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

用于修复污染土壤的芘降解细菌的蛋白质组分析

• 批准号: 15510077
• 负责人: MINEKI Shigeru
• 金额: $ 2.43万
• 依托单位: TOKYO UNIVERSITY OF SCIENCE
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15510077/
• 关键词: SOIL CONTAMINATION; PYRENE; BACTERIA; PROTEOME; BIOREMEDIATION; ピレン分解菌; プロテオーム解析

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.

为了鉴定参与芘降解的蛋白质，研究了从胰蛋白酶大豆肉汤培养基（TSB）和芘作为唯一碳源的矿物培养基（MMP）各自培养基上生长的细胞中提取的蛋白质的差异。 H2-5、No.3、No.4分枝杆菌菌株在各自的培养基上培养后，收获细胞，用1/15M磷酸盐缓冲液(pH 7.0)洗涤，然后超声匀浆，离心排除细胞碎片和未降解的细胞。将所得上清液（S1）超速离心（183,000 x g，60 分钟）并分离成上清液（可溶部分，S2）和沉淀物（膜部分，P2）。 S2 和 P2 的每个级分均通过 2D SDS-PAGE 进行分析。结果发现，在MMP上生长的细胞中特异表达了3种50kDa的蛋白质、4种40kDa的蛋白质和1种20kDa的蛋白质。在 S2 中，还可以发现一些在 MMP 生长的细胞中特异表达的蛋白质。然而，通过这些程序获得的斑点并不那么清楚。因此，尝试了新的方法。 TSB 培养基改为醋酸盐培养基，其斑点比以前的培养基少。为了降低应用于 2D SDS-PAGE 的样品的盐浓度，通过向样品溶液中添加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。结果，在No.3和No.4菌株中分别确认了9种和7种特异性蛋白质。