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研究生： 簡雯棋
Chien, Wen-Chi
論文名稱： 利用定點突變4,5多巴-雙加氧酶探討其受質選擇性及動力學之影響
Effect of site-specific mutations on substrate selectivity and kinetics of 4,5-DOPA-dioxygenase
指導教授： 葉怡均
Yeh, Yi-Chun
口試委員： 徐駿森
Hsu, Chun-Hua
陳頌方
Chen, Sung-Fang
葉怡均
Yeh, Yi-Chun
口試日期： 2022/07/25
學位類別： 碩士
Master
系所名稱： 化學系
Department of Chemistry
論文出版年： 2022
畢業學年度： 110
語文別： 中文
論文頁數： 99
中文關鍵詞： 左旋多巴 、多巴胺 、4,5-多巴雙加氧酶 、蛋白質工程 、定點突變
英文關鍵詞： Dopamine, Levodopa, The kinetic of enzymes, Mutant protein, 4,5-dopa dioxygenase, Betalamic acid, Michaelis-Menten equation
DOI URL： http://doi.org/10.6345/NTNU202201309
論文種類： 學術論文
相關次數： 點閱：96  下載：9 
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    • 本研究主題就是在紫茉莉花中萃取的4,5-多巴雙加氧酶 (DOPA 4,5-dixoygenase, MjDOD)可催化左旋多巴 (Levodopa, L-DOPA)，但對多巴胺 (Dopamine, DA)也會產生相似的催化，然後運用不同位點突變胺基酸得其突變體，分別針對左旋多巴以及多巴胺進行酵素催化反應使其產生兩種不同的甜菜黃素 (Betaxanthin)，而這兩種不同的甜菜黃素在430 nm有最高吸收鋒，因此可以最終產物甜菜黃素在430 nm的吸收值與濃度作圖，再透過Michaelis–Menten equation得出最大反應速率 (Vmax) 和酵素親和力 (Km)。此外，4,5-多巴雙加氧酶中特定的氨基酸位置突變以改變對於基質的選擇性與活性，從中挑選出對於多巴胺或左旋多巴具有獨特專一性的4,5-多巴雙加氧酶突變體，分別有比起野生型對於L-DOPA更專一的F252Y突變體，還有對於DA有良好選擇性的雙點突變體N249D&F252Y。

    The 4,5-dopa dioxygenase purified from Mirabilis jalapa can catalyze levodopa (L-DOPA) and dopamine (DA) to produce two different betaxanthins (Betaxanthin). We thus performed site-directed mutagenesis to generate several mutants, and studied kinetics of mutated enzymes for L-DOPA and DA, respectively. In order to improve specificity, we changed the amino acids near the active sites to alter the hydrophilicity and steric barriers of the entire molecule, which results in changes in the binding ability to dopamine or L-DOPA. The four mutants that undergo site-directed mutagenesis were used for whole-cell bioassays. The detailed enzyme kinetics test was carried out. We totally build over 100 mutate strains, and using whole-cell biosensor for high-throughput screening. Finally, we use protein purification and Michaelis–Menten equation, successfully select the dopamine-specific mutation F252Y and the L-DOPA-specific double mutation N249D&F252Y.

