Thermostable Pyrophosphatase

• Hydrolysis of inorganic pyrophosphate to produce orthophosphate
• Low K_m (5.4 µM)
• Active at pH 7–9
• Optimal temperature at 75°C

Thermostable pyrophosphatase is a recombinant enzyme from Sulfolobus acidocaldarius which catalyzes the Mg-dependent reaction of P₂O₇^-4 + H₂O → 2HPO₄^-2. It is has a low K_m (5.4 µM) for pyrophosphate. In addition it is active between pH 7 and 9, has an optimal temperature for activity at 75°C and is functional under PCR conditions.

This enzyme is supplied in 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 50% glycerol, 1 mM DTT and 0.1 mM EDTA.

SDS available upon request.

Enzyme properties

• Storage temperature: –25°C to –15°C
• Molecular weight: 19,381 Daltons

Source of recombinant enzyme protein
The protein is produced by an E. coli strain carrying the Thermostable Pyrophosphatase gene from S. acidocaldarius.

Unit definition: One unit is the amount of enzyme that will liberate 1 µmol of phosphate per minute from inorganic pyrophosphate at 75°C and pH 8.5

References

1. Leppänen, V.M, Nummelin, H., Hansen, T., Lahti, R., Schäfer, G., and Goldmann A. (1999) Sulfolobus acidocaldarius inorganic pyrophosphatase: structure, thermostability, and effect of metal ion in an archael pyrophosphatase. Protein Sci. 8:1218.
2. Hansen, T., Urbanke, C., Leppänen, V.M., Goldman, A., Brandenburg, K. and Schäfer, G. (1999) The extreme thermostable pyrophosphatase from Sulfolobus acidocaldarius: enzymatic and comparative biophysical characterization. Arch. Biochem. Biophys. 363:135.
3. Meyer, W., Moll,R., Kath, T., Schäfer, G.. (1995) Purification, cloning, and sequencing of archaebacterial pyrophosphatase from the extreme thermoacidophile Sulfolobus acidocaldarius. Arch. Biochem. Biophys. 319:149.
4. Taussky, H.H., Shorr, E. (1953) A microcolorimetric method for the determination of inorganic phosphorus. J. Biol. Chem. 202:675.

Quality control analysis

Enzyme dilutions are added to 30 mM Tris HCl, pH 8.5, 1.5 mM MgCl₂ and 1.5 mM sodium pyrophosphate. After a 10-minute incubation at 75°C, the product formed, 2- orthophosphate, is reacted with ammonium molybdate to form phosphomolybdic acid. The phosphomolybdic acid is then reduced by ferrous sulfate under weak acidic conditions to form a blue color, the absorbance of which is measured at 660 nm. The amount of product formed is extrapolated from a phosphate standard curve generated from the ammonium molydate/ferrous sulfate reaction. The assay is based on that described by Taussky and Shorr (4).

Single-stranded exonuclease is determined in a 50 µl reaction containing a radiolabeled single-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded exonuclease activity is determined in a 50 µl reaction containing a radiolabeled double-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded endonuclease activity is determined in a 50 µl reaction containing 0.5 µg of plasmid DNA and 10 µl of enzyme solution incubated for 4 hours at 37°C.

E. coli contamination is evaluated using 5 µl replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

• Produce orthophosphate