臺灣博碩士論文加值系統

血液中的糖化血紅素濃度可視為體內2-3個月平均血糖的重要指標。分析糖化血素的方法有很多種，本論文選用反應速率較快且不易受變異血紅素影響的酵素呈色法進行分析。利用醣化胜肽氧化酶(fructosyl peptide oxidase, FPOX) 氧化經中性水解酶(Neutral protease)水解HbA1c所得到的FVH (fructosyl valyl histidine)片段產生過氧化氫(H2O2)，再利用酵素呈色法，於過氧化氫酶(horseradish peroxidase, HRP)催化呈色劑DA-64反應，由呈色深淺推算出FPOX活性與HbA1c的濃度。由基因工程合成的fructosyl peptide oxidase (FPOX-C-PnL1)，其對FVH的反應性佳，在大腸桿菌中表現所得的可溶性蛋白含量十分低且活性不穩定，為改善此缺失，因此使用來自熱纖梭菌(Clostridium thermocellum)的纖維素結合功能域(cellulose binding domain, CBD)蛋白的高可溶性表現特性，將之融合於FPOX之N-端；並利用CBD與細菌纖維素(bacterial cellulose, BC)間的高親和性特性進行純化與固定化，藉此增加FPOX可溶性蛋白的表現，為提高其固定化蛋白環境耐受性，將固定化於BC上之CBD-FPOX懸浮液，以抽氣過濾方式進行乾燥成膜，製備出CBD-FPOX-BC膜。此CBD-FPOX-BC膜之活性，不易受環境溫度與pH變異影響，保存於4℃-70℃環境下，活性可維持95%以上，而可承受之pH範圍，也由pH 6-8擴大至pH 5-9。使用CBD-FPOX-BC膜進行酵素呈色法可偵測出血液中5.3-11% HbA1c，所得之檢量線線性回歸值可達0.9834。

The glycated hemoglobin (HbA1c) level in blood is an important indicator for evaluating long-term control of glycemic status in diabetic patients. Enzymatic colorimetric assay is one of the methods for detecting HbA1c. It is based on the enzymatic reaction of fructosyl peptide oxidase (FPOX) toward fructosyl valyl histidine (FVH), a fragment obtained by protease digestion of HbA1c. FVH will be oxidized by FPOX to generate hydrogen peroxide which can further oxidize sodium N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)diphenylamine (DA-64) by using horseradish peroxidase (HRP) are catalyst to develop green color. The color intensity can reflect the concentration of HbA1c. To develop an engineered FPOX which has a higher reactivity toward FVH, we constructed the loop-substitution mutants of FPOX-C (FPOX-C-PnL1). However, most of this mutant FPOX expressed in Escherichia coli existed as inclusion bodies. To improve the solubility of FPOX-C-PnL1, cellulose binding domain (CBD) was fused in N-terminus to generate a soluble CBD-FPOX fusion protein. By taking advantage of CBD’s strong affinity toward crystalline cellulose, CBD-FPOX was easily purified from Escherichia coli cell lysate and immobilized on bacterial cellulose (BC) produced from Gluconacetobacter xylinus. CBD-FPOX immobilized on BC not only increased the stability of FPOX but also could be directly applied for HbA1c detection. CBD fusion FPOX significantly improved the solubility of FPOX without affecting its activity. Approximately 0.24 mg/mg of CBD-FPOX could be tightly bound on bacterial cellulose in 10 minutes. The FPOX activity of CBD-FPOX BC film could be stable maintained at 4-70℃ and pH 5-9. The CBD-FPOX BC film prepared could be applied for HbA1c detection in blood sample. A linear correlation between color developed and HbA1c sample concentration of 5.3-11% was observed by using this FPOX film catalyzed colorimetric method.

