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Determination of the phosphorylated sugars of the Embden‐Meyerhoff‐Parnas pathway in Lactococcus lactis using a fast sampling technique and solid phase extraction
Author(s) -
Jensen Niels B. S.,
Jokumsen Kirsten V.,
Villadsen John
Publication year - 1999
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(19990505)63:3<356::aid-bit12>3.0.co;2-1
Subject(s) - chemistry , lactococcus lactis , chromatography , extraction (chemistry) , biochemistry , lactic acid , yeast , enzyme , quenching (fluorescence) , bacteria , biology , fluorescence , genetics , physics , quantum mechanics
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°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°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%. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 63: 356–362, 1999.