1997 Fiscal Year Final Research Report Summary
Construction and characterization of a novel whole cell biosensor with electrocatalytic function
| Project/Area Number |
08660135
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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 |
Bioproduction chemistry/Bioorganic chemistry
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
IKEDA Tokuji Kyoto Univ., Grad.Sch.Agriculture, Professor, 農学研究科, 教授 (40026422)
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| Co-Investigator(Kenkyū-buntansha) |
KANO Kenji Kyoto Univ., Grad.Sch.Agriculture, Associate Professor, 農学研究科, 助教授 (10152828)
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| Project Period (FY) |
1996 – 1997
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| Keywords | Bacterial cell catalysis / Biosensors / Whole cell electrodes / Ethanol / Nicotinic acid / Nitrate |
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| Research Abstract |
1 The oxidation reactions of D-glucose, nicotinic acid and ethanol by intact cells of Gluconobacter industrius, Pseudomonas fluorescens, and Acetobacter aceti, respectively, are successfully measured by an amperometric method using a membrane-covered electrode in the presence of such compounds as Fe(CN)_6^<3->, p-bsnzoquinone, dichlorophenolindophenol, or 2-methyl-5,6-dimethoxy benzoquinone as an electron acceptor Analysis of the experimental results reveals that the intact cells behave like oxidoreductases whose kinetics follows a Michaelis-Menten type equation. The catalytic constant and the Michaelis constant characterize the catalytic activity of a single cell. The catalytic constant is calculated to be as large as a million per second for the bacterial catalytic reactions.
2 Whole cells of Acetobacter aceti suspended in a buffer solution function as homogeneous catalysts to produce an electrochemical catalytic current for the oxidation of ethanol in the presence of 2-methyl-5,6-dim
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ethoxy benzoquinone(Q_0) ; A.aceti catalyzes the oxidation of ethanol with Q_0, and the reduced form of Q_0 produced by the A.aceti catalysis is electrochemically oxidized to regenerate Q_0. The theory of polarographic kinetic current is available for analyzing the catalytic current, from which the bimolecular rate constant between the bacterial cell and Q_0 can be calculated.
3 Carbon paste electrodes on which intact cells of A.aceti are immobilized produce a catalytic current for the oxidation of ethanol in the presence of Q_0. Analysis of the catalytic current reveals that the catalytic activity of the bacterial cell is due to alcohol dehydrogenase existing in the cytoplasmic membranes. Performance of the electrode as an ethanol sensor is compared with that of an electrode modified with purified enzyme. An alcohol dehydrogenase deficient strain and the mutant harboring alcohol dehydrogenase gene are also examined as biocatalyst in place of A.aceti. Similarly, Pseudomonas fluorescens and Paracoccus denitrificans serves as biocatalysts for constructing cell based biosensors for nicotinic acid and nitrate, respectively. Less
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