| Budget Amount *help |
¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2000: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
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.
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