臺灣博碩士論文加值系統

鳳梨酵素(Bromelain)，屬於半胱胺酸蛋白酶(Cysteine proteinase)，廣泛存在鳳梨全株。本研究利用農業廢棄物的鳳梨皮作為鳳梨酵素的萃取來源，利用超濾濃縮、冷凍乾燥及疏水性層析進行分離純化，得到鳳梨酵素總活性為757 U，比活性為145.11 U/mg，純化倍數為2.55倍，回收率為30.02%。以SDS-PAGE及Zymography對層析樣品進行分析，分子量為24.3 kDa。固定化試驗是將苦茶粕進行前處理，以實驗設計法對固定化因子進行少量實驗組數的排列組合，以Replicate plot、Coefficient plot、Residuals normal probability進行數據優化，得到最適化條件為2.17 mg鳳梨酵素與40 mg載體、4%交聯劑濃度、交聯時間為30分鐘，14C的交聯溫度，其固定化率與相對活性分別為66.81.2%及65.61.1%，鳳梨酵素之單位載體吸附量為29.10.5 g/mg。
游離與固定化鳳梨酵素最適反應pH皆為7，游離酵素在pH 6-9可維持75%以上相對活性，固定化酵素則維持43%之相對活性。游離酵素的最適反應溫度為40C，固定化酵素則提升至50C。固定化酵素重複使用5次後相對活性降為44.62.2%；游離酵素在30-40C水浴1小時後可維持70%以上之相對活性，活化能為259.7 kJ/mole。此外，在1 mM Fe3+、Zn2+會抑制鳳梨酵素活性，其他金屬離子(Al3+、Ca2+、Cu2+、Mg2+、Mn2+、Na+)則具有提高活性能力，在10 mM濃度下僅Mg2+、Ca2+有助於提高酵素活性，適合作為酵素的輔因子。鳳梨酵素在高離子強度(1 M)下會被抑制活性，但在1-50 mM NaCl中可維持99.54.6%之相對活性。

　　Bromelain, which belongs to the Cysteine proteinase, is widely present in whole pineapple plants. In this study, pineapple peel from agricultural waste was used as an extraction source of Bromelain. It was separated and purified by ultrafiltration, freeze-drying, and hydrophobic chromatography. The total activity of purified Bromelain was found to be 757 U, and the specific activity was 145.11 U/mg. The purification fold of 2.55 and 30.02% recovery yield were obtained. The chromatographic purified Bromelain was analyzed by SDS-PAGE and Zymography, and the molecular weight was 24.3 kDa. Immobilization was conducted with pretreatment Camellia oleifera seed shell, and the Design of Experiment method was used to array and combine a small number of experimental groups, and the data was optimized using Replicate plot, Coefficient plot, and Residuals normal probability. The optimal immobilization conditions were 1 mL purified Bromelain (2 mg/mL) and 40 mg Camellia oleifera seed shell, 4% cross-linker, cross-linking time 30 minutes, and cross-linking temperature 14C. The Percentage of immobilization and relative activity of Bromelain were 66.81.2% and 65.61.1%, respectively. Adsorption capacity is 29.10.5 g Bromelain/mg of Camellia oleifera seed shell.
　　The optimum pH for free and immobilized Bromelain is centred at 7, and the free Bromelain can maintain 75% relative activity at pH 6-9, whereas immobilized Bromelain maintain its relative activity about 43%. The optimal temperature were 40C and 50C for free and immobilized Bromelain, respectively. The relative activity of the immobilized Bromelain was retain at 44.62.2% after reused 5 cycles; the free Bromelain can maintain the relative activity of more than 70% after 1 hour between 30C and 40C, and the activation energy calculated for enzyme reaction is 259.7 kJ/mole. In addition, the activity of Bromelain were inhibited by Fe3+ and Zn2+ ions at the concentration of 1 mM, and other metal ions (e.g. Al3+, Ca2+, Cu2+, Mg2+ , Mn2+, Na+) have the ability to increase the activity. Only Mg2+ and Ca2+ could increased the Bromelain activity at the concentration of 10 mM, which is suitable as a cofactor for Bromelain. Bromelian activity could be inhibited at high ionic strength (1 M), but the relative activity may maintain at 99.54.6% in 1-50 mM NaCl.

