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研究生:吳森源
研究生(外文):Sen-Yuan Wu
論文名稱:台稉9號米糠酯酶/脂肪酶之純化及生化特性研究
論文名稱(外文):Purification and Characterization of Esterase/Lipase from Tai Keng 9 Rice Bran
指導教授:李雅琳李雅琳引用關係陳駿季陳駿季引用關係
指導教授(外文):Ya-Lin LeeJunne-Jih Chen
學位類別:碩士
校院名稱:朝陽科技大學
系所名稱:生化科技研究所碩士班
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:2011
畢業學年度:99
語文別:中文
論文頁數:87
中文關鍵詞:基質專一性生化特性酯酶脂肪酶米糠
外文關鍵詞:substrate specificitybiochemical characterizationesteraselipaserice bran
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米糠 (rice bran) 是稻米加工後的副產物,富含油脂、機能性及抗氧化成分,並且存在多種脂質分解酵素 (EC 3.1.1.X.)。酯酶 (carboxylesterase, EC 3.1.1.1) 與脂肪酶 (lipase, EC 3.1.1.3) 為其中重要的水解酵素,由於可能引起米糠氧化酸敗 (rancidification),故本研究以台稉九號米糠為材料,純化分離酵素後,探討其生化特性及基質特異性,期望未來可應用於水稻育種之參考。

米糠粗酵素液經SDS-PAGE及酯酶活性染色分析,顯示分子量~21.5 kDa處,有一個酵素活性條帶,若以native-PAGE分析,則呈現4個活性條帶;以短碳鏈脂肪酸酯 (p-nitrophenyl butyrate) 及長碳鏈脂肪酸酯 (p-nitrophenyl palmitate) 之酯酶及脂肪酶基質分析,分別得到比活性157 U/mg protein及1.66 U/mg protein,推測酵素液同時具有酯酶及脂肪酶,惟前者活性遠高於後者。酵素粗萃液再以20-70%硫酸銨飽和度沉澱劃分,並接續三次管柱層析步驟純化 (Superdex75、Hi-Trap DEAE FF、Superdex75),可分離得到兩個酵素蛋白質,分別命名為lipase I (21.5 kDa) 及lipase II (22 kDa),lipase I純化倍率為86.3倍,回收率0.24%,Lipase II純化倍率為7.74倍,回收率2.96%,經二維電泳分析確定其等電點 (pI) 分別為4.5及8.4;lipase I及lipase II皆可水解α-naphthyl butyrate (C4) 及β-naphthyl oleate (C18:1)基質,其中lipase I對β-naphthyl oleate的活性較lipase II高。生化特性分析顯示lipase I及lipase II之最適溫度和pH值相似,分別是40 ℃與pH 9.0,且lipase II 的耐熱性微幅高於lipase I。在基質特異性方面,lipase I偏好短鏈脂肪酸酯,lipase II則偏好短鏈及中鏈者。使用化學試劑處理二酵素,結果顯示:1 mM PMSF 及1 mM EDTA 對二者均有抑制效果;1 mM DTT及1 mM IAA對於二酵素的抑制效果相近,1 mM SDS 可降低lipase I及lipase II的活性,5 mM SDS 幾乎完全抑制lipase II活性。據此推測二酵素之活性中心可能為絲胺酸或半胱胺酸、蛋白質結構可能含有雙硫鍵、需要二價金屬離子協助催化活性之執行,並且lipase I之結構穩定性較lipase II高。
Rice bran is a by-product during rice milling, containing abundant oils, functional components, antioxidants, and many lipolytic enzymes (EC 3.1.1.X.) as well. Among the enzymes carboxylesterase (EC 3.1.1.1) and lipase (EC 3.1.1.3) are very important, because they may cause rancidification during rice grain storage. In this study, we have used rice cultivar Tai-Keng 9 (TK9) to isolate and purify esterase/lipase. Through studying the biochemical characteristics and substrate specificities of the enzyme, we expect to employ this information on rice breeding in the future.