    Chapter 1 Introduction 1 1.1 蛋白質工程(Protein Engineering) 1 1.1.1 原理 1 1.1.2 定位突變 (Site-directed mutagenesis) 2 1.1.3 定向演化 (Directed evolution) 4 1.2 兒茶酚胺類神經傳導物質 (Neurotransmitter of catecholamine) 7 1.2.1 簡介 7 1.2.2 酪氨酸羥化酶缺乏症 (Tyrosine Hydroxylase Deficiency) 10 1.3 甜菜醛胺酸 (Betalamic acid)與甜菜色素(Betalains) 12 1.3.1 甜菜醛胺酸的合成 12 1.3.2 甜菜色素 13 1.4 4,5-多巴外雙加氧酶 (4,5-DOPA extradiol dioxygenase) 15 1.5 細胞表面表現 (Cell surface display) 17 1.5.1 簡介 17 1.5.2 載體蛋白Carrier protein (anchoring motif) 17 1.5.3 目標蛋白Passenger protein (target protein) 18 1.5.4 宿主菌株 (Host strain) 19 1.6 文獻回顧與探討 (Literature Review) 20 1.6.1 多巴胺檢測技術 20 1.6.2 左旋多巴檢測技術 23 1.6.3 細胞表面表現技術 24 1.7 實驗動機與目的 25 Chapter 2 Materials and Experimental Methods 28 2.1 實驗儀器 28 2.2 實驗藥品 29 2.3 實驗設計 31 2.3.1 突變蛋白質對於多巴胺和左旋多巴的活化效率 31 2.3.2 結合細胞表面表現技術優化突變蛋白質 31 2.4 實驗方法 33 2.4.1 蛋白質表達、純化與定量濃縮實驗流程 33 2.4.2 MjDOD突變蛋白表達之質體設計 33 2.4.3 勝任細胞的製作步驟 34 2.4.4 轉化作用 (Transformation) 35 2.4.5 4,5-多巴雙加氧酶之大量表達及純化 36 2.4.6 鎳樹脂再生 39 2.4.7 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 (SDS-PAGE, Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) 40 2.4.8 定量蛋白質濃度 43 2.4.9 酵素動力學 (The kinetics of enzymes) 44 Chapter 3 Results and Discussions 46 3.1 多巴雙加氧酶對兒茶酚化合物檢測 46 3.2 MjDOD突變蛋白之活性篩選 47 3.2.1 突變胺基酸之挑選 47 3.2.2 單點突變全細胞生物檢測 48 3.3 單點突變蛋白對於左旋多巴及多巴胺檢測 50 3.3.1 紫茉莉4,5-多巴雙加氧酶之純化 50 3.3.2 D11位點突變對於左旋多巴以及多巴胺酵素動力學檢測 51 3.3.3 F252位點突變對於左旋多巴以及多巴胺酵素動力學檢測 55 3.3.4 N249位點突變對於左旋多巴以及多巴胺酵素動力學檢測 59 3.3.5 Y254位點突變對於左旋多巴以及多巴胺酵素動力學檢測 63 3.4 雙點突變蛋白對於左旋多巴及多巴胺檢測 66 3.4.1 雙點突變全細胞檢測 66 3.4.2 雙點突變蛋白之酵素動力學檢測 67 3.5 專一性測試 69 3.5.1 突變全細胞生物檢測之干擾檢測 69 3.5.2 純化突變蛋白之干擾檢測 70 3.6 突變蛋白對於多巴胺和左旋多巴胺檢量線 71 3.6.1 突變全細胞線性檢測 71 3.6.2 純化突變蛋白線性檢測 73 3.7 建立穿膜蛋白的基因工程 75 3.7.1 設計Ag43蛋白之基因工程 75 3.7.2 設計IgA蛋白之基因工程 76 3.7.3 設計eCPX蛋白之基因工程 78 Chapter 4 Conclusions 79 REFERENCE 80 附錄 純化蛋白質之低濃度酵素動力學圖 85 附錄 所有純化蛋白質之SDS-Page膠圖 91 附錄 本研究中所使用之引子與菌種 96

    1. Site-Directed Mutagenesis.
    2. del Prado, A.; Villar, L.; de Vega, M.; Salas, M., Involvement of residues of the ϕ29 terminal protein intermediate and priming domains in the formation of a stable and functional heterodimer with the replicative DNA polymerase. Nucleic acids research 2012, 40 (9), 3886-3897.
    3. Dale, H. H., Otto Loewi, 1873-1961. The Royal Society London: 1962.
    4. Loewi, O., Chemical transmission of nerve impulses. American Scientist 1945, 33 (3), 159-174.
    5. Daubner, S. C.; Le, T.; Wang, S., Tyrosine hydroxylase and regulation of dopamine synthesis. Archives of biochemistry and biophysics 2011, 508 (1), 1-12.
    6. Kuhar, M. J.; Couceyro, P. R.; Lambert, P. D., Catecholamines. Basic neurochemistry: Molecular, cellular and medical aspects 1999, 243-262.
    7. McGeer, P.; McGeer, E. J. J. o. n., Enzymes associated with the metabolism of catecholamines, acetylcholine and GABA in human controls and patients with Parkinson's disease and Huntington's chorea. 1976, 26 (1), 65-76.
    8. Haavik, J.; Toska, K., Tyrosine hydroxylase and Parkinson's disease. Molecular neurobiology 1998, 16 (3), 285-309.
    9. Brautigam, C.; Wevers, R. A.; Jansen, R. J.; Smeitink, J. A.; Andel, J. F. d. R.-v.; Gabreëls, F. J.; Hoffmann, G. F., Biochemical hallmarks of tyrosine hydroxylase deficiency. Clinical chemistry 1998, 44 (9), 1897-1904.
