探討菸鹼醯胺磷酸核糖轉移酶一鍋法合成煙酰胺核糖單核苷酸在不同條件下的影響

以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：42

、訪客IP：3.133.108.48

姓名

盧澤豪(Tse-Hao Lu) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

探討菸鹼醯胺磷酸核糖轉移酶一鍋法合成煙酰胺核糖單核苷酸在不同條件下的影響
(Investigating the Effects of Different Conditions on the One-Pot Synthesis of Nicotinamide Mononucleotide via Nicotinamide Phosphoribosyltransferase)

相關論文

★ 探討菌體形態對於裂褶菌多醣體之影響	★ 探討不同培養方式對猴頭菇抗氧化與抗腫瘤性質的影響
★ 探討不同培養溫度Aspergillus niger 對丹參之機能性影響	★ 光合菌在光生物反應器產氫之研究
★ 探討培養溫度對巴西蘑菇液態醱酵之影響	★ 利用批式液態培養來探討檸檬酸對裂褶菌生長及其多醣體生成影響之研究
★ 探討不同培養基組成對光合菌Rhodobacter sphaeroides生產Coenzyme Q10之研究	★ 利用混合特定菌種生產氫氣之研究
★ 探討氧化還原電位作為Clostridium butyricum連續產氫之研究	★ 探討培養基之pH值與Xanthan gum的添加對巴西蘑菇多醣體生產之影響
★ 探討麩胺酸的添加和供氧量對液態發酵生產裂褶菌多醣體之研究	★ 探討以兩水相系統提昇Clostridium butyricum產氫之研究
★ 探討通氣量對於樟芝醱酵生產生物鹼之影響	★ 探討深層發酵中環境因子對巴西洋菇生產多醣之影響
★ 探討通氣量對於樟芝發酵生產與純化脂解酵素之研究	★ 探討以活性碳吸附酸來提昇Clostridium butyricum產氫之研究

檔案

[Endnote RIS 格式]

[Bibtex 格式]

[相關文章]

[文章引用]

[完整記錄]

[館藏目錄]

至系統瀏覽論文 (2029-7-24以後開放)

摘要(中)

本研究旨在通過一鍋法合成煙酰胺單核苷酸 (Nicotinamide Mononucleotide, NMN) ，並系統性地去找不同反應條件去探討NMN的產率影響。NMN是一種重要的輔酶前體分子，它在人體內轉化為菸鹼醯胺腺嘌呤二核苷酸 (Nicotinamide Adenine Dinucleotide, NAD+)，參與能量代謝、DNA修復及細胞壽命調控等多種生理過程。由於NAD+水平隨著年齡的增長而下降，NMN因其潛在的抗衰老效果而引起了廣泛關注。 NMN的合成路徑起始於D-核糖 (D-Ribose, Ribose) ，經過核糖-5-磷酸 (Ribose-5-Phosphate, R5P) 再到煙酰胺 (Nicotinamide, NAM) 。在這個多步驟的生物合成過程中，涉及到幾種關鍵酶：核糖激酶 (Ribokinase, RbsK) 、磷酸核糖焦磷酸合成酶 (Phosphoribosyl Pyrophosphate Synthetase, PrsA) 、煙酰胺磷酸核糖轉移酶 (Nicotinamide Phosphoribosyltransferase, NAMPT) 和焦磷酸酶 (Pyrophosphatase, PPase) 。其中，NAMPT是合成NMN過程中的關鍵酶，它催化NAM與磷酸核糖焦磷酸 (Phosphoribosyl Pyrophosphate, PRPP) 反應生成NMN，並在維持細胞內NAD+水平中扮演重要角色。由於NAMPT在代謝調控和抗衰老研究中的重要性，對其進行深入研究具有重要意義。為了監控反應的進行，我們通過檢測聚磷酸 (PolyPhosphate, polyP) 的消耗量來確定反應進度。最終生成的NMN產率則通過高效液相色譜法 (High-Performance Liquid Chromatography, HPLC) 來測定。我們的實驗設計是對不同反應條件去探討它們的影響，具體涉及反應溫度、pH值、酶濃度、金屬離子濃度、受質濃度及輔因子濃度等變量。結果表明，這些條件對NMN的合成效率有顯著影響。具體而言，我們發現最佳反應溫度為30℃，最佳pH值為8。在酶濃度方面，NAMPT的最佳濃度為0.1 mg/mL；在金屬離子方面，適量的鎂離子 (Mg²⁺) 可以顯著提高反應效率。此外，酵素濃度和輔因子濃度的也顯示出對產率的重要影響。本研究為NMN的高效生物合成提供了新方法，並在未來的工業化應用中具有潛在的價值。

