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dc.contributorYung-Chuan Liuen_US
dc.contributor.authorLee, I-Tsangen_US
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dc.description.abstract交聯型聚合酵素(Cross-linked enzyme aggregates;CLEAs)是一種新穎簡單之固定化方法,利用酵素交聯聚合,形成生物催化固體物。此酵素固定化方法具低成本、無載體、易製備、高比活性等優勢。因此本論文整合多篇文獻及相關研究結果,分述兩種酵素PGA (penicillin G acylase)和脂肪酵素(Lipase) 用於製備CLEAs,比較其活性、熱穩定性和重覆使用性的影響,由形態學及動力學參數,評估CLEAs的優劣性,並分析不同製備方法於工業應用上之實用性。 經評估得知,利用Lipase固定在磁性載體Dynabeads® M-270,經過交聯反應形成CLEAs,經30天的存放,仍保有80%殘餘活性;30次循環重複使用後,仍保有超過60%殘餘活性,比一般製備Lipase CLEAs方法,有更高的儲存穩定性和重複使用性。此外,Serratia marcescens lipase (SmL),加polyethyleneimine (PEI),製備CLEA-SmL-GA,在50℃下,經10天培養週期後,仍保留64%殘餘活性,顯示高熱穩定性。 另一方面,評估於工業上較具實用性之PGA CLEAs,PGA在乙二醇中,利用離子聚合物Dextran sulfate(DS)和polyethyleneimine(PEI)共聚集製備而成CLEA-GPD,經過30天的存放後,活性增加超過200%,具高儲存穩定性。在50℃下,添加BSA製備PGA CLEAs,經8小時培養,測得剩餘活性約70%。顯示添加BSA做為交聯型聚合酵素的共聚集,能有效提高熱穩定性。zh_TW
dc.description.abstractCross-linked enzyme aggregates (CLEAs) is a novel and simple process for enzyme immobilization, which generates the solid biocatalyst by cross-linking the enzyme to form aggregate. The merits of CLEAs are low-cost, carrier-free, easy preparation, and high specific activity. This study reviewed a series of relative journals and reports, and collected the preparation approaches of CLEAs for two model enzymes, i.e., PGA (penicillin G acylase) and lipase. The activity, reusability, thermal stability, morphology, and kinetic parameters of these two enzymes were compared. The analysis and applications of CLEAs under different methods were also included. As shown in the reports, the storage stability and reusability of immobilized lipase on amino-functionalized magnetic supports (Dynabeads® M-270) via cross-linked enzyme aggregates (CLEA) was the highest as compared to other CLEAs preparations, where 80% of its initial activity could be maintained for 30 days’ storage test, and more than 60% of lipase activity could be reserved after 30 times consecutive use. In addition, the polyethyleneimine (PEI) was added to CLEA-SmL-GA of Serratia marcescens lipase was found to possess the best thermal stability at 50 ℃, where 64% of the activity could be retained for 10 days’ test. On the other hand, the CLEAs preparations for PGA on the industrial applications were reviewed and discussed. The CLEA-GDP, prepared with dextran sulfate (DS) and polyethyleneimine(PEI), gave the highest storage stability. Its activity increased over 200% its original activity for a 30 days’ storage test. PGA CLEAs prepared in the presence of BSA could retain 70% of its original activity for a test at 50℃ for 8 h. From the results, the thermal stability could be effectively improved when using PGA CLEAs prepared with BSA as the co-aggregate cross-linking reaction.en_US
