A novel endopeptidase degrading the pel)tide cross-links in sacculi has been isolated from Escherichia coli and purified to homogeneity. I'lhe enzyme has a molecular weight of 30,000 and, in contrast to already known enzymes of similar specificity, remains fully active in the presence of/3-lactam antibiotics. In adldition, it is exceptional in being inhibited by single-strandetl tleoxyribonucleic acid aild by some polynucleotides. The possible role of the enzymne in cell div-ision is discussed.Hydrolytic enzymes (1,2,6,11,12,18) participate in the metabolism of nmurein sacculi (21) and uindoubtedly p)lay a role in the maintenance of bacterial cell shape. In Escherichia coli, however, the precise biological role of the many murein hydrolases which have been identified remains largely obscure. Among these enzymes, endopeptidases split peptide bridges cross-linking the glycan chains of murein and ar-e strongly inhibited by penicillins, although they are not assumed to be the lethal targets of /3-lactan antibiotics (5,9). I)uring purification of a l)enicillin-sensitive Dr-endopeptidase found to be identical with D-alanine-carboxypeptidase IB (18), we found a novel species of DD-endopeptidase which was totally insensitive to p)enicillin G; some prol)erties of the enzyme have been described previously (20; U. Schwarz, W. Keck, S. Klencke, and H. Mett, Abstr. Svmnip. Finctions Microb. Membranes, 1977). The enzyme thus belongs to a class of hydrolases which remain active in the presence of penicillin and mav be the cause of pericillin-in(duced cell lvsis.In this paper we report on the purification to homogeneity and the p)roperties of the novel endopeptidase. This enzyme is interesting not only because of its biological function but also because it has already been proven to be a powerful analytical tool; we have used it to elucidate the arrangement of the glycan chains within macromolectular sacculi (20).
MATERIALS AND METHODSCell extraction and initial enrichment of endopeptidase. The enizyme was isolated fromii E. colli IPA 3092 (14) (iprn Iys), harvested at mid-exponential growth phase, aindl kept frozein at -70C. Cells (2(10 g, wet weight) were openied by shaking with glass beads in a tell mill in 500 mlt of Tris-miialeate (10 imiM; pH (6.8)-10 nmM MgSOi, containing 4(0 pg of DNase per ml (from bovine panc reas -450 Ktnit.z uInits per nmg, Serva). lThe filteired suspeiision (4) was cetntrifuged (6(1 mtmi at 48,0()) x g), the supernatant was (lialyzed three tiies againlst 5 litei-s Of 1'ris-miialeate htffer (1(1 mlM: pHI 5.2). 10 nmM MgSO, and 0.1 miiM dithioetrthritol, cleared by cntitl iftigatioIn (3(1 mIi, 28,0(1 X g (,aid aplplied On a (-olumn (2.6 h)y 31 (cmt; flow rate, :32 mIl/h) of CM Sepharose (I,-6B (IPharmacia Fine Chemiicals eqLilibrated with dialsis btuffer. T'he colunm was eluted with a (ont aeB giadtient (1380 ml of equilibration buffer, :120 ml of 0.5 M KCI in the sainet buffer).En(lopepti(lase was foundl in a hroatd p)eak letwe1tn ().andl (1.3 M K(CI The pooled fraot-tions with endopeptidase were ...