臺灣博碩士論文加值系統

近年來全球糖尿病患者人數不斷上升，且糖尿病往往會造成重大傷害，因此，對於糖尿病患者之長期血糖監控是非常重要的，HbA1c為葡萄糖接上血紅素β鏈N-端Valine之胺基之產物，能夠反映患者過去二至三個月的平均血糖值。本論文利用糖化胜肽氧化酶(Fructosyl peptide oxidase, FPO)氧化HbA1c之模擬基質Fructosyl valine (FV)產生H2O2，並以辣根過氧化酶(Horseradish peroxidase, HRP)催化呈色劑，利用顏色深淺分析模擬基質FV之濃度。由大腸桿菌生產之可溶性蛋白質含量低且不穩定，將纖維素結合功能域 (CBD)與His-tag融合於FPO上可以改善可溶性不佳的缺點，並使氧化酵素更具應用潛力。然而CBD-FPO-6xHis酵素本身穩定性不佳且反應後回收不易，因此本研究以兩種載體進行融合蛋白CBD-FPO-6xHis酵素固定化，以提高酵素之催化穩定性及重複使用性。
第一種方式為利用包埋法固定化FPO及HRP於鋅離子-腺嘌呤金屬配位凝膠，利用凝膠中鋅離子殘基與FPO酵素之6xHis-tag鍵結，提高CBD-FPO-6xHis包埋效率，同時FPO、HRP雙酵素凝膠之比活性相較於FPO單一酵素凝膠提高了12倍。在固定化於此凝膠後在pH=5-9之間皆能維持80%以上活性，且雙酵素凝膠於4℃下保存一周仍有80%相對活性。由酵素動力學得到自由態CBD-FPO-6xHis及雙酵素凝膠之Vmax/Km分別為0.368及0.029 min-1。
奈米結晶纖維素(Cellulose nanocrystal, CNC)常被作為酵素載體使用，但因CNC在溶液中的高分散性而限制其應用。本研究在FPO之第二種固定化方式是透過靜電作用力以PEI改質之磁性海藻酸鈣凝膠吸附CNC，接著利用融合酵素之纖維素結合功能域(Cellulose binding domain, CBD)與纖維之間的親和力使CBD-FPO-6xHis吸附於具CNC之PEI改質磁性海藻酸鈣凝膠上，結果顯示CBD-FPO-6xHis吸附於具CNC凝膠相較於無CNC之凝膠能提高4.3倍的吸附量，同時比活性也提高12倍。重複使用性方面在使用三次後活性降低至40%，酵素再生後能夠回到原有活性之80%。

In recent years, the amount of global diabetic patients increases. Diabetes can cause significant harm to human body, therefore it is very important to monitor blood sugar in diabetic patient. HbA1c is a product of glucose attached to the amino group of the N-terminal of hemoglobin β chain, which reflects long-term glycemic status of diabetic patients. In this study, fructosyl valine (FV), a simulated fragment of HbA1c will be oxidized by fructosyl peptide oxidase (FPO), that produces hydrogen peroxide (H2O2), and furthermore, color change will be developed when dye was catalyzed by horseradish peroxidase (HRP). The intensity of color can reflect FV concentration that related to HbA1c concentration. Most of this mutant FPO expressed in Escherichia coli existed as inclusion bodies. To improve the solubility and application potential of FPO, Cellulose binding domain (CBD) and His-tag was fused to generate a soluble CBD-FPOX-6xHis fusion protein. However, due to CBD-FPO-6xHis low enzyme stability and reuse efficiency, in this study, we developed two FPO immobilization methods to improve its stability and reusability.
First method is to entrap CBD-FPO-6xHis in Zn-AMP metal-coordination superamolecular gel. Residual Zn ion on the Zn-AMP gel binds with 6xHis-tag protein could improve CBD-FPO-6xHis immobilization efficiency. The FPO and HRP immobilized at same time in Zn-AMP gel enhanced its FPO activity approximately 12 fold. The FPO activity could be stably maintained at pH5-9. The Vmax/Km of the free CBD-FPO-6xHis and the multiple enzyme composite gel were 0.368 and 0.029 min-1, respectively.
