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Thermostable Pyrophosphatase

Product Description

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 Km (5.4 uM) for pyrophosphate, is active between pH 7 and 9, has an optimal temperature for activity at 75°C and is functional under PCR conditions.

Source of Protein
A recombinant 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

Molecular Weight
19,381 Daltons

 

Quality Control Analysis

Unit Activity The assay is based on that described by Taussky and Shorr (4). Briefly, enzyme dilutions are added to 30mM Tris HCl pH 8.5, 1.5 mM MgCl2 and 1.5mM sodium pyrophosphate. After a 10 min incubation at 75&def;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 660nm. The amount of product formed is extrapolated from a phosphate standard curve generated from the ammonium molydate/ferrous sulfate reaction.

Protein Concentration (OD280 ) is determined by OD280 absorbance.

Physical Purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the protein of interest band in the diluted sample.

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 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 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 16S rDNA Contamination is evaluated using 5 µL replicate samples of enzyme solution 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.

Supplied in
10 mM Tris-HCl pH 7.5
50 mM NaCl
50% Glycerol
1 mM DTT
0.1 mM EDTA

Product Information

Thermostable Pyrophosphatase
Part NumberY9370L
PriceInquire
Concentration2,000 U/ml
Unit Size1,250 U
SDS Available on request
This product is only available for bulk orders or OEM orders and not available for individual pack sizes.

Product Specification*

Storage Temperature-25 to -15°C
Test Units Tested Specification
SDS Purityn/a>95%
Specific Activityn/a3,500 U/mg
SS Exonuclease50<1.0% Released
DS Exonuclease50<1.0% Released
DS Endonuclease50No Conversion
E.coli DNA Contamination50<10 copies

* For a detailed summary of assay conditions and data, refer to the Quality Controls Analysis section.

References

  1. Sulfolobus acidocaldarius inorganic pyrophosphatase: structure, thermostability, and effect of metal ion in an archael pyrophosphatase. Leppänen VM, Nummelin H, Hansen T, Lahti R, Schäfer G, Goldman A. Protein Sci. 1999. 8(6):1218- 31.
  2. The extreme thermostable pyrophosphatase from Sulfolobus acidocaldarius: enzymatic and comparative biophysical characterization. Hansen T, Urbanke C, Leppänen VM, Goldman A, Brandenburg K, Schäfer G. Arch Biochem Biophys. 1999. 363(1):135-47.
  3. Purification, cloning, and sequencing of archaebacterial pyrophosphatase from the extreme thermoacidophile Sulfolobus acidocaldarius. Meyer W, Moll R, Kath T, Schäfer G. Arch Biochem Biophys. 1995. 319(1):149-56.
  4. A microcolorimetric method for the determination of inorganic phosphorus. Taussky HH, Shorr E. J Biol Chem. 1953 Jun;202(2):675-85.

Limitations of Use
This product was developed, manufactured, and sold for in vitro use only. The product is not suitable for administration to humans or animals. SDS sheets relevant to this product are available upon request.

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