臺灣博碩士論文加值系統

生質柴油是一種可再生且具有環保性質的替代能源，目前利用生物法生產生質柴油，大多是以固定化酵素或全細胞為觸媒，而這些生物觸媒製備繁複、價格昂貴，會增加生產生質柴油的成本和能量消耗，因此需要開發新的具有經濟效益的生物觸媒及生物催化製程。本研究開發了一種同步整合重組脂肪酶製備與原位生質柴油合成的新製程，此製程以畢氏酵母菌Pichia pastoris表達重組疏棉狀嗜熱絲孢菌脂肪酶Thermomyces lanuginosus lipase ( TLL )，直接利用含有胞外酵素及全細胞的培養液作為觸媒催化生質柴油合成，而重組P. pastoris可以利用生質柴油合成過程中所產生的副產物-甘油做為碳源，持續製造分泌重組TLL至培養基中催化生質柴油合成。我們構築了重組質體pGAPZα-TLL，在P. pastoris X33中成功表達具有活性的TLL並分泌到培養基中，分析此重組TLL性質，顯示在37℃，pH 10具有最佳的水解活性，屬於耐鹼性酵素。以此重組P. pastoris培養液為觸媒，大豆油為原料，進行原位生質柴油合成，並採用兩種不同反應流程，即同步轉酯化與酯化反應(concurrent method) 與先水解再酯化逐步反應(stepwise method)，結果顯示這兩種反應流程，油相與水相(即培養液)混合的最佳體積比例相似(皆為1:3)，最佳的甲醇總添加量也相似(皆為32%)，但是所達到的生質柴油轉化率分別為88.9% (concurrent method) 與76.0% (stepwise method) 。我們嘗試在水相中控制添加相同菌量，即油相與培養液混合的體積比例固定為1:1，再以液體培養基調整水相體積，結果發現兩種反應流程的最佳油相與水相混合體積比例皆改變為1:5，最佳甲醇總添加量皆仍為32%，但是在生質柴油轉化率則顯著提升分別為91.4% (concurrent method) 與94.4% (stepwise method)，酵素活性測試亦顯示，控制添加相同菌量的培養液中含有較高的重組脂肪酶活性。本研究結果顯示，整合型生質柴油合成的產率顯著高於非整合型，而且以油相與培養液混合比例1:1，油相與總水相混合比例1:5，並以stepwise method添加32%甲醇，反應96小時後可得最高94.4%的生質柴油轉化率。這種簡化的製程無需酵素製備的過程，故可適用於工業生產生質柴油。

In previous lipase-catalyzed production of biodiesel, a renewable and environmentally friendly alternative liquid fuel, immobilized enzymes or whole cell catalysts have been extensively used. However, enzymes are prepared in an independent process separated from enzymatic biodiesel production. This would increase the cost and energy consumption. Therefore, there is an urgent need to develop novel cost-effective biocatalysts and biocatalytic processes. In this study, a novel and efficient integrated process with coupled lipase production and in situ biodiesel synthesis has been developed. Based on a recombinant Pichia pastoris culture expressing Thermomyces lanuginosus lipase (TLL), the dual biocatalytic system takes advantage of both cell free enzymes and whole cell catalysts. The recombinant P. pastoris utilizes glycerol, the by-product of biodiesel synthesis, as a carbon source and constitutively secretes recombinant TLL into medium to catalyze the synthesis of biodiesel. We had constructed reconstruct pGAPZα-TLL plasmid into P. pastoris X33. The recombinant P. pastoris strain secreted active TLL into culture medium. Biochemical analysis revealed that the recombinant TLL had an optimal temperature at 37℃ and showed alkalophilic property with an optimal pH at 10. Using recombinant P. pastoris liquid culture as biocatalyst and soybean oil as raw material, in-situ biodiesel synthesis was performed through two different reaction processes, namely concurrent and stepwise methods. The results showed that these two methods had a similar optimal oil-water (i.e. oil-culture) ratio of 1:3, and similar optimal total methanol feeding amounts (32%). The biodiesel yields were 88.9% for concurrent method and 76.0% for stepwise method. When adjusting a same amount of yeast cells (i.e. oil-culture ratio is 1:1) in different oil-water ratios, the optimal oil-water ratio became 1:5 between these two methods, and the optimal total methanol feeding amounts did not change (32%). However, the biodiesel yields were significantly increased to 91.4% for concurrent method and 94.4% for stepwise method. Lipase activity assay revealed the culture from adding same amount of cells contained higher amount of lipase than the culture with different amount of cells. This study suggested that the biodiesel yield of the integrated process was higher than the non-integrated process (i.e. using separately prepared catalysts), and the highest 94.4% biodiesel yield can be achieved under 1:1 oil-culture ratio and 1:5 oil-water ratio, and by feeding 32% methanol using stepwise method for 96-hour reaction. This simplified single-step process could represent a significant advance toward achieving economical production of biodiesel at industrial scale.