Cellulose nanocrystal (CNC) is very useful carrier for enzymes immobilized. However, the CNCs are difficult to recycle from the reaction system due to its nano size. In this work, we described the CNC magnetic colloid as an enzyme carrier using polyethylenimine (PEI) modified magnetic colloid to adsorb CNC through electronic interaction. By taking advantage of CBD's strong affinity toward crystalline cellulose, CBD-FPO-6xHis can be immobilized on the CNC magnetic colloid, compared with non-CNC colloid, the adsorption amount was increased by 4.3 fold, and the specific activity also increased. The activity is reduced to 40% after 3 repeated uses, and the enzyme activity can be recharged to 80% of the original activity after regeneration.

摘要 I
誌謝 III
目錄 IV
圖索引 IX
表索引 XII
第一章 緒論 1
1.1 前言 1
1.2 研究目的及內容簡介 2
第二章 文獻回顧 4
2.1 糖化血紅素(Glycated hemoglobin)的來源與組成 4
2.1.1 HbA1c 6
2.2 糖化血紅素的測定 7
2.3 FPOX(fructosyl peptide oxidase)氧化酵素簡介 8
2.3.1 FAOX(fructosyl amino acid oxidase) 9
2.3.2 FPOX(fructosyl peptide oxidase) 9
2.3.3 纖維素結合功能域(cellulose binding domain) 13
2.4 酵素固定化 16
2.4.1 擔體鍵結法 16
2.4.2 交聯法 16
2.4.3 包埋法 17
2.5 磁性顆粒 17
2.5.1 磁性載體 17
2.5.2 磁性顆粒製備方法 18
2.5.3 磁性顆粒表面修飾 19
2.6 奈米結晶纖維素(cellulose nano-crystalline,CNC) 20
2.6.1 纖維素之來源與組成 20
2.6.2 奈米結晶纖維素製造方法 21
2.6.3 奈米結晶纖維素在酵素固定化應用 22
2.7 海藻酸鈉膠體(Alginate bead) 23
2.7.1 海藻酸鈉(Sodium Alginate) 23
2.7.2 液相-氣相法(liquid-air method) 24
2.7.3 液相-液相法(liquid-liquid method) 25
2.7.4 自組裝法(Self-assemble method) 26
2.7.5 凝膠化 26
2.8 支鏈型聚乙烯亞胺Branch-Polyethylenimine 27
2.9 水凝膠(Hydrogels) 28
2.9.1 超分子聚合物(supermolecular complex) 29
2.9.2 非共價鍵鍵結種類 30
第三章 實驗方法與材料 32
3.1 實驗架構 32
3.1.1 實驗菌株 33
3.1.2 質體 33
3.1.3 實驗藥品 33
3.2 各式溶液配製 35
3.3 實驗設備 39
3.4 實驗方法 41
3.4.1 FPOX蛋白質 41
3.4.1.1 蛋白質純化 42
3.4.1.2 蛋白質濃度分析 42
3.4.2 蛋白質電泳分析 43
3.4.2.1 SDS-PAGE膠片製作 43
3.4.2.2 樣品前處理 44
3.4.2.3 樣品分析 44
3.4.3 合成糖化草胺酸(FV) 45
3.4.4 酵素活性分析 45
3.4.5 消光係數 46
3.4.6 Zinc/Adenosine 5' mono-phosphate(Zn/AMP)金屬配位凝膠 47
3.4.6.1 pH值與鹽類對於Zn/AMP凝膠產率之影響 47
3.4.6.2 配位莫爾濃度對於Zn/AMP凝膠之影響 47
3.4.6.3 Zn/AMP凝膠以包埋法固定化酵素(FPO、HRP) 48
3.4.6.4 FPO、HRP雙酵素凝膠之穩定性 48
3.4.6.5 包埋固定化FPO之反應動力學 49
3.4.7 CBD-FPO-6xHis吸附固定化於CNC 49
3.4.7.1 CBD-FPO-6xHis固定化於CNC之吸附曲線 49
3.4.7.2 CNC固定化CBD-FPO-6xHis之穩定性 50
3.4.7.3 CBD-FPO-6xHis固定化於CNC之反應動力學 50
