[FeFe]-hydrogenases for hydrogen gas production: evolving natural bio-catalysts for artificial devices

In this study, a recombinant expression system for [FeFe]-hydrogenase was used to produce and characterize Chlamydomonas reinhardtii hydrogenase (CrHydA1) and Clostridium acetobutylicum hydrogenase (CaHydA). The suitability of these enzymes as bio-catalysts in artificial devices was tested. In such devices, electrons and protons are supplied to the hydrogenase through nanostructured electrodes. Stability and optimized interaction/electron transfer with electrodes are the key elements to exploit the highly evolved ability of [FeFe]-hydrogenases to catalyze hydrogen production without the need for expensive noble metals. CrHydA1 and CaHydA were expressed in the active form in E. coli and affinity purified with a yield of milligrams of pure protein per litre of culture. The whole process was performed under strict anaerobic conditions to avoid inactivation of the enzymes. Functional characterization confirmed that recombinant CrHydA1 and CaHydA are highly thermo stable: activity is conserved up to 60-70°C with a half-life of several hours at high temperatures. These features will be particularly desirable in artificial devices and other future applications. 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. Directed evolution techniques are being applied to engineer new variants of the recombinant [FeFe]-hydrogenases with improved catalytic properties and enhanced stability to oxygen. To this end a fast screening method for hydrogenase activity was set up.