目錄
中文摘要 I
Abstract III
目錄 V
表目錄 XII
圖目錄 XIII
附錄 XVI
壹、緒論 1
一、生質柴油 1
1、前言 1
2、生質柴油 (Biodiesel) 之簡介 2
3、生質柴油的生產 3
3.1、化學催化法 (Chemical process) 3
3.2、酵素催化法 (Enzymatic process) 4
4、兩種不同的酵素催化策略用於生質柴油的生產 5
4.1、傳統方法 (Conventional method) 5
4.2、整合方法 (Integrated method) 6
5.、生質柴油的反應流程 6
5.1、同步轉酯化與酯化 (Concurrent transesterification-
esterification ) 7
5.2、逐步水解後酯化 (Stepwise hydrolysis-esterification) 7
二、脂肪酶 7
1.、脂肪酶之簡介 7
2、脂肪酶催化反應 8
3、界面活化作用 (Interfacial activation) 8
4、脂肪酶在工業上之應用 9
4.1、家庭清潔劑 9
4.2、食品添加物 10
4.3、造紙工業 11
4.4、其他應用 11
5.、Thermomyces lanuginosus lipase (TLL) 11
6、Pichia pastoris 表達系統 12
7、畢赤酵母菌株 (Pichia pastoris strain) 13
貳、研究目的 15
参、研究材料與方法 17
一、微生物菌種 17
1.、大腸桿菌 ( Escherichia coli ) 17
2.、酵母菌 ( Pichia pastoris ) 17
二、質體與質體DNA的製備 17
1.、質體 17
2.、pGAPZαA & pPICZαA Vector 製備 18
3.、TLL Insert 製備 19
三、質體構築 20
四、質體轉型 (Transformation) 與DNA定序分析 21
1.、E. coli 轉型作用 ( E. coli Transformation ) 21
2.、DNA 定序分析 21
3.、P. pastoris 轉型作用 (P. pastoris Transformation ) 22
3.1、酵母菌 P. pastoris 品系 X33、GS115、KM71、SMD1168勝任
細胞 (Competent cell) 的製備 22
3.2、轉型 DNA 的製備 23
3.3、Phenol/Chloroform 法去除雜蛋白之方法 23
3.4、酒精沉澱之方法 24
3.5、電穿孔轉型 24
五、轉型株的純化 25
六、重組蛋白表達 25
七、蛋白質的定量測定與菌種保存 26
1.、蛋白質的定量 26
2.、菌種保存 27
八、聚丙烯醯胺膠體電泳 (Sodium dodecyl sulfate–polyacrylamide gel electrophoresis, SDS-PAGE) 27
九、酵素生化特性分析 29
1.、脂肪酶的活性測定 29
1.1、Tributyrin (三丁酸甘油酯) agar plate測定 29
1.2、脂肪酶 (lipase) 水解活性測定 29
2.、最適 pH 值 31
3.、pH穩定性分析 32
4、最適作用溫度 32
5、熱穩定分析 32
6、有機溶劑對脂肪酶活性之影響 33
