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研究生： 李泗芃
論文名稱： 探討嗜熱菌 Meiothermus taiwanensis ATCC BAA-400 之半乳糖激酶酵素動力學及應用於合成 Pk 抗原類似物
Kinetics Studies with Galactokinase from Meiothermus taiwanensis ATCC BAA-400 and Application of Galactokinase to Synthesize Pk-antigen analoge
指導教授： 林俊成
口試委員: 吳東昆
林伯樵
學位類別： 碩士
Master
系所名稱： 理學院 - 化學系
Department of Chemistry
論文出版年： 2012
畢業學年度： 100
語文別： 中文
論文頁數： 81
中文關鍵詞： 半乳糖激酶 、半乳糖轉移酶 、Pk 抗原
外文關鍵詞： Galactokinase, Galactosyltransferase, Pk antigen
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    • 在本論文中，選殖、表達台灣本土嗜熱菌 Meiothermus taiwanensis ATCC BAA-400 的半乳糖激酶 （galactokinase，GalK） 並以 IMPACTTM-CN系統純化可得 16 mg/L 的半乳糖激酶重組蛋白。半乳糖激酶重組蛋白最佳化反應溫度為 75 oC、最佳化反應酸鹼值為 9.0。半乳糖激酶重組蛋白對半乳糖的kcat/Km 值為 168.47 s-1mM-1，於 55 oC 和 75 oC 分別測得的比活性為 240 U/mg 和 388 U/mg。半乳糖激酶與實驗室已有的葡萄糖-1-磷酸胸苷轉移酶 （glucose-1-phosphate thymidylyltransferase，RmlA）結合，可進行一鍋化反應製備尿苷二磷酸半乳糖 （UDP-Gal）。
    分別由 Neisseria meningitides 與 Haemophilus influenzae Rd KW20 選殖兩種來源不同的 α-1,4-半乳糖轉移酶。C-端截斷 （truncated） 19 個胺基酸的N. meningitides α-1,4-半乳糖轉移酶（LgtC-19）與N-端截斷 38 個胺基酸的 H. influenzae Rd KW20 α-1,4-半乳糖轉移酶（N-cut-38-Hi0258），皆可運用於階段式一鍋化反應合成 Pk 抗原類似物 （α-D-Gal-(1-4)-β-D-Gal-(1-4)-β-D-Glc -OC6H12N3）。

    In this study, the recombinant galactokinases (GalK) from Meiothermus taiwanensis ATCC BAA-400 was cloned, over-expressed, and purified by IMPACTTM-CN system with a yield of 16 mg/L cell cultures. The optimal reaction conditions for recombinant GalK are at 75 °C, and pH at 9.0. The kcat/Km value of recombinant GalK toward galactose is 168.47 s−1 mM−1 and specific unit at 55 °C and 75 °C are 240 U/mg and 388 U/mg, respectively. The GalK was combined with glucose-1- phosphate thymidylyltransferase (RmlA) to synthesize uridine 5’-diphosphate galactose (UDP-Gal) in one-pot reaction.
    In addition, the genes of α-1,4-galactosyltransferases (GalTs) from Neisseria meningitides (19 residues at C-terminal deleted; LgtC-19) and Haemophilus influenzae Rd KW2 (38 residues of N-terminal was deleted;N-cut-38-Hi0258) were cloned and the corresponding proteins were over-expressed. Both α-1,4-GalTs were respectively combined with GalK and RmlA to synthesize Pk antigen derivatives (α-D-Gal-(1-4)-β-D -Gal-(1-4)-β-D-Glc-OC6H12N3) in sequential addition one-pot reaction manner.

