Purification and partial characterization of the extracellular gamma-D-glutamyl-(L)meso-diaminopimelate endopeptidase I, from Bacillus sphaericus NCTC 9602.
The gamma-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 Vmax of 8.3 mumol min-1 (mg enzyme)-1 with N-acetylmuramyl-L-alanyl-gamma-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 degrees C.
D001407:Bacillus / Q000201:enzymology*; D004355:Drug Stability; D004492:Edetic Acid / Q000494:pharmacology; D010450:Endopeptidases / Q000302:isolation & purification*; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008670:Metals; D010618:Phenanthrolines / Q000494:pharmacology; D011489:Protein Denaturation; D013379:Substrate Specificity
D008670:Metals; D010618:Phenanthrolines; D004492:Edetic Acid; D010450:Endopeptidases; C019404:gamma-D-glutamyl-meso-diaminopimelate peptidase I; C025205:1,10-phenanthroline