Überexpression und Aufreinigung von [FeFe]-Hydrogenasen im Expressionssystem Clostridium acetobutylicum\textit {Clostridium acetobutylicum} und funktionelle Untersuchung

Die [FeFe]-Hydrogenasen HydA1 aus Chlamydomonas reinhardtii und HydA$\tiny {S.o.}$ aus Scenedesmus obliquus konnten mit hohen spezifischen Aktivitäten in Clostridium acetobutylicum heterolog exprimiert und effizient aufgereinigt werden. Die Anpassung der Codon Usage und die Exposition der StrepTagII-Sequenz führten zu einer hundertfachen Steigerung der Proteinausbeute (1mg $\bullet$ 1$_{-1}$). Für HydA aus C. acetobutylicum konnte ein homologes Expressionssystem auf der Grundlage der genomischen Integration durch homologe Rekombination realisiert werden. Für HydA wurde eine neue, besonders hohe spezifische Aktivität für die Protonenreduktion gezeigt. EPR-spektroskopische Untersuchungen an den im Rahmen dieser Arbeit exprimierten [FeFe]-Hydrogenasen ergaben die für intakte H-Kluster erwarteten Spektren. Für HydA1 aus C. reinhardtii gelang zusätzlich die funktionale Immobilisierung auf der Goldoberfläche einer SEIRAS-Messzelle

Immobilization of the [FeFe]-hydrogenase CrHydA1 on a gold electrode: Design of a catalytic surface for the production of molecular hydrogen

Krassen H, Stripp S, von Abendroth G, Ataka K, Happe T, Heberle J. Immobilization of the [FeFe]-hydrogenase CrHydA1 on a gold electrode: Design of a catalytic surface for the production of molecular hydrogen. In: Journal of Biotechnology. JOURNAL OF BIOTECHNOLOGY. Vol 142. ELSEVIER SCIENCE BV; 2009: 3-9.Hydrogenase-modified electrodes are a promising catalytic surface for the electrolysis of water with an overpotential close to zero. The [FeFe]-hydrogenase CrHydA1 from the photosynthetic green alga Chlamydomonas reinhardtii is the smallest [ FeFe]-hydrogenase known and exhibits an extraordinary high hydrogen evolution activity. For the first time, we immobilized CrHydA1 on a gold surface which was modified by different carboxy-terminated self-assembled monolayers. The immobilization was in situ monitored by surface-enhanced infrared spectroscopy. In the presence of the electron mediator methyl viologen the electron transfer from the electrode to the hydrogenase was detected by cyclic voltammetry. The hydrogen evolution potential (-290 mV vs NHE, pH 6.8) of this protein modified electrode is close to the value for bare platinum (-270 mV vs NHE). The surface coverage by CrHydA1 was determined to 2.25 ng mm(-2) by surface plasmon resonance, which is consistent with the formation of a protein monolayer. Hydrogen evolution was quantified by gas chromatography and the specific hydrogen evolution activity of surface-bound CrHydA1 was calculated to 1.3 mu mol H-2 min(-1) mg(-1) (or 85 mol H-2 min(-1) mol(-1)). In conclusion, a viable hydrogen-evolving surface was developed that may be employed in combination with immobilized photosystems to provide a platform for hydrogen production from water and solar energy with enzymes as catalysts. (C) 2009 Elsevier B.V. All rights reserved

Molecular Identification of the Catabolic Vinyl Chloride Reductase from Dehalococcoides sp. Strain VS and Its Environmental Distribution

Reductive dehalogenation of vinyl chloride (VC) to ethene is the key step in complete anaerobic degradation of chlorinated ethenes. VC-reductive dehalogenase was partially purified from a highly enriched culture of the VC-respiring Dehalococcoides sp. strain VS. The enzyme reduced VC and all dichloroethene (DCE) isomers, but not tetrachloroethene (PCE) or trichloroethene (TCE), at high rates. By using reversed genetics, the corresponding gene (vcrA) was isolated and characterized. Based on the predicted amino acid sequence, VC reductase is a novel member of the family of corrinoid/iron-sulfur cluster containing reductive dehalogenases. The vcrA gene was found to be cotranscribed with vcrB, encoding a small hydrophobic protein presumably acting as membrane anchor for VC reductase, and vcrC, encoding a protein with similarity to transcriptional regulators of the NosR/NirI family. The vcrAB genes were subsequently found to be present and expressed in other cultures containing VC-respiring Dehalococcoides organisms and could be detected in water samples from a field site contaminated with chlorinated ethenes. Therefore, the vcrA gene identified here may be a useful molecular target for evaluating, predicting, and monitoring in situ reductive VC dehalogenation