A novel [FeFe]-hydrogenase from C. perfringens. Characterization and immobilization on TiO2 electrodes as catalyst for hydrogen producing devices

Nowadays the research of new energy sources in place of fossil fuels is of paramount importance and hydrogen is considered a promising alternative as renewable, clean and low cost energy carrier. For this purpose, hydrogen producing devices are being developed, including bio-hybrid devices in which [FeFe]-hydrogenases can be used as very efficient catalysts instead of noble metals like platinum [1]. In this work a novel [FeFe]-hydrogenase CpHydA (encoded by gene CPF_2655) from a C. perfringens strain isolated in a pilot plant with high hydrogen productivity [2] was expressed, characterized and immobilized on TiO2 electrodes. The heterologous expression in E. coli was achieved thanks to a previously established system [3] and it allowed to obtain in high yields a pure and active protein. The FT-IR signal recorded in the region from 2100 to 1800 cm-1, typical of the active site H-cluster, confirmed the correct assembly of the enzyme. The kinetic parameters were measured with the artificial electron donor methyl viologen (MV), showing a KM of 6±1 mM and a kcat (or TurnOver Frequency, TOF) of up to 1600 sec-1.Active and correctly orientated hydrogenase on TiO2 takes electrons from the electrode, on which it can be stably adsorbed, and protons from the acqueous medium to produce H2. High current densities, up to 2 mA /cm2, were recorded by cyclic voltammetry for CpHydA on TiO2 (anatase) electrodes (20 nm particle size, 250 nm thickness and 100 nm particle size, 4 m thickness), even with a very low electroactive coverage. Chrono-amperometry experiments performed at an applied potential of -0.741 V demonstrated that the protein remained catalytically active up to 2 hours with a hydrogen production efficiency of about 98% and an estimated TOF of at least 4 sec-1. Therefore this novel enzyme resulted to be a stable catalyst on the bio-electrode, with H2 production accomplished at high efficiency and by applying a small driving force. This work was supported by projects BIOH2 and HyStrEM (EU structural funds N.1083/2006 P.O.R. Regione Piemonte F.E.S.R. 2007/2013). S.M. is supported by Assegno Cofinanziato Biennale from UNITO. The authors are grateful to Dr. P.W. King for the generous gift of the plasmids for expression systems in E.coli. References: 1. Cracknell, J.A., et al. "Enzymes as working or inspirational electrocatalysts for fuel cells and electrolysis" Chem. Rev. 2008; 108: 24392461. 2. La Licata B., et al. "Biohydrogen production from organic wastes in a pilot plant reactor and its use in a SOFC" Int. J. Hydrogen Energy 2011; 36: 78617865. 3. King P.W., et al. "Functional studies of [FeFe] hydrogenase maturation in an Escherichia coli biosynthetic system " J. Bacteriol. 2006; 188: 21632172.