Engineering [FeFe]-hydrogenases for hydrogen bio-gas production by artificial devices

The recent development of recombinant expression systems for [FeFe]-hydrogenases led to availability of good amounts of these enzymes that can be exploited for different purposes. In this study, the recombinant Chlamydomonas reinhardtii hydrogenase (CrHydA1) and Clostridium acetobutylicum hydrogenase (CaHydA) were purified and characterized to confirm their similarity with native forms. Immobilization of the hydrogenase on electrode is the key step to the construction of artificial devices that exploit the highly evolved ability of [FeFe]-hydrogenases to catalyze hydrogen production without the need for expensive noble metals. In such a device, electrons are directly supplied to the hydrogenase that will use water derived protons to produce hydrogen gas. CrHydA1 and CaHydA were expressed in the active form in Escherichia coli and affinity purified with a yield of milligrams per litre of culture. The whole process was performed under strict anaerobic conditions to avoid irreversible inactivation of the enzymes. In these conditions, enzymes could be stored for months without any loss of activity. Purified hydrogenases were highly pure, as estimated by silver stained SDS-PAGE gels. Functional characterization confirmed that recombinant CrHydA1, in particular, is highly thermo stable; activity is conserved up to 60°C and the half-life at 50°C is about 10 hours. These features will be particularly useful in artificial devices and other future applications. Since no crystal structures are available for CrHydA1 and CaHydA, homology models were computed to investigate structural features. Circular dichroism spectroscopy was used to experimentally study the secondary structure of the enzymes; these results showed consistency with models. Preliminary trials of immobilization on different types of electrodes are under study to identify the best targets for the construction of the cathodic hydrogen evolving electrode. In conclusion, the results presented here on the production, purity and stability of recombinant CrHydA1 and CaHydA, together with preliminary immobilization data, confirm the feasibility of using these enzymes for the construction of artificial hydrogen producing devices.