| Project/Area Number |
18580080
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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 |
Applied microbiology
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| Research Institution | University of Miyazaki |
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Principal Investigator |
HIROSE Jun University of Miyazaki, Department of Applied Chemistry, Associate Professor (60264363)
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| Co-Investigator(Kenkyū-buntansha) |
FURUKAWA Kensuke Beppu University, Department of Food and Bioscience, Professor (90221556)
YUI Toshifumi University of Miyazaki, Department of Applied Chemistry, Associate Professor (50230610)
SUGAMOTO Kazuhiro University of Miyazaki, Department of Applied Chemistry, Assistant Professor (10274771)
YOKOI Haruhiko University of Miyazaki, Department of Applied Chemistry, Professor (00253815)
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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 |
¥3,870,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥270,000)
Fiscal Year 2007: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2006: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Keywords | aromatic-ring dioxygenase / combinatorial chemistry / DNA shuffling / chimeric enzyme / molecular dynamics method / biphenyl dioxygenase / ビフェニルジオキシゲナーゼ / オキシゲナーゼ |
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| Research Abstract |
'Aromatic ring dioxygenase' is enzyme which is involved in the initial oxidation of aromatic-ring by bacteria. This enzyme can be used for the synthesis of pharmaceutical and other industrially useful compounds because of its capability in introducing regio- and stereospecific hydroxyl groups into aromatic ring. In this research project, we detected and identified reaction products which were catalyzed by many mutant biphenyl dioxygenases (BphA). Several man-made compounds, for example 1-phenylpyrazole (1PP) or indan were used as substrate. Of the 20 different chimeric BphAs, seven enzymes produced more efficiently 'regio-specifically hydroxylated 1PPs than wild type BphA'. Two products obtained from the reaction catalyzed by BphA (2039) were identified with HRMS and NMR. In the conversion of IPP with all chimeric BphAs, the rate of reaction products between these compounds varied with the chimeric BphAs used; BphA (2029) and BphA (2030) yielded more monohydroxylated compound than the other chimeric enzymes. From computer-aided simulation of the complex between the structure of BphA (2039) and IPP, it was suggested that difference in reaction between chimeric BphA and 1PP is due to change in the manner of binding of 1PP to the active site of BphAs. In the other experiment, another substrate, indan was converted with mutant BphAs. Analysis of the reaction product (1-indanol) by HPLC with chiral separation column revealed the different enantiomeric excess between the reactions by wild-type BphA and mutant BphA. This study demonstrated that mutant BphA can be used to produce rare mono-or dihydroxylated aromatic compounds which deserve to be used as the building blocks of combinatorial chemistry.
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