摘要(英)

This study aims to synthesize Nicotinamide Mononucleotide (NMN) via a one-pot method and systematically optimize the reaction conditions to improve NMN yield. NMN is a crucial coenzyme precursor molecule that converts into Nicotinamide Adenine Dinucleotide(NAD+) in the human body, participating in energy metabolism, DNA repair, and cellular longevity regulation. As NAD+ levels decline with age, NMN has garnered significant attention for its potential anti-aging effects. The synthesis pathway of NMN begins with D-Ribose, proceeding through Ribose-5-phosphate (R5P) to Nicotinamide (NAM). This multi-step biosynthesis process involves several key enzymes: Ribokinase (RBSK),PhosphoribosylPyrophosphate Synthetase (PRSA), Nicotinamide Phosphoribosyltransferase (NAMPT), and Pyrophosphatase (Pyrophosphatase). Among these, NAMPT is the critical enzyme in the NMN synthesis process, catalyzing the reaction between NAM and Phosphoribosyl Pyrophosphate (PRPP) to produce NMN. NAMPT plays a vital role in maintaining intracellular NAD+ levels, making it significant for studies on metabolic regulation and anti-aging. To monitor the reaction progress, we measured the consumption of PolyPhosphate (polyP). The yield of the final NMN product was determined using High-Performance Liquid Chromatography (HPLC). Our experimental design included optimizing various reaction conditions, specifically involving reaction temperature, pH, enzyme concentration, metal ion concentration, substrate concentration, and cofactor concentration. Our results indicate that these conditions significantly affect NMN synthesis efficiency. Specifically, we found that the optimal reaction temperature is 30°C and the optimal pH is 8. For enzyme concentrations, the optimal concentrations for NAMPT are 0.1 mg/mL. Additionally, an appropriate amount of magnesium ions (Mg²⁺) significantly enhances the reaction efficiency. Optimizing substrate and cofactor concentrations also showed a critical impact on the yield. Through these optimizations, we achieved the highest NMN yield. This study provides a new method for the efficient biosynthesis of NMN, holding potential value for future industrial applications. These optimized conditions not only help improve NMN synthesis efficiency but also provide valuable references for studies on enzyme-catalyzed reactions.

關鍵字(中)

★ 煙酰胺單核苷酸
★ D-核糖
★ 核糖-5-磷酸
★ 煙酰胺
★ 核糖激酶
★ 磷酸核糖焦磷酸合成酶
★ 煙酰胺磷酸核糖轉移酶
★ 焦磷酸酶
★ 焦磷酸
★ 高效液相色譜法

關鍵字(英)

★ Nicotinamide Mononucleotide
★ D-Ribose
★ Ribose-5-phosphate
★ Nicotinamide
★ Ribokinase
★ Phosphoribosyl PyrophosphateSynthetase
★ Nicotinamide Phosphoribosyltransferase
★ Pyrophosphatase
★ Pyrophosphate

