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Lactate dehydrogenase from an extreme thermophile was partially purified and its substrate kinetics were investigated.
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The bacteria were grown on two types of media, either a minimal medium or minimal medium supplemented with brain-heart infusion. Lactate dehydrogenase was found to be more thermostable when obtained from cells grown in supplemented medium.
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Lactate dehydrogenase of both low and high thermostability exhibited a Km :for pyruvate of about 30 g2r at 32~ and 125 aM at 63~ At temperatures below 50~ kinetics for both pyruvate and NADIt were hyperbolic, at higher t.emperatures the enzyme showed sigmoidal kinetics. Of several possible regulatory compounds investigated, only ADP and phosphoenolpyruvate were found to be inhibitory; no activators were found.
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Complete and immediate inhibition was observed over a wide temperature range after the addition of!oara-chloromercuribenzoate; the inhibition was reversed by the addition of thiols. Lactate oxidation was not observed withfl,1 the pH range of 5.5 to 9.5. The molecular weight of the enzyme was estimated to be 70,000 daltens by gel filtration.
Three distinct bacteria isolated from hot springs in Yellowstone National Park (U.S.A.) have been described by Heinen and Heinen (1972) who have classified them in the genus Bacillus. One of these, with the suggested name Bacillus caldolyticus is characterized by maximal growth yield at 72~ growth up to 84~ production of extracellular protease, amylase and pyrophosphatase, and ability to grow on a single carbon source such as pyruvate, lactate or glucose without added organic materials. The organism belongs to the group of extremely thermophilic bacteria, characterized by optimal growth above 70~ (Brock and Freeze, 1969;Heinen, 1971). Such extremely thermophflic bacteria present several interesting biological problems since they are able to grow at temperatures at which protein is usually denaturated, and at which nucleic acids melt. (Friedman, 1968 ;Campbell and Pace, 1968). It was decided to investigate an intracellular enzyme of B. catdolyticus to