| Budget Amount *help |
¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2001: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2000: ¥600,000 (Direct Cost: ¥600,000)
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| Research Abstract |
A pyrene-assimilating bacterium ; Mycobacterium sp. H2-5, was cultivated on pyrene as the sole source of carbon. Crude enzyme was reacted with pyrene in phosphate buffer (pH 7) at 30℃ for 1 hr. Pyrene that remained was extracted with CH_2Cl_2 from the reaction mixture, and quantified by, HPLC analyzes. Pyrene-oxidizing activity was determined by. measuring the pyrene that disappeared during the reaction. The activity required NADPH and MgCl_2.The homogenate contained pyrene at a high concentration, even after washing the cells with sufficient buffer, perhaps because it was tightly absorbed to the cells when the cells were harvested. The homogenate was centrifuged at 15, 000 rpm, and the supernatant obtained was further separated into the soluble fraction and the membrane fraction by ultracentrifugation at 130, 000 x g for 1 hr. Pyrene disappearing activity was detected most in the membrane fraction. The possibility of participation of other components to the activity was suggested, because the activity existed at membrane fraction and its total activity was fairly lost compared with that of the homogenate. Therefore, I think this enzyme belongs to the family of cytochrome P450 (CYP450). However, clear results were not obtained in the tests using CYP450-specific inhibitors as aminobenzotriazole and miconazole. If the enzyme belongs to CYP450, the activity depends on other proteins. As an activity-measuring system independent of other proteins, I tested systems of adding oxidizers or electron donors to the reaction mixture, but problems remained to establishing the assay system. However, I got a foothold on establishing it in the test of adding an electron donor to the reaction mixture without NADPH.
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