論文目次

摘要 I
致謝 II
目錄 III
圖目錄 IV
表目錄 V
第一章、前言 1
1-1 研究動機與目的 1
第二章、文獻回顧 2
2.1受質及反應物 2
2.1.1 Nicotinamide(NAM) 2
2.1.2 Adenosine triphosphate(ATP) 2
2.1.3 PolyPhosphate(polyP) 3
2.2 酵素 4
2.2.1 酵素動力學 4
2.2.2 Nicotinamide Phosphoribosyltransferase(NAMPT) 8
2.2.3 PolyPhosphate kinase (PPK) 9
2.2.3 Pyrophosphatase(PPase) 10
2.3 產物 10
2.3.1 Nicotinamide Mononucleotide(NMN) 10
2.4 NMN合成方法 11
2.5 合成方法的選擇 12
2.6 緩衝液 12
2.6.1 緩衝液的功用 12
第三章、材料與方法 13
3.1 實驗架構 14
3.2 實驗材料與設備 15
3.2.1 實驗藥品 15
3.2.2 實驗設備 17
3.3 菌種保存與培養 18
3.3.1 菌種保存 18
3.3.2 菌種培養 18
3.4 蛋白質實驗分析 18
3.4.1 目標蛋白表達 19
3.4.2蛋白質之萃取及純化 20
3.4.3 蛋白質之定性 21
3.4.4 蛋白質之定量 22
3.5 熱力學分析 22
3.6 緩衝液 23
3.6.1 緩衝液的選擇 23
3.7 TOLUIDINE BLUE O (TBO)介紹與配置 24
3.7.1 Toluidine Blue O (TBO)介紹 24
3.7.2 Toluidine Blue O (TBO)配置 24
3.8 POLYP再生ATP反應 24
3.9. NMN一鍋法實驗流程 25
3.9.1 檢量線 25
3.9.2 反應流程 26
3.10 高效液相層析 (HIGH PERFORMANCE LIQUID CHROMATOGRAPHY, HPLC) 26
第4章、結果與討論 27
4.1條件選擇(緩衝液，溫度，PH值) 28
4.1.1緩衝液 28
4.1.2溫度和PH值選擇 29
4.2 NMN一鍋法合成 30
4.2.1 初始條件合成(無KCL和PYROPHOSPHATASE) 30
4.2.2 初始條件合成優化(加KCL) 32
4.2.3 PYROPHOSPHATASE對NMN一鍋法合成的影響 34
4.3 二價金屬離子對NMN合成的影響 35
4.3.1 二價金屬離子對NMN合成反應結果(MG²⁺+ MN²⁺) 36
4.3.2二價金屬離子(MG²+ BA²⁺) 37
4.4 NAMPT濃度的影響 38
4.5 NAM 濃度對NMN合成的影響 40
第5章、結論 41
參考文獻 42

