Expression in Escherichia coli of the thermostable inorganic pyrophosphatase from the Aquifex aeolicus and purification and characterization of the recombinant enzyme.
The gene encoding the inorganic pyrophosphatase from a hyperthermophilic bacterium, Aquifex aeolicus (Aae), was amplified by PCR. Then, the gene was overexpressed in Escherichia coli using a pJR-based expression plasmid, pAIPD. The recombinant Aae pyrophosphatase was purified 16.2-fold with a 53.4% yield and a specific activity of 34 U/mg protein by a combination of heating (to denature E. coli proteins) and two steps of DEAE-Sephacel column chromatography (nonabsorbed enzyme at pH 7.3 and absorbed enzyme at pH 8.0). This enzyme has an approximate molecular mass of 105,000 Da and consists of four subunits, each with a molecular mass of 24,500 Da. The enzyme shows the optimal activity in the pH range 7.5-8.0. The enzyme was stable at 80-95 degrees C. A divalent cation was absolutely required for the enzyme activity, Mg(2+) being most effective.
Hoe
H S
HS
Department of Genetic Engineering, Sungkyunkwan University, 300 Chunchon-Dong, Jangan-Ku, Suwon 440-746, Korea.
Kim
H K
HK
Kwon
S T
ST
D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D010957:Plasmids; D021122:Protein Subunits; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D021122:Protein Subunits; D011994:Recombinant Proteins; D011755:Pyrophosphatases; C442102:pyrophosphatase, Aquifex aeolicus