Purification of active methyle coenzyme M reductance from methanothermobacter marburgensis and probing for cooperation between its active sites

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Kemian tekniikan korkeakoulu | Master's thesis
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Master's Programme in Chemical, Biochemical and Materials Engineering
Biological conversion of CO2 and H2 to CH4 is a fundamental global process carried out by methanogenic archaea. Unfortunately, the catalytic mechanism of its critical enzyme methyl-coenzyme M reductase (MCR) is not yet understood on a mechanistic level. Understanding MCR may enable the efficient in vitro synthesis of CH4 from renewable sources. To study the process of methanogenesis and to obtain insights into the catalytic mech-anism of MCR, an anaerobic laboratory was set up under the guidance of Dr. Silvan Scheller at Aalto University, Finland. The model methanogen Methanothermobacter marburgensis was successfully cultivated under anaerobic conditions, and effects of the temperature, the gassing rate, and the concentration of H2S on its growth were studied. Purification of highly active MCR in the laboratory is a challenging task and has only been acheived by a few laboratories in the world1. In this study, MCR purification in its highly active form (> 90% Ni (I) state) was achieved. In vitro methane formation from its natural substrates was assayed using purified MCR. After establishing in vitro assays, the hypothesis of cooperation between the two active sites of MCR was probed by quantifying the relation of Ni (I) in MCR relative to its catalytic rate. The experiments described in this thesis indicate a non-linear correlation between Ni (I) in MCR and its activity. At 50% Ni (I) in MCR, its activity was less than 50%, which implies that both Ni-centers are required to be in Ni (I) state for the enzyme to be active. This study supports the hypothesis that the two active sites of MCR are coupled. During different in vitro methanogenesis experiments, an unexpected high influence of the temperature on the enzymatic reaction was detected, which requires further investigation.
Scheller, Silvan
Thesis advisor
Bao, Jichen
methane production, anaero- bic growth and cultivation, liquid chromatography, M. marburgensis growth condition optimization, enzyme purification, methyl-coenzyme M reductase (MCR), enzyme mechanism, enzyme assay
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