    10. Lüdecke, B.; Dworniczak, B.; Bartholomé, K., A point mutation in the tyrosine hydroxylase gene associated with Segawa's syndrome. Human genetics 1995, 95 (1), 123-125.
    11. Willemsen, M. A.; Verbeek, M. M.; Kamsteeg, E.-J.; de Rijk-van Andel, J. F.; Aeby, A.; Blau, N.; Burlina, A.; Donati, M. A.; Geurtz, B.; Grattan-Smith, P. J., Tyrosine hydroxylase deficiency: a treatable disorder of brain catecholamine biosynthesis. Brain 2010, 133 (6), 1810-1822.
    12. Hou, Y.; Liu, X.; Li, S.; Zhang, X.; Yu, S.; Zhao, G.-R., Metabolic engineering of Escherichia coli for de novo production of betaxanthins. Journal of Agricultural and Food Chemistry 2020, 68 (31), 8370-8380.
    13. Liveri, M. L. T.; Sciascia, L.; Lombardo, R.; Tesoriere, L.; Passante, E.; Livrea, M. A., Spectrophotometric evidence for the solubilization site of betalain pigments in membrane biomimetic systems. Journal of agricultural and food chemistry 2007, 55 (8), 2836-2840.
    14. Wang, M.; Lopez-Nieves, S.; Goldman, I. L.; Maeda, H. A., Limited tyrosine utilization explains lower betalain contents in yellow than in red table beet genotypes. Journal of Agricultural and Food Chemistry 2017, 65 (21), 4305-4313.
    15. Kovaleva, E. G.; Lipscomb, J. D., Versatility of biological non-heme Fe (II) centers in oxygen activation reactions. Nature chemical biology 2008, 4 (3), 186-193.
    16. Nakatsuka, T.; Yamada, E.; Takahashi, H.; Imamura, T.; Suzuki, M.; Ozeki, Y.; Tsujimura, I.; Saito, M.; Sakamoto, Y.; Sasaki, N., Genetic engineering of yellow betalain pigments beyond the species barrier. Scientific reports 2013, 3 (1), 1-7.
    17. Sasaki, N.; Abe, Y.; Goda, Y.; Adachi, T.; Kasahara, K.; Ozeki, Y., Detection of DOPA 4, 5-dioxygenase (DOD) activity using recombinant protein prepared from Escherichia coli cells harboring cDNA encoding DOD from Mirabilis jalapa. Plant and cell physiology 2009, 50 (5), 1012-1016.
    18. Gandía-Herrero, F.; García-Carmona, F., Escherichia coli protein YgiD produces the structural unit of plant pigments betalains: characterization of a prokaryotic enzyme with DOPA-extradiol-dioxygenase activity. Applied microbiology and biotechnology 2014, 98 (3), 1165-1174.
    19. Wang, Y.; Shin, I.; Fu, Y.; Colabroy, K. L.; Liu, A., Crystal Structures of L-DOPA Dioxygenase from Streptomyces sclerotialus. Biochemistry 2019, 58 (52), 5339-5350.
    20. Walker, D. J.; Martz, E.; Holmes, D. E.; Zhou, Z.; Nonnenmann, S. S.; Lovley, D. R., The archaellum of Methanospirillum hungatei is electrically conductive. MBio 2019, 10 (2).
    21. Stentebjerg-Olesen, B.; Pallesen, L.; Jensen, L. B.; Christiansen, G.; Klemm, P., Authentic display of a cholera toxin epitope by chimeric type 1 fimbriae: effects of insert position and host background. Microbiology 1997, 143 (6), 2027-2038.
    22. Bingle, W. H.; Nomellini, J. F.; Smit, J., Cell‐surface display of a Pseudomonas aeruginosa strain K pilin peptide within the paracrystalline S‐layer of Caulobacter crescentus. Molecular microbiology 1997, 26 (2), 277-288.
    23. Xu, Z.; Lee, S. Y., Display of polyhistidine peptides on the Escherichia coli cell surface by using outer membrane protein C as an anchoring motif. Applied and Environmental Microbiology 1999, 65 (11), 5142-5147.
