Dye-linked L-malate dehydrogenase from thermophilic Bacillus species DSM 465. Purification and characterization.
The distribution of dye-linked L-malate dehydrogenase (L-malate: acceptor oxidoreductase, EC 1.1.99.16) was investigated in many thermophilic bacteria. The enzyme occurred widely in thermophilic spore-forming bacteria like bacilli and thermoactinomycetes. The enzyme was purified to homogeneity from a thermophile, Bacillus sp. DSM 465, with a 2.7% overall recovery by DEAE-Toyopearl column chromatography, Sephacryl S-400 column chromatography and preparative slab PAGE. The enzyme had a molecular mass of about 660 kDa and consisted of about ten subunits all with a molecular mass of 66 kDa. The enzyme retained its full activity upon heating at 55 degrees C for at least 60 min and with incubation at pH 5.0-10.0, 55 degrees C, for 10 min. The enzyme exclusively catalyzed L-malate dehydrogenation in the presence of an electron acceptor such as 2,6-dichloroindophenol. The Michaelis constants for L-malate and 2,6-dichloroindophenol were determined to be 1.67 mM and 0.050 mM, respectively. FAD was identified as a prosthetic group of the enzyme by HPLC.
Department of Chemistry, Kyoto University of Education, Japan.
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Ohshima
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Tanaka
D001407:Bacillus / Q000201:enzymology*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004396:Coloring Agents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D010071:Oxaloacetates / Q000378:metabolism; D010101:Oxygen Consumption; D013379:Substrate Specificity
D004396:Coloring Agents; D010071:Oxaloacetates; D006861:Hydrogen Peroxide; D008291:Malate Dehydrogenase; C081608:malate dehydrogenase (acceptor)