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研究生:林宜聖
研究生(外文):Yi-Sheng Lin
論文名稱:矽藻土擔載二氧化錳對固定化葡萄糖氧化酶操作穩定性之影響
論文名稱(外文):The effect of diatomite-supported manganese dioxide on the operational stability of immobilized glucose oxidase
指導教授:林松池
指導教授(外文):Sung-Chyr Lin
口試委員:楊芳鏘邱信程
口試委員(外文):Fan-Chiang YangSin-Cheng Chiu
口試日期:2016-07-27
學位類別:碩士
校院名稱:國立中興大學
系所名稱:化學工程學系所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:64
中文關鍵詞:固定化葡萄糖氧化酶擔載二氧化錳的矽藻土
外文關鍵詞:immobilized glucose oxidasediatomite-supported manganese dioxide
相關次數:
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由於使用固定化酵素生產海藻糖的方式,無法將麥芽糖完全轉化為海藻糖,同時會產生副產物葡萄糖,因此這次的研究,是以環氧基聚丙烯樹脂作為固定化擔體,進行葡萄糖氧化酶的共價鍵結固定化反應,之後利用固定化葡萄糖氧化酶將葡萄糖轉化為葡萄糖酸以利海藻糖的純化,並且以矽藻土擔載二氧化錳去除葡萄糖氧化酶反應時所產生的副產物過氧化氫,探討葡萄糖氧化酶的操作穩定性。
在研究結果中,二氧化錳擔載量及催化能力方面,得到的最適化的比例為24:1之合成矽藻土。改變固定化葡萄糖氧化酶反應時的合成矽藻土添加量,第一次反應的添加量為60 mg時,固定化酵素有最佳的比活性為645.145 U/mg protein,過氧化氫殘存濃度則為15.205 mM;靜置一天後進行第二次反應,固定化酵素比活性降低至269.000 U/mg protein,過氧化氫殘存濃度提升至39.89 mM。在重複批次操作中,於pH 7下,合成矽藻土大約在第四個批次反應會逐漸失去催化能力,若提升至pH 8,則合成矽藻土大約於第八個批次反應才會失去催化能力。在反應最終pH值測定中,pH 7及pH 8的反應條件下,當進行十五次批次操作後,pH值分別提升至6.9及7.7左右;最後為溶液中錳含量的鑑定,於pH 7及pH 8下,錳含量分別為29.115 mg /L及11.332 mg/L。
綜合以上實驗結果,證實了額外添加表面擔載二氧化錳之矽藻土,是能夠改善固定化葡萄糖氧化酶的操作穩定性。


The employment of immobilized trehalose synthase for the production of trehalose from maltose generally lead to the generation of reaction byproduct, glucose, which needs to be removed. This separation of glucose from trehalose can be facilitated by the converting glucose into gluconic acid with glucose oxidase. In this study, the effect of hydrogen peroxide scavenger, MnO2, on the operation stability of Immobead 150-immobilized glucose oxidase was study. Diatomite-supported manganese dioxide was prepared and characterized. The diatomite with manganese dioxide loading capacity of 100.36 mg/g silica exhibited the optimal catalytic ability, leading to the highest enzyme specific activity of 645.14 U/mg protein and the lowest residual hydrogen peroxide concentration of 15.2 mM. The specific activity of glucose oxidase and the concentration of hydrogen peroxide were 269.00 U/mg protein and 39.8 mM, respectively, after being incubated in phosphate buffer containing glucose for 1 day. In repeated-batch operations, the activity of immobilized enzyme declined after 4 cycles and 8 cycles at pH 7.0 and pH 8.0, respectively, because of the increase in H2O2 concentration possibly resulting from the reduction in scavenging capability of the diamomite-supported MnO2. The results of ICP-MS analysis indicate that the loss in scavenging activity can be attributed to the loss of manganese into the reaction medium. Based on the aboved results, the diatomite-supported manganese dioxide was used for glucose oxidase oxidation, is promising for improving the operational stability.


致謝 i
中文摘要 ii
Abstrate iv
目錄 vi
圖目錄 x
表目錄 xii
第一章 緒論 1
第二章 文獻回顧 2
2.1 酵素介紹 2
2.1.1 酵素之種類 2
2.1.2 酵素之構成 3
2.1.3 酵素的結構與作用力 5
2.1.4 酵素之特性 8
2.1.5 酵素穩定性 8
2.2 酵素固定化 10
2.2.1 酵素固定化優點 10
2.2.2 酵素固定化方法 11
2.2.3 交聯法 12
2.2.4 包埋法 13
2.2.5 鍵結法 13
2.2.6 固定化擔體之種類與選擇 15
2.2.7 共價鍵結法常用之擔體 17
2.3 研究系統簡介 19
2.3.1 Aspergillus niger葡萄糖氧化酶 19
2.3.2 產物的效應 20
2.3.3 環氧基固定化基材 21
2.3.4 矽藻土 23
2.3.5 二氧化錳 23
第三章 實驗藥品與儀器 25
3.1 實驗藥品 25
3.2 實驗儀器與設備 26
第四章 實驗方法 27
4.1 酵素固定化 27
4.1.1 酵素固定於Immobead 150 27
4.1.2 酵素鍵結量分析 27
4.2 矽藻土擔載二氧化錳之製備 28
4.3 二氧化錳擔載量之量測 29
4.4 不同合成比例矽藻土對過氧化氫催化能力之探討 29
4.5 合成矽藻土添加量對固定化酵素活性之探討 30
4.6 合成矽藻土添加量對副產物過氧化氫濃度之探討 31
4.7 過氧化氫與合成矽藻土添加量對固定化酵素活性之探討 31
4.8 固定化酵素之重複批次操作(pH 7) 32
4.9 固定間隔添加合成矽藻土之重複批次操作(pH 7) 33
4.10 固定化酵素之重複批次操作(pH 8) 33
4.11 不同pH值之固定化酵素重複批次操作 34
4.12 批次操作最終pH值之測定 35
4.13 反應溶液之錳含量鑑定 35
4.14 蛋白質濃度分析 36
4.15 過氧化氫濃度分析 37
第五章 結果與討論 39
5.1 表面型態之觀察(Scanning Electron Microscope, SEM) 39
5.2 二氧化錳擔載量之量測 42
5.3 不同合成比例矽藻土對過氧化氫催化能力之探討 43
5.4 合成矽藻土添加量對固定化酵素活性之探討 44
5.5 合成矽藻土添加量對副產物過氧化氫濃度之探討 45
5.6 過氧化氫與合成矽藻土添加量對固定化酵素活性之探討 47
5.7 固定化酵素之重複批次操作(pH 7) 48
5.8 固定間隔添加合成矽藻土之重複批次操作(pH 7) 49
5.9 固定化酵素之重複批次操作(pH 8) 51
5.10 不同pH值之固定化酵素重複批次操作 52
5.11 批次操作最終pH值之測定 53
5.12 反應溶液之錳含量鑑定 55
第六章 結論 56
第七章 參考文獻 58
附錄 63


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