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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 吳蕙芬 | |
dc.contributor.author | Po-Shu Shih | en |
dc.contributor.author | 施博書 | zh_TW |
dc.date.accessioned | 2021-06-17T04:38:02Z | - |
dc.date.available | 2023-08-15 | |
dc.date.copyright | 2018-08-15 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-08-07 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70775 | - |
dc.description.abstract | 微生物所分泌的脂肪酶主要應用於食品工業,清潔劑工業及其他化學工業上,且其分離及純化較動物性脂肪酶及植物性脂肪酶更為容易,因此微生物脂肪酶的生產及研究有其重要性。而其中Bacillus spp.脂肪酶由於有耐熱性或在鹼性環境下酵素作用不受影響,因此特別受到重視,而且其脂肪酶的分子量大小是所有微生物脂肪酶中最小的一群,有利於在生物科技上的應用。然而由於在大規模量產微生物脂肪酶方面,原菌株通常無法生成大量的酵素,因此需透過選殖一個能夠生產大量脂肪酶,且具有特殊性質的轉化株,才能有效應用於工業上。雖然已有許多Bacillus spp.的脂肪酶基因已被定序,也進一步的表現於異源寄主中,但對於Bacillus amyloliquefaciens脂肪酶的研究仍不充分,我們從土壤環境中分離而得的菌株Bacillus amyloliquefaciens中選殖出一段脂肪酶基因,並轉殖到E.coli中表現,得到具有分泌脂肪酶能力的轉殖菌株,並測其分解脂質能力。利用IPTG誘導蛋白質生產並進一步的純化,得到分子量大小約為25 kDa的LipA脂肪酶。此脂肪酶最適反應溫度為35℃、最適pH值為10;此酵素在溫度35℃下酵素活性穩定、且在pH值為9-10時具有良好的pH值穩定性。由受質特異性測試可知此脂肪酶相對於短鏈酯質更偏好長鏈酯質,尤其是在受質為p-nitrophenyl laurate (p-NPC12)時,其水解能力達最佳。10 mM Zn2+、Fe2+、Cu2+、Ca2+、SDS、Triton-X-100 均會強烈的抑制此酵素活性。另外,在10% 有機溶劑存在下,DMSO、Isopropanol均可些微提高酵素活性,但到了有機溶劑濃度為25%時,大部分的有機溶劑會開始抑制酵素活性。以薄層層析法初步分析LipA脂肪酶水解橄欖油後產物的生成,從結果可知其隨著時間的拉長,產物fatty acid及diglyceride的生成量上升,表示此脂肪酶可應用於生質柴油的生產及油脂的改造。以酸鹼滴定法測定原菌株Bacillus amyloliquefaciens、轉殖菌株及LipA脂肪酶對橄欖油及大豆油的降解率,首先在橄欖油方面,在反應96小時後其降解率分別為38.82%、45.33%、53.46%;而三者對大豆油的降解率則分別為35.46%、43.14%、62.2%,表示三者均有能力水解橄欖油,但其作用效率不同。而此油類降解的測定可以進一步應用於廢油回收處理。 | zh_TW |
dc.description.abstract | Microbial lipases are mainly applied in food industry, detergents and the other chemical industry. Because the isolation and purification of lipases from microorganisms is easier than those of animals and plants, the mass production and study of lipases from microorganisms are really important. Lipases of Bacillus spp. are heat and alkaline tolerant, so the lipase activity under heat and alkaline conditions can remain stable. Besides, lipases from the Bacillus spp. belong to those of the smallest molecular weight, and this make it a benefit for application in biotechnology. However, the original bacterial strain usually can not produce large amount of lipases, so it is better to select a transformant carrying a lipase gene in a plasmid which can produce large amount of lipases with special and specific characteristics for effective application in industry. Although many lipase genes form Bacillus spp. have been sequenced, studies on lipases of Bacillus amyloliquefaciens are still not comprehensive. A strain of Bacillus amyloliquefaciens was isolated from the environment and its lipase gene was selected, and cloned into pET21a, for expressing in E.coli by an induction with IPTG. Through the purification of the lipase protein (LipA) by the nickle column, a protein whose molecular weight is about 25kDa was obtained. The optimal temperature and pH value for LipA lipase activity are 35℃ and pH-10, respectively. Also, the LipA lipase is stable under 35℃ and pH value 9-10. The LipA lipase prefers p-nitrophenyl laurate as its substrate. The buffer containing a final concentration of 10 mM Zn2+, Fe2+, Cu2+, Ca2+, SDS or Triton-X-100 would strongly suppress the enzymatic activity. 10% DMSO and isopropanol would slightly enhance the enzyme activity, but 25% of most organic solvent could inhibit LipA activity. The results of the thin-layer-chromatography (TLC) showed that the longer the incubation time, the more degradation product of olive oil, including fatty acid and diglyceride. The degradation rate of olive oil after 96 hrs by B. amyloliquefaciens, the transformant expressing and LipA lipase was 38.8%, 45.3% and 53.5%, respectively, whereas for soybean oil, it was about 35.5%, 43% and 62%, respectively. Taken together, the above results revealed that LipA lipase has the potential to be applied in biodiesel production, recycled oil treatment and oil modification. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T04:38:02Z (GMT). No. of bitstreams: 1 ntu-107-R05623024-1.pdf: 2828987 bytes, checksum: 507dcf674c0703e93a41a0b83b9e89ae (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 目錄
摘要 i Abstract iii 1.緒論 1 1.1研究緣起 1 1.2研究方向和目的 2 2.文獻回顧 4 2.1脂肪酶(lipase) 4 2.1.1脂肪酶的種類 4 2.1.2脂肪酶的構形 15 2.1.3脂肪酶催化形式 19 2.1.4脂肪酶的性質專一性 19 2.1.5脂肪酶與縮酸酯酶相異之處 20 2.1.6 脂肪酶抑制劑 20 2.1.6.1脂肪酶抑制劑種類 21 2.2 Bacillus spp. 在脂肪酶上之發展 27 2.3廢食用油汙染 30 2.3.1廢食用油生產生質柴油 32 2.3.2廢食用油產製廢油甲酯 38 2.3.3廢食用油多元化再利用 40 3.材料與方法 41 3.1實驗器材 41 3.1.1藥品、酵素及相關套組 41 3.1.2.實驗設備 41 3.1.3分析及製圖軟體 42 3.2.培養基 43 3.3可降解油類之菌種來源及篩選降解基因建構轉殖株 45 3.3.1.菌株來源及菌種鑑定 45 3.3.2.初步篩選可降解油類菌株 45 3.3.3抽取Chromosome DNA 45 3.3.4聚合酶鏈鎖反應 (Polymerase chain reaction, PCR) 46 3.3.5 PCR產物純化 47 3.3.6 DNA瓊脂膠體(Agarose gel)電泳 47 3.3.7質體DNA製備 47 3.3.8限制酶截切作用 48 3.3.9接合反應(Ligation) 48 3.3.10勝任細胞(competent cell)製備 49 3.3.11轉型作用(Transformation) 49 3.3.12轉殖質體的篩選及確認 50 3.4蛋白質分析 50 3.4.1蛋白質的誘導表現 50 3.4.2蛋白質純化 50 3.4.3十二浣基硫酸鈉聚丙烯醯胺凝膠電泳(Soduim dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) 51 3.4.4蛋白質濃縮 52 3.4.5蛋白質定量 52 3.5酵素特性之測定 52 3.5.1酵素活性測定 52 3.5.2測定酵素受質特異性 53 3.5.3測定pH值對酵素活性及穩定性影響 53 3.5.4測定溫度對酵素活性及熱穩定性影響 54 3.5.5測定不同金屬離子對酵素活性之影響 54 3.5.6測定不同介面活性劑對酵素活性之影響 54 3.5.7測定不同有機溶劑對酵素活性之影響 55 3.5.8測定脂肪酶抑制劑對酵素活性之影響 55 3.6以不同食用油培養基觀察原菌株、轉殖菌株及LipA脂肪酶降解油脂能力 56 3.7以薄層層析法(Thin-layer chromatography,TLC)初步分析酵素分解橄欖油之情形 56 3.8以酸鹼滴定法分析油類降解率 56 4.結果與討論 58 4.1 Bacillus amyloliquefaciens分解油類基因篩選與分析 58 4.1.1篩選分解油類基因及序列分析 58 4.1.2分解基因蛋白質序列及分析 58 4.2蛋白質分析 59 4.2.1以蛋白質變性膠體電泳(SDS-PAGE)分析酵素誘導表現及純化情形 59 4.2.2酵素特性分析 59 4.3食用油培養基降解 67 4.4以薄層層析法初步分析酵素降解橄欖油情形 68 4.5以酸鹼滴定法分析油類降解率 68 4.5.1原菌株與轉殖菌株對油類的降解率測試 68 4.5.2 LipA脂肪酶對油類的降解率測試 69 5.