Purification and partial characterization of N-acetyl-beta-D-glucosaminidase from Tritrichomonas foetus.
Cells of Tritrichomonas foetus were suspended in buffer (0.1 M phosphate, 0.15 M NaCl, pH 7), sonicated for 2 min on ice, and centrifuged at low speed (500 g/40 min) at 4 degrees C. The resulting supernatant was centrifuged at 100,000 g for 30 min at 4 degrees C. The N-acetyl-beta-D-glucosaminidase activity as assayed by fluorimetric assay using 4-methylumbelliferil beta-D-N-acetylglucosamine (4MU-GlcNAc) was found predominantly (> 95%) in the supernatant. Isolation of the enzyme was achieved by a combination of gel filtration with ion-exchange chromatography. Non-denaturing gel electrophoresis indicated that N-acetyl-beta-D-glucosaminidase activity was present in two bands. When the two fluorescent bands were excised from the non-denaturing gel and rerun on denaturing 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis they exhibited two proteins with molecular masses of 40 and 45 kDa. The pH optimum is approximately 7.5 and the temperature optimum is approximately 37 degrees C.
Cifali
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AP
Laboratório de Fisiologia e Bioquímica de Microrganismos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, Campos, RJ, Brazil.
Dias Filho
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BP
D000118:Acetylglucosaminidase / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D013696:Temperature; D016835:Tritrichomonas foetus / Q000201:enzymology* / Q000254:growth & development
D000118:Acetylglucosaminidase