臺灣博碩士論文加值系統

本研究利用部分改質商業化膠體(Immobead 150) 希望藉由酵素末端含有的 His-tag，選擇性吸附特定的蛋白，進行固定化的同時，同時有純化的效果，省去純化的成本。本實驗利用亞胺乙二酸(IDA)作為螯合劑將基材表面的部分環氧基覆蓋，再利用金屬離子(Cu2+)的含量來判讀亞胺乙二酸(IDA)覆蓋的程度，發現蛋白濃度大約在 2.5-3mg/ml 之間做固定化效果較好。
本實驗所固定的酵素為開關蛋白 RG13 跟 L-N-carbamoylase， RG13 具有辨識麥芽糖的濃度，隨者麥芽糖濃度不同誘導出不同酵素活性，而L-N-carbamoylase 此酵素可以催化多種主 N-carbamoyl-L-amino acid。此酵素可用於催化生產 L-HPA，而 L-HPA 又可做為抗高血壓藥物的前驅物，具有蠻高的經濟價值。
而兩株酵素在選擇性吸附上，發現 L-NCA 這株酵素比 RG13 這株效更明顯，同樣的條件，使用 RG13 的選擇性吸附比 1.078，而 L-NCA 的選擇性吸附比 1.647，選擇性大約多了 53.9%。
在 L-NCA 固定化部分，利用 IMAC 的方法所形成物理吸附大約需要 2-3個小時，而在 4-5 個小時吸附量達到最大，而大約要 6-7 個小時酵素才會與基材形成共價鍵結，而酵素的吸附時間與活性有相當關係，以活性結果來看大約是以 0.3M 亞胺乙二酸(IDA)部分改質的基材，吸附 5 個小時，所測得的活性最大。

It is said that enzyme’s His-tag will absorb certain proteins while immobilization, and save the cost of purification at the same time. In this study, iminodiacetic acid (IDA) is used to modify Immobead 150 as chelating agent to cover the substrate of the epoxy group, and it is concluded that when a protein concentration is between 2.5-3mg/ml, enzyme could be better immobilization.
Two enzymes Protein switch RG13 and L-N-carbamoylase are used for the following two reasons. First, Protein switch RG13 could specify the concentration of maltose, and induce the activity of different enzymes. Second, L-N-carbamoylase could catalyze a variety of primary N-carbamoyl-L -amino acid, and it can be used to catalyze the production of L-HPA. It is known that the L-HPA can be used as precursor of antihypertensive drug, which has high economic value nowadays.
Under the same condition, compared the selectivity adsorption of the two enzymes, the selectivity adsorption of L-NCA is 1.647, and RG13 is 1.07, which is 53.9% more than that of RG13. Therefore, L-NCA is apparently better than RG13. Moreover, in L-NCA immobilization, it took about 2 to 3 hours to form physical adsorption by means of IMAC, and achieved the maximum amount of adsorption after 4 or 5 hours.
To summary, the results show that the time of adsorption is related with the enzyme activity, and by using 0.3M iminodiacetic acid(IDA) to modify Immobead 150 could achieve the maximum activity after five hours of adsorption.

致謝 ........................................................................................................................ I

中文摘要 ............................................................................................................... II

Abstract................................................................................................................. III

目錄 ...................................................................................................................... IV

表目錄 ............................................................................................................... VIII

圖目錄 .................................................................................................................. IX

第一章 緒論 .......................................................................................................... 1

第二章 文獻回顧 .................................................................................................. 2

2-1 認識蛋白質 .................................................................................................. 2

2-1-1 蛋白質的結構 ....................................................................................... 2

2-2 蛋白質純化 .................................................................................................. 4

2-2-1 鹽沈澱法(salting-in/out) ....................................................................... 5

2-2-2 膠體過濾法(gel filtration) .................................................................... 6

2-2-3 離子交換法(ion exchange) ................................................................... 6

2-2-4 親和層析法(affinity chromatography) ................................................. 8