3.4.8 具CNC之PEI修飾磁性海藻酸鈣凝膠(CNC/PEI Magnetic Alginate microbeads) 50
3.4.8.1 檸檬酸包覆磁性奈米顆粒 50
3.4.8.2 電壓液氣法製備磁性海藻酸鈣凝膠 51
3.4.8.3 PEI修飾磁性海藻酸鈣凝膠 51
3.4.8.4 PEI修飾之磁性海藻酸鈣凝膠與戊二醛交聯 51
3.4.8.5 CNC吸附於 PEI修飾之磁性海藻酸鈣凝膠 52
3.4.8.6 具CNC之PEI修飾磁性海藻酸鈣凝膠吸附CBD-FPO-6xHis 52
3.4.9 胺基測定方法 52
3.4.10 粒徑分析儀 (Dynamic Light Scattering) 52
3.4.11 界達電位分析儀 (Zeta-potential) 53
3.4.12 傅立葉紅外光光譜儀 (FTIR) 53
3.4.13 掃描式電子顯微鏡 (SEM) 53
3.4.14 熱重分析儀 (TGA) 53
3.4.15 X光繞射分析 (XRD) 54
第四章 結果與討論 55
4.1 Zinc/Adenosine 5' mono-phosphate (Zn2+/AMP)分子凝膠 55
4.1.1 pH值與鹽類對於Zn/AMP凝膠之產率影響 56
4.1.2 場發射電子顯微鏡 (FE-SEM)觀察Zn/AMP凝膠 58
4.1.3 傅立葉紅外光譜儀分析 (FTIR) 59
4.1.4 包埋法固定化FPO酵素 60
4.1.5 FPO酵素凝膠之保存穩定性 62
4.1.6 FPO酵素凝膠之pH穩定性 63
4.1.7 FPO酵素凝膠之熱穩定性 64
4.1.8 包埋固定化FPO反應動力學 65
4.2 CBD-FPO-6xHis吸附固定化於CNC 67
4.2.1 CNC吸附CBD-FPO-6xHis 67
4.2.2 CBD-FPO-6xHis固定化於CNC之反應動力學 70
4.2.3 pH值與溫度對CNC固定化CBD-FPO-6xHis之影響 72
4.3 具CNC之PEI修飾磁性海藻酸鈣凝膠 73
4.3.1 電噴磁性海藻顆粒製備 73
4.3.2 PEI修飾磁性海藻酸鈣凝膠之膨脹/收縮行為 76
4.3.3 傅立葉紅外光譜儀(FTIR)分析 78
4.3.4 熱重分析儀(TGA)分析 81
4.3.5 場發射電子顯微鏡(SEM)表面分析 83
4.3.6 X-射線繞射(XRD)分析 85
4.3.7 表面胺基分析(Ninhydrin assay) 86
4.3.8 具CNC之PEI修飾之磁性海藻酸鈣吸附CBD-FPO-6xHis 87
第五章 結論. 90
5.1 Zn/AMP水凝膠 90
5.2 具CNC之PEI修飾磁性海藻酸鈣凝膠 91
參考文獻 92

Bugarski1, V. M. J. D. B. o. V. n. B. (2001). immobilization of cells by electrostatic droplet generation: a model system for potential application in medicine.
Campos-Vallette, M. M., Chandía, N. P., Clavijo, E., Leal, D., Matsuhiro, B., Osorio-Román, I. O., & Torres, S. (2009). Characterization of sodium alginate and its block fractions by surface-enhanced Raman spectroscopy. Journal of Raman Spectroscopy, n/a-n/a. doi:10.1002/jrs.2517
Cao, S. L., Huang, Y. M., Li, X. H., Xu, P., Wu, H., Li, N., . . . Zong, M. H. (2016). Preparation and Characterization of Immobilized Lipase from Pseudomonas Cepacia onto Magnetic Cellulose Nanocrystals. Sci Rep, 6, 20420. doi:10.1038/srep20420
Chien, E. Y., Liu, W., Zhao, Q., Katritch, V., Han, G. W., Hanson, M. A., . . . Stevens, R. C. (2010). Structure of the human dopamine D3 receptor in complex with a D2/D3 selective antagonist. Science, 330(6007), 1091-1095. doi:10.1126/science.1197410
D. Poncelet 1, R. L., C. Beaulieu 1, J. P. Halle 3, R. J. Neufeid 2, and A. Fournier 1 (1992). Production of alginate beads by emulsification/internal gelation.
