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Applied and Environmental Microbiology, January 2000, p. 23-28, Vol. 66, No. 1
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Purification and Partial Characterization of a Murein Hydrolase, Millericin B, Produced by Streptococcus milleri NMSCC 061

M. Beukes,1 G. Bierbaum,2 H.-G. Sahl,2 and J. W. Hastings1,*

School of Molecular and Cellular Biosciences, University of Natal, Pietermaritzburg, Scottsville, South Africa,1 and Institute for Medical Microbiology and Immunology, University of Bonn, D-5300 Bonn, Federal Republic of Germany2

Received 24 May 1999/Accepted 28 September 1999

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 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.

* Corresponding author. Mailing address: School of Molecular and Cellular Biosciences, University of Natal, P.O. Box X01, Scottsville 3209, South Africa. Phone: 27 331 260 5434. Fax: 27 331 260 5435. E-mail: Hastings{at}gene.unp.ac.za.

Applied and Environmental Microbiology, January 2000, p. 23-28, Vol. 66, No. 1
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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