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研究生:唐世珍
研究生(外文):Shih-Chen Tang
論文名稱:利用微生物生產不同種類聚羥基烷酸(PHAs)
論文名稱(外文):Producing different polyhydroxyalkanates by microorganisms
指導教授:康世旭
學位類別:碩士
校院名稱:元智大學
系所名稱:生物科技暨生物資訊研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:92
中文關鍵詞:聚羥基烷酸succinate
外文關鍵詞:polyhydroxyalkanoic acids (PHAs)polypropylene (PP)
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先前的研究指出,TCA cycle中的succinyl-CoA可以轉變成methylmalonyl-CoA, methylmalonyl-CoA去掉一個CO2後形成propionyl-CoA,最後經由PhaA與PhaB代謝成PHAs(polyhydroxyvalerate, PHV)(Paulo et al., 2003),但目前並沒有研究指出可以用生物產生高純度PHV的方法。本研究主要是探討提供不同的碳源下Bacillus sp.是否會產生不同種類的PHAs,以及將只合成中長碳鏈PHA的P. putida KT2442利用基因轉質的技術,使其能同時合成短碳鏈及中長碳鏈PHA。
Bacillus sp.在不同的碳源下,如succinate、glucose、sucrose、糖蜜及有機酸,發現除了有機酸不生長外,其他都可以合成胞內的脂質堆積。在氣相層析儀和核磁共振儀的分析中顯示,在以glucose和 sucrose為碳源時胞內的脂質堆積只有poly-ß-hydroxybutyrate (PHB),累積量約25 %,以succinate為碳源時胞內的脂質堆積只有PHV,累積量約15 %,這樣的結果是非常重要的。
我們將Bacillus sp.所產的PHV經純化後,再利用GPC、TGA與DSC作物裡性質及化學性質的分析。又將B. megaterium的PHA合成基因,大小約4.1kb,轉質入帶大桿菌中,以glucose與succinate培養,再以氣相層析儀分析,發現當分別以glucose及succinate為碳源時,改質過的大腸桿菌皆產生PHV。
另外,利用基因轉質技術在P. putida KT2442中放入R. eutropha的PHA operon,在以葡萄糖酸鈉與月桂酸及油酸為碳源培養下,以氣相層析儀分析中顯示,改質過的P. putida KT2442可以同時生成短碳鏈的PHB與中長碳鏈PHA。進一步探討能代謝succinate的細菌是否也會有與Bacillus sp.相同之結果。
Polyhydroxyalkanoic acids (PHAs), are biosynthetic polyester for carbon source storage, produced by a variety of bacteria and archaea under nutrient-unbalance condition with excess carbon source. PHAs have the chemical properties similar to polypropylene (PP) and are biodegradable, insoluble in water, non-toxic, biocompatible, piezoelectric thermoplastic. These features make PHAs are suitable for many applications. According the monomer composition, PHAs are classified into two types, short chain length PHAs (scl-PHAs) with C3–C5 hydroxyacids and medium chain length PHAs (mcl-PHAs) with C6–C16 hydroxyacids. Depending on the monoers compositions, the properties and application of the PHAs vary.
In this study, recombination technology and feeding strategies with different carbon source were used to change the composition for more application. Bacillus sp. can use glucose to produce polyhydroxybutyrate (PHB) when use sodium succinat dibasic only produce polyhydroxyvalarate (PHV) assay by GC and NMR. PHA synthase genes which cloned in B. megaterium transferred to E. coli. Recombinant E. coli use glucose or sodium succinate dibasic as carbon source can produce PHV. The recombinant mcl-PHAs producing bacteria can produce scl-PHAs and mcl-PHAs co-polymers with better properties, but can not use sodium succinate dibasic to produce PHV. Furthermore, some PHAs with brand-new compositions are identified.