Dong, R., Pang, Y., Su, Y., & Zhu, X. (2015). Supramolecular hydrogels: synthesis, properties and their biomedical applications. Biomater Sci, 3(7), 937-954. doi:10.1039/c4bm00448e
Edgar Ong, N. R. G., R. Antony J. Warren, Robert C. Miller Jr. & Douglas G. Kilburn. (1992). Enzyme Immobilization Using the Cellulose-Binding Domain of a Cellulomonas Fimi Exoglucanase.
Eng-Seng Chan a, Boon-Beng Lee a, Pogaku Ravindra a, Denis Poncelet b. (2009). Prediction models for shape and size of ca-alginate macrobeads produced through extrusion–dripping method.
erman Van Tilbeurgh, & Peter Tomme, M. C., Rama Bhikhabhai* and GGran Pettersson*. (1986). Limited proteolysis of the cellobiohydrolase I from Trichuoderma reesei.
Etai Shpigel, A. G., * Gilat Efroni,* Amos Avraham,1 Adi Eshel,* Mara Dekel,1 Oded Shoseyov1. (1999). Immobilization of Recombinant Heparinase I Fused to Cellulose-Binding Domain.
Ferri, S., Miyamoto, Y., Sakaguchi-Mikami, A., Tsugawa, W., & Sode, K. (2013). Engineering fructosyl peptide oxidase to improve activity toward the fructosyl hexapeptide standard for HbA1c measurement. Mol Biotechnol, 54(3), 939-943. doi:10.1007/s12033-012-9644-2
Hirokawa, K., Gomi, K., & Kajiyama, N. (2003). Molecular cloning and expression of novel fructosyl peptide oxidases and their application for the measurement of glycated protein. Biochemical and Biophysical Research Communications, 311(1), 104-111. doi:10.1016/j.bbrc.2003.09.169
Hirokawa, K., Nakamura, K., & Kajiyama, N. (2004). Enzymes used for the determination of HbA1C. FEMS Microbiol Lett, 235(1), 157-162. doi:10.1016/j.femsle.2004.04.027
Ilari Filpponen , D. S. A. (2010). Regular Linking of Cellulose Nanocrystals via Click Chemistry: Synthesis and Formation of Cellulose Nanoplatelet Gels.
Islam, M. S., Chen, L., Sisler, J., & Tam, K. C. (2018). Cellulose nanocrystal (CNC)–inorganic hybrid systems: synthesis, properties and applications. Journal of Materials Chemistry B, 6(6), 864-883. doi:10.1039/c7tb03016a
Jun-Yee Leonga, W.-H. L., Kiang-Wei Hoa, Wan-Ping Vooa, Micky Fu-Xiang Leea, Hui-Peng Lima, Swee-Lu Lima, Beng-Ti Teya,b, Denis Ponceletc, Eng-Seng Chana,b,∗. (2011). Advances in fabricating spherical alginate hydrogels with controlled particle designs by ionotropic gelation as encapsulation systems.
Jun-Yee Leonga, W.-H. L., Kiang-Wei Hoa, Wan-Ping Vooa, Micky Fu-Xiang Leea, Hui-Peng Lima, Swee-Lu Lima, Beng-Ti Teya,b, Denis Ponceletc, Eng-Seng Chana,b,∗. (2015). Advances in fabricating spherical alginate hydrogels with controlled particle designs by ionotropic gelation as encapsulation systems.
Kühbeck, D., Mayr, J., Häring, M., Hofmann, M., Quignard, F., & Díaz Díaz, D. (2015). Evaluation of the nitroaldol reaction in the presence of metal ion-crosslinked alginates. New Journal of Chemistry, 39(3), 2306-2315. doi:10.1039/c4nj02178a
Kabata, M., Hase, E., Kimura, K., Kobayashi, Y., Ueno, Y., & Yoshimune, K. (2016). Assay of hemoglobin A1c using lectin from Aleuria aurantia. AMB Express, 6(1), 119. doi:10.1186/s13568-016-0288-7
Kazuki Maeda, H. O., Masahiro Takinoue, and Shoji Takeuchi*. (2012). Controlled Synthesis of 3D Multi-Compartmental Particles with Centrifuge-Based Microdroplet Formation from a Multi-Barrelled Capillary.
