2001 Fiscal Year Final Research Report Summary
Electroenzymatic Activity of Cytochrome P-450cam and Application to the Organic Synthesis
| Project/Area Number |
11650820
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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 |
生物・生体工学
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| Research Institution | Nagasaki University (2001)
Kyushu University (1999-2000) |
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Principal Investigator |
TANAKA Shuji Nagasaki University, Faculty of Engineering, Associate Professor, 工学部, 助教授 (80217033)
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| Project Period (FY) |
1999 – 2001
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| Keywords | electroenzymatic chemistry / cytochrome P450cam / putidaredoxin / electrochemistry |
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| Research Abstract |
In this project, we constructed a bioreactor where the reduction electron is supplied to immobilized cytochrome P-450cam and/or putidaredoxin and investigated the character of the enzyme electrode.
Putidaredoxin, cytochrome P-450cam and putidaredoxin-cytochrome P-450cam hybrid protein were immobilized on carbon electrode using polyacrylic acid and appropriate linkers, then electrochemical characters of these immobilized proteins were analyzed by cyclic voltammetry. Reversible electron transfer was observed on putidaredoxin immobilized electrode and redox potential of immobilized putidaredoxin was shifted at presence of cytochrome P-450cam in bulk solution. These results indicated interaction between putidaredoxin and cytochrome P-450cam. However, in cytochrome P-450cam electrode reduction peak was only observed on first scan. The elctroenzymatic activity of cytochrome P-450cam immobilized electrode was low and formation hydrogen peroxide was determined. We constructed fusing CDNA of putidaredoxin-cytochrome P-450cam and expressed hybrid protein in Escherichia coli, purified. Cyclic voltammgram of hybrid protein immobilized enzyme electrode was similar to cytochrome P-450cam electrode, but slightly reversible at presence of camphor. Electroenzymatic activity of hybrid protein electrpde was more than 10 times higher than cytochrome P-450cam electrode. Formation of hydrogen peroxide of hybrid protein electrode was little or no. These results suggested that putidaredoxin part of hybrid protein modulate cychrome P-450cam activity and suppressed autooxidation route cytochrome P-450cam reaction cycle.
This present research demonstrated the feasibility of electrochemically driven cytochrome P-450 for syntheses of organic compounds
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