| Project/Area Number |
05044138
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Institution | Toyama Prefectural University |
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Principal Investigator |
ASANO Yasuhisa Associate Professor, Toyama Prefectural University, 工学部, 助教授 (00222589)
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| Co-Investigator(Kenkyū-buntansha) |
YAMADA Hideaki Professor, Toyama Prefectural University, 工学部, 教授 (30027180)
KATO Yasuo Assistant, Toyama Prefectural University, 工学部, 助手 (20254237)
クーパー アーサー.J. 米国コーネル大学, 医学部, 教授
COOPER Arthur j.l. Professor, Cornell University Medical College
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| Project Period (FY) |
1993 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 1994: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1993: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | Amino Acid / Immunotitration / Amino Acid Dehydrogenase / Primary Structure / Enzyme Purification / Phenylalanine Dehydrogenase / Screening / Sequence Homology |
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| Research Abstract |
We discovered for the first time that Bacillus sp.and Sporosarcina sp.produces phenylalanine dehydrogenase. We started our collaboration with Professor Arthur Copper of Cornell University Medical College, New York, U.S.A.We got the following results in this two years collaboration with him.
(1)We have clarified the enzymological properties of phenylalanine dehydrogenase from Bacillus shaericus, B.badius, and Sporosarcina ureae. We synthesized various optically pure L-amino acids from prochiral alpha-keto acids, using the enzyme in combination with Candida formate dehydrogenase. The enzyme was effective for the reductive amination of various alpha-Keto acids. We have been successful in the synthesis of natural and unnatural L-amino acids, including L-homophenylalanine.
(2)We have been successful in the synthesis of ^<13>N-Labelled L-phenylalanine, L-tyrosine, and L-DOPA.
(3)We have cloned the genes for the enzymes and sequenced some of the genes. They showed similarities with leucine dehydrogenase, Streptomyces phenylalanine dehydrogenase, and glutamic acid dehydrogenase.
(4)In the microplate method of PKU screening using the enzyme, L-phe is oxidized with the enzyme to phenylpyruvate, reducing NAD^+ to NADH.In the presence of NADH and the electron carrier 1-methoxy-5-methylphenazium methylsulfate (1-PMS), Co^<3+> is reduced to Co^<2+> which produces a colored chelate. The method is being introduced as a tool of the routine mass screening in Japan.
(5)A spirolactone [3-hydroxy-2-oxo-1-oxaspiro [4.5] -dec-3-ene] and the sodium salts of two new alpha-keto acids [(1-cyclohexenyl) glyoxylate, beta- (1-hydoxycyclohexane) pyruvate (enol form)] were chemically synthesized. These compounds are expected to be useful as prototypes for the design of inhibitors of alpha-keto acid-utilizing enzymes. In fact, one of them was found to be a new type of inhibitor of rabbit muscle lactate dehydrogenase.
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