一鍋法完全酵素生成黃素腺嘌呤二核?酸的研究及第一個PLP依賴性二氨基丙酸消旋?的分子表徵

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高橋拓實(Takumi Takahashi) 查詢紙本館藏

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論文名稱

一鍋法完全酵素生成黃素腺嘌呤二核?酸的研究及第一個PLP依賴性二氨基丙酸消旋?的分子表徵
(Studies on the one-pot complete enzymatic generation of flavin adenine dinucleotide and the molecular characterization of the first PLP-dependent diaminopropionic acid racemase)

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摘要(中)

黃素腺嘌呤二核?酸 (FAD) 是許多生物氧化還原和自由基反應中的關鍵輔助因子。傳統上，核黃素的 FAD 生物合成遵循兩步驟?途徑，需要 ATP，以黃素單核?酸 (FMN) 為中間體。工業規模的 FAD 生產傳統上依賴微生物發酵，但這些方法通常需要繁瑣的淨化步驟。考慮到原子經濟性和產量效率的重要性，酵素體外方法提供了一種更可持續和有效的選擇。然而，由於依賴昂貴的 ATP 作為底物，?促 FAD 合成的廣泛工業應用受到阻礙。為了解決這個限制，我開發了一種雙?級聯繫統，利用來自廢水微藻的聚磷酸鹽作為磷酸鹽供體，從腺?中再生 ATP。此再生機制透過雙功能核黃素激?/FAD合成?和焦磷酸?的共同作用，使核黃素在2小時內完全轉化為FAD，最終濃度達到約1.2g/L（1.5mmol/L）。值得注意的是，唯一的副產品正磷酸鹽可以透過廢水微藻回收來再生聚磷酸鹽，進而可以作為磷酸鹽供體重新融入系統。這種閉環策略有利於實現環境永續的 FAD 合成過程，同時最大限度地減少廢棄物產生。

摘要(英)

Flavin adenine dinucleotide (FAD) serves as a crucial cofactor in numerous biological redox
and radical reactions. Conventionally, FAD biosynthesis from riboflavin follows a two-step
enzymatic pathway requiring ATP, with flavin mononucleotide (FMN) as the intermediate.
Industrial-scale FAD production has traditionally relied on microbial fermentation, but these
methods often entail laborious purification steps. Given the importance of atomic economy and
yield efficiency, an enzymatic in vitro approach presents a more sustainable and effective
alternative. However, the widespread industrial application of enzymatic FAD synthesis is
hindered by its dependence on costly ATP as a substrate. To address this limitation, I have
developed a two-enzyme cascade system that regenerates ATP from adenosine, utilizing
polyphosphate derived from wastewater microalgae as a phosphate donor. This regeneration
mechanism enables the complete transformation of riboflavin into FAD within 2 h, achieving
a final concentration of approximately 1.2 g/L (1.5 mmol/L) through the combined action of
bifunctional riboflavin kinase/FAD synthetase and pyrophosphatase. Notably, the only
byproduct, orthophosphate, can be recycled by wastewater microalgae to regenerate
polyphosphate, which in turn can be reintegrated into the system as a phosphate donor. This
closed-loop strategy facilitates an environmentally sustainable FAD synthesis process with
minimal waste production.

關鍵字(中)

★ 黃素腺嘌呤二核?酸（FAD）
★ ?催化合成
★ 永續生物催化
★ 核黃素激?
★ PLP 依賴型消旋?
★ 綠色化學
★ 生化工程

關鍵字(英)

★ Flavin adenine dinucleotide (FAD)
★ Enzymatic synthesis
★ Sustainable biocatalysis
★ Riboflavin kinase
★ PLP-dependent racemase
★ Green chemistry
★ Biochemical engineering

論文目次

PartI
Abstract ................................................................................................................................... I
Acknowledgements ............................................................................................................... II
Table of Contents .................................................................................................................. III
List of Figures ....................................................................................................................... IV
List of Tables ......................................................................................................................... V
Explanation of Symbols and Abbreviations ......................................................................... VI
1 Introduction ......................................................................................................................... 1
2 Results and Discussion ........................................................................................................ 6
2-1 Biocatalytic ATP synthesis from adenosine using wastewater microalgal polyP ......... 6
2-2 One-pot enzymatic FAD synthesis from riboflavin and adenosine ............................... 9
2-3 Efficiency analysis based on green metrics ................................................................. 14
3 Conclusions ....................................................................................................................... 16
4 Materials and Methods ...................................................................................................... 17
4-1 Heterologous expression of recombinant proteins ...................................................... 17
4-2 Sodium dodecyl sulfate–polyacrylamide gel electrophoresis ..................................... 17
4-3 Microalgae cultivation ................................................................................................ 18
4-4 Quantification of polyP using the toluidine blue O (TBO) method ............................ 18
4-5 Chlorella vulgaris cell lysis and polyP purification .................................................... 20
4-6 ATP regeneration using heterologous microalgal polyP ............................................. 20
4-7 Electrophoretic analysis of polyp using TBO-stained TBE-Urea polyacrylamide gel
electrophoresis ............................................................................................................ 22
4-8 Enzymatic synthesis of FAD from adenosine, riboflavin, and microalgal polyP ....... 22
4-9 Enzymatic cascade for NADH-dependent FAD reduction ......................................... 22
4-10 High-performance liquid chromatography ................................................................ 23
5 References ......................................................................................................................... 24
6 Appendix ............................................................................................................................ 27

