Characterisation of the enzymes involved in methanol dissimilation in Bacillus methanolicus: Assessment of enzyme-based biohydrogen production for a cell-free biofuel cell

Abstract

Hydrogen is a clean, high energy content, low emission energy carrier. The sustainable production of hydrogen from renewable sources is of particular interest in a world with greater emphasis on green credentials for its growing energy use.

The generation of molecular hydrogen by both whole cells and isolated enzymes has been proposed as a means of fuelling conventional fuel cells. The use of isolated enzymes to produce H2 has been the subject of few studies, with the construction od synthetic enzymatic pathways. Here, starting from methanol feedstock, a cheap and high energy density fuel, we propose a three enzyme synthetic pathway built around enzymes from methanol-utilising thermophiles. Methanol requires only three successive dehydrogenase enzymes to remove all of its H atoms. From the thermophilic methanol-utiliser Bacillus methanolicus, a methanol dehydrogenase and a formaldehyde dehydrogenase were identified, cloned and expressed, while a formate dehydrogenase was sourced from Geobacillus thermoglucosidasius. Enzymes were expressed heterologously in E. coli as well as homologously in a Bacillus subtilis-based system.

Soluble expression was achieved for the methanol dehydrogenase and its activator protein. The enzyme was only active in the backwards direction (formaldehyde reductase) in vitro. The kinetics parameters were estimated at KM = 1.2 mM and Vmax = 2.5 U.mg-1 and were similar for the heterologously and homologously expressed enzyme. The formaldehyde dehydrogenase and formate dehydrogenase could not be purified in a soluble form and no kinetics data could be obtained.

Date of Award	1 Nov 2010
Original language	English
Awarding Institution	University of Bath
Supervisor	Michael Danson (Supervisor) & David Hough (Supervisor)