of Mg2+, respectively. Inorganic pyrophosphate was the most specific substrate for PfPPase. The Km and the maximum velocity (Vmax) 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.

The rapid assay of glucose-1-phosphate uridylyltransferase activity from Pyrococcus furiosus by coupling bioreaction with homologous inorganic pyrophosphatase in one well at high temperature.Figure optionsDownload as PowerPoint slide