藉由PRPP合成酶條件的優化增強煙醯胺單核苷酸的一鍋生物合成

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潘治漢(Zhi-Han Pan) 查詢紙本館藏

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化學工程與材料工程學系

論文名稱

藉由PRPP合成酶條件的優化增強煙醯胺單核苷酸的一鍋生物合成
(Optimization of PRPP synthetase conditions for enhanced one-pot biosynthesis of nicotinamide mononucleotide from D-ribose)

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至系統瀏覽論文 (2029-7-24以後開放)

摘要(中)

煙醯胺單核苷酸(Nicotinamide Mononucleotide, NMN)是由 D-核糖使用三種酶合成
的關鍵產物，其中5-磷酸核糖-1-焦磷酸(Phosphoribosyl pyrophosphate, PRPP)合成酶起著
至關重要的作用。PRPP合成酶催化磷酸核糖基焦磷酸(PRPP)的形成，PRPP是NMN生
物合成的關鍵前體。本研究的重點是優化PRPP合成酶以提高NMN生產效率。簡要介
紹了PRPP合成酶，強調了PRPP在細胞代謝中的重要性及其在NMN生物合成中的作
用。優化過程涉及研究各種參數，例如溫度、pH值、受質濃度和金屬輔助因子可用性。
通過系統地優化這些條件，可以改善 D-核糖向 NMN 的酵素合成，從而為通過酶促途
徑增強NMN的產生。
此外，本研究探討了用於NMN生產的一鍋合成方法，利用 D-核糖作為初始受質，，
並用三種酶的組合催化反應：核糖激酶 (Ribokinase, RBSK)、磷酸核糖焦磷酸合成酶
(Phosphoribosyl Pyrophosphate Synthetase, PRPPS)、煙酰胺磷酸核糖轉移酶 (Nicotinamide
Phosphoribosyltransferase, NAMPT)。專門研究了該合成系統中 PRPP 合成酶的優化及其
酶動力學。通過系統地優化反應條件和酶濃度，最大限度地提高D-核糖向NMN的轉化
效率。
總結來說，本研究確認了使用一鍋法生成NMN，，並優化了最金金屬子子濃度為10
mM，使轉化率達到75%。最後，使用8 mM的鎂子子與2 mM的鈷子子搭配，成功將
轉化率提升至88%。

摘要(英)

Nicotinamide Mononucleotide (NMN) is a key product synthesized from D-ribose
using three enzymes, with Phosphoribosyl Pyrophosphate (PRPP) Synthetase playing a crucial
role. PRPP Synthetase catalyzes the formation of PRPP, which is a critical precursor in the
biosynthesis of NMN. This study focuses on optimizing PRPP Synthetase to improve NMN
production efficiency. A brief introduction of PRPP Synthetase highlights its importance in
cellular metabolism and its role in NMN biosynthesis. The optimization process involves
studying various parameters such as temperature, pH, substrate concentration, and the
availability of metal cofactors. By systematically optimizing these conditions, the enzymatic
synthesis of NMN from D-ribose can be enhanced, thereby improving NMN production
through enzymatic pathways.
Additionally, this study explores a one-pot synthesis method for NMN production using
D-ribose as the initial substrate, catalyzed by a combination of three enzymes: Ribokinase
(RBSK), Phosphoribosyl Pyrophosphate Synthetase (PRPPS), and Nicotinamide
Phosphoribosyltransferase (NAMPT). The research specifically focuses on optimizing PRPP
Synthetase within this synthesis system and its enzyme kinetics. By systematically optimizing
the reaction conditions and enzyme concentrations, the conversion efficiency of D-ribose to
NMN can be maximized.
II
In summary, this study confirms the use of a one-pot method to produce NMN and
optimizes the best metal ion concentration to 10 mM, achieving a conversion rate of 75%.
Finally, by using a combination of 8 mM magnesium ions and 2 mM cobalt ions, the conversion
rate was successfully increased to 88%.

關鍵字(中)

★ 酵素工程
★ 磷酸核糖焦磷酸合成酶
★ 煙醯胺單核苷酸
★ 一鍋法生物合成

關鍵字(英)

★ Enzyme Engineering
★ PRPP Synthetase
★ Nicotinamide Mononucleotide
★ One-pot Biosynthesis

論文目次

中文摘要 ................................................................. I
Abstract ................................................................ II
目錄 .................................................................... IV
圖目錄 ................................................................. VII
表目錄 .................................................................. IX
第一章緒論 .............................................................. 1
1 研究背景與動機 ..................................................... 1
第二章文獻回顧 .......................................................... 2
2-1 煙醯胺單核苷酸 ................................................... 2
2-1-1煙醯胺單核苷酸介紹 ......................................... 2
2-1-2 煙醯胺單核苷酸於生物體中的應用 ............................. 3
2-2酵素 .............................................................. 5
2-2-1酵素動力學 ................................................. 5
2-2-2酵素與溫度的關係 ........................................... 7
2-2-3酵素與pH值的關係 .......................................... 8
2-2-4 酵素與金屬輔助因子的關係 ................................... 9
2-2-5 PRPP synthetase(PRPPS) .................................... 11
2-2-6 PolyPhosphate kinase(PPK) ................................. 11
2-3 產物 ............................................................ 14
2-3-1 5-磷酸核糖-1-焦磷酸(Phosphoribosyl Pyrophosphate, PRPP) ... 14
2-3-2 腺苷單磷酸(Adenosine Monophosphate, AMP) ................. 16
第三章研究方法 ......................................................... 18
3-1實驗架構 ......................................................... 18
3-2實驗材料與設備 ................................................... 19
3-2-1實驗藥品 .................................................. 19
3-2-2實驗設備 .................................................. 20
3-3 菌種保存及培養 .................................................. 21
VI

3-3-1 菌種保存 .................................................. 21
3-3-2 菌種培養 .................................................. 21
3-4目標蛋白表達與純化 ............................................... 21
3-4-1 目標蛋白表達 .............................................. 22
3-4-2 目標蛋白純化 .............................................. 23
3-5 蛋白質之分子量 .................................................. 25
3-6 蛋白質之定量 .................................................... 27
3-7 聚磷酸鹽之定量 .................................................. 28
3-8 煙醯胺單核苷酸之定量分析 ........................................ 28
第四章結果與討論 ....................................................... 30
4-1藉由起始反應確認操作條件 ......................................... 30
4-1-1 RBSK活性測試確認使用ph值 ................................ 30
4-1-2 緩衝溶液的選擇 ............................................ 32
4-1-3 操作溫度的確認 ............................................ 33
4-2 一鍋法的生物合成 ................................................ 35
4-2-1 各階段活性測試 ............................................ 36
4-2-2 NMN的定量分析 ............................................ 37
4-2-3 一鍋法之NMN生成 .......................................... 39
4-3 ATP對反應的影響 ................................................. 40
4-3-1 過量ATP .................................................. 41
4-3-2 PPK2循環系統 ............................................. 43
4-4 二價金屬子子對酵素的影響 ........................................ 45
4-4-1 鎂子子對反應活性的影響 .................................... 46
4-4-2 其他二價金屬子子對活性的影響 .............................. 47
4-4-3 不同比例二價金屬子子優化反應 ............................. 49
第五章結論 ............................................................. 50
參考文獻 ................................................................ 51

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指導教授

徐敬衡(David Shu)

審核日期

2024-7-26

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