Open AccessHomologous and Heterologous Overexpression in Clostridium acetobutylicum and Characterization of Purified Clostridial and Algal Fe-Only Hydrogenases with High Specific Activities
Author(s) -
Laurence Girbal,
Gregory von Abendroth,
Martin Winkler,
Paul M. C. Benton,
Isabelle Meynial-Salles,
Christian Croux,
John W. Peters,
Thomas Happe,
Philippe Soucaille
Publication year - 2005
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.71.5.2777-2781.2005
Subject(s) - hydrogenase , clostridium acetobutylicum , biochemistry , clostridium , chlamydomonas reinhardtii , heterologous , clostridiaceae , biology , phosphoenolpyruvate carboxykinase , enzyme , chemistry , bacteria , gene , mutant , butanol , genetics , ethanol
Clostridium acetobutylicum ATCC 824 was selected for the homologous overexpression of its Fe-only hydrogenase and for the heterologous expressions of theChlamydomonas reinhardtii andScenedesmus obliquus HydA1 Fe-only hydrogenases. The threeStrep 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 fromC. 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.