假單胞菌Pseudomonas sp. A46之基因工程菌開發及重金屬之生物累積和生物吸附潛力探討;Development of Recombinant Pseudomonas sp. A46 for heavy metal bioremediation and its potential of bioaccumulation and biosorption

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題名:	假單胞菌Pseudomonas sp. A46之基因工程菌開發及重金屬之生物累積和生物吸附潛力探討;Development of Recombinant Pseudomonas sp. A46 for heavy metal bioremediation and its potential of bioaccumulation and biosorption
作者:	呂哲瑋;Lu, Che-Wei
貢獻者:	生命科學系
關鍵詞:	銅;鎘;微生物整治;金屬硫蛋白;生物累積;生物吸附;Copper;Cadmium;Metallothionein;Bioaccumulation;Biosoprtion
日期:	2018-07-16
上傳時間:	2018-08-31 11:36:57 (UTC+8)
出版者:	國立中央大學
摘要:	生物累積及生物吸附是微生物作為抵抗外界重金屬的兩個重要的機制，可以將外界的重金屬與環境做分離、去毒性並固定在細菌體內，使得細菌進而成為極有潛力的生物整治工具。本研究嘗試在Pseudomonas sp. A46菌株中表現金屬硫蛋白，試圖提升其生物累積之能力。為了增加蛋白質表現之穩定以及避免使用抗生素，使得此菌株能夠應用在野外，本研究開發能於Pseudomonas sp. A46使用之反向篩選系統，在剔除upp基因後，將能表現綠螢光蛋白與金屬硫蛋白融合蛋白之基因片匣整合至染色體中，對染色體做PCR分析確認片匣成功植入染色體中，接著利用反轉錄PCR分析以及西方墨點法證實融合蛋白成功表現，並且連續繼代40次確定基因之穩定性。完成菌株建立後，針對A46、M01及pJBME測定各菌株對於重金屬銅和鎘之最小抑制濃度，銅皆為250ppm，鎘皆為20ppm，在敏感度測試亦得到一樣的結果，菌株對於重金屬之生長抗性沒有因基因改造而有所增加。在銅及鎘之生物累積試驗中，A46、M01及pJBME並沒有明顯差異，顯示金屬硫蛋白可能不能增加生物累積之能力。在EDTA淋洗試驗中，發現大部分的銅累積於胞內，推得此菌對於銅主要行生物累積，但是總體移除率只有2%，推得外送幫浦蛋白及生物膜構造有效阻隔銅的進入。然而，在相同實驗中，大多數之鎘卻吸附於胞外，並且總體移除率達30%，顯示此菌對於鎘主要行生物吸附，生物膜的產生有助於吸附重金屬鎘並且保護菌株。雖然金屬硫蛋白無法提升此株菌之生物累積能力，但本株菌對於重金屬鎘有著強大之生物吸附能力，未來可以針對其機制做更深入之研究。;Bioaccumulation and biosorption are two important mechanisms for some microorganisms to resist heavy metals, which makes bacteria a potential bioremediation tool. In an attempt to enhance the bioaccumulation capacity of Pseudomonas sp. A46 strain, we cloned the gene of metallothionein into Pseudomonas sp. A46. We developed a genetic bacteria with counter selection system that can be used in Pseudomonas sp. A46. After removing the upp gene, the gene fragment that expresses the fusion protein. Thereafter, the minimum inhibitory concentrations (MIC) for the heavy metal copper and cadmium were determined for A46, M01(MT1 expressed by chromosome) and pJBME (expressed MT1 by plasmid). The MIC of copper was 250 ppm and the cadmium was 20 ppm. With these MIC values, the resistance of the genetic strain to heavy metals was not increased. In the copper and cadmium bioaccumulation assay, there was no significant difference among A46, M01, and pJBME, suggesting that metallothionein may not increase the bioaccumulation capacity. In the EDTA leaching test for the biosorption of heavy metals, most of the copper was found to accumulate in the interior of the cells. The bioaccumulation of this bacterium was a key player. However, the overall removal rate was only 2%. n the other hand, most of the cadmium was adsorbed extracellularly, and the overall removal rate was 35%, indicating that the bacterium bioremediate cadmium via biosorption, and the biofilm production was helpful in adsorbing cadmium and protecting the strain. Although metallothionein cannot increase the bioaccumulation capacity of this strain, this strain of bacteria has a strong biosorption capacity for cadmium.
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