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 a nd shown to produce a bacteriolytic cell wall hydrolase, termed millericin B, The enzyme was purified to homogeneity by a four-step purification proce dure that consisted of ammonium sulfate precipitation followed by gel filtr ation, ultrafiltration, and ion-exchange chromatography. The yield followin g ion-exchange chromatography was 6.4%, with a greater-than-2,000-fold incr ease 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 fragme nt which was generated by cyanogen bromide cleavage (NH2 SIQTNAPWGL) were d etermined by automated Edman degradation. Millericin B displayed a broad sp ectrum of activity against gram-positive bacteria but was not active agains t Bacillus subtilis W23 or Escherichia coli ATCC 486 or against the produce r 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 pep tide at the N terminus of glutamic acid as well as the N terminus of the la st residue in the interpeptide cross-link of susceptible strains.