RP-HPLC profile of mutanolysin-digested PGN fragments after incubation with enzyme.
A, S. aureus PGN fragments incubated with elution buffer of AmiA-cat as control. Three peaks around 28 min contain monomer species of one peptide stem with carbohydrates, peaks at 58 min comprise species with two peptide stems, peaks at 73 min cover fragments with three peptide stems, and continuing accordingly. B, S. aureus PGN fragments incubated with AmiA-cat result in completely digested polymers with GlcNAc-MurNAc fragments remaining. C, S. aureus PGN fragments incubated with active site mutant AmiA-H370A show no catalytic activity. D, B. subtilis PGN fragments incubated with elution buffer of AmiA-cat result in a pattern similar to A but with specific retention times for B. subtilis. E, B. subtilis PGN fragments incubated with AmiA-cat exhibit no activity of the staphylococcal amidase for B. subtilis PGN. F, mass analysis of the major product shown in B, which corresponds to the disaccharide GlcNAc-MurNAc (m/z 496).