The rice bran crude enzymes were analyzed by SDS-PAGE and esterase activity staining, showing a single activity band about 21.5 kDa. When analyzed with native-PAGE four active bands were observed. Short-chain fatty acid ester (p-nitrophenyl butyrate) and long-chain fatty acid ester (p-nitrophenyl palmitate) were used for esterase and lipase activities analyses, and their specific activities were 157 and 1.66 U/mg protein, respectively. It indicated the existence of both activities of esterase and lipase in the extract, but the former was more significant than the latter. The crude extract was subjected to 20-70% ammonium sulfate saturation precipitation and three columns of chromatography were conducted, including resins superdex75, Hi-Trap DEAE FF and then superdex75. Two lipolytic enzymes were separated and dubbed as lipase I (21.5 kDa) and lipase II (22 kDa) with pI 4.5 and 8.4, respectively, resolved by two-dimensional gel electrophoresis. The purification fold and recovery were 86.3 and 0.24% for lipase I, and 7.74 and 2.96% for lipase II. Activity stain of electrophoretic gels showed that both enzymes were able to hydrolyze α-naphthyl butyrate (C4) and β-naphthyl oleate (C18:1), while lipase I showed a higher activity toward β-naphthyl oleate (C18:1) than did lipase II. The study of biochemical characteristics showed that both enzymes had similar optimal temperature and pH at 40 ℃ and pH 9.0, respectively. The thermal stability of lipase II was slightly higher than that of lipase I. The study of substrate specificity showed that lipase I preferred short-chain fatty acid esters, and lipase II short- and medium-chain fatty acid esters. The chemical reagent retreatment experiments showed that both enzymes were effectively inhibited by 1 mM PMSF or 1 mM EDTA; 1 mM of DTT and IAA bore similar inhibition effects; 1 mM SDS significantly reduced the activities of lipase I and lipase II, and 5 mM almost completely inhibited the activity of lipase II. These results may suggest that their active centers are composed of serine or cysteine, their proteins structures contain disulfide bond(s), their activity performance needs divalent metal ion(s), and the structure of lipase I is more tolerable than lipase II.
中文摘要......................................................................I
Abstract....................................................................III
致謝..........................................................................V
總目錄.......................................................................VI
表目錄.......................................................................XI
圖目錄......................................................................XII
縮寫表......................................................................XIV
第一章 前言..................................................................1
第二章 文獻回顧..............................................................2
 2.1 稻米介紹...............................................................2
  2.1.1 稻的種類及型態.....................................................2
  2.1.2 台梗9 號...........................................................3
  2.1.3 稻米結構...........................................................3
  2.1.4 米糠組成及應用.....................................................3
  2.1.5 米糠酸敗...........................................................4
  2.1.6 米糠穩定化.........................................................5
 2.2 解脂酵素...............................................................6
  2.2.1 脂肪酶.............................................................6
  2.2.2 脂肪酶之結構特性...................................................6
  2.2.3 脂肪酶之催化機制...................................................7
  2.2.4 脂肪酶之催化反應...................................................8
  2.2.5 脂肪酶之專一性.....................................................9
  2.2.6 酯酶..............................................................10
  2.2.7 脂肪酶與酯酶之特性比較............................................10
  2.2.8 脂肪酶與酯酶之分類................................................11
 2.3 植物脂肪酶............................................................12
  2.3.1 植物脂肪酶之基因家族..............................................13
  2.3.2 植物脂肪酶之蛋白質結構............................................14
  2.3.3 植物脂肪酶之相關研究..............................................14
  2.3.4 米糠脂肪酶之相關研究..............................................15
 2.4 蛋白質純化............................................................16
  2.4.1 硫酸銨沉澱法......................................................17
  2.4.2 管柱層析法........................................................17
 2.5 脂肪酶活性測定........................................................19
第三章 材料與方法...........................................................21
 3.1 材料與藥品............................................................21
 3.2 分析儀器與設備........................................................22
 3.3 實驗流程..............................................................24
 3.4 米糠酵素液製備........................................................25
  3.4.1 米糠之製備........................................................25
  3.4.2 脂肪酶萃取........................................................25
 3.5 脂肪酶之測定..........................................................25