    24. Suzuki, T.; Lett, M.-C.; Sasakawa, C., Extracellular Transport of VirG Protein in Shigella (∗). Journal of Biological Chemistry 1995, 270 (52), 30874-30880.
    25. Stathopoulos, C.; Georgiou, G.; Earhart, C., Characterization of Escherichia coli expressing an Lpp’OmpA (46-159)-PhoA fusion protein localized in the outer membrane. Applied microbiology and biotechnology 1996, 45 (1), 112-119.
    26. Nguyen, T. N.; Gourdon, M.-H.; Hansson, M.; Robert, A.; Samuelson, P.; Libon, C.; Andréoni, C.; Nygren, P.-Å.; Binz, H.; Uhlén, M., Hydrophobicity engineering to facilitate surface display of heterologous gene products on Staphylococcus xylosus. Journal of biotechnology 1995, 42 (3), 207-219.
    27. Lee, S. Y.; Choi, J. H.; Xu, Z., Microbial cell-surface display. Trends in Biotechnology 2003, 21 (1), 45-52.
    28. Feng, J.-J.; Guo, H.; Li, Y.-F.; Wang, Y.-H.; Chen, W.-Y.; Wang, A.-J., Single molecular functionalized gold nanoparticles for hydrogen-bonding recognition and colorimetric detection of dopamine with high sensitivity and selectivity. ACS applied materials & interfaces 2013, 5 (4), 1226-1231.
    29. Huang, Q.; Lin, X.; Tong, L.; Tong, Q.-X., Graphene quantum dots/multiwalled carbon nanotubes composite-based electrochemical sensor for detecting dopamine release from living cells. ACS Sustainable Chemistry & Engineering 2020, 8 (3), 1644-1650.
    30. Lin, J.; Huang, B.; Dai, Y.; Wei, J.; Chen, Y., Chiral ZnO nanoparticles for detection of dopamine. Materials Science and Engineering: C 2018, 93, 739-745.
    31. Goud, K. Y.; Moonla, C.; Mishra, R. K.; Yu, C.; Narayan, R.; Litvan, I.; Wang, J., Wearable electrochemical microneedle sensor for continuous monitoring of levodopa: toward Parkinson management. ACS sensors 2019, 4 (8), 2196-2204.
    32. Ahan, R. E.; Kırpat, B. M.; Saltepe, B.; Şeker, U. Ö. Ş., A Self-Actuated Cellular Protein Delivery Machine. ACS Synthetic Biology 2019, 8 (4), 686-696.
    33. Chou, Y.-C., Functional and structural studies of a 4,5-DOPA dioxygenase involved in betalain pigment biosynthesis from Mirabilis jalapa. 2017.
    34. Dong, H.; Sarkes, D. A.; Rice, J. J.; Hurley, M. M.; Fu, A. J.; Stratis-Cullum, D. N., Living Bacteria–Nanoparticle Hybrids Mediated through Surface-Displayed Peptides. Langmuir 2018, 34 (20), 5837-5848.
    35. Chou, Y.-C.; Shih, C.-I.; Chiang, C.-C.; Hsu, C.-H.; Yeh, Y.-C., Reagent-free DOPA-dioxygenase colorimetric biosensor for selective detection of L-DOPA. Sensors and Actuators B: Chemical 2019, 297, 126717.
    36. Kumar, A.; Maity, H.; Dua, A., Parallel versus off-pathway Michaelis–Menten mechanism for single-enzyme kinetics of a fluctuating enzyme. The Journal of Physical Chemistry B 2015, 119 (27), 8490-8500.
    37. Wang, I.-H., Using protein engineering to modify 4,5-DOPA dioxygenase form Mirabilis jalapa to detect dopamine and L-DOPA. 2020.
    1. Site-Directed Mutagenesis.
    2. del Prado, A.; Villar, L.; de Vega, M.; Salas, M., Involvement of residues of the ϕ29 terminal protein intermediate and priming domains in the formation of a stable and functional heterodimer with the replicative DNA polymerase. Nucleic acids research 2012, 40 (9), 3886-3897.
    3. Dale, H. H., Otto Loewi, 1873-1961. The Royal Society London: 1962.
    4. Loewi, O., Chemical transmission of nerve impulses. American Scientist 1945, 33 (3), 159-174.
    5. Daubner, S. C.; Le, T.; Wang, S., Tyrosine hydroxylase and regulation of dopamine synthesis. Archives of biochemistry and biophysics 2011, 508 (1), 1-12.
