| Project/Area Number |
63850191
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B).
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| Allocation Type | Single-year Grants |
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| Research Field |
発酵工学
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| Research Institution | The Kumamoto Institute of Technology (1990)
Osaka University (1988-1989) |
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Principal Investigator |
OKADA Hirosuke Kumamoto Institute of Technology, Dept. of Appl. Microbiol., Professor, 工学部, 教授 (20028947)
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| Co-Investigator(Kenkyū-buntansha) |
AKAMATSU Takashi Kumamoto Institute of Technology, Dept. of Appl. Microbiol., Assoc. Professor, 工学部, 助教授 (50133567)
SHIMA Yasufumi Osaka University, Dept. of Ferment. Technol Assis. Professor, 工学部, 助手 (50187423)
NEGORO Seiji Osaka University, Dept. of Ferment. Technol Assis. Professor, 工学部, 助手 (90156159)
URABE Itaru Osaka University, Dept. of Ferment. Technol Assoc. Professor, 工学部, 助教授 (60029246)
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| Project Period (FY) |
1988 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥15,400,000 (Direct Cost: ¥15,400,000)
Fiscal Year 1990: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1989: ¥5,200,000 (Direct Cost: ¥5,200,000)
Fiscal Year 1988: ¥7,600,000 (Direct Cost: ¥7,600,000)
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| Keywords | Enzyme-cofactor conjugate / Enzyme-reactor / Dehydrogenase / Covalently linked 5-ethylphenazine / Covalently linked NAD / Coupling of catalytic reactions / NADH-regeneration unit / 連続酵素反応 / 高分子化5ーエチルフェナジン / 酵素・補酵素結合体 / 酵素リアクター / デヒドロゲナーゼ |
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| Research Abstract |
Glucose-dehydrogenase-poly (ethylene glycol) -NAD conjugate (GlcDH-PEG-NAD) was prepared and its kinetic properties as an NADH-rageneration unit were investigated. L-Lactate was continuously produced from pyruvate in a reactor with a PM10 ultrafiltration membrane, and containing GlcDH-PEG-NAD and lactate dehydrogenase (LDH). GlcDH-PEG-NAD proved to be applicable in continuous enzyme reaction as an NADH-regeneration unit with a large molecular size.
Then, we have prepared the following enzyme-cofactor conjugate by covalently linking 5-ethylphenazine (and also NAD) to various dehydrogenases : a covalently linked 5-ethylphenazinepoly (ethylene glycol) -glutamate dehydrogenases conjugate (EP-PEG-GltDH), a covalently linked 5-ethylphenazine-glucose dehydrogenase-NAD conjugate (EP-GlcDH-NAD), and a covalently linked 5-ethylphanazine-lactate dehydrogenase-NAD conjugate (EP-LDH-NAD). These conjugate show new catalytic activities, and the EP moiety works as an artificial catalytic group for the
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oxidation of NADH (or the NADH mojety) with oxygen or a tetrazolium salt such as MTT in the new catalytic reactions.
These conjugates are unique semisynthetic enzymes, and provide us with kinetic basis for artificially designing enzymes. EP-PEG-GltDH works as an NADH oxidase, and the coenzyme-binding site of the GltDH moiety is used as a substrate-binding site. Kinetic analysis of the NADH-oxidase activity shows the effect of the presence of the substrate-binding site near the catalytic group of the EP moiety. EP-GlcDH-NAD works as a glucose oxidase. This activity is due to the coupling of the two catalytic reactions of the GlcDH and the EP moieties, and kinetic analysis shows the presence of the following rate accerelation mechanisms : high effective concentration, intramolecular coupling of succesive catalytic reactions, and multiple connection between the two kinds of the catalytic sites. EP-LDH-NAD works as a lactate oxidase. Kinetic analysis shows the effect of hyding the NADH moiety by the coenzyme-binding site of the LDH moiety. Less
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