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A Moderately Thermostable Alkaline Phosphatase from Geobacillus thermodenitrificans T2: Cloning, Expression and Biochemical Characterization

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Abstract

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 °C, respectively. The enzyme retained a high activity at 45–60 °C, while it could be quickly inactivated by a heat treatment at 80 °C for 15 min, exhibiting a half-life of 8 min at 70 °C. The K m and V max for pNPP were determined to be 31.5 μM and 430 μM/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.

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Acknowledgments

This work is supported by the National Basic Research Program of China (973 Program, 2007CB914304) and New Century Excellent Talents in University (NCET-06-0356).

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Authors and Affiliations

  1. State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, 200433, People’s Republic of China

    Yong Zhang, Chaoneng Ji, Xiaoxiao Zhang, Zhenxing Yang, Jing Peng, Rui Qiu, Yi Xie & Yumin Mao

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  1. Yong Zhang
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  2. Chaoneng Ji
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  3. Xiaoxiao Zhang
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  8. Yumin Mao
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Correspondence to Yumin Mao.

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Zhang, Y., Ji, C., Zhang, X. et al. A Moderately Thermostable Alkaline Phosphatase from Geobacillus thermodenitrificans T2: Cloning, Expression and Biochemical Characterization. Appl Biochem Biotechnol 151, 81–92 (2008). https://doi.org/10.1007/s12010-008-8166-7

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  • DOI: https://doi.org/10.1007/s12010-008-8166-7

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