Homologous and heterologous overexpression in Clostridium acetobutylicum and characterization of purified clostridial and algal Fe-only hydrogenases with high specific activities.
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 micromol H(2) min(-1) mg(-1), while HydA from C. acetobutylicum (HydA(Ca)) shows the highest activity (5,522 micromol H(2) 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 HydA(Ca) protein indicates a characteristic rhombic EPR signal that is typical for the oxidized H cluster of Fe-only hydrogenases.
Girbal
Laurence
L
Laboratoire de Biotechnologie-Bioprocédés, UMR CNRS 5504, UMR INRA 792, INSA, 135 Avenue de Rangueil, 31077 Toulouse Cédex 4, France. girbal@insa-toulouse.fr
von Abendroth
Gregory
G
Winkler
Martin
M
Benton
Paul M C
PM
Meynial-Salles
Isabelle
I
Croux
Christian
C
Peters
John W
JW
Happe
Thomas
T
Soucaille
Philippe
P
D000460:Chlorophyta / Q000201:enzymology*; D046969:Clostridium acetobutylicum / Q000235:genetics*; D004578:Electron Spin Resonance Spectroscopy; D006864:Hydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis*
D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; C118830:iron hydrogenase; D006864:Hydrogenase