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

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：90

、訪客IP：18.191.66.102

姓名

潘治漢(Zhi-Han Pan) 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

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)

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)

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

參考文獻

Cornish-Bowden, A. (2013). Fundamentals of enzyme kinetics. John Wiley & Sons.
Michaelis, L., & Menten, M. L. (1913). Die kinetik der invertinwirkung. Biochem. z, 49(333
369), 352.
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.
Cornish-Bowden, A. (2013). Fundamentals of enzyme kinetics. John Wiley & Sons.
Eriksen, H. M., Jensen, K. F., & Switzer, R. L. (2000). Phosphoribosyl diphosphate (PRPP):
Biosynthesis, Enzymology, and Metabolic Significance. Microbiology and Molecular
Biology Reviews, 64(3), 433-456.
Becker, M. A., & Smith, P. R. (1985). The Regulation of Purine and Pyrimidine Biosynthesis
by PRPP. Trends in Biochemical Sciences, 10(2), 62-65.
Hove-Jensen, B., Andersen, K. R., Kilstrup, M., Martinussen, J., Switzer, R. L., & Willemoës,
M. (2017). Phosphoribosyl diphosphate (PRPP): Biosynthesis, Enzymology, and
Metabolic Significance. Microbiology and Molecular Biology Reviews, 81(1), e00040
16.
Nyhan, W. L. (2001). Nucleotide Synthesis via Salvage Pathway. Inborn Errors of Purine
Metabolism. Springer.
De Brouwer, A. P., & Christodoulou, J. (2022). PRPP Synthetase Superactivity: A Molecular
Basis for Excessive Purine Synthesis. Journal of Molecular Medicine, 100(1), 101
112.
Gots, J. S. (1971). The Regulation of PRPP Synthetase in Yeast. European Journal of
Biochemistry, 57(1), 147-154.
Becker, M. A., & Seegmiller, J. E. (1973). Regulation of Purine and Pyrimidine Biosynthesis.
51
Advances in Enzyme Regulation, 11(1), 79-104.
Seegmiller, J. E. (1979). The Role of PRPP in the Pathogenesis of Gout. Arthritis &
Rheumatism, 22(4), 474-480.
Weber, G., & Kenan, S. (1975). Biochemical Strategy of Cancer Cells and the Role of PRPP.
Advances in Enzyme Regulation, 13(1), 45-67.
Fox, I. H., & Kelley, W. N. (1972). Phosphoribosylpyrophosphate Synthetase Superactivity:
A Molecular Basis for Excessive Purine Synthesis. Proceedings of the National
Academy of Sciences, 69(9), 2467-2471.
Hardie, D. G., Carling, D., & Carlson, M. (2003). The AMP-activated/SNF1 protein kinase
subfamily: Metabolic sensors of the eukaryotic cell? Annual Review of Biochemistry,
66, 971-1002.
Hershfield, M. S. (1979). The Role of PRPP in the Pathogenesis of Gout. Arthritis &
Rheumatism, 22(4), 474-480.
Weber, G. (1983). Biochemical Strategy of Cancer Cells and the Role of PRPP. Advances in
Enzyme Regulation, 21(1), 3-11.
Zalkin, H., & Dixon, J. E. (1992). The Structure and Function of
Phosphoribosylpyrophosphate Synthetase. Annual Review of Biochemistry, 61(1),
171-198.
Marangoni, A. G. (2003). Enzyme Kinetics: A Modern Approach. Wiley-Interscience.
Michaelis, L., & Menten, M. L. (1913). "Die Kinetik der Invertinwirkung". Biochem. Z., 49,
333–369.
Dixon, M., & Webb, E. C. (1979). Enzymes. Longmans.
Somero, G. N. (1995). Proteins and temperature. Annual Review of Physiology, 57(1), 43-68.
Petsko, G. A. (2001). Structural basis of temperature adaptation in proteins. Journal of
Biological Chemistry, 276(29), 26087-26090.
52
Daniel, R. M., Dines, M., & Petach, H. H. (2001). The denaturation and degradation of stable
enzymes at high temperatures. Biochemical Journal, 317(1), 1-11.
Chhetri, G., Kalita, P., & Tripathi, T. (2015). An efficient protocol to enhance recombinant
protein expression using ethanol in Escherichia coli. MethodsX, 2, 385-391.
Mukherjee, C., & Ray, K. (2015). An improved method for extraction and quantification of
PolyPhosphate granules from microbial cells. Protoc. Exch, 10.
Rashid, M. H., & Kornberg, A. (2000). Inorganic PolyPhosphate is needed for swimming,
swarming, and twitching motilities of Pseudomonas aeruginosa. Proceedings of the
National Academy of Sciences, 97(9), 4885-4890.
Ahn, K., & Kornberg, A. (1990). PolyPhosphate kinase from Escherichia coli. Journal of
Biological Chemistry, 265(20), 11734-11739.
Rao, N. N., Gómez-García, M. R., & Kornberg, A. (2009). Inorganic PolyPhosphate: essential
