| Project/Area Number |
13460045
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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 | 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 | Toyama Prefectural University |
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Principal Investigator |
ASANO Yasuhisa Biotechnology Research Center, Toyama Prefectural University, Professor, 工学部, 教授 (00222589)
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| Co-Investigator(Kenkyū-buntansha) |
KOMEDA Hidenobu Biotechnology Research Center, Toyama Prefectural University, Instructor, 工学部, 助手 (50285160)
KATO Yasuo Biotechnology Research Center, Toyama Prefectural University, Associate Professor, 工学部, 助教授 (20254237)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥9,500,000 (Direct Cost: ¥9,500,000)
Fiscal Year 2002: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 2001: ¥6,700,000 (Direct Cost: ¥6,700,000)
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| Keywords | Aldoxime Dehydratase / Opine dehydrogenase / Site-directed mutagenesis / Phenylalanine dehydrogenease / Gene cloning / X-ray structural analysis / ニトリラーゼ / 酵素的合成 / Rhodococcus sp / アルドキシム・ニトリル経路 |
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| Research Abstract |
Discoveries of an accumulation of amides by the use of new bacterial enzyme "nitrile hydratase" led to the successful application of nitrile hydratase to the industrial production of various amides. Nitrile hydratase e has become one of the most important industrial enzymes. We have been successful in the enzymatic synthesis of nitriles from aldoximes by using the new microbial enzyme aldoxime dehydratase, which works in newly proposed "Aldoxime-Nitrile Pathway".
(1) The distribution of aldoxime dehydratase in microorganisms was shown to be relatively wide among microorganisms.
(2) Phenylacetaldoxime dehydratase has been purified, characterized and its gene cloned from Bacillus sp. Strain OxB-1 and expressed in E. coli JM 109.
(3) We screened for microorganisms to convert E-pyridine-3-aldoxime to the corresponding nitrile and isolated Rhodococcus sp. YH3-3 from soil. The cells containing the inducible enzyme catalyzed the stoichiometric dehydration of aldoximes to form nitriles. Various nitriles were synthesized at preparative scales from 10-100 mM aldoximes under optimized reaction conditions. We also optimized conditions for large-scale synthsis of aryl-and alkyl-nitriles in high yields from the corresponding aldoximes by Escherichia coli JM109/poxd90F cells expressing phenylacetaldoxime dehydratase from Bacillus sp. strain OxB-1. We synthesized various ary1- and alky1-nitriles from the corresponding aldoximes by the transformant cells.
(4) Aldoxime dehydratase has been purified, characterized and its gene cloned and sequenced from Rhodococcus globerulus A-4 and expressed in E. coli JM 109. The gene for the enzyme was shown to be linked with the nitrile hydratase gene.
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