Construction of functinal device with molecular oriented immobilization using genetic engineering
| Project/Area Number |
18510097
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Nanomaterials/Nanobioscience
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| Research Institution | University of Fukui |
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Principal Investigator |
SUYE Shin-ichiro University of Fukui, Applied Chemistry and Biotechnology, Associate Professor (90206376)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥3,290,000 (Direct Cost: ¥2,900,000、Indirect Cost: ¥390,000)
Fiscal Year 2007: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2006: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | molecular orientation / oriented control / biosensing / electron transfer / 電極 / バイオセンサ / L-プロリン脱水素酵素 / 耐熱性酵素 / L-プロリン |
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| Research Abstract |
Hiperthermostable L-proline dehydrogenase (L-ProDH) from hyperthermophilic Achaean (Therococcus profundus) catalyzes the oxidation of L-proline in the presence of artificial electron acceptors such as 2,6-dichloroindophenol (DCIP) and ferricyanide. L-ProDH consists of a heterotetrameric αβγδ (52, 43, 19, and 11 kDa) structure. The p subunit catalyzed the dye-linked L-proline dehydrogenase reaction by itself and that, unexpectedly, the αsubunit exhibited dye-linked NADH dehydrogenase activity. Both α and β subunits have FAD, and γ subunit contains ferredoxine). For oriented immobilization of L-ProDH, His-tag was introduced on the C-terminal site of each subunit and oriented enzyme was found to Ni complex on the electrode.
We cloned the gene cording for the L-ProDH foreign DNA into pET15b plasmid vector that provides the enzyme with a C-terminal His-tag. His-tagged pdh α and His-tagged pdh D and His-tagged pdh γ were expressed in E. coli. L-ProDH was purified using heat treatment and immobilized metal-ion affinity chromatography from cell free extract. His-tagged enzymes were immobilized on the gold electrode via NTA-Ni.
His tagged α subunit L-ProDH immobilized electrode showed higher electrochemical response compared with other His-tagged subunits enzyme immobilized electrodes. Efficient electron transfer between substrate and electrode thorough the enzyme molecule was found between β and α subunits. The results in this study show that an enzyme immobilization system has the potential to be applied in a sensitive biosensing system.
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Report
(3 results)
Research Products
(39 results)
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[Book] Smart Biosensor2007
Author(s)
S. Suye and H. Zheng
Publisher
Molecular film assembled detection system for biosensors bylayer-by-layer adsorption, G. K. Knopf, ed., Marcell Deckker, NY
Description
「研究成果報告書概要(和文)」より
Related Report
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