Molecular Biological Study on Nitrite-Oxidizing Enzyme of Heterotophic Bacteria

本研究的目的是从生物化学和分子生物学的角度阐明异养细菌亚硝酸盐氧化酶的功能。从活性污泥中分离到一株具有较高亚硝酸盐氧化活性的芽孢杆菌（Bacillusbadius）I-73，经纯化和鉴定，该酶具有较高的亚硝酸盐氧化活性。对亚硝酸氧化酶的N-末端氨基酸序列进行了分析，得到了23个氨基酸残基的N-末端氨基酸序列，并据此对地高辛标记的寡核苷酸混合物进行了化学合成。该菌株与ca. 6.4来自芽孢杆菌I-73的Hind III消化的染色体DNA的kbp片段。然后将Hind Ⅲ酶切后的6.4kbp片段与经磷酸酶处理的pUC 18连接，构建了芽孢杆菌I-73的染色体DNA文库，转化大肠杆菌JM 109。经菌落杂交筛选，获得6株阳性转化子。另一方面，通过PCR反应扩增出约1.3kbp的16 SrDNA片段，并将其克隆到大肠杆菌中。序列分析表明，该菌与B.badius ATCC 14574有较近的亲缘关系。因此，有可能设计一种特定的证明来追踪开放系统中的有机体，例如活性污泥和土壤。

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.