PURIFICATION AND PARTIAL CHARACTERIZATION OF THE EXTRACELLULAR GAMMA-D-GLUTAMYL-(L)MESO-DIAMINOPIMELATE ENDOPEPTIDASE-I, FROM BACILLUS-SPHAERICUS NCTC-9602
PURIFICATION AND PARTIAL CHARACTERIZATION OF THE EXTRACELLULAR GAMMA-D-GLUTAMYL-(L)MESO-DIAMINOPIMELATE ENDOPEPTIDASE-I, FROM BACILLUS-SPHAERICUS NCTC-9602
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DOI：
10.1111/j.1432-1033.1985.tb08873.x
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发表时间：
1985-01-01
期刊：
影响因子：
--
通讯作者：
MICHEL, G
中科院分区：
文献类型：
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作者：
GARNIER, M;VACHERON, MJ;MICHEL, G
γ-来自B的D-谷氨酰基-（L）-内消旋-二氨基庚二酸内肽酶或内肽酶I。将Sphaericus 9602纯化至表观蛋白质均一性。经硫酸铵分级分离、苯基-Sepharose柱层析、DEAE-Trisacryl柱层析、聚焦柱层析和Sephacryl S-200柱层析6步纯化。该酶纯化5000倍，裂解活性回收率为38%。它是一种疏水性酸性蛋白质（pI 5.4）。动力学研究表明，用N-乙酰胞壁酰-L-丙氨酰-γ-氨基苯甲酸酯，Km值为0.57 mM，表观Vmax为8.3 μ mol min-1（mg酶）-1。D-谷氨酰-（L）-内消旋-二氨基庚二酰（L）-D-[14 C]丙氨酸作为底物。该酶被邻菲咯啉和EDTA抑制，并被锌，钴和锰离子重新激活，因此内肽酶I是一种金属酶，可能是锌酶。它是一种热稳定蛋白质，表观失活温度为80 ℃。C.
The .gamma.-D-glutamyl-(L)-meso-diaminopimelate endopeptidase, or endopeptidase I, from B. sphaericus 9602 was purified to apparent protein homogeneity. The purification was achieved by a 6-step procedure: ammonium sulfate fractionation, phenyl-Sepharose chromatography, 2 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 .mu.mol 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 Zn, Co and Mn ions; thus endopeptidase I is a metallo enzyme, probably a Zn enzyme. It is a heat-stable protein with an apparent inactivation temperature of 80.degree. C.