Chaperone in methanotroph and its application for bioremediation

为了利用生物修复技术(生物处理)净化地下水污染，我们分离到了一株降解三氯乙烯的甲烷硝化菌--甲基胞囊藻。这株表达可溶性甲烷单加氧酶(SMMO)的菌株已被用于TCE的生物修复。但在TCE降解过程中，甲壳藻的初始降解速度较慢。认为TCE降解活性半衰期较短是由于TCE降解所形成的有毒中间体的酶失活所致。这是甲藻的一个亟待解决的问题。M用于生物修复过程，因为甲壳藻具有持续的降解活性。M在TCE降解过程中是必需的。作为对有害环境条件的反应，细胞可能会产生额外的蛋白质，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，我们研究了一种应激蛋白--伴侣因子在甲壳藻中的表达。M，位于sMMO基因上游约450bp处。在该地区发现了两个ORF，暂定名为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之处。相反，cpn60具有分子伴侣60标志，与其他分子伴侣有很高的同源性。为了检测cpn10和cpn60对sMMO基因表达的影响，我们尝试在E.colt和恶臭假单胞菌中过表达这些基因，但没有成功，其原因尚不清楚，可能是由于包涵体的形成和大肠杆菌不能组装活性复合蛋白所致。此外，还预测了sMMO基因调控存在未知的机制。

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.