dc.description.tableofcontents目次 中文摘要 I Abstract II 總目錄 III 表目錄 VI 圖目錄 VII 一、緒論 1 1-1 前言 1 1-2 研究動機 2 二、文獻回顧 5 2-1 酵素固定化 5 2-1-1 交聯鍵結型酵素的發展 5 2-1-2 聚集型交聯鍵結酵素的發展 6 2-1-3 製備CLEAs沉澱劑的改良 7 2-1-4 製備CLEAs使用牛血清蛋白(BSA)與蛋白質的共聚集 8 2-1-5 製備CLEAs交聯劑的改良 9 2-1-6 使用DS和PEI共聚合發展CLEA-GDP 12 2-1-7 酵素固定化的優缺點 14 2-1-8 酵素在工業上之應用 14 2-2 固定化方法 17 2-2-1 擔體鍵結型(Carrier-binding) 17 2-2-2 交聯型(Cross linking) 17 2-2-3 包埋型(Entrapping) 18 2-3 酵素的簡介 24 2-3-1 酵素催化機制 24 2-3-2 脂肪酵素(Lipase)之介紹 24 2-3-3 脂肪酵素在工業上的應用 26 2-3-4 Penicillin之介紹 27 2-3-5 Penicillin之結構 27 2-3-6 penicillin醯胺酵素之催化作用機制 28 2-3-7 penicillin醯胺酵素之用途 28 2-3-8 交聯劑戊二醛之特性(Glutaraldehyde) 30 三、製備與分析方法 32 3-1 CLEAs製備方法 32 3-1-1 製備Lipase CLEAs方法一: 32 3-1-2 製備Lipase CLEAs方法二: 32 3-1-3 製備Lipase CLEAs方法三: 33 3-1-4 製備Lipase CLEAs方法四: 34 3-1-5 製備Lipase CLEAs方法五: 34 3-1-6 製備Lipase CLEAs方法六: 35 3-1-7 製備Lipase CLEAs方法七: 36 3-1-8 製備PGA CLEAs方法一: 37 3-1-9 製備PGA CLEAs方法二: 38 3-1-10 製備PGA CLEAs方法三: 39 3-1-11 製備PGA CLEAs方法四: 39 3-1-12 製備PGA CLEAs方法五: 40 3-2 分析方法 41 3-2-1 利用自動電位儀器,測量Lipase活性 42 3-2-2 利用自動電位儀器,測量PGA活性 43 3-2-3 利用分光光度計儀器,測量Lipase活性 43 3-2-4 利用分光光度計儀器,測量PGA活性 44 3-2-5 利用高效液相層析儀(HPLC)分析penicillin G acylase活性 44 3-2-6 比活性(Specific activity)和水解度(Hydrolysis degree)的分析 45 3-2-7 Lipase CLEAs熱穩定性和儲存穩定性測量 45 3-2-8 PGA CLEAs熱穩定性和儲存穩定性測量 45 3-2-9 動力學常數(Km和Vmax) 測定 46 3-2-10 利用氣相層析儀(GC),偵測轉酯化產物 46 四、結果與討論 47 4-1 交聯劑的影響 47 4-1-1 Lipase CLEAs活性的影響 47 4-1-2 不同交聯劑的影響 49 4-1-3 PGA CLEAs活性的影響 52 4-2 沉澱劑的影響 54 4-2-1 沉澱劑對Lipase CLEAs活性回收率的影響 54 4-2-2 不同菌株來源 Lipase對沉澱劑的影響 56 4-2-3 沉澱劑對PGA CLEAs活性的影響 58 4-2-4 沉澱劑對蛋白質溶解度的影響 60 4-3 儲存穩定性 61 4-3-1 各種製備方法與共聚合物對Lipase CLEAs儲存穩定性 61 4-3-2 各種有機溶劑對PGA CLEAs儲存穩定性 64 4-4 熱穩定性的比較 66 4-4-1 Lipase CLEAs熱穩定性 66 4-4-2 PGA CLEAs熱穩定性 68 4-5 重複使用性 70 4-6 動力學參數Km與Kcat 值 72 4-7 CLEAs的形態學 74 五、結論 78 六、未來與展望 79 參考文獻 80 附錄 88zh_TW
dc.subjectCross-linked enzyme aggregatesen_US
dc.titleEvaluation on the preparation, characterization, and application of cross-linked enzyme aggregates for enzymes immobilizationen_US
dc.typeThesis and Dissertationzh_TW
item.openairetypeThesis and Dissertation-
item.fulltextno fulltext-
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