Abstract
The perchloric acid extraction method for measuring concentrations of intracellular intermediary metabolites was adapted and optimized forCandida tropicalis. Cells were harvested by filtration and rapidly frozen in liquid nitrogen in order to image anin vivo situation. Intracellular metabolites were determined with NAD/NADH linked enzymatic assays and detected spectrofluorimetrically, which allowed determinations in the mM range. The assays allow measurements of 50–60 samples at the same time. Six different metabolites (fructose 1,6-diphosphate, pyruvate, phosphoenolpyruvate, citrate, malate and fumarate) could be determined in the same sample. The method was applied to study the relation of product formation and the intracellular intermediary metabolite concentrations inCandida tropicalis at varying degrees of oxygen limitation in xylose fermenting cells. Maximum metabolite concentrations as well as maximum ratio of Krebs cycle to glycolysis metabolites were found at 5 mM/h OTR, at which point maximum ethanol production was observed.
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