Kineosphaera limosa 菌株中 phaC 基因之序列分析

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葉傳山(Chuan-Shan Yeh) 查詢紙本館藏

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論文名稱

Kineosphaera limosa 菌株中 phaC 基因之序列分析
(Sequence analysis of the phaC gene of Kineosphaera limosa)

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摘要(中)

利用微生物系統生產可分解性的材質，來解決全球普遍性的塑膠廢棄物污染問題，是日益重要的課題。PHA 是一種聚合性的酯類，常發現於不同種類的革蘭氏陽性菌、革蘭氏陰性菌以及古細菌的細胞內。這類聚酯化合物以一種不可溶的聚合體沈積在細胞質中，作為額外碳源與能量來源的儲存物質。由於 PHA 具有生物可分解性、熱塑性等特性，被認為是生物科技工業重要產業之一。最近，劉文佐博士實驗室由活性汙泥系統分離出一株名為 Kineosphaera limosa (代號Lpha5) 的革蘭氏陽性菌，此菌具有合成大量 PHA 的能力。值得令人注意的是 Lpha5 在無氧、有氧的環境下皆能合成 PHA 。
我們的研究結果顯示，在 Lpha5 菌株中的確存有 phaC 基因(PHA 合成酵素)；我們使用低專一性序列引子 (degenerate DNA primer) 成功的將部份 Lpha5 的 phaC 基因選殖出來，經過 DNA 和蛋白質序列的比對，發現我們選殖 Lpha5 的 phaC 基因片段與其他菌種的 phaC 基因有很高比例的相似性；現在我們利用此 phaC 基因片段作為探針，使用 DNA 雜交的方式篩選 Lpha5 的基因庫 (genomic library)，希望能更進一步找到完整的 phaC 基因叢，以便能進一步研究 Lpha5菌株中有哪些蛋白質或酵素牽涉到 PHA 的代謝。

摘要(英)

The utilization of biological systems for production of biodegradable materials is becoming more important as a solution of the problems concerning plastic waste and the global environment. Polyhydroxyalkanoic acids (PHA) represent a complex class of storage polyesters that are synthesized by a wide range of different gram-positive and gram-negative bacteria as well as by some archaea. These polyesters are deposited as a form of insoluble cytoplasmic inclusions, and can be used as intracellular carbon and energy-storage materials. Since these bacterial PHAs are biodegradable thermoplastics, they have attracted industrial attention as possible candidates for large-scale biotechnological products. Recently, a gram-positive bacterium, named Lpha5, capable of accumulating high levels of PHA, was isolated in Professor WT Liu’s lab from an activated sludge system fed with acetate. An especially intriguing feature regarding the metabolism of this PHA-producing strain is that it can make the polyesters in aerobic as well as anaerobic environments.
Using a set of degenerate primers, I was able to amplify a partial sequence of the phaC gene (PHA synthase) from Lpha5 chromosomal DNA. Protein sequence deduced from this segment of DNA revealed a high similarity to phaC genes from other organisms, suggesting an evolutionarconservation for this class of enzymes. A genetic screening is underway to clone the whole PHA gene cluster, which will profoundly further our understanding of PHA matabolism is this particular bacterium.

關鍵字(中)

★ 聚羥丁酸
★ 生物可分解塑膠

關鍵字(英)

★ kineosphaera limosa
★ phaC
★ PHB
★ PHA

論文目次

目錄
文中摘要-----------------------------------------------------------1
英文摘要-----------------------------------------------------------2
第一章序論-----------------------------------------------------4
壹、生物可分解塑膠---------------------------------------------4
貳、微生物的聚羥丁酸------------------------------------------5
參、PHA 聚合物存在的生理意義----------------------------6
肆、PHA 的分類-------------------------------------------------8
伍、PHA 生產菌群----------------------------------------------8
陸、PHA 合成酵素---------------------------------------------10
柒、研究材料: Kineosphaera limosa (Lpha5)---------------11
第二章研究目的---------------------------------------------------13
第三章材料與方法------------------------------------------------14
壹、培養基的製備-----------------------------------------------14
貳、Lpha5 的培養----------------------------------------------17
參、Lpha5 染色體 DNA 的分離----------------------------17
肆、聚合酵素鏈反應-------------------------------------------18
伍、Lpha5 染色體基因庫的構築----------------------------22
陸、染色體基因庫的篩選-------------------------------------26
柒、實驗方法流程圖-------------------------------------------30
捌、染色體徒步-------------------------------------------------31
第四章結果與討論----------------------------------------------35
壹、Lpha5 的培養----------------------------------------------35
貳、Lpha5 染色體 DNA 的分離----------------------------36
參、聚合酵素鏈反應-------------------------------------------37
肆、PCR 產物之選殖------------------------------------------38
伍、PCR 產物之 DNA 序列---------------------------------39
陸、核酸序列與資料庫比對之結果-------------------------41
柒、DNA 探針雜交後顯影之結果--------------------------43
捌、染色體徒步之結果----------------------------------------45
第五章未來展望---------------------------------------------51
參考文獻---------------------------------------------------------------52
圖目錄------------------------------------------------------------------IV
圖目錄
圖一、PHB 化學結構圖----------------------------------------------5
圖二、微生物可將代謝中間產物轉變PHA-----------------------7
圖三、Alcaligenes eutrophus 的 PHA 合成酵素---------------10
圖四、Lpha5 在電子顯微鏡下的形態----------------------------12
圖五、Lpha5 的培養-------------------------------------------------35
圖六、Lpha5 染色體 DNA 的分離-------------------------------36
圖七、Semi-nasted PCR 放大phaC 基因------------------------37
圖八、PCR 產物之選殖結果---------------------------------------38
圖九、DNA 序列與資料庫比對之結果--------------------------41
圖十、蛋白質序列與資料庫比對之結果-------------------------42
圖十一、DNA探針雜交後顯影結果------------------------------43
圖十二、DNA探針雜交後顯影結果------------------------------44
圖十三、染色體徒步之 PCR 產物--------------------------------45

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指導教授

王健家(Chien-Chia Wang)

審核日期

2003-7-14

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