臺灣博碩士論文加值系統

二苯乙烯類二聚體化合物之特性與相關產品研發廣受重視，由於此類化合物在自然界中含量通常很低且純化獲得成本高，雖然目前此類化合物大部分可以利用有機合成，若以天然物與有機合成產物相比較，消費大眾普遍對天然來源之化合物具有較高接受性，因此，利用酵素進行仿生合成值得深入探討，整體而言，如何取得關鍵性酵素與最適反應製程是重要的技術建立，本研究基於花生仁中二苯乙烯類化合物包括白藜蘆醇及其衍生物之複雜生化合成反應，會伴隨發芽而被活化，因此選擇花生仁作為生物資材，並以發芽誘發產生可催化白藜蘆醇二聚體合成之過氧化酶為主軸，其基本製備過程與探討步驟係將花生仁經復水後進行培養發芽，經脫膜後將胚(包含胚芽、胚軸與胚根)與子葉分離，再分別凍結乾燥並粉碎成粉作為酵素源。二聚體反應係以酵素粉配置於磷酸鹽緩衝溶液，攪拌混入白藜蘆醇丙酮溶液作為基質，再於反應液中添加過氧化氫促進酵素性氧化聚合反應，並以HPLC分析檢測白藜蘆醇與新生成物之變化。以反應液HPLC分析圖譜中生成物與白藜蘆醇兩者波峰高度比值作為酵素活性評估依據，顯示花生仁經發芽後，其胚與子葉酵素之催化活性皆隨發芽時間之增加而明顯提高，在相同發芽程度條件下，胚之活性比子葉者高。進一步利用發芽5天花生胚凍結乾燥粉作為酵素源，於反應系統中同步添加白藜蘆醇與過氧化氫溶液，可獲得將白藜蘆醇近100%之轉換率，該生成物經半製備型HPLC分離純化及NMR二維分析並與文獻資料比對後，確定其結構為δ-viniferin。為分離純化花生胚過氧化酶，經比較發芽5天花生胚芽、胚軸與胚根重量與過氧化酶活性之差異，顯示胚軸之活性比另兩者略低但所佔質量比率最高，因此，以發芽5天僅去除子葉之花生胚為材料，經均質萃取離心取得上清液後，先以硫酸銨40-75%飽和度鹽析，再以分子篩過濾膜區分濃縮 (Molecular Cut-off 300,000 and 10,000)，並以膠體過濾管柱層析法(Sephacry S300)及DEAE Sephadex A25管柱層析分離，可純化分子量約32,000 Dalton之過氧化酶，此酵素可催化白藜蘆醇仿生合成δ-viniferin。整體而言，經適當發芽之花生胚可直接作為酵素源，應用於仿生合成反應系統中進行催化白藜蘆醇轉換為δ-viniferin，而且所建立以連續饋料添加反應基質及過氧化氫之系統，具有被相關產業進一步發展進行量產δ-viniferin之潛力。

