Intracellular glucosaminidase of the bacterium Xanthomonas campestris IBPM B-124: purification and properties.
A system of intracellular autolytic enzymes of the bacterium Xanthomonas campestris IBPM B-124 was found to include enzymes with muramidase and glucosaminidase activities, while a system of extracellular bacteriolytic enzymes of the same bacterium includes muramidase, muramoylalanine amidase, and endopeptidase. Using a purification technique including fractional precipitation with ammonium sulfate, gel-filtration on Toyopearl HW-55F, and FPLC ion-exchange chromatography on Mono Q, a preparation of intracellular glucosaminidase 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, Km = 5.6 x 10(-4) M with p-nitrophenyl-2-acetamido-2-deoxy-beta-D-glucopyranoside as the substrate, 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-chloromercuribenzoate (PCMB), phenylmethylsulfonyl fluoride (PMSF), and the disodium salt of ethylenediamine tetraacetic acid (EDTA). The properties of this glucosaminidase markedly differ from those of all extracellular bacteriolytic enzymes of Xanthomonas campestris. These findings indicate that the system of autolytic enzymes of this bacterium functions independently and is not connected with the system of extracellular bacteriolytic enzymes.
Tsfasman
I M
IM
Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region, 142292, Russia.
Stepnaya
O A
OA
Bazhanova
N V
NV
Kulaev
I S
IS
D000645:Ammonium Sulfate / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006596:Hexosaminidases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009113:Muramidase / Q000378:metabolism; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013696:Temperature; D016959:Xanthomonas campestris / Q000201:enzymology*; D020245:p-Chloromercuribenzoic Acid / Q000494:pharmacology
D002614:Chelating Agents; D004791:Enzyme Inhibitors; D013439:Sulfhydryl Reagents; D010664:Phenylmethylsulfonyl Fluoride; D020245:p-Chloromercuribenzoic Acid; D004492:Edetic Acid; D006596:Hexosaminidases; D009113:Muramidase; D000645:Ammonium Sulfate