グルタミン酸生産菌Brevibacterium flavumからのリンゴ酸脱水素酵素の精製と分子形態

概要

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Malate dehydrogenase (MDH) [EC 1.1.1.37] was extracted from Brevibacterium flavum cells by disruption in a blender containing glass beads. The enzyme was purified to homogeneity by using ammonium sulfate precipitation, DEAE-Toyopearl and AF-Blue Toyopearl column chromatographies. MDH was purified 200-fold with 11% yield. Its amino-terminal sequence (residues 1 to 8) matched to the sequence (residues 2 to 9) of the MDH from Corynebacterium glutamicum (GenBank accession number, CAC83073). The molecular weight of the native enzyme was 130,000. The protein was a homotetramer, with a subunit molecular weight of 33,000. Lineweaver-Burk plots of the MDH reactions at pH 6.5 were linear with respects to NADH, NADPH and oxalacetate, indicating no allosteric effect in this enzyme. The enzyme has almost equal kcat values in the both reaction systems of NADH-oxalacetate and NADPH-oxal acetate. But Km values of the former with respect to the substrates were much smaller than the latter.
2003-01-31