| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2004: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2003: ¥3,300,000 (Direct Cost: ¥3,300,000)
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| Research Abstract |
To identify the proteins participating the degradation of pyrene, the differences of proteins extracted from the cells grown on respective media of Tryptic soy broth medium(TSB) and pyrene as a sole carbon source in the mineral medium(MMP), were investigated. After Mycobacterium sp.strains of H2-5,No.3,and No.4, were cultivated on respective media, the cells were harvested and washed with 1/15M phosphate buffer (pH 7.0), then they were homogenized by ultra sonication, and cell debris and undegraded cells were excluded by centrifugation. Resulting supernatant solution (S1) was ultra centrifuged (183,000 x g,60 min) and separated into supernatant (soluble fraction, S2) and precipitate (membrane fraction, P2). Each fraction of S2 and P2 was analyzed by 2D SDS-PAGE. As a result, it was found that 3 proteins of 50 kDa, 4 proteins of 40kDa, and 1 protein of 20 kDa were specifically expressed in the cells grown on MMP. In S2 also, some proteins specifically expressed in MMP-grown cells could be found. However, spots got by these procedures were not so clear. Therefore, new methods were tried. TSB medium was changed to acetate medium, which gave fewer spots than the former medium. To reduce the salt concentration of the sample applying on 2D SDS-PAGE, the protein was precipitated by adding trichloroacetic acid to the sample solution and the precipitate was directly dissolved with Lysis Buffer. Consequently, 9 and 7 specific proteins were confirmed in No.3 and No.4 strains, respectively.
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