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研究生:黃怡樺
研究生(外文):Yi-Hua Huang
論文名稱:植物乳汁來源脂肪分解酵素篩選及生質柴油生產應用
論文名稱(外文):Screening of plant lipases and applications to biodiesel production
指導教授:蔡少偉蔡少偉引用關係
指導教授(外文):S. W. Tsai
學位類別:碩士
校院名稱:長庚大學
系所名稱:生化與生醫工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
論文頁數:110
中文關鍵詞:生質柴油木瓜脂肪分解酵素轉酯
外文關鍵詞:biodieselpapaya lipasetransesterification
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由於石油枯竭與環境污染的問題,替代能源的重要性與日俱增。生質柴油之物性、化性與化石柴油相近,且具備可再生、生物可分解性與環保等特點而十分有應用潛力。政府已規劃自2008年起化石柴油中必須添加1%生質柴油,2010年為2%,2020年升高至5%,進而達到2010年生質柴油年使用量10萬公秉,2020年15萬公秉的目標。目前工業化生產生質柴油的方法為鹼催化法。強鹼製程之優點為反應速率快、技術也較成熟,但此法有耗能、甘油回收困難、催化劑須從產物中移除、鹼廢水需處理以及游離脂肪酸和水會干擾反應等缺點。相反的,酵素催化法反應條件溫和、副產物甘油易分離,且適用於游離脂肪酸和水含量較高的原料;然其催化劑較昂貴而目前尚無商業化應用實例。因此本研究之主要目標為利用價格較低廉之植物乳汁來源脂肪分解酵素來進行生質柴油之生產,期望能夠降低酵素製程之成本。
實驗結果發現植物乳汁來源脂肪分解酵素中以木瓜脂肪分解酵素最具應用之潛力,然而其具有1,3位置選擇性,且對於MG之反應性較差;在無法提升醯基轉移速率的情況下,本研究便採用兩階段的雙酵素法合成生質柴油。當第一階段使用20 wt% pCPL、第二階段為2.5 wt% Novo 435於45˚C下進行反應,反應物中甲醇與大豆油中估計酯鍵之莫耳比為1.7:1時,於反應24小時後可達到80.9%的甲酯產率。此外,當基質為廢食用油時,本反應系統亦不受基質中的FFA及水分干擾,於相同條件下(除了甲醇與廢油中假設酯鍵之莫耳比為1.3:1),反應24小時後同樣達到80%的甲酯產率,顯示雙酵素法相當適合用來將廢食用油轉化為生質柴油。
Alternative fuels are becoming increasingly important due to the diminishing of petroleum reserves and the environmental pollution of exhaust gases from petroleum-fuelled engines. Biodiesel has much applying potential because of its similar physical and chemical properties to diesel, aside from the advantages such as renewable, biodegradable, and environment-friendly. The government has planned to combine fossil diesel with 1% biodiesel starting from 2008, following blend with 2% from 2010, and finally go up to 5% from 2020. Presently, the method to produce biodiesel industrially is the alkali-catalyzed process. This process is more developed and gives high conversion of esters in short reaction time. However, the reaction has several drawbacks: it is energy intensive, recovery of glycerol is difficult, the alkaline catalyst has to be removed from the product, alkaline wastewater requires treatment, and free fatty acids and water interfere with the reaction. On the contrary, the enzymatic method is performed in mild conditions, and the by-product, glycerol, can be easily recovered, and also can apply to raw material containing higher free fatty acids and water. However, the cost of biocatalyst is much more high and there are still no commercial applications. Therefore, in this research, we will use cheaper lipases from plant latex to produce biodiesel, expecting to lower the cost of enzymatic process.
According to the experimental results, papaya lipase has the most potential applying to biodiesel production. However, pCPL has 1,3- regioselectivity, besides its reaction ability with monoacylglycerol is bad. In the situation that the rate of acyl migration couldn’t be improved easily, we adopted the method using two lipases in series for biodiesel production. While the reaction was carried out with 20 wt% pCPL in the first step and 2.5 wt% Novo 435 in the second step, where the molar ratio of methanol and ester bonds of triglyceride was 1.7 to 1, a methyl ester yield of 80.9% was obtained after 24 hours at 45˚C. Furthermore, when using waste oil as the substrate, the reaction was not interfer by free fatty acids and water contained in the substrate. Under the same reaction conditions (except for the molar ratio of methanol and ester bonds contained in the waste oil as 1.3 to 1), a methyl ester yield of 80% was obtained after 24 hours. The result showed that bi-enzyme method was quite suitable for converting the waste oil into biodiesel.