  3.5.1 蛋白質定量........................................................25
  3.5.2 脂肪酶活性分析....................................................26
 3.6 米糠脂肪酶之純化......................................................28
  3.6.1 第一階段硫酸銨沉澱劃分.............................................28
  3.6.2 第二階段Superdex 75 管柱層析.......................................29
  3.6.3 第三階段Hi-Trap DEAE FF管柱層析....................................29
  3.6.4 第四階段Superdex 75 管柱層析.......................................30
 3.7 蛋白質電泳分析........................................................30
  3.7.1 膠體製備..........................................................30
  3.7.2 電泳分析..........................................................31
  3.7.3 酵素膠體活性染色..................................................31
  3.7.4 蛋白質染色........................................................32
 3.8 蛋白質二維電泳........................................................32
  3.8.1 樣品前處理........................................................33
  3.8.2 第一維:等電點焦集膠..............................................33
  3.8.3 第二維:硫酸十二酯納聚丙烯胺膠體電泳..............................34
  3.8.4 酵素活性染色......................................................34
  3.8.5 蛋白質銀染.........................................................35
 3.9 蛋白質質譜分析........................................................35
 3.10 脂肪酶生化特性分析...................................................35
  3.10.1 最適pH 值........................................................36
  3.10.2 最適溫度.........................................................36
  3.10.3 脂肪酶之基質特異性...............................................36
  3.10.4 化學試劑對脂肪酶活性之影響.......................................37
第四章 結果與討論...........................................................38
 4.1 米糠酵素粗萃取.........................................................38
 4.2 米糠酯酶/脂肪酶之蛋白質純化............................................39
  4.2.1 第一階段硫酸銨劃...................................................39
  4.2.2 第二階段膠過濾管柱層析.............................................40
  4.2.3 第三階段陰離子交換管柱層析.........................................41
  4.2.4 第四階段管柱膠體過濾管柱層析.......................................41
  4.2.5 米糠酯酶/脂肪酶蛋白質純化純度分析.................................42
 4.3 米糠酯酶/脂肪酶蛋白質生化特性分析.....................................43
  4.3.1 基質特異性........................................................43
  4.3.2 脂肪酶之最適pH 值.................................................44
  4.3.3 脂肪酶之最適溫度..................................................44
  4.3.4 化學試劑對酵素活性的影響..........................................45
 4.4 二維電泳...............................................................46
  4.4.1 蛋白質質譜分析....................................................46
第五章 結論.................................................................48
參考文獻.....................................................................50
附錄.........................................................................83
表目錄
表一、米糠脂肪酶/酯酶/磷脂酶研究.............................................55
表二、純化方法之差異比較.....................................................56
表三、米糠酵素粗萃液之脂肪酶活性分析.........................................57
表四、米糠脂肪酶之純化總表...................................................58
表五、硫酸銨劃分之脂肪酶活性分析.............................................59
表六、化學試劑對米糠脂肪酶之活性影響.........................................60
圖目錄
圖一、 三種主要的亞洲稻米種類...............................................61
圖二、台梗9 號...............................................................62
圖三、稻米結構圖.............................................................62
圖四、米糠...................................................................63
圖五、米糠酸敗關係圖.........................................................63
圖六、脂肪酶催化三酸甘油脂之反應式...........................................64
圖七、α/β-Hydrolase superfamily 之二級結構.................................64
圖八、脂肪酶之蓋子(lid)結構..................................................65
圖九、脂肪酶蓋子關閉及打開之3D 結構..........................................65
圖十、BSA 標準曲線...........................................................66
圖十一、硝基苯酚標準曲線.....................................................66
圖十二、4-甲基繖形酮標準曲線.................................................67
圖十三、快速蛋白質液相層析儀.................................................67
圖十四、蛋白質純化流程.......................................................68
圖十五、二維電泳流程.........................................................69
圖十六、酵素粗萃液之SDS/native PAGE 電泳分析.................................70
圖十七、米糠酯酶/脂肪酶之硫酸銨劃分..........................................71
圖十八、米糠粗萃液經過硫酸銨劃分之電泳分析圖.................................72
圖十九、米糠酯酶/脂肪酶之Superdex 75 HR 管柱膠體過濾層.......................73
圖二十、米糠脂肪酶之Hi-Trap DEAE FF管柱陰離子交換層析........................74
圖二十一、Lipase I 之Superdex 75 HR 管柱膠體過濾層析.........................75
圖二十二、 Lipase II之Superdex 75 HR管柱膠體過濾層析........................76
圖二十三、米糠酯酶/脂肪酶純化過程之SDS-PAGE 電泳分析圖.......................77
圖二十四、米糠脂肪酶之基質特異性.............................................78
圖二十五、pH 值對於米糠脂肪酶活性之影響......................................79
圖二十六、溫度對米糠脂肪酶之活性影響.........................................80
圖二十七、米糠脂肪酶二維電泳圖譜.............................................81
圖二十八、Lipase II 之LC/MS/MS 序列分析......................................82
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