Purification and Characterization of D-Glucosaminitol Dehydrogenase from Agrobacterium radiobacter
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概要
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D-Glucosaminitol dehydrogenase, which catalyzes the conversion of D-glucosaminitol to 3-keto-D-glucosaminitol, was purified to apparent homogeneity from extracts of Agrobacterium radiobacter. This organism has constitutively depressed levels of the enzyme but expression of the enzyme is induced by addition of D-glucosamine to the medium. Purification included ammonium sulfate fractionation and chromatography on columns of DEAE-Sephacel, Octyl-Sepharose CL-4B, and Cellulofine. The purified enzyme migrated as a single band, coinciding with dehydrogenase activities specific for D-glucosaminitol and ethanol, when electrophoresed on a 7.5% polyacrylamide gel at pH 8.0. Electrophoresis on a 12.5% PAGE in the presence of 1% SDS also yielded a single band. The enzyme had an apparent molecular mass of 79 kDa, as measured by the pattern of elution from a column of Cellulofine. The results indicated that the enzyme was a dimer of identical (or nearly identical) subunits of 39.5 kDa. D-Glucosaminitol dehydrogenase required NAD^+ as a cofactor and used ethanol as the preferred substrate, as well as aliphatic alcohols with 2 to 4 carbon atoms, D-glucosaminitol, D-glucosaminate, DL-allothreonine, glycerol, and erythritol as additional substrates. In 50 mM Tris-HCl buffer (pH 9.0) at 25℃, the K_m for D-glucosaminitol, ethanol, and NAD^+ were 2.2,2.0,and 0.08 mM, respectively. The enzyme had a pH optimum of 10 for D-glucosaminitol and 8.5 for ethanol. The enzyme lost substantial activity when treated with pyrazole, with certain reagents that react with sulfhydryl groups and with Zn^2+ ion. The various results together suggest that the enzyme exploits different amino acid residues for the dehydrogenation of ethanol and of D-glucosaminitol.
- 社団法人日本農芸化学会の論文
- 1999-05-23
著者
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MIKATA Yuji
Department of Chemistry, Faculty of Science, Nara Women's University
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Tamura Keiko
Department Of Chemistry Faculty Of Science Nara Women's University
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Tanaka M
Department Of Chemistry Faculty Of Science Nara Women's University
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IWAMOTO Ryoko
Department of Chemistry, Faculty of Science, Nara Women's University
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SAKAMOTO Chieko
Department of Chemistry, Faculty of Science, Nara Women's University
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TANAKA Mieko
Department of Chemistry, Faculty of Science, Nara Women's University
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Mikata Yuji
Department Of Chemistry Faculty Of Science Nara Women's University
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Tanaka Mieko
Department Of Chemistry Faculty Of Science Nara Women's University
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Iwamoto Ryoko
Department Of Chemistry Faculty Of Science Nara Women's University
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Sakamoto Chieko
Department Of Chemistry Faculty Of Science Nara Women's University
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