for growth and survival. Annual Review of Biochemistry, 78, 605-647.
Nelson, D. L., & Cox, M. M. (2008). Lehninger Principles of Biochemistry (5th ed.). W.H.
Freeman.
Berg, J. M., Tymoczko, J. L., & Stryer, L. (2002). Biochemistry (5th ed.). W.H. Freeman.
Holm, R. H., Kennepohl, P., & Solomon, E. I. (1996). Structural and functional aspects of
metal sites in biology. Chemical Reviews, 96(7), 2239-2314.
Steitz, T. A. (1999). DNA polymerases: structural diversity and common mechanisms.
Journal of Biological Chemistry, 274(25), 17395-17398.
Reece, J. B., Taylor, M. R., Simon, E. J., Dickey, J. L., & Hogan, K. A. (2011). Campbell
Biology (9th ed.). Benjamin Cummings.
Coleman, J. E. (1992). Zinc proteins: enzymes, storage proteins, transcription factors, and
replication proteins. Annual Review of Biochemistry, 61(1), 897-946.
Maret, W. (2013). Zinc biochemistry: from a single zinc enzyme to a key element of life.
Advances in Nutrition, 4(1), 82-91.
53
Ohtomo, R., Sekiguchi, Y., Mimura, T., Saito, M., & Ezawa, T. (2004). Quantification of
PolyPhosphate: different sensitivities to short-chain PolyPhosphate using enzymatic
and colorimetric methods as revealed by ion chromatography. Analytical
biochemistry, 328(2), 139-146.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature, 227(5259), 680-685.
Weber, K., & Osborn, M. (1969). The reliability of molecular weight determinations by
dodecyl sulfate-polyacrylamide gel electrophoresis. Journal of Biological Chemistry,
244(16), 4406-4412.
Shapiro, A. L., Vinuela, E., & Maizel, J. V. (1967). Molecular weight estimation of
PolyPeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochemical and
Biophysical Research Communications, 28(5), 815-820.
Walker, J. M. (2002). The protein protocols handbook. Springer Science & Business Media.
Hames, B. D. (1998). Gel electrophoresis of proteins: a practical approach. Oxford University
Press.
Good, N. E., et al. (1966). Hydrogen ion buffers for biological research. Biochemistry, 5(2),
467-477.
Gomori, G. (1955). Preparation of Buffers for Use in Enzyme Studies. Methods in
Enzymology, 1, 138-146.
Robinson, N. C., et al. (1980). The effect of HEPES-K buffer on the activity of membrane
bound enzymes. Journal of Biological Chemistry, 255(13), 5895-5897.
Vallee, B. L., & Auld, D. S. (1990). Zinc coordination, function, and structure of zinc
enzymes and other proteins. Biochemistry, 29(24), 5647-5659.
Berg, J. M., Tymoczko, J. L., & Stryer, L. (2002). Biochemistry (5th ed.). W H Freeman.
Nelson, D. L., & Cox, M. M. (2008). Lehninger Principles of Biochemistry (5th ed.). W H
Freeman.
54
Steitz, T. A. (1999). "DNA polymerases: structural diversity and common mechanisms." J
Biol Chem, 274(25), 17395-17398.
Good, N. E., et al. (1966). "Hydrogen ion buffers for biological research." Biochemistry, 5(2),
467-477.
Robinson, N. C., et al. (1980). "Metal ion interactions with HEPES-K buffer." Biochim
Biophys Acta, 630(3), 467-473.
Vallee, B. L., & Auld, D. S. (1990). "Zinc coordination, function, and structure of zinc
enzymes and other proteins." Biochemistry, 29(24), 5647-5659.
Good, N. E., Winget, G. D., Winter, W., Connolly, T. N., Izawa, S., & Singh, R. M. M.
(1966). Hydrogen ion buffers for biological research. Biochemistry, 5(2), 467-477.
Zhang, H., Ishige, K., & Kornberg, A. (2011). PolyPhosphate kinase (PPK2), a potent,
PolyPhosphate-driven generator of GTP. Proceedings of the National Academy of
Sciences, 99(26), 16670-16675.
Fox, I. H., & Kelley, W. N. (1971). The role of phosphoribosylpyrophosphate and the specific
activity of phosphoribosylpyrophosphate synthetase in human tissues. The Journal of
Biological Chemistry, 246(22), 5892-5895.
Ishige, K., Hamamoto, T., & Kurane, R. (2002). Enzymatic synthesis of ATP using
PolyPhosphate kinase. Journal of Bioscience and Bioengineering, 94(5), 389-392.
Revollo, J. R., Grimm, A. A., & Imai, S. (2004). The NAD biosynthesis pathway mediated by
nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells.
The Journal of Biological Chemistry, 279(49), 50754-50763.
Zhang, H., Ishige, K., & Kornberg, A. (2011). A PolyPhosphate kinase (PPK2) widely
conserved in bacteria. Proceedings of the National Academy of Sciences, 99(26),
16678-16683.

指導教授

徐敬衡(David Shu)

審核日期

2024-7-26

推文