2-3 固定化金屬親和層析法(Immobilized metal ion affinity chromatography)9

2-3-1 吸附基材(matrix) ................................................................................ 10

2-3-2 延伸臂(space-arm) .............................................................................. 12

2-3-3 金屬螯合劑(chelating agent) .............................................................. 12

2-3-4 金屬離子(metal ion) ........................................................................... 13

2-3-5 影響固定化金屬離子親和層析法的因素探討 ................................. 14

2-4 酵素固定化 ................................................................................................ 15

2-4-1 固定化方法 ......................................................................................... 16

2-4-2 包埋法(encapsulation) ........................................................................ 16

2-4-3 交聯法(cross-linking) ......................................................................... 17

2-4-4 鍵結法(binding to a support or carrier) .............................................. 17

2-4-3 酵素穩定性 ......................................................................................... 18

2-5 研究系統簡介 ............................................................................................ 20

2-5-1 多功能性的環氧基固定化基材 ......................................................... 22

2-5-2 固定化之酵素 ..................................................................................... 24

第三章 藥品與儀器 ............................................................................................ 28

3-1 實驗材料 .................................................................................................... 28

3-2 實驗藥品 .................................................................................................... 28

3-3 實驗儀器 .................................................................................................... 30

第四章 實驗方法 ................................................................................................ 31

4-1 酵素 L-NCA 的製備 .................................................................................. 31

4-1-1 L-N-carbamoylas 之菌種來源 ............................................................ 31

4-1-2 LB 固態洋菜培養基的製備 ............................................................... 31

4-1-3 LB 液態培養基的製備 ....................................................................... 31

4-1-4 菌種的培養方式 ................................................................................. 32

4-1-5 菌體回收與粗酵素液的製備 ............................................................. 32

4-2 酵素 RG13 的製備 ..................................................................................... 33

4-2-1 菌種的來源 ......................................................................................... 33

4-2-2 LB 固態培養基的製備 ....................................................................... 33

4-2-3 LB 液態培養基的製備 ....................................................................... 33

4-2-4 菌種的培養方式 ................................................................................. 34

4-2-5 菌體回收與粗酵素液的製備 ............................................................. 34

4-2-6 酵素純化 ............................................................................................. 34

4-3 酵素固定化基材製備 ................................................................................ 36

4-3-1 改質 Immobead 150 ............................................................................ 36

4-4 酵素固定化 ................................................................................................ 36

4-4-1 於不同酵素濃度進行酵素固定化 ..................................................... 36

4-5 不同條件 RG13 吸附動力學..................................................................... 37

4-6 分析方法 .................................................................................................... 38

4-6-1 SDS-PAGE 電泳分析 ......................................................................... 38

4-6-2 銅離子濃度分析 ................................................................................. 40

4-6-3 蛋白質濃度分析 ................................................................................. 41

4-7 活性分析 .................................................................................................... 41

4-7-1 RG13 活性分析................................................................................... 41

4-7-2 N-Carbamoyl-L-HPA 製備 ................................................................. 42

4-7-3 L-N-carbamoylase 活性分析 ............................................................. 43

第五章 結果與討論 ............................................................................................ 45

5-1 開關蛋白 RG13 的製備 ............................................................................. 45

5-2 銅離子的含量 ............................................................................................ 46

5-3 不同酵素濃度固定化 ................................................................................ 47

5-4 RG13 吸附動力學 ...................................................................................... 49

5-5 IDA 所佔含量之密度探討 ....................................................................... 56

5-6 RG13 酵素固定化 ...................................................................................... 57

5-7 L-N-carbamoylase 的製備 .......................................................................... 60
5-8 L-NCA 酵素固定化 .................................................................................. 60

5-9 L-NCA 活性分析 ....................................................................................... 63

5-10 固定化 L-NCA 重複批次操作試驗 ........................................................ 65

第六章 結論與未來展望 .................................................................................... 67

第七章 參考文獻 ................................................................................................ 68

附錄 計算式定義 ................................................................................................ 72

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