亞磷酸鹽（H₃PO₃）是一種重要的化學物質，在農業和工業領域皆具有廣泛應用。在農業上，亞磷酸鹽常作為殺菌劑與植物生長促進劑使用，能直接被植物吸收並迅速發揮功效，有效促進植物生長並增強植物對環境胁迫的抵抗能力。然而，在實際應用過程中，亞磷酸鹽若過度使用或處理不當，則可能導致環境污染問題，對生態系統造成負面影響。
本研究旨在透過酵素催化反應，以亞磷酸鹽作為受質，將β-煙醯胺單核苷酸（NMN）轉換成具有重要生理功能的還原型菸鹼醯胺腺嘌呤二核苷酸（NADH）及還原型菸鹼醯胺腺嘌呤二核苷酸磷酸（NADPH）。其中，NADH與NADPH在人體內是極為重要的輔酶，分別在細胞能量代謝與抗氧化防禦系統中扮演不可或缺的角色。然而，商業化的NADPH價格昂貴且化學性質不穩定，容易受到氧化而失去活性，因此透過酵素催化的方式合成NADPH，具有經濟及實用上的顯著優勢。
此外，本研究發現NMN轉換為NADH的效率幾乎完全，利用NADH在340 nm處具特殊吸光性的特點，即可快速且準確地定量NMN的濃度。與傳統利用高效液相層析儀（HPLC）進行檢測相比，此方法更加快速便捷，並可顯著降低有機溶液的使用量，有效減少分析過程中所產生的廢棄物，達到環保且永續的效益。
目前，亞磷酸鹽在土壤或水體中的去除方法主要分為物理、化學及生物三種方式，但使用物理或化學方法去除時，往往會產生二次污染問題。本研究提出以酵素工程的方式，將廢棄的亞磷酸鹽轉化為人體所需且昂貴的輔酶（NADH及NADPH），不僅可降低亞磷酸鹽對環境的負擔，亦能避免HPLC分析所需有機流動相造成的環境污染，符合聯合國永續發展目標（SDGs）第12項指標「Responsible Consumption and Production」的理念，兼具經濟與環保的雙重效益。

Phosphite (H₃PO₃) is an important chemical compound with extensive applications in both agricultural and industrial fields. In agriculture, phosphite is frequently utilized as a fungicide and plant growth promoter due to its ability to be directly absorbed by plants and rapidly exert its beneficial effects, enhancing growth and improving plant resistance against environmental stress. However, improper handling or excessive use of phosphite can lead to environmental contamination, negatively impacting ecosystems.
This study aims to enzymatically catalyze the conversion of β-nicotinamide mononucleotide (NMN) into biologically essential reduced coenzymes, nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), using phosphite as a substrate. NADH and NADPH are critical coenzymes within the human body, significantly involved in cellular metabolism and antioxidative defense systems, respectively. Commercially available NADPH, however, is costly and chemically unstable due to its susceptibility to oxidation, resulting in loss of activity. Therefore, enzymatic synthesis of NADPH presents significant economic and practical advantages.
Furthermore, this study demonstrates that the conversion efficiency from NMN to NADH is nearly complete. By exploiting the distinctive absorbance characteristic of NADH at 340 nm, this method allows for rapid and accurate quantification of NMN concentration. Compared to traditional detection methods using high-performance liquid chromatography (HPLC), this enzymatic approach is quicker, more convenient, and significantly reduces the use of organic solvents, thereby minimizing analytical waste and achieving environmental sustainability.Currently, the removal of phosphite from soil or water typically employs physical, chemical, or biological methods. However, physical and chemical methods often generate secondary pollutants. This research proposes an enzymatic engineering approach to transform waste phosphite into valuable and costly coenzymes (NADH and NADPH), effectively reducing environmental impact. Additionally, this method avoids the environmental issues associated with organic solvents used in HPLC analysis, aligning with the United Nations Sustainable Development Goals (SDGs), particularly Goal 12: "Responsible Consumption and Production," achieving both economic and environmental benefits.

Authority, E. F. S., Bellisai, G., Bernasconi, G., Brancato, A., Carrasco Cabrera, L., Ferreira, L., Giner, G., Greco, L., Jarrah, S., & Kazocina, A. (2021). Reasoned opinion on the joint review of maximum residue levels (MRLs) for fosetyl, disodium phosphonate and potassium phosphonates according to Articles 12 and 43 of Regulation (EC) No 396/2005. EFSA Journal, 19(8), e06782.
Alsafran, M., Saleem, M. H., Al Jabri, H., Rizwan, M., & Usman, K. (2023). Principles and applicability of integrated remediation strategies for heavy metal removal/recovery from contaminated environments. Journal of Plant Growth Regulation, 42(6), 3419-3440.
