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Analysis of Peptidoglycan structureAnalysis of Peptidoglycan structure

Analysis of peptidoglycan structure

Isolation and purification of peptidoglycan

For peptidoglycan analysis 3-4 g wet weight of cells (centrifuge pellet) is needed. The wet biomass can be sent either in frozen state on dry ice or suspended in isopropanol/water (1:1, v/v) at ambient temperature. The use of lyophilized biomass may cause problems in the purification of the peptidoglycan preparations from contaminating proteins.

The peptidoglycan is isolated after disruption of the cells by shaking with glass beads in a Vibrogen cell mill (Johanna Otto GmbH, Germany), subsequent trypsin digestion and treatment with sodium dodecylsulfate according to the method of Schleifer (1985).

Peptidoglycan structure

Analysis of the peptidoglycan structure is a requirement for all members of novel Gram-positive genera when they are described and at least the amino acid composition should
be provided for every novel Gram-positive species described (Tindall et al., 2010).

Depending on the complexity of the peptidoglycan structure, the elucidation may be rather tedious and time-consuming as different derivatization procedures and analytical techniques (TLC, 2D-TLC, GC, MS) must be applied. Information on the genus affiliation of the strain under study is helpful for the selection of appropriate techniques and standards. The peptidoglycan structure is concluded from the detected products of hydrolyses. A complete elucidation of hitherto unknown peptidoglycan structures by the DSMZ Identification Service is not always possible.

The peptidoglycan is hydrolyzed in 3 experiments:

  1. Total hydrolysis: 4N HCl, 100°C, 16 hours; results in amino acids and hydrolytically relatively resistant peptides (as e.g. L-Lys - D-Asp)
  2. Super total hydrolysis: 6N HCl, 120 °C, 16 hours; results in amino acids and cleaves even resistant peptides
  3. Partial hydrolysis: 4N HCl, 100 °C, 45 minutes; results in short peptides and amino acids

The hydrolysates are analyzed by TLC and 2D-TLC on cellulose plates by using the solvent systems of Rhuland et al. (1955) and Schleifer & Kandler (1972), respectively. The approximate molar ratio of amino acids is analyzed by gas chromatography/mass spectrometry (MacKenzie, 1987). In special cases it is necessary to determine the N-terminus of the interpeptide by dinitrophenylation according to Schleifer (1985) and/or to analyze the enantiomers of amino acids by gas chromatography using a chiral column (Frank et al., 1980).

The peptidoglycan analyses are performed by the DSMZ Identification Service according to published protocols (Schumann, 2011).


  • Rhuland, L. E., Work, E., Denman, R. F. & Hoare, D. S. (1955). The behaviour of the isomers of 2,6-diaminopimelic acid on paper chromatograms. J Am Chem Soc 77, 4844-4846.
  • Schleifer, K. H. (1985). Analysis of the chemical composition and primary structure of murein. Methods Microbiol 18, 123-156.
  • Schleifer, K. H. & Kandler, O. (1972). Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36, 407-77.
  • Schumann, P. (2011). Peptidoglycan structure. Methods Microbiol 38, 101-129.
  • Tindall, B. J., Rosselló-Móra, R., Busse, H.-J., Ludwig, W. & Kämpfer, P. (2010 ). Notes on the characterization of prokaryote strains for taxonomic purposes. Int J Syst Evol Microbiol 60, 249-266.

Photo of Peter  Schumann
Dr. Schumann, Peter
Phone: +49-531/2616-106

MALDI-TOF mass spectrometry of bacteria, RiboPrinting, peptidoglycan analysis, determination of DNA base composition