COMPARISON OF DIFFERENT METHODS FOR METABOLITE EXTRACTION FROM ASPERGILLUS NIGER MYCELIUM
National Institute of Chemistry, Laboratory of Biotechnology, Hajdrihova 19, SI 1001 Ljubljana, Slovenia
Two ways of quenching metabolism and four procedures for extraction of intracellular metabolites were elaborated with the filamentous fungus Aspergillus niger. Inactivation of metabolism by rapid quenching of the mycelium in liquid nitrogen or in 60% methanol solution kept at –40 °C gave similar, yet too low results after extraction with boiled buffered ethanol. Stability of analytical grade metabolites in solutions of known concentrations after acid and alkaline treatments resulted in minor deviations of 4-7%. Results obtained by extraction in buffered boiling ethanol were up to 30% too low, suggesting the reliability of the method questionable. Concentrations of metabolites present in fungal mycelia during fermentation varied with time. Amounts of pyruvate in Aspergillus niger mycelia varied from 0.5 to 2.0 mmol/g dry biomass, 2-oxoglutarate ranged between 0.2 and 0.8 mmol/g dry mycelium and malate from 3.0 to 16.0 mmol/g respectively, depending on time of cultivation. Both, intracellular and extracellular metabolites could be measured. Extractions of Aspergillus niger mycelia by perchloric acid and by sodium hydroxide were efficient and reliable, whereas treatment with buffered boiling ethanol returned too low amounts of metabolites examined.
Key words: quenching ofmetabolism, Aspergillus niger, extraction of metabolites, enzymes, <<a niger-2-1v3.jpg>>fermantation