臺灣博碩士論文加值系統

中文摘要
本論文研究開發出另一種促進silica沉澱的方式，在酸水解後的tetramethylorthosilicate (TMOS)中加入酸水解後的3-aminopropyl triethoxysilane(APTS)及酵素溶液會促成silica沉澱，在silica沉澱的過程中酵素會被包埋在silica顆粒中而形成固定化酵素。經電子顯微鏡(SEM)觀察silica顆粒粒徑約為400~500nm，在包埋固定化牛血清蛋白時，最適固定化的條件為pH7~9的Tris-HCl緩衝液，牛血清蛋白濃度為2.5mg/mL，可全部被包埋。
D-p-hydroxyphenylglycine( D-p-HPG )為抗生素Amoxicillin的前驅物之一，而D-hydantoinase為製備D-p-HPG的必要酵素。因此本論文以基因重組技術建構從Agrobacterium radiobacter來的D-hydantoinase表現質體，在E. coli BL21(DE)中大量表現，在直接固定化D-hydantoinase粗萃液時，每克silica乾重約可包埋189.3mg的蛋白質，包埋率約為82.3%，活性回收率約為91.86%，silica顆粒粒徑約為50~80nm。因silica顆粒在pH>8的環境中會被水解，在經戊二醛表面處理後可達到保護silica效果，防止酵素漏失，其活性回收率約為71.32%，最適戊二醛濃度為2%，酵素最適反應溫度為55℃~60℃，在55℃下，固定化後可使半生期增加3倍，固定化後之酵素重複使用七次後，殘餘活性仍可達92%。

ABSTRACT
D-hydantoinase, encoded from recombinant Escherichia coli BL21 (DE3) harbouring plasmid pET30b derived from Agrobacterium radiobacter, was used as an immobilization enzyme to catalyst the production of N-carbamoyl-D-phenylglycine from DL-p-phenylhydantoin, an intermediate substance for the production of D-phenylglycine. The encapsulation of enzyme is an attractive approach to prevent the enzyme from leakage. Enzyme-encapsulated in silica is the commenly used and the matrix is usually formed by sol-gel processing, by using polypeptides (polyamines), or biomimicking catalysts, i.e. cysteamine, to initiate silica polycondensation. In this study, silica matrix are formed from silicic acid by acid hydrolyzed of 3-aminopropyl triethoxysilane (APTS) under neutral pH and at ambient temperature. It was observed by scanning electron microscopy (SEM) that the diameter of silica spheres formed after silification is approximately 500 nm. BSA is used as test protein to be immobilized into the silica before D-hydantoinase immobilization is taken place. It was shown that BSA concentration as high as 2.5mg/mL could be totally encapsulation. D-hydantoinase of 189.3 mg was immobilized in one gram of dry silica matrix and the efficiency of encapsulated D-hydantoinase in silica matrix was 82.3%. As compared to its free enzyme, its activity was 91.86% and the half-life was 3 times longer at 55 oC. However, upon extended operation the enzyme catalytic activity was decreased significantly due to enzyme leakage and the silica matrix was degraded due to alkaline condition applied during the enzymatic reaction. Glutaraldehyde was then used to form a protection film covering the silica matrix by a reaction with the amine group on silica which is derived from APTS. The enzyme activity yield was reduced to 70% after glutaraldehyde modification and its half life was 3.5 times longer at 55 oC than free enzyme. Moreover, immobilized D-hydantoinase could be reused for 7 times to maintain 92% residual activity. The enzymatic reaction followed Michaelis-Menten equation and the kinetics of the enzyme will be discussed.

