| Project/Area Number |
09556018
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
OGAWA Jun (1998) Kyoto Univ.Agr.Assistant Prof., 農学研究科, 助手 (70281102)
小林 達彦 (1997) 京都大学, 農学研究科, 講師 (70221976)
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| Co-Investigator(Kenkyū-buntansha) |
FURUYA Yuji Ikeda Toka Industries, Researcher, 研究室, 次長(研究職)
EMDO Ryuichi Nitto Chemical Industries, Researcher, 中央研究所, 首席主任研究員
SHIMIZU Sakoyu Kyoto Univ.Agr.Prof., 農学研究科, 教授 (70093250)
KOBAYASHI Michihiko Kyoto Univ.Agr.Senior Lecturer, 農学研究科, 講師 (70221976)
古谷 裕治 池田糖化工業(株), 研究室, 次長(研究職)
小川 順 京都大学, 農学研究科, 助手 (70281102)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥8,700,000 (Direct Cost: ¥8,700,000)
Fiscal Year 1998: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1997: ¥6,200,000 (Direct Cost: ¥6,200,000)
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| Keywords | Cyanide / C1 compound / nitrile hydratase / beta-cyanoalanine synthase / nitrilase / beta-oyanoalanine / Cl化合物 / Pseudomonas ovalis / PLP |
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| Research Abstract |
The final purpose of this project is the establishment of bioconversion process for the production of useful compounds from nitriles or that of nitriles from cyanide. Various types of microorganisms that metabolize cyanide were obtained from soil by an enrichment culture technique. They have been found to contain enzymes that synthesize nitriles from various amino acids and cyanide. beta-Cyanoalanine synthases were purified and characterized from Pseudomonas ovalis. The amino acid sequences of N-terminal and internal peptide fragments of the beta-cyanoalanine synthase from P.ovalis were determined in order to prepare synthetic oligonucleotides as primers of the polymerase chain reaction. A 700-bp DNA fragment thus amplified was used as the probe to clone a 3.0-kb BcII fragment coding for the partial beta-cyanoalanine synthase, and the 680-bp EcoRI-BcII from 3.0-kb BcII fragment was used as the probe to clone a 2.2-kb BssHII-EcoRI fragment coding for the whole beta-cyanoalanine synthase. The beta-cyanoalanine synthase gene modified in the sequence upstream of the presumed ATG start codon was expressed to about 40% of the total soluble protein in E, coli. The P.ovalis beta-cyanoalanine synthase and Rhodococcus rhodochrous K22 nitrilase were co-expressed in E.coli. The cell-free extracts were used for the production of aspartic acid from KCN.beta-cyanoalanine was produced from KCN and O-acetyl-serine immediately, and the resultant beta-cyanoalanine was transformed into aspartic acid. On the other hand, P.ovalis beta-cyanoalanine synthase and R.rhodochrous J1 nitrile hydratase were co-expressed in R.rhodochrous ATCC12674. The cell-free extracts were used in the production of asparagine. beta-Cyanoalanine was produced immediately, and the resultant beta-cyanoalanine was converted into asparagine.
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