Reliable and sensitive analysis of amino acids in the peptidoglycan of actinomycetes using the advanced Marfey's method

For the chemotaxonomic classification of actinomycetes, we developed a new method for the detection of the 2, 6-diaminopimelic acid (A2pm) stereoisomers and 3-hydroxy diaminopimelic acid (3-OH A2pm) in the cell wall peptidoglycan of actinomycetes using “the advanced Marfey's method”, which consists of a chromatographic technique for the separation of amino acids into each of its enantiomers by derivatization with 1-fluoro-2, 4-dinitrophenyl-5-l-luecinamide (l-FDLA) and d-FDLA, and a detection method using liquid chromatography/mass spectrometry (LC/MS). This method was successfully applied to determine the absolute configuration of the A2pm and detect the 3-OH A2pm included in the cell wall peptidoglycan of the standard strains. Because the procedure can be performed with very small amounts of an amino acid, it was possible to use one colony from an agar plate of the actinomycetes for the acid hydrolysis. In addition, the constituent amino acids and their absolute configurations in the cell wall of the actinomycetes could be simultaneously determined. Thus, a reliable, sensitive and rapid analytical method for amino acids including A2pm in the peptidoglycan of the microorganisms was established.