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.
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
|Storage Temperature||-25 to -15°C|
|Specific Activity||n/a||3,500 U/mg|
|SS Exonuclease||50||<1.0% Released|
|DS Exonuclease||50||<1.0% Released|
|DS Endonuclease||50||No Conversion|
|E.coli DNA Contamination||50||<10 copies|
* For a detailed summary of assay conditions and data, refer to the Quality Controls Analysis section.
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.
10 mM Tris-HCl pH 7.5
50 mM NaCl
1 mM DTT
0.1 mM EDTA
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.