A moderately thermostable alkaline phosphatase from Geobacillus thermodenitrificans T2: cloning, expression and biochemical characterization.
A gene-encoding alkaline phosphatase (AP) from thermophilic Geobacillus thermodenitrificans T2, termed Gtd AP, was cloned and sequenced. The deduced Gtd AP protein comprises 424 amino acids and shares a low homology with other known AP (<35% identity), while it exhibits the conservation of the active site and structure element of Escherichia coli AP. The Gtd AP protein, without a predicted signal peptide of 30 amino acids, was successfully overexpressed in E. coli and purified as a hexa-His-tagged fusion protein. The pH and temperature optima for purified enzyme are 9.0 and 65 degrees C, respectively. The enzyme retained a high activity at 45-60 degrees C, while it could be quickly inactivated by a heat treatment at 80 degrees C for 15 min, exhibiting a half-life of 8 min at 70 degrees C. The K(m) and V (max) for pNPP were determined to be 31.5 muM and 430 muM/min at optimal conditions. A divalent cation is essential, with a combination of Mg2+ and Co2+ or Zn2+ preferred. The enzyme was strongly inhibited by 10 mM ethylenediaminetetraacetic acid (EDTA) and vanadate but highly resistant to urea and dithiothreitol. The properties of Gtd AP make it suitable for application in molecular cloning or amplification.
Zhang
Yong
Y
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, 200433, People's Republic of China.
Ji
Chaoneng
C
Zhang
Xiaoxiao
X
Yang
Zhenxing
Z
Peng
Jing
J
Qiu
Rui
R
Xie
Yi
Y
Mao
Yumin
Y
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D045302:Geobacter / Q000201:enzymology* / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics; D013696:Temperature
D011994:Recombinant Proteins; D000469:Alkaline Phosphatase