英文摘要................................................. V
中文摘要............................................... VII
目錄.................................................... IX
表目錄................................................. XIV
圖目錄.................................................. XV
第一章 前言...............................................1
1.1 可分解生物材料的種類...............................2
1.2 聚羥基烷酸(PHAs)的介紹.............................5
1.2.1 PHAs 的基本結構..............................5
1.3 PHAs 的合成........................................8
1.3.1 PHAs合成的相關代謝..........................10
1.3.2 SCL-PHA的合成...............................11
1.3.3 MCL-PHA的合成...............................12
1.3.4 PTE的形成...................................13
1.4 PHAs的性質........................................15
1.4.1 PHAs性質的鑑定..............................15
1.5 PHAs的應用........................................18
1.6 PHAs的分解........................................19
1.7 PHAs的工業生產....................................20
1.8 生物可分解塑膠的標準檢驗規範......................21
第二章 實驗目標與實驗架構................................23
第三章 實驗材料與方法....................................27
3.1 菌株及質體…......................................27
3.2 培養基............................................28
3.3 藥品..............................................28
3.4 Bacillus megaterium PHA synthase基因的選殖..........29
3.4.1 引子的設計..................................29
3.4.2 以PCR技術增殖B. megaterium 的PHA合成基因..29
3.4.3 B. megaterium 的PHA合成基因的選殖...........29
3.4.4 質體的純化與分析............................30
3.5 大腸桿菌的轉型作用............................... 30
3.6 假單胞菌的轉型作用............................... 31
3.6.1勝任細胞的備製.................................31
3.6.2 轉型作用......................................32
3.7 枯草桿菌的培養....................................32
3.8 重組大腸桿菌的培養................................33
3.9 假單胞菌的培養....................................33
3.10 PHAs之萃取....................................... 34
3.10.1 SDS-次氯酸鈉萃取法...........................34
3.10.2 氯仿-次氯酸鈉萃取法..........................35
3.11 分析方法.........................................35
3.11.1 以氣相層析技術(GC, Gas Chromatography)分析PHA產物.......................................35
3.11.2 NMR(Nuclear Magnetic Resonance)分析PHA產物 ........................................36
3.11.3 穿透式顯微鏡(Transmission Electron Microscopy)................................36
3.11.4 熱重量分析儀(Thermogravimetric Analysis,TGA)38
3.11.5示差掃瞄熱分析儀(Differential Scanning Calorimetry, DSC)..........................38
3.11.6膠體滲透層析儀(Gel-Permeation Chromatography, GPC).......................................38
3.11.7 熱壓成膜...................................39
第四章 結果..............................................26
4.1不同碳源對枯草桿菌累積PHAs的影響..................40
4.1.1 PHAs的組成分析…………........................42
4.1.1.1 PHB與PHV標準品的分析.....................42
4.1.1.2 Bacillus sp.體內所累積的PHAs..............43
4.1.2 以NMR技術進行結構分析........................46
4.1.3以穿透式電子顯微鏡觀察Bacillus sp. KC046與KC056........................................49
4.1.4 PHV的外觀與性質探討...........................50
4.1.5利用GPC測定PHAs的分子量......................51
4.1.6利用TGA測定PHAs的裂解溫度....................53
4.1.7利用DSC測定PHAs..............................54
4.2 基因改質對假單胞菌累積PHAs的影響.................55
4.2.1利用GC分析P. putida KT2442所合成的PHAs........56
4.2.2假單胞菌不同的培養時間對所合成PHAs的影響......59
4.2.3 不同培養時間及碳源對PHAs累積的影響...........69
4.2.4 利用TGA測定PHAs的裂解溫度....................70
4.2.5 利用DSC測定PHAs..............................72
4.3其他PHAs生產株是否可以succinate為碳源產生高純度PHV..............................................74
4.4 利用基因改質技術測試其他菌株是否產生PHV...........77
4.4.1選殖B. megaterium 的PhaP-PhaQ-PhaR-PhaB-PhaC基因並進行功能分析...............................77
4.4.2基因改質對大腸桿菌DH5α累積PHAs的影響........78
4.4.3重組大腸桿菌DH5α進行基因改質.................79
第五章 討論..............................................83
5.1 枯草桿菌所產生的PHAs............................ 83
5.2 假單胞菌所產生的PHAs............................ 84
5.3 PHV的合成途徑................................... 85
5.4 利用穿透式電子顯微鏡觀察PHV在菌體內的累積........ 86
5.5 利用Bacillus megaterium 的PHA synthase基因在大腸桿菌DH5α中累積PHAs..................................87
第六章 參考文獻..........................................88
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