Abali, E. E., Skacel, N. E., Celikkaya, H., & Hsieh, Y. C. (2008). Regulation of human dihydrofolate reductase activity and expression. Vitamins & Hormones, 79, 267-292.
Babior, B. M. (2004). NADPH oxidase. Current opinion in immunology, 16(1), 42-47.
Belenky, P., Bogan, K. L., & Brenner, C. (2007). NAD+ metabolism in health and disease. Trends in biochemical sciences, 32(1), 12-19.
Barron, K., Ogretmen, B., & Krupenko, N. (2021). Dietary folic acid alters metabolism of multiple vitamins in a CerS6-and sex-dependent manner. Frontiers in Nutrition, 8, 758403.
Babior, B. M. (1999). NADPH oxidase: an update. Blood, The Journal of the American Society of Hematology, 93(5), 1464-1476.
Bailey, S. W., & Ayling, J. E. (2009). The extremely slow and variable activity of dihydrofolate reductase in human liver and its implications for high folic acid intake. Proceedings of the National Academy of Sciences, 106(36), 15424-15429.
Bieganowski, P., & Brenner, C. (2004). Discoveries of nicotinamide riboside as a nutrient and conserved NRK genes establish a Preiss-Handler independent route to NAD+ in fungi and humans. Cell, 117(4), 495-502.
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, 72(7), 3302-3313.
Covarrubias, A. J., Perrone, R., Grozio, A., & Verdin, E. (2021). NAD+ metabolism and its roles in cellular processes during ageing. Nature reviews Molecular cell biology, 22(2), 119-141.
Deichmann, U. (2012). Beyond Popper and Polanyi: Leonor Michaelis, a critical and passionate pioneer of research at the interface of medicine, enzymology, and physical chemistry. Perspectives in Biology and Medicine, 55(4), 612-626.
Eriksson, J., Karamohamed, S., & Nyrén, P. (2001). Method for real-time detection of inorganic pyrophosphatase activity. Analytical biochemistry, 293(1), 67-70.
Ernst, O., & Zor, T. (2010). Linearization of the Bradford protein assay. Journal of visualized experiments: JoVE(38), 1918.
Fukamizu, Y., Uchida, Y., Shigekawa, A., Sato, T., Kosaka, H., & Sakurai, T. (2022). Safety evaluation of β-nicotinamide mononucleotide oral administration in healthy adult men and women. Scientific reports, 12(1), 14442.
García-Contreras, R., de la Mora, J., Mora-Montes, H. M., Martínez-Álvarez, J. A., Vicente-Gómez, M., Padilla-Vaca, F., Vargas-Maya, N. I., & Franco, B. (2024). The inorganic pyrophosphatases of microorganisms: A structural and functional review. PeerJ, 12, e17496.
Ginting, E., & Arcot, J. (2004). High-performance liquid chromatographic determination of naturally occurring folates during tempe preparation. Journal of Agricultural and Food Chemistry, 52(26), 7752-7758.
Hachisuka, S.-i., Sato, T., & Atomi, H. (2018). Hyperthermophilic archaeon Thermococcus kodakarensis utilizes a four-step pathway for NAD+ salvage through nicotinamide deamination. Journal of Bacteriology, 200(11), 10.1128/jb. 00785-00717.
Havlin, J., & Schlegel, A. (2021). Review of phosphite as a plant nutrient and fungicide soil systems. 5 (3): 52-71. In.
Havlin, J. L., & Schlegel, A. J. (2021). Review of phosphite as a plant nutrient and fungicide. Soil systems, 5(3), 52.
Hirota, R., Motomura, K., & Kuroda, A. (2019). Biological phosphite oxidation and its application to phosphorus recycling. Phosphorus recovery and recycling, 499-513.
Hoxhaj, G., Ben-Sahra, I., Lockwood, S. E., Timson, R. C., Byles, V., Henning, G. T., Gao, P., Selfors, L. M., Asara, J. M., & Manning, B. D. (2019). Direct stimulation of NADP+ synthesis through Akt-mediated phosphorylation of NAD kinase. Science, 363(6431), 1088-1092.
Islam, P. (2023). Creating bacterial glycerol stocks for long-term storage. In.
Johannes, T. W., Woodyer, R. D., & Zhao, H. (2007). Efficient regeneration of NADPH using an engineered phosphite dehydrogenase. Biotechnology and Bioengineering, 96(1), 18-26.