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摘要 i
英文摘要 ii
誌謝 iv
目錄 v
圖目錄 viii
表目錄 xi
符號說明 xii

目錄
第一章 緒論 1
1-1 再生能源之發展背景 1
1-1-1 化石能源枯竭 1
1-1-2 環境污染 1
1-1-3 再生能源 2
1-1-4 各種替代燃料之評價比較 3
1-2 生質柴油之簡介 4
1-3 生質柴油與化石柴油之特性比較 6
1-4 生質柴油之優缺點 8
1-5 生質柴油之發展 10
1-6 生質柴油之製備 10
1-6-1 轉酯化反應 11
1-6-2 鹼催化法 13
1-6-3 酸催化法 14
1-6-4 酵素催化法 15
第二章 文獻回顧 17
2-1 酵素催化法生產生質柴油之研究現況 17
2-2 酵素催化法生產生質柴油之機制 22
2-3 研究動機 25
第三章 實驗方法 27
3-1 藥品與材料 27
3-2 儀器設備 28
3-3 分析條件建立 29
3-4 酵素製備 32
3-5 生質柴油合成 32
3-6 雙酵素系統合成生質柴油 32
3-6-1 酵素量之選擇 33
3-6-2 兩階段合成生質柴油 33
3-7 利用廢食用油合成生質柴油 34
3-8 pCPL之再生 34
第四章 結果與討論 35
4-1 植物脂肪分解酵素之篩選 35
4-2 酵素量之選擇 35
4-3 酵素之前處理 39
4-4 pCPL之除水 43
4-5 不同第三丁醇溶劑添加量對反應的影響 44
4-6 不同醇類基質影響 47
4-7 TOG、1,3-DOG、1,2-DOG、1-MOG及2-MOG之轉酯速率比較 48
4-8 添加擔體之測試 52
4-9 添加擔體之前處理 55
4-10 雙酵素系統催化生產生質柴油 66
4-10-1 酵素量之選擇 66
4-10-2 兩階段合成生質柴油 67
4-11 利用廢食用油合成生質柴油 78
4-12 pCPL之再生 78
第五章 結論與未來工作 83
5-1結論 83
5-2 酵素量之選擇 84
參考文獻 88
附錄一:現行各國生質柴油標準規範 94

圖目錄
圖1-1 使用生質能之碳循環流程圖 3
圖1-2 TG與醇類進行轉酯反應產生烷基酯(生質柴油)和甘油之總反應與中間
反應 13
圖1-3 鹼催化、酸催化及酵素催化法之流程 16
圖2-1 TG經酵素法合成ME之機制 23
圖2-1 (續上頁)TG經酵素法合成ME之機制 24
圖2-2 實驗架構示意圖 26
圖3-1 未經轉酯之大豆油HPLC分析圖 29
圖3-2 以Novo 435進行轉酯反應24小時之大豆油HPLC分析圖 29
圖3-3 各成分之校正曲線圖 31
圖4-1 以不同量之Novo 435進行反應,溶液中各成分含量之變化 37
圖4-2 以不同量之pCPL進行反應,溶液中各成分含量之變化 38
圖4-3 以不同前處理方式之pCPL進行反應,溶液中各成分含量之變化 40
圖4-4 以第三丁醇含浸1小時之5 wt% Novo 435進行反應,溶液中各成分含
量之變化 42
圖4-5 以第三丁醇預含浸1小時之20 wt% pCPL於不同體積第三丁醇溶劑中進
行反應,溶液中各成分含量之變化 45
圖4-5 (續上頁)以第三丁醇預含浸1小時之20 wt% pCPL於不同體積第三丁醇
溶劑中進行反應,溶液中各成分含量之變 46
圖4-6 以不同醇類進行反應,溶液中各成分含量之變化 49
圖4-7 以乙酸乙酯進行反應,溶液中各成分含量之變化 50
圖4-8 以第三丁醇配製10 mM之基質利用20 wt% pCPL進行轉酯反應之轉化
率變化 51
圖4-9 2-MG之醯基轉移機制(類似酸催化法) 53
圖4-10 2-MG之醯基轉移機制(類似鹼催化法) 53
圖4-11 Silica gel之表面結構 54
圖4-12 添加5 wt% 聚丙醯酸樹脂進行反應,溶液中各成分含量之變化 56
圖4-13 添加5 wt% 離子交換樹脂進行反應,溶液中各成分含量之變化 57