參考文獻

Adrio, J. L., & Demain, A. L. (2014). Microbial Enzymes: Tools for Biotechnological
Process. Biomolecules, 4(1), 117-139.
Bergman, J. (1999). ATP: the perfect energy currency for the cell. Creation Research Society Quarterly, 36(1), 2-9.
Chen, J., Brevet, A., Fromant, M., Leveque, F., Schmitter, J., Blanquet, S., & Plateau, P. (1990). Pyrophosphatase is essential for growth of Escherichia coli. Journal of bacteriology, 172(10), 5686-5689.
Cheng, F., Li, K.-X., Wu, S.-S., Liu, H.-Y., Li, H., Shen, Q., Xue, Y.-P., & Zheng, Y.-G. (2024). Biosynthesis of Nicotinamide Mononucleotide: Synthesis Method, Enzyme, and Biocatalytic System. Journal of Agricultural and Food Chemistry.
Gehring, W. (2004). Nicotinic acid/niacinamide and the skin. Journal of cosmetic dermatology, 3(2), 88-93.
Hua, W., Jiang, N., Wu, Y., Zhou, C., Chen, K., & Wang, X. (2023). Synthesis of NMN by cascade catalysis of intracellular multiple enzymes. RSC advances, 13(40), 28131-28138.
Kornberg, A. (1962). On the metabolic significance of phosphorolytic and pyrophosphorolytic reactions. Horizons in biochemistry, 251-264.
Kornberg, A., Rao, N. N., & Ault-Riche, D. (1999). Inorganic polyphosphate: a molecule of many functions. Annual review of biochemistry, 68(1), 89-125.
Lipmann, F. (1941). Metabolic generation and utilization of phosphate bond energy, in, eds FF Nord. CH Werkman, New York, 1, 100-162.
Mills, K. F., Yoshida, S., Stein, L. R., Grozio, A., Kubota, S., Sasaki, Y., Redpath, P., Migaud, M. E., Apte, R. S., & Uchida, K. (2016). Long-term administration of nicotinamide mononucleotide mitigates age-associated physiological decline in mice. Cell metabolism, 24(6), 795-806.
Morrissey, J. H., Choi, S. H., & Smith, S. A. (2012). Polyphosphate: an ancient molecule that links platelets, coagulation, and inflammation. Blood, The Journal of the American Society of Hematology, 119(25), 5972-5979.
Motomura, K., Hirota, R., Okada, M., Ikeda, T., Ishida, T., & Kuroda, A. (2014). A new subfamily of polyphosphate kinase 2 (class III PPK2) catalyzes both nucleoside monophosphate phosphorylation and nucleoside diphosphate phosphorylation. Applied and environmental microbiology, 80(8), 2602-2608.
Nadeeshani, H., Li, J., Ying, T., Zhang, B., & Lu, J. (2022). Nicotinamide mononucleotide (NMN) as an anti-aging health product–promises and safety concerns. Journal of advanced research, 37, 267-278.
Neville, N., Roberge, N., & Jia, Z. (2022). Polyphosphate kinase 2 (PPK2) enzymes: structure, function, and roles in bacterial physiology and virulence. International journal of molecular sciences, 23(2), 670.
Nocek, B. P., Khusnutdinova, A. N., Ruszkowski, M., Flick, R., Burda, M., Batyrova, K., Brown, G., Mucha, A., Joachimiak, A., & Berlicki, Ł. (2018). Structural insights into substrate selectivity and activity of bacterial polyphosphate kinases. ACS Catalysis, 8(11), 10746-10760.
Parnell, A. E., Mordhorst, S., Kemper, F., Giurrandino, M., Prince, J. P., Schwarzer, N. J., Hofer, A., Wohlwend, D., Jessen, H. J., & Gerhardt, S. (2018). Substrate recognition and mechanism revealed by ligand-bound polyphosphate kinase 2 structures. Proceedings of the National Academy of Sciences, 115(13), 3350-3355.
Ren, L., Wang, X., Qi, Y., Han, G., Han, T., Gui, Y., Zhang, M., & Li, X. (2021). Research progress on function and synthesis of β-nicotinamide mononucleotide. Biotic. Resources, 43(2), 127-132.
Rodríguez‐Laguna, N., Rojas‐Hernández, A., Ramírez‐Silva, M. T., Moya‐Hernández, R., Gómez‐Balderas, R., & Romero‐Romo, M. A. (2017). The Conditions Needed for a Buffer to Set the pH in a System. In Advances in Titration Techniques. IntechOpen.
Yoshino, J., Baur, J. A., & Imai, S.-i. (2018). NAD+ intermediates: the biology and therapeutic potential of NMN and NR. Cell metabolism, 27(3), 513-528.
Yoshino, M., Yoshino, J., Kayser, B. D., Patti, G. J., Franczyk, M. P., Mills, K. F., Sindelar, M., Pietka, T., Patterson, B. W., & Imai, S.-I. (2021). Nicotinamide mononucleotide increases muscle insulin sensitivity in prediabetic women. Science, 372(6547), 1224-1229.
Zhou, C., Feng, J., Wang, J., Hao, N., Wang, X., & Chen, K. (2022). Design of an in vitro multienzyme cascade system for the biosynthesis of nicotinamide mononucleotide. Catalysis Science & Technology, 12(4), 1080-1091.

指導教授

徐敬衡(Chin-Hang Shu)

審核日期

2024-7-29

推文