| Project/Area Number |
06650924
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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 |
生物・生体工学
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| Research Institution | Saitama Institute of Technology |
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Principal Investigator |
UCHIYAMA Shunichi Saitama Inst.of Technol., Faculty of Engineering, Professor, 工学部, 教授 (80129163)
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| Co-Investigator(Kenkyū-buntansha) |
HASEBE Yasushi Saitama Inst.of Technol., Faculty of Engineering, Lecturer, 工学部, 講師 (20212144)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1995: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1994: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Keywords | enzyme amplification / biosensor / histamine sensor / antibody sensing / conversion of enzymatic function / 酵素機能変換 / 酵素増幅法 |
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| Research Abstract |
Copper-containing monoamine oxidase (MAO) generates a novel ascorbate oxidase activity upon the addition of L-histidine (His) and histamine (Hm) in Tris/HCl buffer at slightly alkaline condition (pH>8.0). In this reaction, L-ascorbate (AsA) was enzymatically oxidized dehydroascorbate via a monodehydroascorbate radical, and the consumed oxygen was revealed to be finally converted to hydrogen peroxide according to Michaelis-Menten kinetics. Km of ascorbate oxidase activity of MAO induced by 1*10^<-3> M of Hm was calculated to ca. 0.62 mM at 35ﾟC and pH 8.5. ESR signal of coppe (II) in active site of MAO at 77 K was apparently changed upon the addition of His and Hm in a concentration depending manner, indicating that ligands of copper center partly exchanged to exogenous His and Hm. The amount of oxygen consumption in MAO-catalyzed AsA oxidation was dependent on the concentration of His and Hm added, and the typical step-wise current responses were obtained by using immobilized MAO-coupling oxygen electrode in Tris buffer containing AsA.Tris buffer is essential to generate this activity and the response was completely disappeared in other buffers. The calibrtion curves of His and Hm obtained with 5 mM AsA show a linear line over the concentration range form 2*10^<-7> M to 1*10^<-4> M of His and 3*10^<-6> M to 1*10^<-3> M of Hm, respectively.
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