Krizkova, S., Dostalova, S., Michalek, P., Nejdl, L., Kominkova, M., Milosavljevic, V., Moulick, A., Vaculovicova, M., Kopel, P., & Adam, V. (2015). SDS-PAGE as a tool for hydrodynamic diameter-dependent separation of quantum dots. Chromatographia, 78, 785-793.
Kawai, S., & Murata, K. (2008). Structure and function of NAD kinase and NADP phosphatase: key enzymes that regulate the intracellular balance of NAD (H) and NADP (H). Bioscience, biotechnology, and biochemistry, 72(4), 919-930.
Loescher, W. H., Tyson, R. H., Everard, J. D., Redgwell, R. J., & Bieleski, R. L. (1992). Mannitol synthesis in higher plants: evidence for the role and characterization of a NADPH-dependent mannose 6-phosphate reductase. Plant Physiology, 98(4), 1396-1402.
Lovatt, C., & Mikkelsen, R. (2006). Phosphite fertilizers: What are they? Can you use them? What can they do. Better crops, 90(4), 11-13.
Li, B.-B., Wang, X., Tai, L., Ma, T.-T., Shalmani, A., Liu, W.-T., Li, W.-Q., & Chen, K.-M. (2018). NAD kinases: metabolic targets controlling redox co-enzymes and reducing power partitioning in plant stress and development. Frontiers in Plant Science, 9, 379.
Luo, S., Zhao, J., Zheng, Y., Chen, T., & Wang, Z. (2023). Biosynthesis of nicotinamide mononucleotide: Current metabolic engineering strategies, challenges, and prospects. Fermentation, 9(7), 594.
Mayevsky, A., & Rogatsky, G. G. (2007). Mitochondrial function in vivo evaluated by NADH fluorescence: from animal models to human studies. American journal of physiology-Cell physiology, 292(2), C615-C640.
McComb, R. B., Bond, L. W., Burnett, R. W., Keech, R., & Bowers Jr, G. (1976). Determination of the molar absorptivity of NADH. Clinical chemistry, 22(2), 141-150.
Marbach, A., & Bettenbrock, K. (2012). lac operon induction in Escherichia coli: Systematic comparison of IPTG and TMG induction and influence of the transacetylase LacA. Journal of biotechnology, 157(1), 82-88.
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.
Oka, S.-i., Titus, A. S., Zablocki, D., & Sadoshima, J. (2023). Molecular properties and regulation of NAD+ kinase (NADK). Redox Biology, 59, 102561.
Offer, T., Ames, B. N., Bailey, S. W., Sabens, E. A., Nozawa, M., & Ayling, J. E. (2007). 5‐Methyltetrahydrofolate inhibits photosensitization reactions and strand breaks in DNA. The FASEB journal, 21(9), 2101-2107.
Poddar, S. K., Sifat, A. E., Haque, S., Nahid, N. A., Chowdhury, S., & Mehedi, I. (2019). Nicotinamide mononucleotide: exploration of diverse therapeutic applications of a potential molecule. Biomolecules, 9(1), 34.
Pavani, P., Kumar, K., Rani, A., Venkatesu, P., & Lee, M.-J. (2021). The influence of sodium phosphate buffer on the stability of various proteins: Insights into protein-buffer interactions. Journal of Molecular Liquids, 331, 115753.
Passos, M. L., & Saraiva, M. L. M. (2019). Detection in UV-visible spectrophotometry: Detectors, detection systems, and detection strategies. Measurement, 135, 896-904.
Petrelli, R., Felczak, K., & Cappellacci, L. (2011). NMN/NaMN adenylyltransferase (NMNAT) and NAD kinase (NADK) inhibitors: chemistry and potential therapeutic applications. Current medicinal chemistry, 18(13), 1973-1992.
Relyea, H. A., & Van Der Donk, W. A. (2005). Mechanism and applications of phosphite dehydrogenase. Bioorganic Chemistry, 33(3), 171-189.
Robinson, P. K. (2015). Enzymes: principles and biotechnological applications. Essays in biochemistry, 59, 1.
Schweiger, M., Hennig, K., Lerner, F., Niere, M., Hirsch-Kauffmann, M., Specht, T., Weise, C., Oei, S. L., & Ziegler, M. (2001). Characterization of recombinant human nicotinamide mononucleotide adenylyl transferase (NMNAT), a nuclear enzyme essential for NAD synthesis. FEBS letters, 492(1-2), 95-100.
Schink, B., & Friedrich, M. (2000). Phosphite oxidation by sulphate reduction. Nature, 406(6791), 37-37.