7、脂肪酶轉酯化反應 (Transesterification reaction) ━ 生質柴油的合成 33
7.1、油脂的酸價測定 35
7.2、脂肪酸甲酯測定 35
十、原位生質柴油合成 37
1、含脂肪酶的菌株培養液製備 37
2、兩種原位生質柴油的反應流程 38
2.1、同步轉酯化與酯化反應(Concurrent transesterification-
esterification) 38
2.1.1、不同含水量的影響 38
2.1.1.1、添加不同量的P.psatoris培養液 (無控制菌量) 38
2.1.1.2、添加相同量的P.psatoris培養液 (控制菌量) 39
2.1.2不同甲醇濃度的影響 39
2.2、先水解再酯化逐步反應(Stepwise hydrolysis-
esterification) 40
2.2.1、不同含水量的影響 40
2.2.1.1、添加不同量的P.psatoris培養液 (無控制菌量) 40
2.2.1.2、添加相同量的畢酵母培養液 (控制菌量) 40
2.2.2不同甲醇濃度的影響 41
3、檢測生質柴油反應後pGAPZα-TLL/ P. pastoris X33培養液之酵素
活性 41
肆、研究結果 42
一、pGAPZαA-TLL 和 pPICZαA-TLL重組質體的構築 42
二、重組pGAPZαA-TLL 和 pPICZαA-TLL蛋白質的表現 42
三、重組酵素生化性質分析—pGAPZαA-TLL脂肪酶和 pPICZαA-TLL脂肪
酶兩種表達系統 43
1、脂肪酶活性的測定 43
1.1、Tributyrin (三丁酸甘油酯) agar plate活性測定 43
1.2、脂肪酶 (lipase) 水解活性測定 44
2、最適 pH 值 44
3、pH穩定性分析 45
4、最適作用溫度 46
5、熱穩定分析 47
6、有機溶劑對脂肪酶活性之影響 47
7、脂肪酶轉酯化反應 (Transesterification reaction) ━ 生質
柴油的合成 49
四、原位生質柴油合成 50
1、兩種原位生質柴油的反應流程 50
1.1.、不同含水量的影響 50
1.1.1、添加不同量的P.psatoris培養液 (無控制菌量) 50
1.1.2、添加相同量的P.psatoris培養液 (控制菌量) 51
1.2、不同甲醇濃度的影響 52
1.2.1、不同甲醇濃度對於添加不同量的P.psatoris培養液之原位生質
柴油生產的影響 (無控制菌量) 52
1.2.2、不同甲醇濃度對於添加相同量的P.psatoris培養液之原位生質
柴油生產的影響 (控制菌量) 53
2、檢測生質柴油反應後pGAPZα-TLL/ P. pastoris X33培養液之酵素
活性 54
伍、討論 56
一、pGAPZαA質體和 pPICZαA質體 56
二、重組酵素生化性質分析—pGAPZαA-TLL 和 pPICZαA-TLL兩種表達
系統之比較 56
三、兩種原位生質柴油的反應流程比較 58
1、不同含水量的影響 58
1.1、添加不同量的P.psatoris培養液 (無控制菌量) 59
1.2、添加相同量的P.psatoris培養液 (控制菌量) 59
2、不同甲醇濃度的影響 60
四、檢測生質柴油反應後pGAPZα-TLL/ P. pastoris X33培養液之酵
素活性 60
陸、參考文獻 62

