Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Cloning and Expression of the Gene for Inorganic Pyrophosphatase of Thermus caldophilus GK24 and Properties of the Enzyme

  • Hoe, Hyang-Sook (Department of Genetic Engineering, Sungkyunkwan University) ;
  • Jo, In-Geun (Department of Genetic Engineering, Sungkyunkwan University) ;
  • Shin, Hea-Jin (Department of Genetic Engineering, Sungkyunkwan University) ;
  • Jeon, Hyo-Jeong (Department of Genetic Engineering, Sungkyunkwan University) ;
  • Kim, Hyun-Kyu (Genetic Resource R＆D Institute, Super Bio Co., Ltd.) ;
  • Lee, Jin-Sung (Korea Research Institute of Bioscience and Biotechnology) ;
  • Kim, Yong-Sung (Korea Research Institute of Bioscience and Biotechnology) ;
  • Lee, Dae-Sil (Korea Research Institute of Bioscience and Biotechnology) ;
  • Kwon, Suk-Tae (Department of Genetic Engineering, Sungkyunkwan University)
  • Published : 2002.04.01
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Abstract

The gene (ppaT) encoding Thermus caldophilus GK24 pyrophosphatase (Tca pyrophosphatase) was cloned and sequenced. The gene was found to contain an open reading frame encoding 175 amino acids with a calculated mass of 19,155 Da. The ppaT gene was expressed under the control of the tac promoter in Escherichia coli. The recombinant Tca pyrophosphatase was purified 21.4-fold with $56\%$ yield and specific activity of 25.7 U $mg^-1$, following a combination of heating (to denature the E. coli proteins) and one step of DEAE-Sephacel column chromatography. The native enzyme was found to have an approximate molecular mass of 110,000 Da and consisted of six subunits. The enzyme exhibited maximal activity at pH of 8.0-8.5 and was stable at $80-90^{\circ}C$. A divalent cation was absolutely required for the enzyme activity, with $Mg^2+$. being the most effective.

Keywords

References

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