Open AccessPurification and partial characterization of the extracellular γ‐D‐glutamyl‐(L) meso ‐diaminopimelate endopeptidase I, from Bacillus sphaericus NCTC 9602
Author(s) -
GARNIER Michèle,
VACHERON MarieJeanne,
GUINAND Micheline,
MICHEL Georges
Publication year - 1985
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1985.tb08873.x
Subject(s) - bacillus sphaericus , endopeptidase , extracellular , microbiology and biotechnology , chemistry , biology , bacillales , biochemistry , bacteria , enzyme , genetics , bacillus subtilis
The γ‐D‐glutamyl‐(L) meso ‐diaminopimelate endopeptidase, or endopeptidase I, from Bacillus sphaericus 9602 was purified to apparent protein homogeneity. The purification was achieved by a six‐step procedure: ammonium sulfate fractionation, phenyl‐Sepharose chromatography, two consecutive DEAE‐Trisacryl chromatographies, chromatofocusing and Sephacryl S‐200 permeation chromatography. The enzyme was purified 5000‐fold with a 38% recovery of lytic activity. It is an acidic protein (pI 5.4) of hydrophobic nature. Kinetic studies have shown a K m value of 0.57 mM and an apparent K max of 8.3 μmol min −1 (mg enzyme) −1 with N ‐acetylmuramyl‐ l ‐alanyl‐γ‐D‐glutamyl‐(L) meso‐diaminopimelyl (L)‐D‐[ 14 C]alanine as substrate. The enzyme was inhibited by o ‐phenanthroline and EDTA and was reactivated by zinc, cobalt and manganese ions; thus endopeptidase I is a metallo enzyme, probably a zinc enzyme. Moreover it is a heat‐stable protein with an apparent inactivation temperature of 80°C.