臺灣博碩士論文加值系統

本研究針對小麥草汁中所含過氧化進行性質分析，並比較純化前後之差異。結果發現，經由熱處理、硫酸銨劃分以及等電焦集層析等連續步驟，可提高過氧化之純度約87倍，活性收率約為4.8%。而過氧化之RZ值（A403/A280）則為0.5。將純化之酵素通入Superose 12 HR管柱後發現其分子量為23 kDa。活性染色分析結果顯示，小麥草過氧化之等電點分別為8.88及8.43，屬鹼性過氧化。而小麥草粗酵素液中之過氧化於30-50℃保溫60分鐘或60℃保溫40分鐘皆很穩定，活性幾乎無喪失，於60℃下靜置60分鐘活性始明顯降低，殘留活性約為67%。而純化之過氧化於30-50℃保溫60分鐘，活性亦幾乎無喪失，然當溫度提高至60℃時，活性開始明顯降低，於60℃下靜置10分鐘之殘留活性約為51%。由殘留酵素活性百分率之對數值對保溫靜置時間作圖發現，小麥草過氧化之熱失活反應在反應初期為一級反應，且溫度對熱失活速率之影響符合阿瑞尼亞方程式(Arrhenius equation)，粗酵素液之熱失活活化能為2.2×105 J/mol；純化酵素液之熱失活活化能則為4.2×104 J/mol。化學修飾劑N-bromosuccinimide（0.5 mM）顯著抑制過氧化活性，但p-hydroxymercuribenzoate（0.5 mM）無顯著影響，因此色胺酸可能與酵素催化活性有關。由基質專一性分析顯示過氧化催化四種供氫體氧化之速率依序為鄰苯二胺(o-phenylenediamine)＞焦五倍子酸(pyrogallol)＞兒茶酚(catechol)＞癒創木酚(guaiacol)。

This study aimed at characterizing the peroxidase present from wheat grass before and after purification. It was found that after heat treatment, ammonium sulfate fractionation and chromatofocusing, about 87 purification fold for peroxidase was achieved and the enzyme yield was about 4.8%. The RZ value of peroxidase was 0.5. The molecular mass of the peroxidase from wheat grass was about 23 kDa as estimated by Superose 12 HR gel filtration. As estimated by zymogram staining, the isoelectric points of the peroxidase were found to be 8.88 and 8.43, respectively, indicating that the peroxidase from wheat grass belonged to alkali peroxidase. The crude extract of peroxidase from wheat grass was thermally stable after holding at 30-50℃ for 60 min or at 60℃ for 40 min, nevertheless the peroxidase activity started to drop significantly after holding at 60℃ for 60 min. Similarly, the purified peroxidase from wheat grass was thermally stable after holding at 30-50℃for 60 min. However, as the temperature was higher than 60℃, the enzyme activity decreased significantly. The residual peroxidase activity was only about 51% after holding at 60℃ for 10 min. The thermal inactivation reaction of peroxidase during initial thermostat stage followed first-order reaction kinetics, and the temperature dependence of rate constants was in agreement with the Arrhenius equation. The activation energy of thermoinactivation of the crude extract and the purified peroxidase from wheat grass was found to be 2.2×105 J/mol and 4.2×104 J/mol, respectively. Chemical modification agent N-bromosuccinimide（0.5 mM）significantly inhibited the activity of peroxidase but p-hydroxymercuribenzoate（0.5 mM）had no significantly effect. Tryptophan probably correlated with the activity of peroxidase. From the substrate specificity examined, relative activity of peroxidase for four hydrogen donors were o-phenylenediamine > pyrogallol > catechol > guaiacol.

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中文摘要………………………………………………………………. Ⅰ
英文摘要………………………………….…………………………… Ⅱ
目錄………………………………………..……………...…………… Ⅳ
圖目錄……………………………………………..…………………... Ⅷ
表目錄…………………………………………………..………..……. Ⅹ
第一章 前言….…..…………………………………………………… 1
第二章 文獻回顧……………...……………………………………… 3
第一節 小麥草(wheat grass)之簡介……...……………… 3
第二節 小麥草汁之混濁現象……………..…………… 6
一、影響果汁混濁、沉澱的因子…………………. 7
二、增加果汁混濁安定的方法………...………… 11
第三節 過氧化之簡介……………………………….. 14
第四節 過氧化之生化學性質…..………………..…… 19
一、異構………………………………………... 19
二、過氧化之抽取和純化……………...……… 22
三、過氧化之基質……………………………... 22
四、最適pH和溫度………………………………. 26
五、催化機制……………………………...……… 26
第五節 過氧化與植物生理之關係………………….... 29
一、木質素之生合成……………………………... 29
二、Indole acetic acid（IAA）的代謝…………….. 30
三、乙烯生合成之調控…………………………... 30
四、單寧與黑色素之合成與防禦機制……...…… 31
五、細胞壁多醣體蛋白質之結合………………... 32
第六節 過氧化之應用……………………………..….. 34
一、食品方面……………………………………... 34
二、臨床醫學方面………………………………... 36
三、工業方面………………………………...…… 39
第三章 研究目的……………………………………………………. 41
第四章 材料與方法…………………………………………………... 42
第一節 實驗材料及主要儀器設備…………………….. 42
一、化學藥品……………………………………... 42
二、儀器設備……………………………………... 44
三、植物材料………………………………...…… 46
第二節 實驗方法…………………………...……………. 48
一、小麥草成分分析…………………...………… 48
二、小麥草粗酵素液之製備…………...………… 48
三、小麥草粗過氧化之性質測定…...………… 48
四、小麥草過氧化之純化……………………... 54
五、小麥草純化之過氧化的性質測定……...… 57
六、統計分析…………………………………....... 66
第四章 結果與討論…………………………………………………... 68
第一節、小麥草之成分分析……………………………….. 68
第二節、小麥草過氧化之純化………………………….. 70
第三節、小麥草過氧化之生化性質…………………….. 75
一、過氧化酵素純度…………………………... 75
二、原態分子量測定……………………………... 76
三、pI值測定……………………………………... 76
四、最適pH測定…………………………………. 80
五、最適溫度測定………………………………... 80
六、熱穩定性測定……………...………………… 83
七、活化能測定…………………………………... 85
八、金屬離子及鹽類對酵素活性之影響………... 89
九、化學修飾劑對酵素活性之影響……………... 91
第四節、小麥草過氧化之催化特性……………………… 94
一、緩衝液pH及濃度對過氧化催化各種供氫體活性之影響…………………………. 94
二、基質濃度對反應速率之影響………………... 97
三、過氧化催化不同供氫體之比活性………... 103
第五章 結論………………………………………………… 110
第六章 參考文獻……………………………………………. 112

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