Molecular Biological Study on Nitrite-Oxidizing Enzyme of Heterotophic Bacteria

异养细菌亚硝酸盐氧化酶的分子生物学研究

• 批准号: 08660116
• 负责人: SAKAI Kenji
• 金额: $ 1.09万
• 依托单位: Oita University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08660116/
• 关键词: Nitrification; Nitrite-oxidizing enzyme; Hetero trophic bacteria; 亜硝酸酸化; バチルス; 従属栄養微生物; 生物学的窒素除去

The aim of this study was on elucidation of nitirite-oxidizing enzyme of heterotrophic bacteria from biochemical and molecular biological aspects.First, nitrite-oxidizing enzyme system of various heterotrophic bacteria was investigated widely. The enzyme was purified and characterized from Bacillus badius I-73, which has been isolated from activated-sludge and showed relatively higer nitrite-oxidizing activity in bacteria. We also analyzed the N-terminal amino acid sequence of the nitrite-oxidizing enzyme and showed its N-teminal amino acid sequence of 23 residues, from which information the mixture of digoxigenin-labeled oligonucleotide prove was cynthesized chemically. This prove was specifically hybridized with ca. 6.4 kbp fragments of Hind III-digest chromosomal DNA from Bacillus badius I-73. Then the chromosomal DNA library of Bacillus badius I-73 was constructed by ligasing Hind III-and phosphatase-treated pUC18 with 6.4 kbp fragments digested by Hind III and extracted from agarose gel electrophoresed, by which Escherichia coli JM 109 was transformed. As a result, 6 positive transformants were obtained after screening by colony hybridization test. The chimeraplasmids from these transformants are analyzing now.On the other hand, the 16SrDNA gene of Bacillus badius I-73 was obtained by amplifying ca.1.3kbp fragment by PCR reaction and cloned in Escherichia coli. The sequence analysis with RDP databases indicated that the microorganism was the close relative of B.badius ATCC 14574. So that it would be possible to design a specific prove to trace the organism in an open system such as in activated sludge and soil.

本研究的目的是从生化和分子生物学角度阐明异养细菌的亚硝酸盐氧化酶。首先，对各种异养细菌的亚硝酸盐氧化酶系统进行了广泛的研究。该酶是从Bacillus badius I-73 中纯化和表征的，该酶是从活性污泥中分离出来的，在细菌中表现出较高的亚硝酸盐氧化活性。我们还分析了亚硝酸盐氧化酶的N端氨基酸序列，并显示了其23个残基的N端氨基酸序列，由此信息证明地高辛标记的寡核苷酸混合物是化学合成的。该证明与 ca 专门杂交。 Hind III 消化 Bacillus badius I-73 染色体 DNA 的 6.4 kbp 片段。然后将Hind III和磷酸酶处理的pUC18与Hind III酶切的6.4 kbp片段连接，并从琼脂糖凝胶电泳中提取，构建Bacillus badius I-73染色体DNA文库，转化大肠杆菌JM 109。经菌落杂交试验筛选，获得6个阳性转化子。这些转化子的嵌合质粒正在分析中。另一方面，通过PCR反应扩增约1.3kbp片段，获得Bacillus badius I-73的16SrDNA基因，并将其克隆到大肠杆菌中。 RDP数据库的序列分析表明该微生物是B.badius ATCC 14574的近亲。因此可以设计特定的证明在开放系统（例如活性污泥和土壤）中追踪该微生物。

项目成果

K.Sakai, Y.Ikenaga, M.Wakayama and M.Moriguchi: "Nitrite Oxidation by Heterotrophic Bacteria under Various Nutritional and Aerobic Conditions." J.Ferment.Bioeng. 82. 613-617 (1996)

K.Sakai、Y.Ikenaga、M.Wakayama 和 M.Moriguchi：“异养细菌在各种营养和有氧条件下的亚硝酸盐氧化”。