Beta-glucuronidases of clostridium perfringens.
GAM broth was cultured for 5 days at 37 degrees C to obtain maximum yields of extracellular beta-glucuronidase from smooth colonies of Clostridium perfringens (Hobbs' type 4) isolated from the feces of a patient. A crude enzyme preparation was obtained by 20-80% ammonium sulfate precipitation of the broth. The beta-glucuronidase was purified using DEAE cellulose column chromatography, gel filtration on Sephadex G-200, and affinity chromatography on Sepharose 4B-bound glucuronolactone. We obtained two kinds of beta-glucuronidase. Properties of the purified beta-glucuronidase I were an optimum pH of 7.2, a pH stability range below 7.0, and a molecular weight of 115,000. The purified beta-glucuronidase II had an optimum pH of 6.0, pH stability at around 6.0, and a molecular weight of 195,000. Cu++ and Hg++ were strong inhibitors, which inhibition was restored by cysteine. EDTA did not influence enzyme activity. The Michaelis constants of beta-glucuronidase I and beta-glucuronidase II for p-nitrophenyl glucuronide were 1.25 X 10(-3) M and 4.17 X 10(-4) M, for naphthol AS-BI glucuronide 1.35 X 10(-4) M and 1.14 X 10(-4) M, and for phenolphthalein glucuronide 7.46 X 10(-5) M and 2.50 X 10(-4) M.
D003016:Clostridium perfringens / Q000166:cytology / Q000201:enzymology*; D005966:Glucuronidase / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D013379:Substrate Specificity
D007527:Isoenzymes; D008670:Metals; D005966:Glucuronidase