目錄
摘要 I
Abstract II
致謝 IV
目錄 VI
圖目錄 X
表目錄 XII
第一章 緒論 1
1-1 前言 1
1-2 研究目的 1
第二章 文獻回顧 3
2-1 鳳梨簡介 3
2-2 鳳梨廢棄物處置及利用探討 6
2-2-1 鳳梨葉與葉冠萃取物應用 6
2-2-2 鳳梨莖萃取物應用 7
2-2-3 鳳梨果皮與果核萃取物應用 8
2-3 鳳梨酵素簡介 11
2-4 鳳梨酵素發展與應用潛力 14
2-5 酵素固定化簡介 19
2-6 酵素固定化技術 21
2-7 實驗設計法簡介 24
第三章 鳳梨酵素分離純化與分析 27
3-1 前言 27
3-2 酵素之分離純化 28
3-3 分析方法 31
3-3-1 總蛋白濃度測定 31
3-3-2 酵素活性測定 31
3-3-3 SDS膠體電泳分析 32
3-3-4 酵素圖譜分析(Zymography) 34
3-4 結果與討論 36
3-4-1 鳳梨皮前處理探討 36
3-4-2 鳳梨酵素之疏水性作用層析 36
3-4-3 SDS膠體電泳分析 41
3-4-4 酵素圖譜分析 41
第四章 苦茶粕載體製備及固定化最適條件 45
4-1前言 45
4-2 苦茶粕載體製備與固定化 46
4-2-1 苦茶粕前處理 46
4-2-2 鳳梨酵素固定化 46
4-3 酵素固定化效率與相對活性分析 48
4-3-1 載體之固定化率測定 48
4-3-2 相對活性測定 48
4-4 固定化最適條件分析 49
4-5 結果與討論 50
4-5-1 苦茶粕前處理 50
4-5-2 固定化因子之實驗設計 50
第五章 游離與固定化鳳梨酵素特性分析 57
5-1前言 57
5-2 實驗方法 58
5-2-1 最適反應pH探討 58
5-2-2 最適反應溫度探討 58
5-2-3 熱穩定性測定 58
5-2-4 金屬離子對游離酵素活性之影響 59
5-2-5 鹽離子強度對游離酵素活性之影響 59
5-2-6 固定化酵素之反應重複性測定 60
5-3 結果與討論 61
5-3-1 最適反應pH探討 61
5-3-2 最適反應溫度探討 61
5-3-3 熱穩定測試 67
5-3-4 金屬離子對酵素活性之影響 67
5-3-5 鹽離子強度對酵素活性之影響 68
5-3-6 固定化酵素之反應重複性測定 73
第六章 結論與未來展望 75
參考文獻 77

圖目錄
圖2-1 從葉冠至莖部之鳳梨全株圖 5
圖2-2 鳳梨果實發育的三個主要階段 10
圖2-3 鳳梨酵素結構模型預測 13
圖2-4 PLA2生成花生四烯酸以調節下游的環氧合酶和脂氧合酶之途徑 16
圖2-5 促進脂肪形成之轉錄因子 18
圖2-6 不同之酵素固定化方法 20
圖2-7 以多亞基共價與交聯固定聚合酶 22
圖3-1 鳳梨皮中鳳梨酵素之分離純化流程圖 30
圖3-2 鳳梨酵素活性測定流程圖 33
圖3-3 Zymography分析流程圖 35
圖3-4 鳳梨皮粗萃液之疏水性管柱層析圖 38
圖3-5 層析分離之波峰P1、P2於活性測定之曲線圖 39
圖3-6 各階段純化樣品之SDS-PAGE 43
圖3-7 鳳梨酵素之酵素圖譜分析 44
圖4-1 鳳梨酵素固定於苦茶粕之流程圖 47
圖4-2 以實驗設計法分析鳳梨酵素於固定化率模型 53
圖4-3 以實驗設計法分析鳳梨酵素於相對活性模型 54
圖4-4 固定化模型之殘差常態機率 55
圖5-1 pH值對游離之鳳梨酵素活性影響 63
圖5-2 pH值對固定化之鳳梨酵素活性影響 64
圖5-3 反應溫度對游離之鳳梨酵素活性影響 65
圖5-4 反應溫度對固定化之鳳梨酵素活性影響 66
圖5-5 游離之鳳梨酵素熱穩定性影響 69
圖5-6 游離之鳳梨酵素於不同溫度下反應速率常數(k)之關係圖 70
圖5-7 溶液中鹽離子強度對游離之鳳梨酵素活性影響 72
圖5-8 固定化酵素之反應重複性測定 74

表目錄
表2-1 2017年鳳梨生產國家之產量統計 4
表3-1 鳳梨酵素各階段純化結果 40
表4-1 固定化因子之實驗設計法組合表 52
表4-2 鳳梨酵素固定化率與相對活性之實驗組與DoE計算之理論值比較 56
表5-1 不同濃度之金屬離子對游離之鳳梨酵素活性影響 71

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