臺灣博碩士論文加值系統

大汕山藥塊莖多酚氧化(polyphenol oxidase：PPO)之純化，採用新鮮山藥經丙酮乾燥處理，所得丙酮粉末再以緩衝液抽取、硫酸銨劃分、離子交換層析以及膠體過濾層析等連續步驟純化。純化之酵素以5~12.5 ﹪梯度電泳，配合多酚氧化活性染色，發現純化之酵素含有3種同功異構，測得原態分子量為66.9、86.0及91.6 kDa。純化之大汕山藥塊莖多酚氧化，以單元酚、二元酚、三元酚及其衍生物等十三種酚類化合物為基質，經由基質專一性測定，顯示純化之多酚氧化僅具有氧化鄰位二元酚之活性，不具氧化單元酚之活性。多酚氧化氧化多帕(L-DOPA)之最適pH為6.5-6.8，最適溫度為40 ℃，在pH 5.0~8.0及4~30 ℃頗為穩定，氧化L-DOPA之Km值為4.3 mM，氧化兒茶酚之Km值為1.6 mM。
純化之大汕山藥塊莖多酚氧化，經添加鹽類、有機酸、金屬離子螯合劑、還原劑以及芳香族酸(aromatic acid)等作為山藥塊莖多酚氧化之抑制劑，發現銅離子螯合劑DEDTC(0.1 mM)，金屬離子錯合物形成劑thiourea(5.1 mM)、硫醇化合物β-mercaptoethanol(3.5 mM)、dithiothreitol(1.1 mM)以及還原劑Na2S2O5 (1.9 mM)、NaHSO3(2.7 mM)和ascorbic acid(0.08 mM)等均在極低濃度下，即可完全抑制酵素活性。尤其芳香族酸等天然抗氧化劑如4-hexylresorcinol、kojic acid、nordihydroguaiaretic acid等作為山藥塊莖多酚氧化之抑制劑，效果相當良好，其中以4-hexylresorcinol(0.26 mM)及nordihydroguaiaretic acid(0.02 mM)的抑制效果最佳，因此在食品工業中，可以用來取代硫化物作為山藥褐變的抑制劑。

The properties of polyphenol oxidase were studied in this thesis. First of all, polyphenol oxidase (PPO) was isolated and purified from acetone-pretreated Darsan yam powder by salting, ionic exchange and gel chromatography. Subjecting to 5 - 12.5 % gradient polyacrylamide gel electrophoresis, PPO was shown to consist of three subunits with molecular weight 66.9, 86.0 and 91.6 KDa, respectively. Upon testing with 13 different phenolic substrates including monophenolic, diphenolic, and triphenolic compounds such as p-cresol, L-DOPA, and pyrogallol et al., only o-diphenolic compound exhibited enzymatically active to PPO while mono- and triphenolic compounds being inactive. The optimal condition for PPO reacting with L-DOPA was pH 6.5-6.8 at 40 oC or pH 5.0-8.0 at 30 oC. Under this condition, kinetic studies showed that the Km value for L-DOPA and pyrocatechol was 4.3 mM and 1.6 mM, respectively.
Both synthetic and natural inhibitants were applied to inhibit PPO activity. It showed that the activity of PPO was inhibited effectively in the presence of salts, organic acids, metallic chelating compounds, aromatic acids, and reducing agents at limited concentration; for instance, DEDTC (0.1 mM), thiourea (5.1 mM),β-mecarptoethanol (3.5 mM), dithiothreital (1.1 mM), Na2S2O5 (1.9 mM), NaHSO3 (2.7 mM), and ascorbic acid (0.08 mM). Interestingly, the natural antioxidant such as 4-hexylresorcinol, kojic acid, and nordihydroguaiaretic acid exhibited apparent inhibition on PPO activity suggesting that natural inhibitants, particularly 4-hexylresorcinol, kojic acid, and nordihydroguaiaretic acid were potential agents for lessoning yam’s browning.

