Espressione, purificazione e caratterizzazione funzionale dell'idrogenasi ricombinante HydA1 da Chlamydomonas reinhardtii

Hydrogenases are a class of enzymes responsible for hydrogen gas production in many microorganisms. Their study is considered of great interest for biotechnological applications to support a fossil fuel-free world. To this end, the construction of artificial devices that are able to use solar energy to produce hydrogen (the so- called ¿nano-leaf¿) has been proposed. Because of its relatively simple structure and high specific activity, Chlamydomonas reinhardtii HydA1 hydrogenase (CrHydA1) is an enzyme of outstanding interest for this purpose. Thus, the aim of this study is to produce and characterize CrHydA1. Since CrHydA1 is quickly inactivated by oxygen, this study required the development of protocols for CrHydA1 production, manipulation and storage in strictly anoxic conditions. The recombinant expression system in Escherichia coli developed at the National Renewable Energy Laboratory (NREL), USA, was used to produce and purify CrHydA1 to homogeneity with a specific activity of about 60 μmol H2 ¿ min-1 ¿ mg protein-1 and a yield of 0.4 mg protein per litre of culture. Produced samples were stored at -20°C for months without any loss of activity. The recombinant expression system was further characterized in terms of recombinant proteins expression levels; various modifications of the purification protocol were evaluated. CrHydA1 hydrogen oxidation activity was characterized at different pH and temperature. Besides the specific information on hydrogen oxidation activity, these experiments demonstrated that CrHydA1 is not only very stable in alkaline conditions, up to pH 11, but also very stable in terms of temperature, up to 60°C. Moreover, 50% activity was conserved after 10 hours incubation at 50°C. This intrinsic thermo stability of CrHydA1 is an important factor in its biotechnological application in the construction of the ¿nano-leaf¿ due to the fact that when exposed to solar light, this device will reach high temperatures and the availability of thermo stable components will be essential. In the absence of a crystal structure for CrHydA1, a computer model, based on homology, was produced and evaluated. Also, for the very first time, the CrHydA1 protein structure was experimentally studied by circular dichroism spectroscopy and the far UV spectrum led to the prediction of the secondary structure content that is in good accordance with that predicted with the model. Preliminary trials of CrHydA1 immobilization on different electrodes were carried out. The obtained results suggest the possibility of immobilizing the enzyme on TiO2 or carbon based electrodes. Finally, the data presented in this work on the production, purity and stability of CrHydA1 together with preliminary immobilization results, confirm the feasibility of using this enzyme in the construction of artificial ¿nano-leaf¿ device.