    目錄 第一章 序論 1.1. 酵素在有機合成的應用 1.2. 酵素於醣類合成之應用 1.2.1. 醣基水解酶 1.2.2. 醣基轉移酶 1.3. 酵素合成尿苷二磷酸半乳糖方法 1.4. 半乳糖激酶 （GalK） 1.4.1. GalK 的受質忍受度 1.4.2. 基因工程突變 GalK 1.5. 葡萄糖-1-磷酸胸苷轉移酶 （RmlA） 1.6. 半乳糖轉移酶 （GalT） 1.6.1. α-1,3-GalT 1.6.2. β-1,3- GalT 1.6.3. α-1,4- GalT 1.6.4. β-1,4- GalT 1.7. Pk 抗原 1.8. 研究動機與目標 第二章 實驗結果與討論 2.1. 分析 GalK 胺基酸序列 2.2. 以大腸桿菌誘導表現 GalK 2.2.1. 建構含 galk 基因重組載體 2.2.2. galk 基因之聚合酶連鎖反應 2.2.3. 誘導 galk 基因產物之表現 2.3. 最佳化 GalK 反應條件 2.3.1. 測試 DNS 法是否適用於檢測 GalK 產率 2.3.2. GalK 反應活性測試 2.3.3. 最佳化 GalK 反應溫度 2.3.4. 最佳化 GalK 二價金屬離子輔助因子 2.3.5. 最佳化 GalK 反應酸鹼值 2.4. 探討 GalK 動力學特性 2.4.1. 測定 GalK 比活性 2.4.2. 測定 GalK 的 kcat 和對 Gal 的 Km 值 2.4.3. GalK 的受質忍受度 2.4.4. GalK 的熱穩定性 2.5. GalK 的結構預測與分析 2.5.1. 預測 GalK 結構 2.5.2. 以 Ellman 法定量 GalK 中的硫醇基 2.5.3. 測量 GalK CD 光譜與 Tm 值 2.6. 合成 Gal-1-P 2.6.1. 製備 Gal-1-P 2.6.2. 一鍋化合成 UDP-Gal 2.7. 建構 hi0258 基因之重組載體與活性測試 2.7.1. 分析 hi0258 基因序列 2.7.2. hi0258 基因之聚合酶連鎖反應 2.7.3. 誘導 hi0258 基因與 N-cut-38-hi0258 基因產物 之表現 2.7.4. N-cut-38-Hi0258 重組蛋白活性測試 2.8. α-1,4-GalT （LgtC-19） 合成 Pk-C6N3 2.8.1. 表現 α-1,4-GalT（LgtC-19）重組蛋白 2.8.2. 階段性一鍋化反應合成三醣體 Pk-C6N3 第三章 結論與未來展望 3.1. GalK 3.2. α-1,4-GalT 3.3. 階段性一鍋化合成三醣體 Pk 類似物 第四章 實驗材料與方法 4.1. Materials 4.1.1. The Gene Source of Galactokinase 4.1.2. The Gene Source of α-1,4-galactosyltransferase 4.1.3. Enzymes 4.1.4. Chemicals 4.1.5. Machines 4.1.6. Electrophoresis Systems for DNA and Protein 4.2. Cloning, Overexpression, and Purification 4.2.1. Cloning, Overexpression, and Purification of the Full-Length Galactokinase from Meiothermus tai- wanensis ATCC BAA-400 (galk) by IMPACT system 4.2.2. Cloning, Overexpression and Purification of the α-1,4-galactosyltransferase with C-terminal trunca- ted residues of 19 amino acids from Neisseria meni- ngitidis (lgtC-19) by IMPACT system 4.2.3. Cloning, Overexpression and Purification of the α-1,4-galactosyltransferase with N-terminal trunc- ated residues of 38 amino acids from Haemophilus influenzae Rd KW20 (n-cut-38-hi0258) by IMPA- CT system 4.2.4. Cloning the full-length α-1,4-galactosyltransferase from Haemophilus influenzae Rd KW20 (hi0258) by IMPACT system 4.2.5. Cloning the full-length α-1,4-galactosyltransferase (hi0258) and the α-1,4-galactosyltransferase with N-terminal truncated residues of 38 amino acids (n-cut-38-hi0258) from Haemophilus influenzae Rd KW20 by pET system 4.3. Experiments of Galactokinase 4.3.1. DNS Assay 4.3.1.1. The Procedure of DNS Assay 4.3.2. Galactokinase Activity Assay 4.3.3. Determinations of Physicochemical Properties 4.3.3.1. Determination of the Optimal Reaction Tempera- ture of GalK 4.3.3.2. Determination optimum metal ion cofactor of GalK 4.3.3.3. Determination of Optimal pH for GalK Catalyzed Reaction 4.3.4. Determination of Kinetic Parameters of GalK Cata- lyzed Reaction 4.3.5. Determination of Thermostability of GalK 4.3.6. Evaluation of Substrate Tolerance of GalK 4.3.7. Ellman’s Test 4.3.8. Synthesis of Galactose-1-phosphate 4.4. Experiment of α-1, 4-galactosyltransferase 4.4.1. Synthesis of Pk antigen-C6H12N3

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