頁次
目錄……………………………………………………………………I
表目錄 ………………………………………………………………IV
圖目錄 ………………………………………………………………V
中文摘要……………………………………………………………VI
英文摘要 …………………………………………………………VII
第一章 前言…………………………………………………………1
第二章 文獻整理
第一節 山藥之簡介 ……………………………………………………4
第二節 多酚氧化之特性……………………………………………7
第三節 山藥塊莖多酚氧化活性之貯藏變化 ……………………19
第四節 多酚氧化之理化性質 ………………………………………20
第五節 多酚氧化之反應動力學 ……………………………………25
第六節 酵素性褐變的控制 ……………………………………………26
第七節 PPO對食品加工的影響…… …………………………………35
第八節 研究目的………………………………………………………36
第三章 材料與方法
第一節 實驗材料………………………………………………………37
第二節 實驗方法………………………………………………………37
壹、多酚氧化活性測定………………………………………………37
貳、蛋白質之定量………………………………………………………38
參、硫酸銨劃分預備試驗………………………………………………39
肆、多酚氧化之純化…………………………………………………40
伍、多酚氧化之性質測定
一、分子量測定………………………………………………………43
二、聚丙烯醯胺膠體電泳……………………………………………43
三、多酚氧化活性染色……………………………………………47
四、最適酸鹼度………………………………………………………47
五、對酸鹼度的穩定性………………………………………………47
六、最適溫度…………………………………………………………48
七、熱穩定性…………………………………………………………48
八、基質特異性測定…………………………………………………48
九、pH值對酵素基質特異性之影響 ………………………………49
十、抑制劑對多酚氧化的影響…………………………………49 十一、反應動力學測定………………………………………………50
十二、Km值 …………………………………………………………51
第四章 結果與討論
第一節 大汕山藥塊莖多酚氧化之純化……………………………52
第二節 大汕山藥塊莖多酚氧化之生化性質 ……………………56
一、分子量測定……………………………………………………………56
二、多酚氧化活性染色…………………………………………………56
三、最適酸鹼度……………………………………………………………60
四、對酸鹼度的穩定性……………………………………………………61
五、最適溫度………………………………………………………………61
六、熱穩定性………………………………………………………………65
七、多酚氧化的熱變性機構……………………………………………65
八、基質特異性……………………………………………………………65
九、pH值對酵素基質特異性之影響 ……………………………………69
十、抑制劑對多酚氧化的影響…………………………………………72
十一、反應動力學測定……………………………………………………81
十二、Km值…………………………………………………………………82
第五章 結 論………………………………………………………89
第六章 參考文獻 ……………………………………………………90
表 目 錄
頁次
表一、不同植物材料純化多酚氧化之生化性質………………23
表二、不同植物材料純化多酚氧化之多分子型態或異構……24
表三、不同植物材料純化多酚氧化之Km值………………………27
表四、大汕山藥塊莖多酚氧化之純化結果………………………55
表五、純化之多酚氧化經5~12.5 ﹪梯度電泳及活性染色結果…59
表六、大汕山藥塊莖多酚氧化之基質專一性………………………70
表七、pH值對大汕山藥塊莖多酚氧化基質專一性之影響…………73
表八、金屬離子對大汕山藥塊莖多酚氧化活性之影響……………75
表九、不同化合物對大汕山藥塊莖多酚氧化活性之影響…………76
表十、大汕山藥塊莖多酚氧化之理化性質…………………………87
表十一、大汕山藥與其他品種山藥塊莖多酚氧化之性質比較……88
圖 目 錄
頁次
圖一、酵素性褐變之生成途徑………………………………………10
圖二、抗壞血酸還原酵素性褐變所產生氧化物之機制……………29
圖三、大汕山藥塊莖多酚氧化之離子交換層析……………………53
圖四、大汕山藥塊莖多酚氧化之膠體過濾層析……………………54
圖五、大汕山藥塊莖多酚氧化之5~12.5 ﹪聚丙烯醯胺膠體
梯度電泳及活性染色結果 ……………………………………………57
圖六、大汕山藥塊莖多酚氧化之5~12.5 ﹪聚丙烯醯胺膠體
梯度電泳分子量測定……………………………………………………58
圖七、pH值對大汕山藥塊莖多酚氧化活性之影響 …………………62
圖八、pH值對大汕山藥塊莖多酚氧化穩定性之影響 ………………63
圖九、溫度對大汕山藥塊莖多酚氧化活性之影響…………………64
圖十、溫度對大汕山藥塊莖多酚氧化熱穩定性之影響………………66
圖十一、大汕山藥塊莖多酚氧化的熱變性機構(一)…………………67
圖十二、大汕山藥塊莖多酚氧化的熱變性機構(二)…………………68
圖十三、氯化鈉對大汕山藥塊莖多酚氧化活性之影響………………79
圖十四、不同pH值下、氯化鈉對大汕山藥塊莖多酚氧化活性之影響80
圖十五、大汕山藥塊莖多酚氧化氧化L-DOPA之動力學 ……………84
圖十六、L-DOPA濃度對大汕山藥多酚氧化活性之影響 ……………85
圖十七、L-DOPA對大汕山藥多酚氧化活性之動力學的雙倒數圖形 86

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