| 摘要: | 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。;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. |
