Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Cloning, Expression, and Characterization of a Hyperalkaline Phosphatase from the Thermophilic Bacterium Thermus sp. T351

  • Choi Jeong-Jin (Department of Genetic Engineering, Sungkyunkwan University) ;
  • Park Jong-Woo (Department of Genetic Engineering, Sungkyunkwan University) ;
  • Shim Hye-Kyung (Department of Genetic Engineering, Sungkyunkwan University) ;
  • Lee Suk-Chan (Department of Genetic Engineering, Sungkyunkwan University) ;
  • Kwon Moo-Sik (Department of Genetic Engineering, Sungkyunkwan University) ;
  • Yang Joo-Sung (Department of Genetic Engineering, Sungkyunkwan University) ;
  • Hwang Heon (Department of Biomechatronic Engineering, Sungkyunkwan University) ;
  • Kwon Suk-Tae (Department of Genetic Engineering, Sungkyunkwan University)
  • Published : 2006.02.01
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Abstract

The gene encoding Thermus sp. T351 alkaline phosphatase (T351 APase) was cloned and sequenced. The gene consisted of 1,503 bp coding for a protein with 500 amino acid residues including a signal peptide. The deduced amino acid sequence of T351 APase showed relatively low similarity to other Thermus APases. The T351 APase gene was expressed under the control of the T7lac promoter on the expression vector pET-22b(+) in Escherichia coli BL21 (DE3). The expressed enzyme was purified by heat treatment, and $UNO^{TM}$ Q and $HiTrap^{TM}$ Heparin HP column chromatographies. The purified enzyme exhibited high activity at extremely alkaline pHs, reaching a maximum at pH 12.0. The optimum temperature of the enzyme was $80^{\circ}C$, and the half-life at $85^{\circ}C$ was approximately 103 min. The enzyme activity was found to be dependent on metal ions: the addition of $Mg^{2+}$ and $CO^{2+}$ increased the activity, whereas EDTA inhibited it. With p-nitrophenyl phosphate as the substrate, T351 APase had a Michaelis constant ($K_{m}$) of $3.9{\times}10^{-5}M$. The enzyme catalyzed the hydrolysis of a wide variety of phosphorylated compounds.

Keywords

References

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