Milligram to gram scale purification and characterization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F |
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| Authors: | A W Miller S H Eklund J F Robyt |
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| Institution: | 1. Division of Nutrition, St. John’s Research Institute, A Recognized Research Centre of University of Mysore, Bangalore, India;2. Department of Pathology, St John’s Medical College Hospital, Bangalore, India;3. Department of Obstetrics and Gynaecology, St John’s Medical College Hospital, Bangalore, India;4. Department of Biostatistics, St. John’s Medical College Hospital, Bangalore, India;1. Pharmaceutical Biotechnology Research Group, Faculty of Pharmacy, Universitas Indonesia, Kampus UI Depok, Depok 16424, Indonesia;2. Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama, Ikoma, Nara 630-0101, Japan;1. Department of Physics, University of Ioannina, GR 45110 Ioannina, Greece;2. Department of Physics, University of Crete, P.O. Box 2208, GR 71003 Heraklion, Greece;3. Tandem Accelerator Laboratory, Institute of Nuclear and Particle Physics, NCSR Demokritos, GR 15310 Ag. Paraskevi, Greece;1. Department of Chemistry and Biomolecular Sciences, University of Ottawa, D’Iorio Hall, 10 Marie Curie, Ottawa, Ontario K1N 6N5, Canada;2. National Research of Council Canada, 100 Sussex Drive, Ottawa, Ontario K1A 0R6, Canada;3. From the Departments of Neurosciences and;4. Pathology, College of Medicine and Life Sciences, University of Toledo, Toledo, Ohio 43614 and;5. the Department of Chemical Engineering, College of Engineering, University of Toledo, Toledo, Ohio 43607 |
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| Abstract: | A sequence of dextranase treatment, DEAE-cellulose chromatography, affinity chromatography on Sephadex G-200, and chromatography on DEAE-Trisacryl M has been optimized to give a dextransucrase preparation with low carbohydrate content (1-100 micrograms/mg protein) and high specific activity (90-170 U/mg protein) relative to previous procedures, in 30-50% yield. Levansucrase was absent after DEAE-cellulose chromatography, and dextranase was undetectable after Sephadex G-200 chromatography. The method could be scaled up to produce gram quantities of purified enzyme. The purified dextransucrase had a pH optimum of 5.0-5.5, a Km of 12-16 mM, and produced the same lightly branched dextran as before purification. The purified enzyme was not activated by added dextran, but the rate of dextran synthesis increased abruptly during dextran synthesis at a dextran concentration of approximately 0.1 mg/mL. The enzyme had two major forms, of molecular weight 177,000 and 158,000. The 177,000 form predominated in fresh preparations of culture supernatant or purified enzyme, whereas the amount of the 158,000 form increased at the expense of the 177,000 form during storage of either preparation. |
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