| Project/Area Number |
05660115
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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 |
Bioproduction chemistry/Bioorganic chemistry
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| Research Institution | The University of Tokyo |
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Principal Investigator |
OKUBO Akira Univ.of Tokyo.Faculty of Agriculture.Associate Professor, 農学部, 助教授 (20111479)
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| Co-Investigator(Kenkyū-buntansha) |
YOSHIMURA Etsuro Univ.of Tokyo.Faculty of Agriculture.Associate Professor, 農学部, 助教授 (10130303)
YAMAZAKI Sunao Univ.of Tokyo.Faculty of Agriculture.Professor, 農学部, 教授 (00011982)
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| Project Period (FY) |
1993 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1994: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1993: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | capillary electrophoresis / DMSO reductase / CZE / molybdenum containing enzyme / micellar electrokinetic chromatography / MEKC |
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| Research Abstract |
Dimethyl sulfoxide reductase was obtained by anaerobic cultivation of a green mutant of Rhodobacter sphaeroides f.s.denitrificans under visible light in the presence of DMSO.The purified enzyme through several column techniques gave a single band in SDS PAGE.A novel assay method for DMSO reductase was developed using capillary electrophoresis. A micellar electrokinetic chromatography mode was quite effective for a quantitative determination below the nanogram level without any pretreatment of the reaction mixture of the enzyme. About 6nl of sample solution was introduced in the hydrostatic mode (20sec, 5cm height) from the anodic end of the capillary. A boratephosphate-SDS buffer, pH7, was used for MEKC.The sample solution was electrophoresed at 15kV with an electric current of around 25 uA,on-column detection being done at an absorbance of 200nm. The enzymatic activity calculated by MEKC was compared with that by conventional optical absorption method. Both methods was in close agreement, indicating that MEKC is useful for a quantitative assay of DMSO reductase. This novel assay method established enabled us to search the electron donor for DMSO reductase. In the water soluble fraction of the cell homogenate, DMSO dependent oxidation activity was found. The active compound was now being purified.
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