Agrobacterium radiobactor之N-Carbamoyl-D-amino acid amidohydrolase（DCase）是一種胺基水解酵素，對於N-Carbamoyl-D-amino acids有專一的旋光選擇性，能水解成相對應的D型胺基酸。此酵素存在於細菌細胞中，本論文利用基因重組菌株Escherichia coli BL21(DE3)/pDCase-ChBD生產DCase-ChBD，將此酵素固定化於幾丁質顆粒（chitin beads）。
1g濕重幾丁質顆粒可固定化DCase-ChBD 44.66mg，得到比活性為11.33 U/wet-g之幾丁質顆粒。固定化DCase會被Zn、Ni、Mn、Cu、Co等二價離子抑制活性，而會被二價的Fe離子提升其活性兩倍。然而固定化DCase最適反應pH範圍不變，範圍為pH 7.0~pH 8.0，400C之半衰期，則由34小時提升至86小時。於400C 0.1M 磷酸緩衝溶液, pH＝7.0反應3小時，基質CpHPG濃度19mM，可以將CpHPG完全轉化為pHPG，再利用簡單的過濾方式可將固定化DCase與溶解於溶液中的產物分離如此重複操作五次後，仍保持有90％的活性。
S10為組成性的啟動子利用其表現重組蛋白，則不用添加任何的誘導劑，所要表現的異源蛋白會隨著E. coli的生長而會被表現出來。在搖瓶培養下比較，不論在370C或300C下培養，以0.1 mM IPTG誘導T7啟動子驅動，DCase的產量皆以T7啟動子驅動產量較多。如利用乳糖誘導T7啟動子驅動時，T7啟動子容易因葡萄糖濃度而抑制其表現量，而饋料批次醱酵培養亦然，S10啟動子驅動生產不會受葡萄糖濃度影響其產量且較不易產生出不可溶之DCase。但醱酵槽培養，370C以0.1 mM IPTG誘導結果則與搖瓶培養相符合，如以乳糖誘導T7啟動子驅動DCase且降低誘導溫度則會有較高產量。以S10啟動子驅動生產DCase，只要將菌體濃度提高，不需考慮其誘導時機、誘導濃度。

Chitin-binding Domain fused N-Carbamoyl-D-amino acid amidohydrol- ase (DCase) of Agrobacterium radiobactor was expressed in Escherichia coli. This fusion protein can be easily immobilized onto chitin beads by incubating cells crude extract. DCase-ChBD fusion protein of 44.66 mg was immobilized on one gram of wet chitin beads. The activity toward CpHPG was 11.33 U/g-wet chitin beads. Once immobilized on chitin beads, the stability of DCase was increased. Its half-life at 40oC increased from 34 h to 86 h. The immobilized was repeatedly used for 5 time to convert CpHPG to pHPG and 90% of its initial activity was maintained. Constitutive promoter S10 and inducible promoter T7 were employed to drive the expression of DCase in E. coli. Most of DCase expressed under the control of T7 was insoluble form. Promoter S10 can drive the DCase expression along with cell growth and most of expressed DCase was in a soluble form.

目錄
中文摘要……………………………………………………………………………Ⅰ
英文摘要……………………………………………………………………………Ⅲ
目錄…………………………………………………………………………………Ⅳ
圖目錄……………………………………………………………………………Ⅵ
表目錄………………………………………………………………………………Ⅷ
第一章 緒論…………………………………………………………………… 1
1.1 研究背景………………………………………………………………………1
1.2 研究目的………………………………………………………………………4
第二章 文獻回顧…………………………………………………………………6
2.1 N-Carbamoyl-D-amino acid amidohydrolase………………………6
2.2 固定化酵素…………………………………………………………………12
2.2.1 載體-幾丁質……………………………………………………12
2.2.2 幾丁質區域結合蛋白 chitin binding domain………………16
2.2.3 DCase固定化…………………………………………………18
第三章 實驗…………………………………………………………………22
3.1 實驗流程……………………………………………………………………22
3.2 實驗材料…………………………………………………………………22
3.2.1 菌株………………………………………………………………………22
3.2.2 質體………………………………………………………………………22
3.2.5 DNA操作試液套件………………………………………………………22
3.2.6 標準分子量溶液…………………………………………………………23
3.2.8 其他………………………………………………………………………23
3.3 實驗藥品……………………………………………………………………23
3.4 實驗培養基及試劑…………………………………………………………25
3.4.1 培養基……………………………………………………………………25
3.4.2 試劑………………………………………………………………………27
3.5 實驗設備……………………………………………………………………31
3.6 實驗方法……………………………………………………………………33
3.6.1 質體轉殖入宿主細胞……………………………………………………33
3.6.2 質體純化…………………………………………………………………34
3.6.3 搖瓶培養………………………………………………………………35
3.6.4 生長曲線測定……………………………………………………………36
3.6.5 質體穩定度………………………………………………………………36
3.6.6 重組蛋白之取得…………………………………………………………37
3.6.7 重組蛋白之純化………………………………………………………37
3.6.8 蛋白質之濃度分析(protein assay)…………………………………38
3.6.9 DCase活性分析………………………………………………………39
3.6.10 蛋白質電泳分析………………………………………………………40
3.6.11 固定化酵素……………………………………………………………41
3.6.11.1 載體chitin beads的製備……………………………………41
3.6.11.2 酵素的製備……………………………………………………42
3.6.11.3 酵素的固定化…………………………………………………43
3.6.11.4 固定化酵素活性分析…………………………………………43
3.6.12 醱酵槽饋料批次培養…………………………………………………44
3.6.12.1 pH電極的校正與保存…………………………………………44
3.6.12.2 DO電極的校正與保存…………………………………………46
3.6.12.3 菌體濃度測定…………………………………………………47
3.6.12.4 乾菌重測定……………………………………………………47
第四章 結果與討論………………………………………………………49
4.1 固定化酵素…………………………………………………………………49
4.1.1 表現融合酶之培養基選用及誘導溫度選擇…………………………49
4.1.2 DCase-ChBD的固定化…………………………………………………51
4.1.3 幾丁質顆粒對DCase-ChBD的吸附量及活性影響…………………53
4.1.4 緩衝溶液的pH值與濃度及金屬離子對DCase活性時的影響………54
4.1.5 固定化DCase的半衰期及重複性……………………………………57
4.1.6 固定化DCase總結……………………………………………………59
4.2 組成性啟動子S10與誘導性啟動子T7驅動時DCase表現量的影響……60
4.2.1 搖瓶培養比較組成性啟動子S10與誘導性啟動子T7表現DCase的表現量………………………………………………………………………60
4.2.2 醱酵槽批式培養生產DCase……………………………………………70
4.2.3 指數型饋料批式………………………………………………………76
參考文獻……………………………………………………………………………81

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