Intracellular glucosaminidase of the bacterium Xanthomonas campestris IBPMB-124: Purification and properties

A system of intracellular autolytic enzymes of the bacterium Xanthomonas ca mpestris IBPM B-124 was found to include enzymes with muramidase and glucos aminidase activities, while a system of extracellular bacteriolytic enzymes of the same bacterium includes muramidase, muramoylalanine amidase, and en dopeptidase. Using a purification technique including fractional precipitat ion with ammonium sulfate, gel-filtration on Toyopearl HW-55F, and FPLC ion -exchange chromatography on Mono Q, a preparation of intracellular glucosam inidase was purified 435-fold with 16%; yield (SDS-PAGE data indicated the presence of minor protein contaminants). Some physicochemical properties of the purified enzyme were determined: molecular mass 26 kD, K-m = 5.6.10(-4 ) M with p-nitrophenyl-2-acetamido-2-deoxy-beta-D-glucopyranoside as the su bstrate, and pH optimum 8.0-8.5. The enzyme is active over a wide range of Tris-HCl buffer concentrations (0.01-0.5 M) and has temperature optimum at 37-40 degrees C. The glucosaminidase activity is sensitive to p-chloromercu ribenzoate (PCMB), phenylmethylsulfonyl fluoride (PMSF), and the disodium s alt of ethylenediamine tetraacetic acid (EDTA). The properties of this gluc osaminidase markedly differ from those of all extracellular bacteriolytic e nzymes of Xanthomonas campestris. These findings indicate that the system o f autolytic enzymes of this bacterium functions independently and is not co nnected with the system of extracellular bacteriolytic enzymes.