Abstract:
Hydrogenases for the production of hydrogen are found in many microorganisms. The isolation of a stable hydrogenase enzyme would be beneficial for the development of an in vitro system for hydrogen production as a fuel. In this project two approaches were taken for the expression, purification and isolation of [FeFe]-hydrogenase. Firstly, native [FeFe]-hydrogenase expression, purification and isolation from the green alga Chlamydomonas reinhardtii (124+) was attempted. Hydrogen production was successfully achieved in sealed, anaerobic C. reinhardtii cultures and verified by gas chromatography thereby confirming hydrogenase was successfully expressed. However, due to the high oxygen sensitivity of hydrogenase and the subsequent inhibition of this enzyme at low levels of oxygen the native [FeFe]-hydrogenase could not be successfully isolated from C. reinhardtii cultures. To partially circumvent this problem the second main focus of this thesis was the expression, purification and isolation of recombinant [FeFe]-hydrogenase from C. reinhardtii and Clostridium acetobutylicum. However, transformation of a full complement of genes encoding recombinant [FeFe]-hydrogenase from C. reinhardtii into E. coli proved difficult. This was probably due to incompatibilities arising from the expression of eukaryotic hydrogenase, and the co-expression of essential assembly proteins that are required for a functioanl enzyme in C. reinhardtii, in the E. coli prokaryotic expression system. To overcome this limitation the genes encoding hydrogeanse from the bacterium C. acetobutylicum were introduced into E. coli which resulted in the succesful expression and isolation of hydrogenase as judged by SDS-PAGE and western blotting. However, as isolation methods could not be performed under anaerobic conditions, catalytic activity of the purified enzymes was not demonstrated. Nonetheless, this work has established a procedure to obtain an isolated hydrogenase for future use in synthetic systems for the production of hydrogen that will ultimately be economically viable and competitive in a future hydrogen economy.