目錄
中文摘要………………………………………………………………...Ⅰ
英文摘要.................................................II
致謝……………………………………………………………………..III
目錄……………………………………………………………………..V
表目錄…………………………………………………………………. VIII
圖目錄………………………………………………………………....IX

第一章 緒論……………………………………………………………..1
1-1 研究背景……………………………………………………………...1
1-2 研究內容簡介………………………………………………………...3

第二章 文獻回顧………………………………………………………..4
2.1 D-hydantoinase………………………………………………………..4
2.2 D-hydantoinase固定化..........................................................................7
2.3 溶膠凝膠技術背景與發展沿革……………………………………..11
2.4 溶膠凝膠理論………………………………………………………..11
2.5 影響溶膠凝膠反應的因素…………………………………………..13
2.6 生物礦化(biomineralization) ………………………………………..17

第三章 實驗內容…………………………………………………...…..21
3.1實驗流程…………………………………………………………..…..21
3.2 實驗材料………………………………………………………...……25
3.2.1 菌株……………………………………………………………......25
3.2.2 載體……………………………………………………………......25
3.2.3 D-hydantoinase酵素之引子……………………………………….25
3.2.4 酵素…………………………………………………………….......25
3.2.5 DNA操作試液套件………………………………………………. 25
3.2.6標準分子量試劑……………………………………………………26
3.2.7 HIS-tagged蛋白質純化試劑…………………………………........26
3.3 實驗藥品………………………………………………………………26
3.4實驗培養基及試劑…………………………………………………….27
3.4.1 培養基………………………………………………………….......27
3.4.2 試劑……………………………………………………………... ...27
3.5實驗設備…………………………………………………………...…..29
3.6實驗方法………………………………………………………….…....30
3.6.1 pET30b-hyt質體之建構……………………………………….…...30
3.6.2 質體轉殖入宿主細胞…………………………………………..….35
3.6.3 菌株培養及重組蛋白之生產及取得……………………………...36
3.6.4 重組蛋白之純化…………..……………………………………….36
3.6.5 蛋白質之濃度分析……………………………………………….37
3.6.6 蛋白質電泳分析………………………………………………….37
3.6.7 silica顆粒製備……………………………………………………38
3.6.8 鹽類及pH值緩衝液之牛血清蛋白包埋率……………………..39
3.6.9 不同濃度牛血清蛋白包埋率..…..……………………………….39
3.6.10 D-hydantoinase活性分析(呈色法) ……………………………..40
3.6.11 產物分析………………………………………………………...41
3.6.12 imidazole對D-hydantoinase的影響……………………………...41
3.6.13 固定化D-hydantoinase..................................................................41
3.6.14 pH對蛋白質從silica-hyt溶解出的影響…………………...…..42
3.6.15 戊二醛修飾濃度對活性之影響…………………………….. ....43
3.6.16 固定化酵素活性分析……………………………………..… ....44
3.6.17 最適反應溫度測定………………………………………...…....44
3.6.18 熱穩定性之測定………………………………………...….…...44
3.6.19 重複使用性之測定……………………………………...….…...44
3.6.20 酵素動力學分析…………………………………..……..……...45
3.6.21 電子掃描式顯微鏡（SEM）樣品製備程序…..……………….45

第四章 結果與討論
4.1 D-hydantoinase表現載體之確認………………….………………..47
4.2 D-hydantoinase之表現……………………………………………..48
4.3 HPLC產物分析…………………………………………………….50
4.4 imidazole對D-hydantoinase的影響………………………………52
4.5 silica顆粒的製備…………………………………………………...53
4.6 鹽類及pH值對牛血清蛋白包埋的影響………………………….56
4.7 蛋白濃度對包埋濃度之影響………………………………………57
4.8 D-hydantoinase的固定化...................................................................58
4.9 pH對蛋白質從silica-hyt溶解出的影響……………………………. 62
4.10 戊二醛修飾濃度對活性之影響…………………………………..63
4.11 D-hyt固定化對反應速率之影響………………………………….64
4.12最適反應溫度……………………………………………………...65
4.13 熱穩定性之測定…………………………………………………..66
4.14 重複使用性之測定………………………………………………..66
4.15 酵素動力學分析…………………………………………………..68

