臺灣博碩士論文加值系統

本研究使用菌株Comamonas sp. L8U篩選自台灣環境土壤，透過剔除PHA合成酶phaC1L8U基因和可水解3-hydroxybutyryl-CoA (3HBCoA) 的硫酯酶 (thioesterase) TE2、TE4基因，並同時表現下列三個基因，分別為來自Pseudomonas putida H9的廣泛基質專一性PHA合成酶phaC1H9基因、來自Aeromonas caviae的 (R)-specific enoyl-CoA hydratase (phaJ) 可將β-氧化路徑中間體trans-2-enoyl-CoA轉化成 (R)-3-hydroxyhexanoyl-CoA、Vitreoscilla sp. 之細菌血紅蛋白vgb基因。本研究所使用的碳源果糖酸 (levulinic acid) 為可再生性碳源，可由稻稈、蔗渣和麥稈等農業廢棄物中的纖維素，在酸性加熱條件下轉換獲得，能達到不與人類搶糧食與耕地之目的。果糖酸對於一般的細菌具有毒性，Comamonas sp. 可利用不同比例的果糖酸和己酸 (hexanoic acid) 為複合碳源，獲得不同單體組成的poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) 三元共聚物。此聚合物的組成以3HV單體為主3HB、3HHx為輔。使用GC、NMR、GPC、DSC和奈微拉伸試驗機等多種儀器測量，分析單體組成、物理性質如玻璃轉換溫度 (Tg)、熔點 (Tm)、結晶溫度 (Tc) 和機械性質如抗拉強度 (tensile strength)、楊氏模量 (Young's modulus)、斷裂伸長率 (elongation at break) 等。

Comamonas sp. L8U is isolated form environmental soils in Taiwan. Deletion PHA synthase phaCL8U gene and the thioesterase genes, TE2 and TE4, which can hydrolyze 3-hydroxybutyryl-CoA (3HBCoA). Exhibiting phaC1 gene of a broad range substrate specificity from Pseudomonas putida H9, (R)-specific enoyl-CoA hydratase (phaJ) of Aeromonas caviae which converts the internediate β-oxidation pathway, trans-2-enoyl-CoA, into (R)-3-hydroxyhexanoyl-CoA, and the hemoglobin vgb of Viteloscilla sp. of. The carbon source levulinic acid used in this study is a renewable carbon source. It can be converted from cellulose in agricultural waste such as rice straw, bagasse and wheat straw under acidic heating conditions, and can achieve the purpose of not robbing humans of grain and cultivated land. Levulinic acid is toxic to bacteria. Comamonas sp. can use different ratios of levulinic acid and hexanoic acid as composite carbon sources to obtain poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) terpolymers with different monomer compositions. The composition of this polymer is dominated by 3HV monomers, while 3HB and 3HHx are auxiliary. Using a variety of instruments to measure such as GC, NMR, GPC, DSC and Micro/Nano tensile tester. Analysis of monomer composition, physical properties such as glass transition temperature (Tg), melting temparature (Tm), crystallization temparature (Tc) and mechanical properties for tensile strength, Young's modulus, and elongation at break.

摘 要 i
Abstract ii
目 錄 iii
表目錄 vi
圖目錄 vii
附圖目錄 viii
第一章 前 言 1
一、石化塑膠與環境汙染 1
二、聚羥基烷酸酯（polyhydroxyalkanoates, PHA） 1
1. 來源 1
2. 聚羥基烷酸酯的化學結構 2
3. 聚羥基烷酸酯的物理性質 3
4. 聚羥基烷酸酯的生合成路徑 4
5. 聚羥基烷酸酯的應用 5
三、果糖酸 (levulinic acid) 6
四、Comamonas sp. L8U 7
五、研究目的與策略 8
1. 研究目的 8
2. 研究策略 8
第二章 材料與方法 10
一、實驗菌株及培養基 10
二、器材與儀器 10
三、基因剔除與基因置換 11
1. 基因剔除之PCR擴增 11
2. 基因置換之PCR擴增 11
3. 接合作用 11
4. 轉形作用 12
5. 質體DNA的萃取與定序 12
6. 接合生殖 (conjugation) 12
7. 基因剔除與置換 (doube crossover) 13
四、基因互補實驗 13
五、PHA之累積 14
六、PHA含量和單體組成之分析 14
1. PHA之轉酯化 14
2. 氣相層析儀分析 14
七、PHA之饋料批式醱酵實驗 14
八、PHA之半連續醱酵試驗 (semi-continous fermerntation) 16
九、PHA之萃取與純化 16
十、PHA之分子量分析 16
十一、PHA之聚合物化學結構分析 17
十二、PHA之膜片觀察與顯微鏡分析 17
十三、PHA之物理性質分析 17
十四、PHA之機械性質分析 18
第三章 結 果 19
一、置換Comamonas sp. L8U phaC基因對PHA累積的影響 19
二、剔除Comamonas sp. L8U多種基因並表現phaC1H9基因對於PHA累積的影響 19
三、Comamonas sp.重組菌株表現phaJ基因和vgb基因對PHA累積的影響 20
四、Comamonas sp.重組菌株之饋料批式醱酵試驗 20
五、Comamonas sp.重組菌株之半連續醱酵試驗 22
六、醱酵試驗之PHA單體組成分析 (NMR) 22
七、醱酵試驗之PHA聚合物分子量分析 (GPC) 22
八、醱酵試驗之PHA膜片外觀和顯微鏡觀察 23
九、PHA聚合物熱性質分析 23
十、醱酵試驗之PHA聚合物機械性質分析 (拉伸試驗) 24
第五章 結 論 28
第六章 參考文獻 29

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