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Literature summary for 2.7.7.7 extracted from

  • Cho, S.S.; Yu, M.; Kim, S.H.; Kwon, S.T.
    Enhanced PCR efficiency of high-fidelity DNA polymerase from Thermococcus waiotapuensis (2014), Enzyme Microb. Technol., 63, 39-45.
    View publication on PubMed

Application

Application Comment Organism
molecular biology the H633R mutant DNA polymerase may be useful in high-fidelity DNA amplification and various PCR-based applications Thermococcus waiotapuensis

Cloned(Commentary)

Cloned (Comment) Organism
overexpression of the wild-type and mutant enzymes in Escherichia coli Thermococcus waiotapuensis

Protein Variants

Protein Variants Comment Organism
H633D DNA polymerase activity of the mutant enzyme is higher than the polymerase activity of the wild-type enzyme. PCR efficiency of the H633D mutant is higher than that of the N565K mutant enzyme Thermococcus waiotapuensis
H633R significantly improved polymerase function compared to wild-type enzyme in terms of processivity (2-fold), extension rate (1.5fold) and PCR efficiency. The kcat value of the Twa H633R mutant is similar to that of wild-type, but the Km of the Twa H633R mutant is about 1.6fold lower than that of the wild-type Thermococcus waiotapuensis
N565K DNA polymerase activity of the mutant enzyme is higher than the polymerase activity of the wild-type enzyme. PCR efficiency of the H633D mutant is higher than that of the N565K mutant enzyme Thermococcus waiotapuensis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0000066
-
DNAn pH 8.6, 75°C, mM of template, in the presence of an excess of annealed primer, mutant enzyme H633R Thermococcus waiotapuensis
0.0000104
-
DNAn pH 8.6, 75°C, mM of template, in the presence of an excess of annealed prime, wild-type enzyme Thermococcus waiotapuensis
0.003
-
deoxynucleoside triphosphate pH 8.6, 75°C, mM of each nucleotide in an equimolar mixture of the four nucleotides, mutant enzyme H633R Thermococcus waiotapuensis
0.003
-
deoxynucleoside triphosphate pH 8.6, 75°C, mM of each nucleotide in an equimolar mixture of the four nucleotides, wild-type enzyme Thermococcus waiotapuensis

Organism

Organism UniProt Comment Textmining
Thermococcus waiotapuensis H9CW54
-
-

Purification (Commentary)

Purification (Comment) Organism
-
Thermococcus waiotapuensis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
deoxynucleoside triphosphate + DNAn
-
Thermococcus waiotapuensis diphosphate + DNAn+1
-
?

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
75
-
assay at Thermococcus waiotapuensis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.4
-
DNAn pH 8.6, 75°C, mM of template, in the presence of an excess of annealed primer, mutant enzyme H633R Thermococcus waiotapuensis
2.6
-
DNAn pH 8.6, 75°C, mM of template, in the presence of an excess of annealed primer, wild-type enzyme Thermococcus waiotapuensis
3.9
-
deoxynucleoside triphosphate pH 8.6, 75°C, mM of each nucleotide in an equimolar mixture of the four nucleotides, wild-type enzyme Thermococcus waiotapuensis
4.2
-
deoxynucleoside triphosphate pH 8.6, 75°C, mM of each nucleotide in an equimolar mixture of the four nucleotides, mutant enzyme H633R Thermococcus waiotapuensis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
assay at Thermococcus waiotapuensis