第五章 結論…………………………………………………………….71
參考文獻………………………………………………………………….72

表目錄
表2-1 D-hydantoinase的固定化研究……………………………………..10
表2-2 酸性條件下烷氧基矽烷(tetraalkoxysilanes,Si(OR)4)水解常數
……………………………………………………………………………..15
表4.1固定化D-hydantoinase酵素反應動力學參數……………..….….70
圖目錄
圖 2-1 由D-p-HPG and 6-APA生產Amoxicillin…...……………………...5
圖2-2 酵素法生成D-p-Hydroxyphenylglycine…………………………...6
圖2-3 pH值對膠體(colloid)silica-water系統膠體影響…………………14
圖2-4 氧化矽聚合之性質………………………………………………...14
圖2-5 以氫原子為基準的相對誘導效應大小…………………………...15
圖2-6 不同silane酸水解隨時間變化的相對silane濃度………………….16
圖2-7 silaffins胺基酸序列………………………………………………..18
圖2-8 natSil-1A化學結構…………………………………………………18
圖2-9 long-chain polyamines化學結構…………………………………...18
圖2-10(a) poly-L-lysine化學結構………………………………………...19
圖2-10(b) poly(allylamine hydrochloride) 化學結構…………………….19
圖2-12 小分子催化劑結構圖…………………………………………….20
圖2-13 3-aminopropyl triethoxysilane結構式……………………………...20
圖3.1 實驗流程第一部份D-hydantoinase 之生產表現…………………22
圖3.2 實驗流程第二部份以BSA測試silica沉澱法固定化蛋白質……23
圖3.3 實驗流程第三部份silica沉澱包埋固定化D-hydantoinase………24
圖3.4 pET30b(+)之載體…………………………………………………...25
圖3.5 pET30b-hyt質體之建構流程………………………………………31
圖3.6 silica 顆粒製備流程圖……………………………………………..38
圖3.7 C-p-HPG與C-PG之間的差異……………………………………….40
圖3.8 酵素固定化流程圖………………………………………………...43
圖3.9以Coomassie brilliant blue G-250做出之蛋白質濃度檢量線
……………………………………………………………………………...46
圖3.10 以Dimethylaminobenzaldehyde做出之NC-D-p-HPG濃度檢量線
.......................................................................................................................46
圖4.1 colony PCR for pET30b-hyt................................................................48
圖4.2.1 E. coli BL21(DE3)/pET30b-hyt 30℃SDS-PAGE電泳結果...........49
圖4.2.2加入IPTG、15℃SDS-PAGE電泳結果............................................50
圖4.3 HPLC產物分析結果...........................................................................51
圖4.4 粗酵素溶液內含不同imidazole濃度對產物生成濃度影響............52
圖4.5.1 酵素催化sol-gel的形成機制.......................................................53
圖4.5.2 酸鹼催化sol-gel的形成機制…………………………………...53
圖4.5.3水解後的TMOS加水解後的APTS產生silica顆粒的電子顯微鏡
……………………………………………………………………………...54
圖4.5.4 有添加矽酸誘導劑APTS，形成silica 顆粒…………………..55
圖4.5.5 無添加矽酸誘導劑APTS形成gel………………………………55
圖4.6鹽類及pH值對矽酸沉澱包埋牛血清蛋白的影響………………..56
圖4.7牛血清蛋白濃度的包埋的影響……………………………………57
圖4.8.1 直接包埋的示意圖………………………………………………58
圖4.8.2 間接包埋的示意圖………………………………………………58
圖4.8.3添加酵素後所得的silica電子顯微鏡圖…………………………59
圖4.8.4 cysteamine催化TMOS形成silica的電子子顯微鏡圖...............60
圖4.8.5 Polypeptides(R5)催化TMOS形成silica.......................................60
圖4.8.6 APTS所沉澱之矽膠顆粒的X光射線能量散佈分析儀（EDAX）
.......................................................................................................................61
圖4.9 pH對蛋白質從silica-hyt溶解出的影響..........................................62
圖4.10戊二醛修飾濃度對活性之影響………………………………......63
圖4.11 D-hyt固定化對反應速率之影響………………………………....64
圖4.12 D-hyt最適反應溫度……………………………………………....65
圖4.13 固定化對D-hydantoinase熱穩定的影響………………………..66
圖4.14 固定化hyt(silica-hyt；silica-ga-hyt)重複操作反應曲線…………66
圖4.15.1 D-hyt反應與基質濃度之關係………………………………….69
圖4.15.2 D-hyt Lineweaver-Burk 雙倒數作圖…………………………...70

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