表目錄
表1、兩種原位生質柴油的合成反應 66

圖目錄
圖1、pGAPZαA-TLL& pPICZαA-TLL重組質體圖與限制酶 mapping 電
泳圖 67
圖2、pGAPZαA -TLL DNA 定序之序列比對 68
圖3、pPICZαA -TLL DNA 定序之序列比對 69
圖4、pGAPZαA-TLL 和 pPICZαA-TLL質體轉型到P. pastoris X33、
GS115、KM71、SMD1168宿主細胞實驗結果 70
圖5、帶有pGAPZαA-TLL 之P. pastoris X33、GS115、KM71、
SMD1168轉型株的蛋白電泳分析 71
圖6、帶有pPICZαA-TLL之P. pastoris X33、GS115、KM71、
SMD1168轉型株的蛋白電泳分析 72
圖7、pGAPZαA-TLL 和 pPICZαA-TLL 在P. pastoris X33、GS115、
KM71、SMD1168轉型株的蛋白表達電泳分析 73
圖8、pGAPZαA-TLL 和 pPICZαA-TLL 在P. pastoris X33、GS115、
KM71、SMD1168轉型株於Tributyrin (三丁酸甘油酯) agar
plate活性測定 74
圖9、重組TLL (pGAPZα-TLL / Pichia X33-1)與商業化 ET之最適pH
值 75
圖10、重組TLL (pGAPZα-TLL / Pichia X33-1)與商業化 ET之pH穩
定性分析 76
圖11、重組TLL (pGAPZα-TLL / Pichia X33-1)與商業化 ET之最適
作用溫度 77
圖12、重組TLL (pGAPZα-TLL / Pichia X33-1)與商業化 ET之熱穩
定分析 78
圖13、重組TLL (pGAPZα-TLL / Pichia X33-1)與商業化 ET之有機
溶劑對脂肪酶活性的影響 80
圖14、重組pGAPZαA-TLL脂肪酶與重組pPICZαA-TLL脂肪酶酯化反應
81
圖15、重組pGAPZαA-TLL利用Concurrent與Stepwise系統進行不同
P.psatoris培養液含量對原位生質柴油生產的影響之比較 82
圖16、重組pPICZαA-TLL利用Concurrent與Stepwise系統進行不同
P.psatoris培養液含量對原位生質柴油生產的影響之比較 83
圖17、重組pGAPZαA-TLL利用Concurrent與Stepwise系統添加相同
P.psatoris培養液含量並調節培養基體積來控制水的比例進行對
原位生質柴油生產的影響之比較 84
圖18、重組pPICZαA-TLL利用Concurrent與Stepwise系統添加相同
P.psatoris培養液含量並調節培養基體積來控制水的比例進行對
原位生質柴油生產的影響之比較 85
圖19、重組pGAPZαA-TLL利用Concurrent與Stepwise系統進行不同甲
醇濃度對於添加不同量的P.psatoris培養液之原位生質柴油生產
的影響之比較 86
圖20、重組pPICZαA-TLL利用Concurrent與Stepwise系統進行不同甲
醇濃度對於添加不同量的P.psatoris培養液之原位生質柴油生產
的影響之比較 87
圖21、重組pGAPZαA-TLL利用Concurrent與Stepwise系統進行不同甲
醇濃度對於添加相同量的P.psatoris培養液之原位生質柴油生產
的影響之比較 88
圖22、重組pPICZαA-TLL利用Concurrent與Stepwise系統進行不同甲
醇濃度對於添加相同量的P.psatoris培養液之原位生質柴油生產
的影響之比較 89
圖23、生質柴油合成後，檢測pGAPZα-TLL/ P. pastoris X33培養液
之脂肪酶活性。 90


附錄
附錄1、轉酯化反應之反應式 91
附錄2、批次加入甲醇對於轉酯化的影響 92
附錄3、生產生質柴油的化學催化法與酵素法的比較 93
附錄4、兩種不同的策略用於生質柴油的生產 94
附錄5、『同步轉酯化與酯化』反應和『逐步水解後酯化』反應脂肪酶
生產生質柴油的主要過程 95
附錄6、脂肪酶水解作用 96
附錄7 脂肪酶催化反應 97
附錄8、pGAPZαA, B, C質體圖 98
附錄9、pPICZαA, B, C質體圖 99

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