綜合討論 71 6.結論 75 7.參考文獻 76 8.圖表集 84 9.附錄 108 表目錄 表2-1、產生脂肪酶的微生物種類 6 表2-2、細菌脂肪酶來源 7 表2-3、嗜熱及嗜冷微生物脂肪酶 9 表2-4、商業用微生物脂肪酶之來源、應用與供 10 表2-5、脂肪酶發酵條件 11 表2-6、微生物脂肪酶特性 12 表2-7、Bacillus脂肪酶之生化特性 29 表2-8、鹼性催化劑與脂解媒催化劑之比較 35 表2-9、生質柴油與石化柴油標準之比較 36 表2-10、生質柴油(B100、B20)排放量與石化柴油之比較 37 表一、食用油培養基降解 84 圖目錄 圖2-1、脂肪酶蓋子結構 16 圖2-2、α/β水解酶構型 17 圖2-3、脂肪酶構型改變3-D結構 18 圖2-4、脂肪酶及脂肪酶抑制劑與介面活性劑及脂質之交互作用 26 圖2-5、B. amyloliquefaciens E1PA 及其他相近的Bacillus脂肪酶之親緣關係樹 28 圖2-6、廢油甲酯生產 39 圖一、轉殖株結果確認 85 圖二、Bacillus amyloliquefaciens之lipA基因核苷酸序列及胺基酸序列 86 圖三、轉殖菌株接種於tributyrin agar 87 圖四、Bacillus amyloliquefaciens之LipA基因核苷酸序列與Bacillus amyloliquefaciens subsp. plantarum NAU-B3(HG514499.1)之核苷酸序列比對 88 圖五、胺基酸比對。 89 圖六、以蛋白質變性膠體電泳分析LipA脂肪酶誘導表現及純化情形 90 圖七、LipA脂肪酶於三丁酸甘油酯培養基之測試結果 91 圖八、LipA脂肪酶之受質特異性測試 92 圖九、LipA脂肪酶於不同pH值環境中之酵素活性 93 圖十、LipA脂肪酶於不同pH值環境中之酵素穩定性 94 圖十一、LipA脂肪酶於不同溫度下酵素活性測試 95 圖十二、LipA脂肪酶於不同溫度中之酵素穩定性 96 圖十三、LipA 脂肪酶於不同金屬離子存在下之酵素活性測試 97 圖十四、LipA 脂肪酶於不同介面活性劑存在下之酵素活性測試 98 圖十五、LipA脂肪酶於不同有機溶劑存在下之酵素活性測試 99 圖十六、LipA脂肪酶於不同濃度脂肪酶抑制劑存在下之酵素活性測試 100 圖十七、LipA脂肪酶於終濃度5mM脂肪酶抑制劑(Orlistat)存在下之加入不同量之酵素活性測試 101 圖十八、LipA脂肪酶於終濃度10mM脂肪酶抑制劑(Orlistat)存在下之加入不同量之酵素活性測試 102 圖十九、以薄層層析法初步分析橄欖油降解產物 103 圖二十、橄欖油降解率 104 圖二十一、LipA脂肪酶之橄欖油降解率 105 圖二十二、大豆油降解率 106 圖二十三、LipA脂肪酶大豆油降解率 107 附錄目錄 附錄一、 LipA脂肪酶之受質特異性活性測定 108 附錄二、LipA脂肪酶於不同pH值下之酵素活性測定 109 附錄三、LipA脂肪酶於不同pH值下之酵素穩定性測定 110 附錄四、LipA脂肪酶於不同溫度下之酵素活性測定 111 附錄五、LipA脂肪酶於不同溫度下之酵素穩定性測定 112 附錄六、LipA脂肪酶於不同金屬離子存在下之酵素活性測試 113 附錄七、LipA脂肪酶於不同介面活性劑存在下之酵素活性測試 114 附錄八、LipA脂肪酶於不同介面活性劑存在下之酵素活性測試 115 附錄九、LipA脂肪酶於不同有機溶劑存在下之酵素活性測試 116 附錄十、LipA脂肪酶於不同濃度脂肪酶抑制劑(Orlistat)存在下之酵素活性測試 117 附錄十一、LipA脂肪酶於終濃度5 mM脂肪酶抑制劑(Orlistat)存在下之加入不同量之酵素活性測試 118 附錄十二、LipA脂肪酶於終濃度10 mM脂肪酶抑制劑(Orlistat)存在下之加入不同量之酵素活性測試 119 附錄十三、B.amyloliquefaciens對橄欖油降解率 120 附錄十四、LipA-21轉殖菌株對橄欖油降解率 121 附錄十五、LipA脂肪酶對橄欖油降解率分析 122 附錄十六、B.amyloliquefaciens對大豆油降解率 123 附錄十七、LipA-21轉殖菌株對大豆油降解率 124 附錄十八、LipA脂肪酶對大豆油降解率分析 125 附錄十九、BSA蛋白質之檢量線 126 附錄二十、對硝基苯酚之檢量線 126 附錄二十一、本論文所使用的菌株 127 附錄二十二、本論文所使用之質體 127 附錄二十三、本試驗所使用之引子 128 | |
dc.language.iso | zh-TW | |
dc.title | Bacillus amyloliquefaciens之LipA分離純化及性質分析 | zh_TW |
dc.title | Purification and characterization of LipA from Bacillus amyloliquefaciens | en |
dc.type | Thesis | |
dc.date.schoolyear | 106-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 林乃君,羅凱尹,徐駿森,陳建德 | |
dc.subject.keyword | LipA脂肪?;枯草桿菌屬;廢油處理;生質柴油;油脂改造, | zh_TW |
dc.subject.keyword | Bacillus amyloliquefaciens;LipA lipase;biodiesel production;oil modification;recycled oil treatment, | en |
dc.relation.page | 128 | |
dc.identifier.doi | 10.6342/NTU201802715 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2018-08-08 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 農業化學研究所 | zh_TW |
顯示於系所單位: | 農業化學系 |
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