Purification and partial characterization of a murein hydrolase, millericin B, produced by Streptococcus milleri NMSCC 061

Streptococcus milleri NMSCC 061 was screened for antimicrobial substances and shown to produce a bacteriolytic cell wall hydrolase, termed millericin B, The enzyme was purified to homogeneity by a four-step purification procedure that consisted of ammonium sulfate precipitation followed by gel filtration, ultrafiltration, and ion-exchange chromatography. The yield following ion-exchange chromatography was 6.4%, with a greater-than-2,000-fold increase in specific activity, The molecular weight of the enzyme was 28,924 as determined by electrospray mass spectrometry, The amino acid sequences of both the N terminus of the enzyme (NH2 SENDFSLAMVSN) and an internal fragment which was generated by cyanogen bromide cleavage (NH2 SIQTNAPWGL) were determined by automated Edman degradation. Millericin B displayed a broad spectrum of activity against gram-positive bacteria but was not active against Bacillus subtilis W23 or Escherichia coli ATCC 486 or against the producer strain itself, N-Dinitrophenyl derivatization and hydrazine hydrolysis of free amino and free carboxyl groups liberated from peptidoglycan digested with millericin B follow ed by thin-layer chromatography shelved millericin B to be an endopeptidase with multiple activities. It cleaves the stem peptide at the N terminus of glutamic acid as well as the N terminus of the last residue in the interpeptide cross-link of susceptible strains.