Sung, Y.-J., Takahashi, T., Lin, Y.-H., Jia, T. Z., Chiang, Y.-R., & Wang, P.-H. (2023). Microalgal polyphosphate drives one-pot complete enzymatic generation of flavin adenine dinucleotide from adenosine and riboflavin. ACS Sustainable Chemistry & Engineering, 12(2), 680-686.
Sharma, A., Chabloz, S., Lapides, R. A., Roider, E., & Ewald, C. Y. (2023). Potential synergistic supplementation of NAD+ promoting compounds as a strategy for increasing healthspan. Nutrients, 15(2), 445.
Sikk, P., Käämbre, T., Vija, H., Tepp, K., Tiivel, T., Nutt, A., & Saks, V. (2009). Ultra performance liquid chromatography analysis of adenine nucleotides and creatine derivatives for kinetic studies. Proceedings of the Estonian Academy of Sciences, 58(2).
Srivastava, S. (2016). Emerging therapeutic roles for NAD+ metabolism in mitochondrial and age-related disorders. Clinical and translational medicine, 5, 1-11.
S Askari, B., & Krajinovic, M. (2010). Dihydrofolate reductase gene variations in susceptibility to disease and treatment outcomes. Current genomics, 11(8), 578-583.
Spriestersbach, A., Kubicek, J., Schäfer, F., Block, H., & Maertens, B. (2015). Purification of his-tagged proteins. In Methods in enzymology (Vol. 559, pp. 1-15). Elsevier.
Sauer, U., Canonaco, F., Heri, S., Perrenoud, A., & Fischer, E. (2004). The soluble and membrane-bound transhydrogenases UdhA and PntAB have divergent functions in NADPH metabolism of Escherichia coli. Journal of Biological Chemistry, 279(8), 6613-6619.
Trinchera, A., Parisi, B., Baratella, V., Roccuzzo, G., Soave, I., Bazzocchi, C., Fichera, D., Finotti, M., Riva, F., & Mocciaro, G. (2020). Assessing the origin of phosphonic acid residues in organic vegetable and fruit crops: the biofosf project multi-actor approach. Agronomy, 10(3), 421.
Tola, F. S. (2024). The concept of folic acid supplementation and its role in prevention of neural tube defect among pregnant women: PRISMA. Medicine, 103(19), e38154.
Thao, H. T. B., & Yamakawa, T. (2009). Phosphite (phosphorous acid): fungicide, fertilizer or bio-stimulator? Soil science and plant nutrition, 55(2), 228-234.
Tjong, E., Dimri, M., & Mohiuddin, S. S. (2023). Biochemistry, tetrahydrofolate. In StatPearls [Internet]. StatPearls Publishing.
Tropea, J. E., Cherry, S., & Waugh, D. S. (2009). Expression and purification of soluble His 6-tagged TEV protease. High throughput protein expression and purification: methods and protocols, 297-307.
Van Beilen, J. B., & Li, Z. (2002). Enzyme technology: an overview. Current opinion in biotechnology, 13(4), 338-344.
Wang, P.-H., Fujishima, K., Berhanu, S., Kuruma, Y., Jia, T. Z., Khusnutdinova, A. N., Yakunin, A. F., & McGlynn, S. E. (2019). A bifunctional polyphosphate kinase driving the regeneration of nucleoside triphosphate and reconstituted cell-free protein synthesis. ACS synthetic biology, 9(1), 36-42.
Woodyer, R., Zhao, H., & Van Der Donk, W. A. (2005). Mechanistic investigation of a highly active phosphite dehydrogenase mutant and its application for NADPH regeneration. The FEBS journal, 272(15), 3816-3827.
Wingfield, P. T. (2014). Preparation of soluble proteins from Escherichia coli. Current protocols in protein science, 78(1), 6.2. 1-6.2. 22.
Wang, X., Saba, T., Yiu, H. H., Howe, R. F., Anderson, J. A., & Shi, J. (2017). Cofactor NAD (P) H regeneration inspired by heterogeneous pathways. Chem, 2(5), 621-654.

Ying, W. (2008). NAD+/NADH and NADP+/NADPH in cellular functions and cell death: regulation and biological consequences. Antioxidants & redox signaling, 10(2), 179-206.
Yoshino, J., Mills, K. F., Yoon, M. J., & Imai, S.-i. (2011). Nicotinamide mononucleotide, a key NAD+ intermediate, treats the pathophysiology of diet-and age-induced diabetes in mice. Cell metabolism, 14(4), 528-536.
Zhang, L., King, E., Black, W. B., Heckmann, C. M., Wolder, A., Cui, Y., Nicklen, F., Siegel, J. B., Luo, R., & Paul, C. E. (2022). Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform. Nature Communications, 13(1), 5021.