    6. Kuhar, M. J.; Couceyro, P. R.; Lambert, P. D., Catecholamines. Basic neurochemistry: Molecular, cellular and medical aspects 1999, 243-262.
    7. McGeer, P.; McGeer, E. J. J. o. n., Enzymes associated with the metabolism of catecholamines, acetylcholine and GABA in human controls and patients with Parkinson's disease and Huntington's chorea. 1976, 26 (1), 65-76.
    8. Haavik, J.; Toska, K., Tyrosine hydroxylase and Parkinson's disease. Molecular neurobiology 1998, 16 (3), 285-309.
    9. Brautigam, C.; Wevers, R. A.; Jansen, R. J.; Smeitink, J. A.; Andel, J. F. d. R.-v.; Gabreëls, F. J.; Hoffmann, G. F., Biochemical hallmarks of tyrosine hydroxylase deficiency. Clinical chemistry 1998, 44 (9), 1897-1904.
    10. Lüdecke, B.; Dworniczak, B.; Bartholomé, K., A point mutation in the tyrosine hydroxylase gene associated with Segawa's syndrome. Human genetics 1995, 95 (1), 123-125.
    11. Willemsen, M. A.; Verbeek, M. M.; Kamsteeg, E.-J.; de Rijk-van Andel, J. F.; Aeby, A.; Blau, N.; Burlina, A.; Donati, M. A.; Geurtz, B.; Grattan-Smith, P. J., Tyrosine hydroxylase deficiency: a treatable disorder of brain catecholamine biosynthesis. Brain 2010, 133 (6), 1810-1822.
    12. Hou, Y.; Liu, X.; Li, S.; Zhang, X.; Yu, S.; Zhao, G.-R., Metabolic engineering of Escherichia coli for de novo production of betaxanthins. Journal of Agricultural and Food Chemistry 2020, 68 (31), 8370-8380.
    13. Liveri, M. L. T.; Sciascia, L.; Lombardo, R.; Tesoriere, L.; Passante, E.; Livrea, M. A., Spectrophotometric evidence for the solubilization site of betalain pigments in membrane biomimetic systems. Journal of agricultural and food chemistry 2007, 55 (8), 2836-2840.
    14. Wang, M.; Lopez-Nieves, S.; Goldman, I. L.; Maeda, H. A., Limited tyrosine utilization explains lower betalain contents in yellow than in red table beet genotypes. Journal of Agricultural and Food Chemistry 2017, 65 (21), 4305-4313.
    15. Kovaleva, E. G.; Lipscomb, J. D., Versatility of biological non-heme Fe (II) centers in oxygen activation reactions. Nature chemical biology 2008, 4 (3), 186-193.
    16. Nakatsuka, T.; Yamada, E.; Takahashi, H.; Imamura, T.; Suzuki, M.; Ozeki, Y.; Tsujimura, I.; Saito, M.; Sakamoto, Y.; Sasaki, N., Genetic engineering of yellow betalain pigments beyond the species barrier. Scientific reports 2013, 3 (1), 1-7.
    17. Sasaki, N.; Abe, Y.; Goda, Y.; Adachi, T.; Kasahara, K.; Ozeki, Y., Detection of DOPA 4, 5-dioxygenase (DOD) activity using recombinant protein prepared from Escherichia coli cells harboring cDNA encoding DOD from Mirabilis jalapa. Plant and cell physiology 2009, 50 (5), 1012-1016.
    18. Gandía-Herrero, F.; García-Carmona, F., Escherichia coli protein YgiD produces the structural unit of plant pigments betalains: characterization of a prokaryotic enzyme with DOPA-extradiol-dioxygenase activity. Applied microbiology and biotechnology 2014, 98 (3), 1165-1174.
    19. Wang, Y.; Shin, I.; Fu, Y.; Colabroy, K. L.; Liu, A., Crystal Structures of L-DOPA Dioxygenase from Streptomyces sclerotialus. Biochemistry 2019, 58 (52), 5339-5350.
    20. Walker, D. J.; Martz, E.; Holmes, D. E.; Zhou, Z.; Nonnenmann, S. S.; Lovley, D. R., The archaellum of Methanospirillum hungatei is electrically conductive. MBio 2019, 10 (2).
    21. Stentebjerg-Olesen, B.; Pallesen, L.; Jensen, L. B.; Christiansen, G.; Klemm, P., Authentic display of a cholera toxin epitope by chimeric type 1 fimbriae: effects of insert position and host background. Microbiology 1997, 143 (6), 2027-2038.
