PHA合成基因選殖及重組體表現

Cloning of polyhydroxyalkanoate synthesis genes and expression in recombinants

郭俐櫻(Li-Ying Kuo)

指導教授 ： ARRAY(0xbcbffec)

中原大學/工學院/土木工程研究所/碩士(2009年)

https://doi.org/10.6840/cycu200900748

摘要

利用活性污泥法處理廢污水具有經濟效益，但廢水處理後的廢棄污泥處理及處置對於環境為一重大課題，因此廢棄物減量及再生資源化為現今研究之趨勢。本研究利用基因轉殖技術將活性污泥中特定菌種Ralstonia eutropha之PHA合成基因選殖至大腸桿菌中，控制其最佳生長條件使重組菌體內PHA含量最大化，以達到廢棄污泥減量及資源化之目的。本研究結果顯示野生型以葡萄糖、果糖、醋酸為碳源，合成的PHB含量分別為25.9％、30.2％、17.8％。本研究建構重組大腸桿菌BL21含有質體pET-phaCABRe，在未添加誘導劑條件下以葡萄糖、果糖、醋酸為碳源進行培養，30℃為最佳PHB合成溫度，其PHB含量分別為37.7％、37％、14.6％。添加誘導劑下培養，特別是以葡萄糖為碳源，其合成PHB含量可達73.4％。因此本研究的重組大腸桿菌BL21具有合成PHA之能力，在未來將可應用於以廢棄污泥經處理後之上澄液為碳源合成PHA。

關鍵字

Ralstonia eutropha ； 基因轉殖 ； 重組體 ； polyhydroxyalkanoate (PHA)

並列摘要

It is economically efficiency to treat wastewater by activated sludge. However, disposal of waste sludge which provide by activated sludge treatment is still a major issue for environment. Recently, many research focus on the study of waste reduction and generation . In this study, PHA synthesis genes of specific strains Ralstonia eutropha in activated sludge system were cloned to Escherichia coli. Next, different temperature and carbon source were optimized to maximize the PHA content of recombinant bacteria. The results showed that the highest PHB contents of wild-type fed with glucose, fructose and, acetic acid as carbon source were 25.9％, 30.2％, and 17.8％, respectively. Moreover, this study constructed recombinant E.coli harboring plasmid pET-phaCABRe and cultured at the optimize temperature 30℃ with glucose, fructose and, acetic acid. The PHB content were 37.7％, 37％,and 14.6％, respectively. Furthermore, inducer was added and PHB content of recombinant E.coli was the highest, 73.4%, while glucose was used as carbon source. The recombinant E.coli constructed in this study have ability to synthesize PHA, we will apply this recombinant by feeding supernatant of sludge treatment as the carbon source to synthesize PHA in the future.

並列關鍵字

Gene clone ； Ralstonia eutropha ； Recombinants ； polyhydroxyalkanoate (PHA)

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