Clonaggio, espressione, purificazione e caratterizzazione della [FeFe]-idrogenasi da Clostridium tyrobutyricum

The decrease in fossil energy resources will lead to the emergence of new energy sources. Among the new fuel to come, hydrogen gas is one of the most promising, since its combustion yields water and water can be used to regenerate H2 (Vignais 2004). Molecular hydrogen is a key intermediate in the metabolic interactions of a wide range of micro-organisms. Among the microorganisms capable of fermentative H2 production, strict anaerobes such as members of the genus Clostridium are the most widely studied (Calusinska 2010). The key enzyme involved in the metabolism of H2 is the hydrogenase (Vignais 2004). Hydrogenases are redox metalloenzymes divided into three main groups based on their metallocenter composition: [NiFe]-hydrogenases, [FeFe]-hydrogenases and [Fe]-hydrogenases (Meyer 2007). They catalyze the activation of molecular H2 through the reversible reaction H2 ⇄ 2H+ + 2e−. Physiologically, they can function to couple H2 oxidation to energy yielding processes, to evolve H2 at high turnover rates as a means to dispose of reducing equivalents that accumulate during fermentation (Mulder 2013). The aim of this study was to clone, express, purify and characterize the [FeFe]-hydrogenase from Clostridium tyroburtyricum, which is a Gram positive, anaerobic bacterium, that was previously isolated from an agricultural waste biomass during fermentative processes. C. tyrobutyricum expresses a [FeFe]-hydrogenase, which is 577 amino acids long and has the typical M3 structure. Since little was known about this enzyme, starting from the genomic DNA, its gene was cloned into two plasmids in order to produce two versions of the protein: one simple with the streptavidin tag at the C terminal and another one with an amino acidic spacer at the C terminal followed by the tag. The cloning was performed in the E. coli Dh5α strain cells. The two recombinant proteins (without and with the spacer) were expressed with a heterologous recombinant expression in the E.coli Rosetta 2 (DE3) strain cells and then purified by affinity chromatography (Strep-Tag system). First, the yield, purity and hydrogen evolution and uptake of the two versions of the [FeFe]-hydrogenase were compared to decide which one was the best. As a result, the simple protein without the spacer showed the higher yield (5.41 mg/l), purity and activity (kcat H2 evolution: 1788 s-1, kcat H2 uptake: 2364 s-1) so it was further characterized studying its activity at different pH in H2 evolution (more active at pH 8) and uptake (more active at pH 9), FTIR spectra to see the H-cluster, absorption and circular dichroism spectra to see the FeS centers. Moreover the homology modeling of the [FeFe]-hydrogenase and the holoprotein maturation vitro were done. In conclusion, this seemed to be a promising [FeFe]-hydrogenase, with high efficiency of catalysis and with high yields of expression, potentially applicable to biotechnological and industrial use for the production of molecular hydrogen.