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研究生:謝宛珊
研究生(外文):Hsieh, Wan-Shan
論文名稱:生物分解性塑膠(PHA)合成酵素基因之結構分析
論文名稱(外文):Structural Analysis of Polyhydroxyalkanoates Synthase Genes
指導教授:洪淑嫻
指導教授(外文):Hung, Shu - Hsien
學位類別:碩士
校院名稱:大葉大學
系所名稱:食品工程研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:63
中文關鍵詞:生物分解性塑膠聚羥基烷酸酯PHA合成酵素假單胞菌(P. resinovorans)
外文關鍵詞:Biodegradable polymerspoly(hydroxyalkanoates)PHAPHA synthaseP. resinovorans
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Poly(hydroxyalkanoates) (PHAs)是細菌合成的聚酯類高分子,物性接近石化塑膠,可為生物分解性塑膠之材料,但由於成本比石化塑膠高出許多而限制其用途。因此,提高產量以降低成本,改造菌株以生產不同特性之PHA,應當可提昇此生物分解性塑膠之實用性及普及性。PHA有多種型式,依組成單體之碳鏈長度可分成短鏈(PHASCL)、中鏈(PHAMCL)及長鏈(PHALCL)三種。PHAMCL由於碳鏈長度較長,使其具有比PHASCL較好的熔點穩定性,且因其熔點較低,因而增加其可塑性。
Pseudomonas resinovorans為一具有PHAMCL生產能力之菌株,其會依碳源、菌種之不同而堆積出不同鏈長之PHAMCL,進而增加PHA之開發性及實用性。本研究即以此菌株為對象,先利用不同分析方法(Nile red染色、萃沖、gas chromatography)建立PHA生產菌的鑑定方法,並進一步對PHA合成基因進行選殖。
由GC分析結果得知,當P. resinovorans以Octanoic acid 或Hexanoic acid為碳源時,生產之PHA以中鏈PHO和PHD為主。另外在基因選殖方面,我們從P. resinovorans基因庫中篩選出包含完整phaC1-phaZ-phaC2 operon,並可能涵蓋啟動子區域之phage,未來的研究,除了進一步將其它與PHA生合成相關的基因選殖出來,並將對可能之啟動子區域,其活性及調控方式做進一步的探討。

Poly(hydroxyalkanoates) (PHAs) are microbial polyesters that can be used as completely biodegradable polymers. Because of the high production cost, the application of PHA is limited. There are many types of PHAs. According to the chain length of carbon atoms of the monomers, they are classified to three groups: PHASCL, PHAMCL, and PHALCL.
Pseudomonas resinovorans has the ability to produce PHAMCL copolymer when grown on tallow or fatty acid. However, the producing strains as well as the carbon substrates employed give variety to the features of PHAMCL, increasing their potentiality in exploitation.
In this study, the PHA polymers were characterized by Nile red colony staining, Soxtec extraction, and gas chromatography. It was showed that C8 and C10 were the major monomer repeat-unit of the polymer. To ivestigate the genes for the synthesis of PHA in P. resinovorans, we constructed a genomic library from this strain. After several times of screening, a phage clone with a 15 kb EcoRI fragment was found to contain PHA synthase (phaC2). The structures of the genes will be revealed and their functions, such as expression and substrate specificity, will be further investigated.

目 錄
封面內頁
簽名頁
授權書 iii
中文摘要 iv
英文摘要 v
誌謝 vi
目錄 vii
圖目錄 xi
表目錄 xii
附錄圖目錄 xiii
附錄表目錄 xiv
縮寫表 xv
壹、緒論 1
一、PHA之發現 1
二、PHA之化學結構 2
三、PHA之物理特性 2
四、PHA相關酵素基因之分類命名 3
五、PHA合成酵素( PHA synthase ) 4
(一) PHA synthases一級結構 4
(二) PHA 生合成基因分類 5
六、PHA代謝途徑 6
(一) PHASCL合成途徑 6
(二) PHAMCL合成途徑 8
貳、材料與方法 10
一、實驗材料 10
(一)菌株 10
(二)載體(vector) 10
(三)藥品 10
(四)培養基 11
(五)緩衝液及試劑 11
(六)實驗中使用之套組(Kit) 14
二、實驗步驟 15
(一)菌株分析 15
1.菌株培養 15
2.生長曲線 15
3.PHA生產菌鑑定 15
4.PHA生產累積量分析 16
(1)萃沖分析 16
(2) GC分析PHA組成 16
(二) phaC2基因選殖 17
1.Genomic DNA之製備 17
2.大腸桿菌勝任細胞(competent cell)之製備 17
3.轉形作用(transformation) 18
4.質體(Plasmid)DNA之製備 18
5.Partial digestion 19
6.選殖DNA片段之製備 19
7.接合作用(ligation)及建立基因庫 20
8.PCR技術 20
9.DNA產物純化 21
10.南方吸漬法(Southern blotting) 21
11.探針之製備及標定 22
12.雜交反應 22
13.Plaque lifts hydridization 23
14.偵測及顯影技術 23
15.噬菌體DNA之製備 24
16.序列之分析比對 24
參、結果與討論 25
一、生長曲線圖之建立 25
二、Nile red staining 25
三、PHA生產累積量分析 26
四、PHA組成分析 26
五、PHA鑑定及分析方法之建立 26
六、基因選殖 27
(一) phaC-phaA-phaB基因選殖 27
(二) phaC1-phaZ-phaC2基因選殖 27
1. 以PCR擴增genomic DNA中phaC1及phaC2基因 28
2. 基因庫之製備 28
3. 篩選phaC2基因 29
肆、結論 30
參考文獻 31
圖表 41
附錄 55

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