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Chaperone in methanotroph and its application for bioremediation

甲烷氧化菌中的分子伴侣及其在生物修复中的应用

基本信息

批准号：
11660098
负责人：
UCHIYAMA Hiroo
金额：
$ 0.77万
依托单位：
National Institute for Environmental Studies
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11660098/
关键词：
methanotroph methane monooxygenase chaperone stress protein trichloroethylene バイオレメディエーション

项目摘要

In order to clean up the groundwater contamination by bioremediation technology (biological treatment), we isolated a TCE-degrading methanotroph, Methylocystis sp. M.The use of this strain, which expresses sMMO (soluble methane monooxygenase), has been assessed for the bioremediation of TCE.However, during TCE degradation, the initial degradation rate by Methylocystis sp. M declined.It has been suggested that the short half-life of TCE-degradation activity was due to enzyme inactivation by toxic intermediates formed by TCE degradation. This is a major problem to be solved if Methylocystis sp. M is to be used in a bioremediation process since constant degradation activity by Methylocystis sp. M is required during TCE degradation.As a response to harmful environmental conditions, the cell may produce additional proteins, referred to as stress proteins. We expect that enhancing the amounts of stress proteins induced by TCE may increase the short half-life of TCE degradation activity and, therefore, facilitate enhanced degradation ability.The first, we investigated chaperonelike factor, a kind of stress protein, in Methylocysttis sp. M, and found in about 450bp upstream region of sMMO gene. In this region, two orfs were located and named as cpn10 and cpn60, tentatively. Although cpn10 contained leucine-zipper motif, no similarity was observed to chaperones. Contrary, cpn60 had chaperonin 60 signiture and high homology to other chaperones. To examine the effect of cpn10 and cpn60 to sMMO gene expression, over-expression of these genes in E.colt and Pseudomonas putida was attempted.However, attempts were not successful, reasons for which were unknown but could have been due to the formation of inclusion body and the inability of E.coli to assemble active complex proteins.Furthermore, the existence of unknown mechanism in sMMO gene regulation was predicted.

为了利用生物修复技术（生物处理）对地下水污染进行治理，我们分离了一株降解TCE的甲烷营养菌Methylocystis sp. m .，并利用该菌株对TCE进行了生物修复。然而，在TCE降解过程中，Methylocystis sp. M的初始降解速率下降。有人认为，TCE降解活性的半衰期短是由于TCE降解形成的有毒中间体使酶失活所致。如果要在生物修复过程中使用Methylocystis sp. M，这是一个需要解决的主要问题，因为在TCE降解过程中需要Methylocystis sp. M的持续降解活性。作为对有害环境条件的反应，细胞可能产生额外的蛋白质，称为应激蛋白。我们预计，增加TCE诱导的应激蛋白的数量可能会增加TCE降解活性的短半衰期，从而促进降解能力的增强。首先，我们研究了Methylocysttis sp. M中的一种应激蛋白伴侣蛋白样因子（chaperonelike factor），发现其位于sMMO基因上游约450bp的区域。在这一地区发现了两个山口，暂定命名为cpn10和cpn60。虽然cpn10含有亮氨酸-拉链基序，但与伴侣蛋白没有相似性。相反，cpn60具有伴侣蛋白60特征，与其他伴侣蛋白具有高度同源性。为了研究cpn10和cpn60对sMMO基因表达的影响，我们尝试在大肠杆菌和恶臭假单胞菌中过表达这两个基因。但未能成功，其原因尚不清楚，可能与大肠杆菌包涵体的形成和无法组装活性复合体蛋白有关。进一步预测sMMO基因调控存在未知机制。