1997 Fiscal Year Final Research Report Summary
Molecular Biological Study on Nitrite-Oxidizing Enzyme of Heterotophic Bacteria
| Project/Area Number |
08660116
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | Oita University |
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Principal Investigator |
SAKAI Kenji Oita Univ. Fac.Engineering, Associate Professor, 工学部, 助教授 (50205704)
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| Co-Investigator(Kenkyū-buntansha) |
WAKAYAMA Mamoru Oita Univ.Fac.Engineering, Research Associate, 工学部, 助手 (70240455)
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| Project Period (FY) |
1996 – 1997
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| Keywords | Nitrification / Nitrite-oxidizing enzyme / Hetero trophic bacteria |
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| Research Abstract |
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.
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