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KMID : 0545120020120020301
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Journal of Microbiology and Biotechnology
2002 Volume.12 No. 2 p.301 ~ p.305
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Cloning Expression of the Gene for Inorganic Pyrophosphatase of Thermus caldophilus GK24 and Properties of the Enzyme
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Hoe, Hyang Sook
Jo, In Geun/Shin, Hea Jin/Jeon, Hyo Jeong/Kim, Hyun Kyu/Lee, Jin Sung/Kim, Yong Sung
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Abstract
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The gene (ppaT) encoding Thermos 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 ㎎^-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℃. A divalent cation was absolutely required for the enzyme activity, with Mg^2+ being the most effective.
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