    22. Bingle, W. H.; Nomellini, J. F.; Smit, J., Cell‐surface display of a Pseudomonas aeruginosa strain K pilin peptide within the paracrystalline S‐layer of Caulobacter crescentus. Molecular microbiology 1997, 26 (2), 277-288.
    23. Xu, Z.; Lee, S. Y., Display of polyhistidine peptides on the Escherichia coli cell surface by using outer membrane protein C as an anchoring motif. Applied and Environmental Microbiology 1999, 65 (11), 5142-5147.
    24. Suzuki, T.; Lett, M.-C.; Sasakawa, C., Extracellular Transport of VirG Protein in Shigella (∗). Journal of Biological Chemistry 1995, 270 (52), 30874-30880.
    25. Stathopoulos, C.; Georgiou, G.; Earhart, C., Characterization of Escherichia coli expressing an Lpp’OmpA (46-159)-PhoA fusion protein localized in the outer membrane. Applied microbiology and biotechnology 1996, 45 (1), 112-119.
    26. Nguyen, T. N.; Gourdon, M.-H.; Hansson, M.; Robert, A.; Samuelson, P.; Libon, C.; Andréoni, C.; Nygren, P.-Å.; Binz, H.; Uhlén, M., Hydrophobicity engineering to facilitate surface display of heterologous gene products on Staphylococcus xylosus. Journal of biotechnology 1995, 42 (3), 207-219.
    27. Lee, S. Y.; Choi, J. H.; Xu, Z., Microbial cell-surface display. Trends in Biotechnology 2003, 21 (1), 45-52.
    28. Feng, J.-J.; Guo, H.; Li, Y.-F.; Wang, Y.-H.; Chen, W.-Y.; Wang, A.-J., Single molecular functionalized gold nanoparticles for hydrogen-bonding recognition and colorimetric detection of dopamine with high sensitivity and selectivity. ACS applied materials & interfaces 2013, 5 (4), 1226-1231.
    29. Huang, Q.; Lin, X.; Tong, L.; Tong, Q.-X., Graphene quantum dots/multiwalled carbon nanotubes composite-based electrochemical sensor for detecting dopamine release from living cells. ACS Sustainable Chemistry & Engineering 2020, 8 (3), 1644-1650.
    30. Lin, J.; Huang, B.; Dai, Y.; Wei, J.; Chen, Y., Chiral ZnO nanoparticles for detection of dopamine. Materials Science and Engineering: C 2018, 93, 739-745.
    31. Goud, K. Y.; Moonla, C.; Mishra, R. K.; Yu, C.; Narayan, R.; Litvan, I.; Wang, J., Wearable electrochemical microneedle sensor for continuous monitoring of levodopa: toward Parkinson management. ACS sensors 2019, 4 (8), 2196-2204.
    32. Ahan, R. E.; Kırpat, B. M.; Saltepe, B.; Şeker, U. Ö. Ş., A Self-Actuated Cellular Protein Delivery Machine. ACS Synthetic Biology 2019, 8 (4), 686-696.
    33. Chou, Y.-C., Functional and structural studies of a 4,5-DOPA dioxygenase involved in betalain pigment biosynthesis from Mirabilis jalapa. 2017.
    34. Dong, H.; Sarkes, D. A.; Rice, J. J.; Hurley, M. M.; Fu, A. J.; Stratis-Cullum, D. N., Living Bacteria–Nanoparticle Hybrids Mediated through Surface-Displayed Peptides. Langmuir 2018, 34 (20), 5837-5848.
    35. Chou, Y.-C.; Shih, C.-I.; Chiang, C.-C.; Hsu, C.-H.; Yeh, Y.-C., Reagent-free DOPA-dioxygenase colorimetric biosensor for selective detection of L-DOPA. Sensors and Actuators B: Chemical 2019, 297, 126717.
    36. Kumar, A.; Maity, H.; Dua, A., Parallel versus off-pathway Michaelis–Menten mechanism for single-enzyme kinetics of a fluctuating enzyme. The Journal of Physical Chemistry B 2015, 119 (27), 8490-8500.
    37. Wang, I.-H., Using protein engineering to modify 4,5-DOPA dioxygenase form Mirabilis jalapa to detect dopamine and L-DOPA. 2020.

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