Kim, S., Ferri, S., Tsugawa, W., Mori, K., & Sode, K. (2010). Motif-based search for a novel fructosyl peptide oxidase from genome databases. Biotechnol Bioeng, 106(3), 358-366. doi:10.1002/bit.22710
Liang, H., Zhang, Z., Yuan, Q., & Liu, J. (2015). Self-healing metal-coordinated hydrogels using nucleotide ligands. Chem Commun (Camb), 51(82), 15196-15199. doi:10.1039/c5cc06824j
Lindén, J. B., Larsson, M., Kaur, S., Skinner, W. M., Miklavcic, S. J., Nann, T., . . . Nydén, M. (2015). Polyethyleneimine for copper absorption II: kinetics, selectivity and efficiency from seawater. RSC Advances, 5(64), 51883-51890. doi:10.1039/c5ra08029k
Lorenzo Capretto, a. S. M., a Cosimo Balestra,a Azzura Tosib and Claudio Nastruzzi*a. (2008). Effect of the gelation process on the production of alginate microbeads by
microfluidic chip technology.
Markus Linder, T. T. T. (1997). The roles and function of cellulose-binding domains.
Miura, S., Ferri, S., Tsugawa, W., Kim, S., & Sode, K. (2008). Development of fructosyl amine oxidase specific to fructosyl valine by site-directed mutagenesis. Protein Eng Des Sel, 21(4), 233-239. doi:10.1093/protein/gzm047
Nanjo, T., Sakurai, T., Totoki, Y., Toyoda, A., Nishiguchi, M., Kado, T., . . . Shinohara, K. (2007). Functional annotation of 19,841 Populus nigra full-length enriched cDNA clones. BMC Genomics, 8, 448. doi:10.1186/1471-2164-8-448
Pasquale Del Gaudio∗, P. C., & Gaia Colombo, P. R., Fabio Sonvico. (2005). Mechanisms of formation and disintegration of alginate beads obtained by prilling.
Ro´bert Me´sza´ros, †,‡ Laurie Thompson,† Imre Varga,‡ and Tibor Gila´nyi‡. (2002). Adsorption Properties of Polyethyleneimine on Silica Surfaces in the Presence of Sodium Dodecyl Sulfate.
Sheikholeslami, M., Ashorynejad, H. R., Ganji, D. D., & Kolahdooz, A. (2011). Investigation of Rotating MHD Viscous Flow and Heat Transfer between Stretching and Porous Surfaces Using Analytical Method. Mathematical Problems in Engineering, 2011, 1-17. doi:10.1155/2011/258734
SHOSEYOV*, C. K. A. O. (2000). Novel Methodology for Enzymatic Removal of Atrazine from Water by CBD-Fusion Protein Immobilized on Cellulose.
Skj~k-Braek, O. S. d. a. G. (1990). Alginate as immobilization matrix for cells.
Stefano Ferri, P. D., 1 Seungsu Kim, M.Eng.,1 Wakako Tsugawa, Ph.D.,1,2 and Koji Sode, Ph.D. (2009). Review of Fructosyl Amino Acid Oxidase Engineering Research: A Glimpse into the Future of Hemoglobin A1c Biosensing.
Terpe, K. (2003). Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol, 60(5), 523-533. doi:10.1007/s00253-002-1158-6
Tong, W., Gao, C., & Möhwald, H. (2008). Poly(ethyleneimine) microcapsules: glutaraldehyde‐mediated assembly and the influence of molecular weight on their properties. Polymers for Advanced Technologies, 19(7), 817-823. doi:10.1002/pat.1040
Tsuyoshi Tanaka, T. M. (2001). Detection of HbA1c by boronate affinity immunoassay using
bacterial magnetic particles.
Xiong, B., Wang, N., Chen, Y., & Peng, H. (2018). Self-assembly of alginate/polyethyleneimine multilayer onto magnetic microspheres as an effective adsorbent for removal of anionic dyes. Journal of Applied Polymer Science, 135(7). doi:10.1002/app.45876
YÄ rr A. Mørch, § Ivan Donati,‡ Berit L. Strand,§ and Gudmund Skja˚k-Bræk§. (2006). Effect of Ca2+, Ba2+, and Sr2+ on Alginate Microbeads.
Zhang, F., Wang, R., Zhen, C., & Li, B. (2016). Magnetic cellulose nanocrystals: Synthesis by electrostatic self-assembly approach and efficient use for immobilization of papain. Journal of Molecular Catalysis B: Enzymatic, 134, 164-171. doi:10.1016/j.molcatb.2016.11.017