Determination of the phosphorylated sugars of the Embden-Meyerhoff-Parnas Pathway in Lactococcus lactis using a fast sampling technique and solid phase extraction

Niels B.s. Jensen, Kirsten Væver Jokumsen, John Villadsen

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    Abstract

    An experimental procedure for the determination of intracellular concentrations of the phosphorylated sugars in the lactic acid bacterium Lactococcus lactis is presented. The first step of the procedure is a rapid sampling of a small volume of the growth medium into 60% (v/v) methanol precooled to -35 degrees C, bringing about a fast and complete stop of all metabolic activity. In contrast to yeast the metabolites leak out of the cells when these are brought into contact with methanol and are present in the medium and in the biomass after the quenching. A liquid-liquid extraction with chloroform at -25 degrees C ensures a total permeability of the cellular membrane towards the metabolites of interest as well as the inactivation of enzymes liable to alter their levels. The final step of the procedure consists in a solid phase extraction using columns with a high affinity for phosphorylated components. The internal standard was recovered to an extent of 85-95%. (C) 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 63: 356-362, 1999.
    Original languageEnglish
    JournalBiotechnology and Bioengineering
    Volume63
    Issue number3
    Pages (from-to)356-362
    ISSN0006-3592
    DOIs
    Publication statusPublished - 1999

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