Open AccessExpression and Characterization of Recombinant Thermostable Alkaline Phosphatase from a Novel Thermophilic Bacterium Thermus thermophilus XM
Author(s) -
LI Jianbo,
XU Limei,
YANG Feng
Publication year - 2007
Publication title -
acta biochimica et biophysica sinica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.771
H-Index - 57
eISSN - 1745-7270
pISSN - 1672-9145
DOI - 10.1111/j.1745-7270.2007.00347.x
Subject(s) - thermus thermophilus , thermostability , alkaline phosphatase , thermophile , biochemistry , phosphatase , recombinant dna , thermus , escherichia coli , enzyme , biology , chemistry , microbiology and biotechnology , gene
A gene ( tap ) encoding a thermostable alkaline phosphatase from the thermophilic bacterium Thermus thermophilus XM was cloned and sequenced. It is 1506 bp long and encodes a protein of 501 amino acid residues with a calculated molecular mass of 54.7 kDa. Comparison of the deduced amino acid sequence with other alkaline phosphatases showed that the regions in the vicinity of the phosphorylation site and metal binding sites are highly conserved. The recombinant thermostable alkaline phosphatase was expressed as a His 6 ‐tagged fusion protein in Escherichia coli and its enzymatic properties were characterized after purification. The pH and temperature optima for the recombinant thermostable alkaline phosphatases activity were pH 12 and 75 °C. As expected, the enzyme displayed high thermostability, retaining more than 50% activity after incubating for 6 h at 80 °C. Its catalytic function was accelerated in the presence of 0.1 mM Co 2+ , Fe 2+ , Mg 2+ , or Mn 2+ but was strongly inhibited by 2.0 mM Fe 2+ . Under optimal conditions, the Michaelis constant ( K m ) for cleavage of p ‐nitrophenyl‐phosphate was 0.034 mM. Although it has much in common with other alkaline phosphatases, the recombinant thermostable alkaline phosphatase possesses some unique features, such as high optimal pH and good thermostability.