PartII
Abstract ................................................................................................................................... I
Acknowledgements ............................................................................................................... II
Table of Contents .................................................................................................................. III
List of Figures ....................................................................................................................... IV
List of Tables ......................................................................................................................... V
Explanation of Symbols and Abbreviations ......................................................................... VI
1 Introduction ......................................................................................................................... 1
2 Results ................................................................................................................................. 7
2-1 Expression, purification, and physicochemical characterization of Lf-Orf6 ................. 7
2-2 Quantitative evaluation of the stereoinversion of Dap catalyzed by Lf-Orf6 ............... 9
2-3 Cofactor dependence of the stereoinversion of Dap catalyzed by Lf-Orf6 ................. 11
2-4 Biochemical characterization of recombinant Lf-Orf6 as Dap racemase .................... 13
2-5 Phylogenetic analysis of PLP-dependent DapR .......................................................... 20
2-6 Structural prediction of Lf-Orf6 .................................................................................. 27
3 Discussion .......................................................................................................................... 32
4 Materials and methods ....................................................................................................... 40
4-1 Plasmids, bacterial strains, and growth conditions ..................................................... 40
4-2 Expression and purification of recombinant Lf-Orf6 and Bs-ThrC ............................ 40
4-3 Physicochemical characterization of Lf-Orf6 .............................................................. 41
4-4 Racemase assays ......................................................................................................... 42
4-5 Threonine synthase assay ............................................................................................ 43
4-6 In silico analyses including phylogenetic tree analysis and molecular modeling ....... 43
5 References ......................................................................................................................... 44

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Normal and Tumor Stem Cells. Adv. Cancer Res. 2014, 122, 1?67.
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Metabolites Control Physiology and Disease. Nat. Commun. 2020, 11
(1), 1?11.
(3) Nolfi-Donegan, D.; Braganza, A.; Shiva, S. Mitochondrial
Electron Transport Chain: Oxidative Phosphorylation, Oxidant
Production, and Methods of Measurement. Redox Biol. 2020, 37,
101674.
(4) Heine, T.; van Berkel, W. J. H.; Gassner, G.; van Pee, K.-H.;
Tischler, D. Two-Component FAD-Dependent Monooxygenases:
Current Knowledge and Biotechnological Opportunities. Biology
2018, 7 (3), 42.
(5) Mansoorabadi, S. O.; Thibodeaux, C. J.; Liu, H.-W. The Diverse
Roles of Flavin Coenzymes?Nature’s Most Versatile Thespians. J.
Org. Chem. 2007, 72 (17), 6329?6342.
(6) Adeva-Andany, M. M.; Carneiro-Freire, N.; Seco-Filgueira, M.;
Fernandez-Fernandez, C.; Mourin?o-Bayolo, D. Mitochondrial β Oxidation of Saturated Fatty Acids in Humans. Mitochondrion 2019,
46, 73?90.
(7) Kim, J.-J. P.; Miura, R. Acyl-CoA Dehydrogenases and Acyl-CoA
Oxidases. Structural Basis for Mechanistic Similarities and Differ ences. Eur. J. Biochem. 2004, 271 (3), 483?493.
(8) Kavakli, I. H.; Ozturk, N.; Gul, S. DNA Repair by Photolyases.
In Advances in Protein Chemistry and Structural Biology; Academic
Press, 2019; Vol. 115, pp 1?19. DOI: 10.1016/bs.apcsb.2018.10.003.
(9) Liu, S.; Hu, W.; Wang, Z.; Chen, T. Production of Riboflavin
and Related Cofactors by Biotechnological Processes. Microb. Cell
Fact. 2020, 19 (1), 31.
(10) Giancaspero, T. A.; Colella, M.; Brizio, C.; Difonzo, G.;
Fiorino, G. M.; Leone, P.; Brandsch, R.; Bonomi, F.; Iametti, S.;
Barile, M. Remaining Challenges in Cellular Flavin Cofactor
Homeostasis and Flavoprotein Biogenesis. Front Chem. 2015, 3, 30.
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Flavine-Adenine Dinucleotide from Riboflavine by a Mutant of
Sarcina Lutea. Appl. Microbiol. 1974, 27 (3), 531?536.
(12) Hagihara, T.; Fujio, T.; Aisaka, K. Cloning of FAD Synthetase
Gene from Corynebacterium Ammoniagenes and Its Application to
FAD and FMN Production. Appl. Microbiol. Biotechnol. 1995, 42 (5),
724?729.
(13) Yatsyshyn, V. Y.; Fedorovych, D. V.; Sibirny, A. A. Metabolic
and Bioprocess Engineering of the Yeast Candida Famata for FAD
Production. J. Ind. Microbiol. Biotechnol. 2014, 41 (5), 823?835.
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Modular Engineering of the Flavin Pathway in Escherichia Coli for
Improved Flavin Mononucleotide and Flavin Adenine Dinucleotide
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Regulation Strategy for Microalgae-Based Sugar Industry Wastewater
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Long-Chain Polyphosphates from Microalgal Biomass. Green Chem.
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指導教授

王柏翔(Po-hsiang Wang)

審核日期

2025-3-26

推文