PROTEOME ANALYSIS OF PYRENE-DEGRADING BACTERIA FOR REMEDIATION OF CONTAMINATED SOIL

为了鉴定参与芘降解的蛋白质，研究了在胰蛋白酶大豆肉汤培养基（TSB）和以芘为唯一碳源的无机培养基（MMP）上生长的细胞提取的蛋白质的差异。将H2- 5、No.3和No.4分枝杆菌菌株在各自的培养基上培养后，收获细胞，用1/15 M磷酸盐缓冲液（pH7.0）洗涤，然后用超声处理将它们匀浆，离心除去细胞碎片和未降解的细胞。将所得上清液（S1）超离心（183，000 x g，60 min）并分离成上清液（可溶性级分，S2）和沉淀物（膜级分，P2）。通过2D SDS-PAGE分析S2和P2的每个级分。结果发现，在MMP上生长的细胞中特异性表达3种50 kDa的蛋白，4种40 kDa的蛋白和1种20 kDa的蛋白。在S2细胞中也能检测到MMP特异性表达的蛋白质，但斑点不明显。因此，尝试了新的方法。TSB培养基改为醋酸盐培养基，得到的斑点比以前的培养基少。为了降低应用于2D SDS-PAGE上的样品的盐浓度，通过向样品溶液中加入三氯乙酸来沉淀蛋白质，并将沉淀物直接用裂解缓冲液溶解。因此，在3号和4号菌株中分别确认了9个和7个特异性蛋白。

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.