中文摘要…………………………………………………………………..………..I
英文摘要……………………………………………………………….…….…….III
謝誌………………………………………………………………………………....V
目錄………………………………………………………………….……….…….VI
表次………………………………………………………………….……….…….XI
圖次………………………………………………………………………….…….XII
壹、 前言…………………………………………………………………..…….1
貳、 文獻回顧………………………………………………………….….…….4
一、 落花生簡介…………………………………………….………….……..4
(一) 落花生分類及品種……………………………………………..……...4
1. 台南選9號…………………………………………………..……..5
2. 台南11號…………………………………………………..……....5
3. 台南14號…………………………………………………..…..…..5
4. 黑金剛花生………………………………………………….……..6
(二) 落花生營養成分………………………………………………….…....6
(三) 種子萌芽過程型態變化………………………………………….……7
(四)種子萌芽期間生理生化之改變……………………………………….7
(五)落花生中二苯乙烯類化合物………………………………………….8
二、 過氧化酶(Peroxidase)之簡介……………………………………………8
(一) 過氧化酶之功能………………………………………………………8
(二) 陽電性花生過氧化酶(Cationic peanut peroxidase)………11
(三) Horseradish peroxidase (HRP)的應用………………………11
三、 仿生合成(Biomimetic synthesis) …………………………14
(一) 仿生合成之簡介……………………………………………………...14
(二) δ-Viniferin不同合成路徑……………….…………………………..14
參、 材料與方法……………………………………………………………….16
一、 試驗材料………………………………………………………………..16
(一)原料…………………………………………………………………...16
(二)化學藥品……………………………………………………………...16
(三)儀器設備……………………………………………………………...16
二、試驗架構…………………………………………………………….........19
三、實驗方法…………………………………………………………….........20
(一) 花生仁材料與復水培養發芽………………………………………..20
(二) 不同發芽程度花生胚及子葉催化白黎蘆醇聚合反應之測定……..22
(三) 不同酵素濃度對白藜蘆醇聚合反應及酵素催化反應之確認……..24
(四) 以間續方式添加H2O2溶液對白藜蘆醇聚合反應之影響………….24
(五) 連續式添加H2O2水溶液進行白藜蘆醇氧化聚合反應……………25
(六) 連續式同步添加基質與H2O2氧化催化水溶液進行白藜蘆醇聚合反應……25
(七) 連續溢流式同步添加基質與H2O2氧化催化白藜蘆醇聚合反應....27
(八) 白藜蘆醇聚合物之回收與結構鑑定………………………………..28
1. Solid phase extraction (SPE)前處理白藜蘆醇聚合物…28
2. 以半製備型HPLC純化白藜蘆醇聚合物………………………..28
3. 聚合物結構鑑定與旋光測定……………………………………..29
(九) 白黎蘆醇及其二聚體抗氧化活性之測定………………………….29
(十) 花生胚不同部位之過氧化酶活性測定……………………………30
(十一) 花生胚過氧化酶之分離純化…………………………………….31
1. 粗萃液萃取、過氧化酶活性與蛋白質含量測定………………..31
2. 硫酸銨區段鹽析…………………………………………………..32
3. Ultrafiltration (UF) molecular cut-off 區段分離……………33
4. 膠體過濾管柱層析法……………………………………………..33
5. 以8-10區段和33-36區段回收液進行白藜蘆醇聚合反應…….34
6. 離子交換樹脂管柱層析…………………………………………..34
7. DEAE-Sephadex A25回收液進行白藜蘆醇聚合反應............34
8. 過氧化酶比活性及酵素純度測定………………………………..35
9. SDS-聚丙稀醯胺凝膠電泳分析…………………………….…….35
10. 電泳膠及相關溶液之配置……………………………………...35
(十二) 統計分析…………………………………………………………37
肆、 結果與討論……………………………………………………………….38
一、 花生仁材料與復水培養發芽…………………………………………..38
二、 不同發芽程度花生胚及子葉催化白黎蘆醇聚合反應之測定………..40
三、 不同酵素濃度對白藜蘆醇聚合反應及酵素催化反應之確認………..47
四、 以間續方式添加H2O2溶液對白藜蘆醇聚合反應之影響…………….47
五、 連續式添加H2O2水溶液進行白藜蘆醇氧化聚合反應………………50
六、 連續式同步添加基質與H2O2氧化催化水溶液進行白藜蘆醇聚合反應……50
七、 連續溢流式同步添加基質與H2O2氧化催化白藜蘆醇聚合反應…….53
八、 白藜蘆醇聚合物之回收與結構鑑定…………………………………..55
九、 白黎蘆醇及其二聚體抗氧化活性之測定……………………………..64
十、 花生胚不同部位之過氧化酶活性測定…………………………….….67
十一、 蛋白質定量………………………………………………….….…….67
十二、 花生胚過氧化酶之分離純化………………………………………...69
(一) 粗萃液萃取、過氧化酶活性與蛋白質含量測定………………….69
(二) 硫酸銨區段鹽析…………………………………………………….69
(三) Ultrafiltration (UF) molecular cut-off 區段分離……71
(四) 膠體過濾管柱層析法(SepHacryl S-300)和以8-10區段和33-36區段回收液來進行白藜蘆醇聚合反應………………………………….71
(五) 陰離子交換樹脂管柱層析(DEAE SepHadex A25 )……………….74
(六) DEAE-Sephadex回收液進行白藜蘆醇聚合反應………………….74
(七) 過氧化酶比活性及酵素純度測定………………………………….76
(八) SDS-聚丙稀醯胺凝膠電泳分析…………………………………….76
伍、 結論……………………………………………………………………….80
參考文獻…………………………………………………………………………...82
作者簡介…………………………………………………………………………...88

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