Partial purification and characterization of N-acetylmuramyl-l-alanine amidase from human and mouse serum

The enzyme N-acetylmuramyl-l-alanine amidase (mucopeptide amidohydrolase, EC 3.5.1.28) has been detected in human, mouse, rabbit, bovine and sheep sera. A method for detection of amidase activity using [14C]peptidoglycan monomer as the substrate has been developed. Partial purification of human and mouse amidase was achieved by gel chromatography on Bio-Gel A-l.5 m, DEAE-Sephadex A-50 and Sephadex G-100. Both amidase preparations exhibited maximal activity at pH 9.0 in Tris-HCl buffer and required Mg2+ for maximal activity. Following digestion of peptidoglycan monomer, GlcNAc-MurNAc-l-Ala-d-isogiutaminyl-meso-diaminopimelyl-d-Ala-d-Ala, the disaccharide GleNAc-MurNAc and the corresponding pentapeptide l-Ala-o-isoglutaminyl-meso-diaminopimelyl-o-Ala-o-Ala were formed and subsequently isolated and chemically characterized. The enzyme therefore acts as an N-acetylmuramyl-l-alanine amidase by cleaving the bond between N-acetylmuramic acid and l-alanine. The glycan linked, peptide-not-cross-linked peptidoglycen dimer was also shown to be a substrate for human and mouse amidase.