Purification and partial characterization of the extracellular gamma-D-glutamyl-(L)meso-diaminopimelate endopeptidase I, from Bacillus sphaericus NCTC 9602
Open Access
- 1 May 1985
- journal article
- research article
- Published by Wiley in European Journal of Biochemistry
- Vol. 148 (3) , 539-543
- https://doi.org/10.1111/j.1432-1033.1985.tb08873.x
Abstract
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 Km value of 0.57 mM and an apparent Kmax of 8.3 μmol min−1 (mg enzyme)−1 with N‐acetylmuramyl‐l‐alanyl‐γ‐D‐glutamyl‐(L)meso‐diaminopimelyl (L)‐D‐[14C]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.This publication has 25 references indexed in Scilit:
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