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Applied and Environmental Microbiology, May 2005, p. 2777-2781, Vol. 71, No. 5
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.5.2777-2781.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Homologous and Heterologous Overexpression in Clostridium acetobutylicum and Characterization of Purified Clostridial and Algal Fe-Only Hydrogenases with High Specific Activities

Laurence Girbal,1* Gregory von Abendroth,2 Martin Winkler,2 Paul M. C. Benton,3 Isabelle Meynial-Salles,4 Christian Croux,1 John W. Peters,3 Thomas Happe,2 and Philippe Soucaille1

Laboratoire de Biotechnologie-Bioprocédés, UMR CNRS 5504, UMR INRA 792, INSA, 135 Avenue de Rangueil,1 CRT/CRITT-Bioindustries, INSA, DGBA, 31077 Toulouse Cedex 4, France,4 Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717,3 Lehrstuhl Biochemie der Pflanzen, AG Photobiotechnologie, Ruhr-Universitaet-Bochum, 44780 Bochum, Germany2

Received 1 July 2004/ Accepted 21 November 2004

Clostridium acetobutylicum ATCC 824 was selected for the homologous overexpression of its Fe-only hydrogenase and for the heterologous expressions of the Chlamydomonas reinhardtii and Scenedesmus obliquus HydA1 Fe-only hydrogenases. The three Strep tag II-tagged Fe-only hydrogenases were isolated with high specific activities by two-step column chromatography. The purified algal hydrogenases evolve hydrogen with rates of around 700 µmol H2 min–1 mg–1, while HydA from C. acetobutylicum (HydACa) shows the highest activity (5,522 µmol H2 min–1 mg–1) in the direction of hydrogen uptake. Further, kinetic parameters and substrate specificity were reported. An electron paramagnetic resonance (EPR) analysis of the thionin-oxidized HydACa protein indicates a characteristic rhombic EPR signal that is typical for the oxidized H cluster of Fe-only hydrogenases.

* Corresponding author. Mailing address: Laboratoire de Biotechnologie-Bioprocédés, UMR CNRS 5504, UMR INRA 792, INSA, 135 Avenue de Rangueil, 31077 Toulouse Cédex 4, France. Phone: 33 5 61 55 94 19. Fax: 33 5 61 55 94 00. E-mail: girbal{at}insa-toulouse.fr.

Applied and Environmental Microbiology, May 2005, p. 2777-2781, Vol. 71, No. 5
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.5.2777-2781.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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