圖4-13 (續上頁)添加5 wt% 離子交換樹脂進行反應,溶液中各成分含量之
變化 58
圖4-14 添加不同量silica gel進行反應,溶液中各成分含量之變化 59
圖4-14 (續上頁) 添加不同量silica gel進行反應,溶液中各成分含量之變
化 60
圖4-15 添加15 μl HClO4進行反應,溶液中各成分含量之變化 61
圖4-16 以第三丁醇預含浸1小時之20 wt% pCPL於65˚C下進行反應,溶液
中各成分含量之變化 62
圖4-17 添加5 wt% 經第三丁醇含浸一小時之DEAE Sephadex A-25擔體進
行反應,溶液中各成分含量之變化 63
圖4-18 添加10 wt% 經pH值等於8之磷酸緩衝溶液處理之silica gel擔體進
行反應,溶液中各成分含量之變化 65
圖4-19 以5 wt% Novo 435進行反應,溶液中各成分含量之變化 68
圖4-20 結合不同酵素量Novo 435與20 wt% pCPL進行反應,溶液中各成分
含量之變化 69
圖4-20 (續上頁)結合不同酵素量Novo 435與20 wt% pCPL進行反應,溶液
中各成分含量之變化 70
圖4-21 結合不同酵素量Novo 435與20 wt% pCPL進行反應,溶液中ME含量
之變化 71
圖4-22 結合不同酵素量pCPL與2.5 wt% Novo 435進行反應,溶液中各成分
含量之變化 72
圖4-23 結合不同酵素量pCPL與2.5 wt% Novo 435進行反應,溶液中ME含
量之變化 73
圖4-24 結合20 wt% pCPL與2.5 wt% Novo 435,加入不同量甲醇進行反
應,溶液中各成分含量之變化 74
圖4-25 結合20 wt% pCPL與5 wt% Novo 435進行反應,溶液中各成分含量
之變化 76
圖4-26 使用含有pCPL雜質之甲醇為基質,並以5 wt% Novo 435進行反
應,溶液中各成分含量之變化 77
圖4-27 以廢食用油為基質進行反應,溶液中各成分含量之變化 80
圖4-28 以甲醇為基質,並使用再生酵素進行反應,溶液中各成分含量之變
化 81
圖4-29 以正丁醇為基質,並使用再生酵素進行反應,溶液中各成分含量之變
化 82
圖5-1 酵素催化甘油酯轉酯反應機構 85

表目錄
表1-1 世界各國原油蘊藏量、生產量與可採年限 1
表1-2 各種替代燃料之評價比較 4
表1-3 各種植物油脂肪酸甲酯(ME)的理化特性比較 6
表1-4 生質柴油與化石柴油特性比較 7
表1-5 使用B20及純生質柴油減少空氣污染物質之比率 8
表1-6 鹼催化、酸催化及酵素催化法之比較 15
表2-1 酵素法生產生質柴油之研究成果 20
表2-1 (續上頁)酵素法生產生質柴油之研究成果 21
表3-1 不同油酸衍生物之fj值 30
表4-1 以不同植物乳汁來源之脂肪分解酵素進行反應24小時後反應物中所含
各成分之含量 35
表4-2 以5 wt% 及10 wt% Novo 435進行反應150小時後反應物中各成分之
含量 36
表4-3 以10 wt% 以及20 wt% pCPL進行反應48小時後反應物中各成分之含
量 36
表4-4 以不同方法前處理之pCPL進行反應24小時後反應物中各成分含量 41
表4-5 真空除水不同時間之pCPL進行反應24小時後反應物中各成分含量 43
表4-6 以第三丁醇預含浸1小時之20 wt% pCPL於不同量第三丁醇中進行反應
24小時後反應物中各成分之含量 47
表4-7 以不同基質進行反應24小時後反應物中各成分之含量 47
表4-8 各成分之初始反應速率 48
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蘇俊吉,〈利用微胞電動力毛細管層析法分離同化性類固醇〉,國立成功大學,碩士論文,民國96年。
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