D型胺基酸氧化酶 (D-Amino acid oxidase；DAO) 工業上被應用於製造頭孢菌素類抗生素的前驅物7-amino cephalosporanic acid (7-ACA)。但DAO易因溫度、pH值改變和副產物H2O2氧化傷害，造成蛋白質結構改變、或FAD (flavin adenine dinucleotide)輔酶的脫落，而甚至失去活性。
本研究將Bacillus circulans WL-12幾丁質酶之幾丁質鍵結區域chitin-binding domain (CBD) 的對應基因片段和Rhodosporidium toruloides DAO (RtDAO) cDAO基因融合，並置於大腸桿菌表現生產具有C端CBD的RtDAO，以固定於幾丁質顆粒增加其穩定性。此RtDAO-CBD融合酵素因具有N端His tag，故可以金屬螯合層析純化後進行生化性質檢測。最適反應溫度和TM分別為35和45oC，最適反應pH則為8.0，最佳pH穩定度於pH 7.0，皆與RtDAO相似。當與10 mM H2O2反應時，RtDAO和RtDAO-CBD的半衰期分別為105和75分鐘，當以0.01 mg/ml濃度儲存於4 oC下時，兩者的半衰期分別為7、6天；經過15次重複結凍，RtDAO和RtDAO-CBD分別保有20和50%的殘餘活性。

D-Amino acid oxidase (DAO) has been applied industrially to produce the precusor for cephalosporin antibiotic, 7-amino cephalosporanic acid. However, DAO is vulnerable to changes in temperature, pH as well as oxidative damages by hydrogen peroxide, thus resulting in alteration of the protein structure, the loss of coenzyme FAD (flavin adenine dinucleotide), and even inactvation.
In this study, the corresponding gene fragment for chitinase from Bacillus circulans WL-12 chitin-binding domain (CBD) was fused to Rhodosporidium toruloides DAO (RtDAO) cDAO gene, and was then expressed in E coli to generate the RtDAO fusion protein with C-terminal CBD for immobilization on chitin beads to obtain better stability. Because of having N-terminal His tag, RtDAO-CBD fusion protein was purified by metal chelation chromatography for the analysis of biochemical properties. The optimal temperature and the Tm value were 35 and 45oC, respectively, while the optimal pH was at 8.0 and the best pH stability appeared at pH 7.0. These properties were all similar to those of RtDAO. When reaction with 10 mM hydrogen peroxide, the half-lives of RtDAO and RtDAO-CBD were 105 and 75 min, respectively. At the concentration of 0.01 mg/ml, their half-lives of storage at 4 oC were 7and 6days, respectively. After 15-cycles of freeze-and-thaw, the residual activities of RtDAO and RtDAO-CBD were 20 and 50%, respectively.

目錄
誌謝I
中文摘要I
AbstractIII
目錄V
表索引IX
圖索引X
第一章 前言1
1.1. D型胺基酸氧化酶1
1.1.1 簡介1
1.1.2 生化特性2
1.1.3 生理功能3
1.1.3.1 調控D-serine3
1.1.3.2 調控D-alanine3
1.1.3.3 調節激素分泌4
1.1.4 DAO的應用4
1.1.4.1 D型胺基酸的定量與分析4
1.1.4.2 生產7-ACA5
1.1.4.3 診斷與預防身心失調的疾病與癌症6
1.2.酵素固定化7
1.2.1 固定化技術7
1.2.2 親和力標籤(affinity tag) 8
1.3.幾丁質及幾丁質酶9
1.3.1 幾丁質9
1.3.1.1 簡介9
1.3.1.2 幾丁質的應用11
1.3.2 幾丁質酶11
1.3.2.1 簡介11
1.3.2.2 幾丁質酶之種類12
1.3.2.3 Bacillus circulans WL-12 ChiA114
1.4.研究目的16
第二章 實驗材料與方法17
2.1 實驗材料17
2.1.1 菌種17
2.1.2 質體17
2.2 實驗方法18
2.2.1 重組質體的建構18
2.2.1.1 基因的複製放大18
2.2.1.2 DNA的剪切和黏合19
2.2.1.4 Competent Cell的製備20
2.2.1.5 E. coli 的轉形作用20
2.2.1.6 抽取質體21
2.2.2 DAO於E. coli 中的表現22
2.2.3 以金屬螯合層析法純化22
2.2.4 DAO活性檢測24
2.2.4.1 測定H2O2產量24
2.2.4.2 Pyruvic acid的產量測定25
2.2.5 蛋白質定量 (Bradford assay) 26
2.2.6 SDS-PAGE分析27
2.2.7 固定於幾丁質微珠30
2.2.8 生化性質檢測30
2.2.8.1 溫度對DAO活性的影響30
2.2.8.2 pH質對DAO活性之影響31
2.2.8.3 H2O2耐受性的檢測32
2.2.9 儲存穩定性32
2.2.9.1 重複結凍解凍檢測32
2.2.9.2 保存期32
第三章實驗結果與討論33
3.1重組表現質體的建構33
3.2 HRtDAO-CBD於E.coli中的異源表現33
3.3 HRtDAO-CBD SDS-PAGE分析33
3.4 HRtDAO-CBD的固定化34
3.5 溫度對HRtDAO-CBD活性的影響34
3.6 pH對HRtDAO-CBD活性的影響35
3.7 HRtDAO-CBD的過氧化氫耐受性35
3.8 HRtDAO-CBD重複結凍解凍檢測36
3.9 HRtDAO-CBD儲存穩定性36
第四章 結論37
第五章 圖表38
第六章 參考文獻61
表索引
表1 DAO於生物體內的各種功能38
表2 各種親和力標籤的胺基酸序列和大小39
表3 各種幾丁質酶的特性40
圖索引
圖1 DAO催化反應機制41
圖2 7-ACA的生成機制42
圖3 H2O2生成量測定法之反應式.43
圖4 HRtDAO-CBD表現重組質體的建構流程44
圖5 pRtDAO-CBD 的建構確認45
圖6 HRtDAO cDNA基因的核苷酸和推演出的對應胺基酸序列46
圖7 HRtDAO-CBD cDNA基因的核苷酸和推演出的對應胺基酸序列47
圖8 H2O2標準曲線48
圖9 Pyruvic acid標準曲線49
圖10 BSA標準曲線50
圖11 HRtDAO與HRtDAO-CBD於TB中的活性表現51
圖12 HRtDAO和HRtDAO-CBD SDS-PAGE 分析52
圖13 HRtDAO和HRtDAO-CBD之最適反應溫度53
圖14 HRtDAO和HRtDAO-CBD的熱穩定性54
圖15 HRtDAO和HRtDAO-CBD之最適反應pH55
圖16 HRtDAO和HRtDAO-CBD的pH穩定性56
圖17 HRtDAO和HRtDAO-CBD之H2O2耐受性57
圖18 HRtDAO和HRtDAO-CBD的重複結凍解凍測試58
圖19 HRtDAO和HRtDAO-CBD於4 oC之儲存穩定性59
圖20 HRtDAO和HRtDAO-CBD於25 oC之儲存穩定性60

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