Cloning and expression of the gene for inorganic pyrophosphatase of Thermus caldophilus GK24 and properties of the enzyme

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Title
Cloning and expression of the gene for inorganic pyrophosphatase of Thermus caldophilus GK24 and properties of the enzyme
Author(s)
H S Hoe; I G Jo; H J Shin; H J Jeon; H K Kim; Jin Sung Lee; Yong Sung Kim; Dae Sil Lee; S T Kwon
Bibliographic Citation
Journal of Microbiology and Biotechnology, vol. 12, no. 2, pp. 301-305
Publication Year
2002
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°C. A divalent cation was absolutely required for the enzyme activity, with Mg2+ being the most effective.
Keyword
gene expressionppaT genepyrophosphataseTca pyrophosphatasethermus caldophilus GK24
ISSN
1017-7825
Publisher
Korea Soc-Assoc-Inst
Type
Article
Appears in Collections:
1. Journal Articles > Journal Articles
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