臺灣博碩士論文加值系統

D型胺基酸氧化酶(D-amino acid oxidase; EC 1.4.3.3；DAO)是以flavin adenine dinucleotide(FAD)作為輔酶的氧化酶。D型胺基酸氧化酶以D型胺基酸為基質，進行氧化脫胺作用(oxidative deamination)，產生相對應的α酮酸和氨氣，並伴隨過氧化氫的產生。先前研究顯示，固定在聚多巴胺包覆磁珠的Rhodosporidum toruloides D型胺基酸氧化酶(RtDAO)具有較佳的熱和氧化穩定性。本研究使用磁性奈米粒子(MNP)或經聚多巴胺包覆的磁性奈米粒子(PDMNP)，將RtDAO和商業購買的催化酶進行固定化，以評估其消除DAO反應產生的過氧化氫，減少DAO受氧化傷害，而增加酵素活性的效能。催化酶固定於MNP或PD-MNP的固定率約為50%以及40%，但分別保有80和40%的原始比活性。將RtDAO和催化酶固定於磁珠，RtDAO的活性分別上升1.9倍和2.1倍。

The flavoenzyme D-Amino acid oxidase(EC 1.4.3.3;DAO)can utilize various D-Amino acids as substrates to catalyze oxidative deamination to produce the corresponding α-keto acids and release ammonia, and concurrent production of hydrogen peroxidase. Previous studies demonstrated that yeast Rhodosporidum toruloides D-amino acid oxidase (RtDAO) immobilized onto polydopamine-coated magnetic nanoparticles (PD-MNP) exhibited better thermal stability and resistance against hydrogen peroxide. In this study, magnetic nanoparticles (MNP) and polydopamine-coated MNP (PD-MNP)were used for immobilization of RtDAO and commercial catalase to evaluate the efficacy of immobilized catalase to eliminate hydrogen peroxide generated during DAO catalysis, thus alleviating the oxidative damage and improving the DAO activity. The immobilization efficiencies of catalases onto MNP and PD-MNP were 50% and 40%, while retaining 80% and 40% of the original specific activities, respectively. In the presence of catalase immobilized onto MNP and PD-MNP, DAO activities were improve by 1.9 and 2.1, respectively.

致謝 I
摘要 II
Abstract III
目錄 IV
第一章 前言 1
1.1 D型胺基酸氧化酶 1
1.1.1 催化反應 1
1.1.2 來源 2
1.1.3 蛋白質結構 3
1.1.4 基質特異性 4
1.1.5 應用 5
1.2 催化酶 6
1.2.1 催化反應 6
1.2.2 蛋白質結構 7
1.2.3 來源 8
1.2.4 應用 9
1.3 酵素固定化 9
1.3.1 固定化使用的載體 10
1.3.2 固定的方法 10
1.3.3 利用多巴胺進行表面修飾 11
1.4研究目的 12
第二章 材料與方法 13
2.1實驗架構 13
2.2 菌體 13
2.3 質體 14
2.4 DAO的誘導表現 14
2.5 DAO純化 15
2.6 DAO的活性測定 17
2.6.1 利用過氧化氫的生成量測定DAO的活性 17
2.6.2 利用丙酮酸的生成量測定DAO的活性 18
2.7 DAO的定量 18
2.8 DAO固定化 19
2.8.1 製備磁性奈米粒子 19
2.8.2 磁性奈米粒子表面修飾 20
2.8.3. RtDAO固定化 20
2.8.4 固定效率評估 21
2.8.5過氧化氫耐受性檢測 21
2.10催化酶固定化 22
2.10.1催化酶的定量 22
2.10.2催化酶的活性測定 22
2.10.3催化酶固定化 23
2.11 催化酶消除DAO產生的過氧化氫 23
2.11.1 催化酶消除過氧化氫檢測 23
2.11.2 催化酶消除過氧化氫對DAO活性的影響 24
第三章 結果與討論 25
3.1 RtDAO固定化 25
3.1.1 磁性奈米粒子和酵素比例 25
3.1.2固定化反應體積 26
3.1.3過氧化氫耐受性檢測 27
3.2催化酶固定化 28
3.2.1磁性奈米粒子和酵素比例 28
3.2.2 固定化反應時間及溫度 29
3.2.3 固定化反應體積 30
3.3以催化酶消除DAO反應產生的過氧化氫 31
3.3.1 催化酶消除過氧化氫檢測 31
3.3.2 催化酶消除過氧化氫對DAO活性影響 32
第四章 結論 33
第五章 圖表 35
第六章 參考資料 57
附錄 67





 
圖表索引
表一 不同來源的DAO參與的生理作用 35
表二 不同來源D型胺基酸氧化酶的基質特異性 36
表三 磁珠和RtDAO比例對固定化DAO比活性和固定化效率的影響 37
表四 不同反應體積對固定化DAO比活性和固定化效率影響 38
表五 磁珠和催化酶比例對固定化催化酶比活性和固定化效率的影響 39
表六 不同反應時間及溫度對固定化催化酶比活性和固定化效率影響 40
表七 不同反應體積對固定化催化酶比活性和固定化效率影響 41
表八 催化酶消除RtDAO產生的過氧化氫對DAO活性的影響 42
圖1-1 DAO反應機制 44
圖1-2 不同來源的D型胺基酸氧化酶一級結構的比較 45
圖1-3 不同DAO的單體和接合方式 46
圖1-4 人類D型胺基酸氧化酶 47
圖1-5 7-ACA的製備 48
圖1-6 過氧化氫酶三種不同的結構 49
圖1-7 由胺基酸片段將113種過氧化氫酶分類的譜系學分析樹 50
圖1-8 多巴胺的機制 51
圖1-9 兩步驟固定脂質酶於PD-MNP 52
圖2-1 DAO的活性檢測原理 53
圖3-1 過氧化氫耐受性檢測 54
圖3-2 以未固定催化酶消除固定DAO反應產生的過氧化氫 55
圖3-3 以固定催化酶降解固定RtDAO產生的過氧化氫 56

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