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 (NH(2) SENDFSLAMVSN) and an internal fragment which was generated by cyanogen bromide cleavage (NH(2) 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 followed by thin-layer chromatography showed 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.
Beukes
M
M
School of Molecular and Cellular Biosciences, University of Natal, Pietermaritzburg, Scottsville, South Africa.
Bierbaum
G
G
Sahl
H G
HG
Hastings
J W
JW
D000595:Amino Acid Sequence; D002473:Cell Wall / Q000378:metabolism; D006094:Gram-Positive Bacteria / Q000187:drug effects / Q000254:growth & development; D016982:Micrococcus luteus / Q000187:drug effects; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D010457:Peptidoglycan / Q000378:metabolism; D013211:Staphylococcus aureus / Q000187:drug effects; D013291:Streptococcus / Q000201:enzymology*
D010457:Peptidoglycan; D009238:N-Acetylmuramoyl-L-alanine Amidase