| Project/Area Number |
05660100
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | Kochi University |
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Principal Investigator |
MISONO Haruo Kochi University, Agriculture, Professor, 農学部, 教授 (30027073)
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| Co-Investigator(Kenkyū-buntansha) |
NAGATA Shinji Kochi University, Agriculture, Asistant Prof., 農学部, 助教授 (60180494)
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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 |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1994: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1993: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | D-Amino acid dehydrogenase / D-Threonine dehydrogenase / D-phenylserine dehydrogenase / Pseudomonas syringae / Pseudomonas cruciviae / NADP-binding site / Cloning |
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| Research Abstract |
A soil bacterium which produced an inducible NADP-dependent D-phenylserine dehydrogenase was identified as Pseudomonas syringae. The enzyme catalyzed the oxidation of the 3-hydroxyl group of D-threo-3-phenylserine. The enzyme was purified to homogeneity from a crude extract of P.syringae NK-15 and its properties were compared with those of a constitutive NADP-dependent D-threonine dehydrogenase from P.cruciviae. The properties such as molecular weight, subunit composition, and optimum pH were similar to those of D-threonine dehydrogenase. However, thermal stability, substrate specificity, cofactor specificity, and inhibitors were defferent in both enzymes. The N-terminal 27 amino acids sequence suggested that the NADP-binding site was located in the N-terminal region of the enzyme. To determine primary structure of both enzymes, cloning of both enzyme genes was carried out using colony hybridization. Clones having both enzyme geneswere not obtained. PCR was carried out using primers synthesized according to the amino acid sequence of the isolated peptide and N- and C-terminal amino acid sequences. The amplified DNA fragments of D-threonine dehydrogenase gene was cloned into E.coli JM109 using pUC18. The PCR product of D-phenylserine dehydrogenase gene was cloned into E.coli JM109 and the sequence of the inserted DNA was determined. Although the complete primary structure of both enzymes could not be determined, N-terminal and C-terminal amino acid sequences were not similar in both enzymes.
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