Abstract
A thl gene encoding the thiolase (EC 2.3.1.9) of Clostridium pasteurianum was cloned by thermal asymmetric interlaced (TAIL) PCR. It consists of 1179 bp with 36.8% GC content and encodes 392 amino acids with a deduced molecular mass of 40,954 Da and shows 77% identity and 88% similarity to that of Clostridium tetani E88 and should be classified as a biosynthetic thiolase with three conserved residues Cys89, Cys382 and His352. The gene was over-expressed in Escherichia coli and the thiolase was purified with Ni-NTA agarose column to homogeneity. The K m of this thiolase for acetoacetyl-CoA is 0.13 mM with 0.06 mM CoASH at pH 8.2, 25°C and a V max value of 46 µmol min−1 mg−1.
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Acknowledgements
We thank Dr. Jiesheng Tian, Xiaoyu Zhou, Jiangning Liu, Nining Hong, Miao Pan and Feng Guan for helpful discussions. Thanks also to Dr. Yaoping Zhang for critical reading of the manuscript.
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Meng, Y., Li, J. Cloning, Expression and Characterization of a Thiolase Gene from Clostridium pasteurianum . Biotechnol Lett 28, 1227–1232 (2006). https://doi.org/10.1007/s10529-006-9089-4
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DOI: https://doi.org/10.1007/s10529-006-9089-4