Purification and characterization of fumarase from the syntrophic propionate-oxidizing bacterium strain MPOB |
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Authors: | Bernardina L M Van Kuijk Nico-Dirk Van Loo Alexander F Arendsen Wilfred R Hagen A J M Stams |
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Institution: | (1) Department of Microbiology, Agricultural University, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands Tel. +31-317-483101; Fax +31-317-483829 e-mail: fons.stams@algemeen.micr.wau.nl, NL;(2) Department of Biochemistry, Agricultural University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands, NL |
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Abstract: | Fumarase from the syntrophic propionate-oxidizing bacterium strain MPOB was purified 130-fold under anoxic conditions. The
native enzyme had an apparent molecular mass of 114 kDa and was composed of two subunits of 60 kDa. The enzyme exhibited maximum
activity at pH 8.5 and approximately 54° C. The K
m
values for fumarate and l-malate were 0.25 mM and 2.38 mM, respectively. Fumarase was inactivated by oxygen, but the activity could be restored by
addition of Fe2+ and β-mercaptoethanol under anoxic conditions. EPR spectroscopy of the purified enzyme revealed the presence of a 3Fe-4S]
cluster. Under reducing conditions, only a trace amount of a 4Fe-4S] cluster was detected. Addition of fumarate resulted
in a significant increase of this 4Fe-4S] signal. The N-terminal amino acid sequence showed similarity to the sequences of
fumarase A and B of Escherichia coli (56%) and fumarase A of Salmonella typhimurium (63%).
Received: 15 September 1995 / Accepted: 13 November 1995 |
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Keywords: | Fumarase Syntrophy Propionate oxidation Fumarate fermentation Anaerobic oxidation Iron-sulfur cluster |
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