摘要 I
Abstract II
誌謝 III
目錄 IV
圖目錄 VII
表目錄 IX
第一章 緒論 1
1.1 前言 1
1.2 研究目的及內容簡介 1
第二章 文獻回顧 3
2.1 糖化血紅素 (Glycated hemoglobin) 之來源與組成 3
2.1.1 HbA1c 糖化血紅素 5
2.2 糖化血紅素之測定與分析 7
2.2.1 陽離子交換樹指層析法 ( Cation exchange HPLC) 8
2.2.2 硼酸親和性層析法 (Boronate affinity HPLC) 8
2.2.3 免疫比濁法 (Immunoassay) 9
2.2.4 電化學法 (Electrochemical method) 9
2.2.5 酵素比色法 (Enzyme colorimetric assay) 10
2.3 酵素比色法所使用之氧化酵素 10
2.3.1 FAOX (fructosyl amino acid oxidase) 11
2.3.2 FPOX (fructosyl peptide oxidase) 12
2.4 纖維素結合功能域 (cellulose binding domain, CBD) 15
2.4.1 纖維素結合功能域的分類 16
2.4.2 融合纖維素功能域之應用 17
2.5 細菌纖維素(bacterial cellulose, BC) 19
2.5.1 細菌纖維素之應用 20
第三章 實驗材料與方法 21
3.1 實驗流程 21
3.2 實驗材料 22
3.2.1 實驗菌株 22
3.2.2 質體 22
3.2.3 其它 22
3.3 實驗藥品 22
3.4 溶液配製 24
3.5 實驗儀器與設備 28
3.6 實驗方法 29
3.6.1 E.coli重組基因菌株培養及FPOX重組蛋白之生產 29
3.6.2 FPOX重組蛋白之純化 30
3.6.3 細菌纖維素(BC)吸附CBD-FPOX之吸附動力測定 31
3.6.4 細菌纖維素(BC)吸附CBD-FPOX含量測定 32
3.6.5 CBD-FPOX重組蛋白之純化與固定化 32
3.6.6 製備CBD-FPOX-BC薄膜 32
3.6.7 合成糖化纈草胺酸(FV) 33
3.6.8 蛋白質電泳分析 33
3.6.9 FPOX重組蛋白質之濃度分析 35
3.6.10 CBD-FPOX-BC膜之蛋白質含量 36
3.6.11 FPOX活性分析 36
3.6.12 CBD-FPOX-BC膜活性分析 37
3.6.13 FPOX之酵素動力學測定 37
3.6.14 溫度與pH值對FPOX活性的影響 38
3.6.15 溫度與pH值對固定化CBD-FPOX-BC膜活性的影響 38
3.6.16 溶血與偵測HbA1c中fructosyl valyl histidine(FVH)的含量 38
3.6.17 CBD-FPOX-BC懸浮液偵測HbA1c中fructosyl valyl histidine(FVH)含量 40
3.6.18 CBD-FPOX-BC膜偵測HbA1c中fructosyl valyl histidine(FVH)含量 40
第四章 結果與討論 41
4.1 含pET24a – FPOX質體之大腸桿菌表現生產FPOX 41
4.1.1 搖瓶培養pET24a-FPOX@E.coli BL21 (DE3)之生長曲線 41
4.1.2 培養條件 42
4.2 DA-64之消光係數(ε)與FPOX活性分析及純化 46
4.2.1 消光係數(ε) 46
4.2.2 FPOX-6xHis、CBD-FPOX-6xHis活性分析與純化 47
4.3 CBD-FPOX固定化 49
4.3.1 固定化基材BC的特性 49
4.3.2 細菌纖維素(BC)吸附CBD-FPOX 52
4.4 溫度與pH值對FPOX活性之影響 54
4.4.1 FPOX之熱穩定性 54
4.4.2 FPOX之pH穩定性 55
4.5 固定化CBD-FPOX-BC膜之保存穩定性與重複使用性 56
4.6 FPOX酵素動力學 57
4.7 酵素呈色法偵測糖化血紅素 (HbA1c) 60
第五章 結論 62
第六章 建議 64
參考資料 65

Akazawa, S.-i., Karino, T., Yoshida, N., Katsuragi, T., & Tani, Y. (2004). Functional Analysis of Fructosyl-Amino Acid Oxidases of Aspergillus oryzae. Applied and Environmental Microbiology, 70(10), 5882-5890. doi:10.1128/AEM.70.10.5882-5890.2004
Bunn, H. F., Haney, D. N., Kamin, S., Gabbay, K. H., & Gallop, P. M. (1976). The biosynthesis of human hemoglobin A1c. Slow glycosylation of hemoglobin in vivo. Journal of Clinical Investigation, 57(6), 1652-1659. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC436825/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC436825/pdf/jcinvest00149-0266.pdf
Chen, J.-H. (2015). Paper-based Glycosylated Hemoglobin (HbA1C) Determination Based on Interaction with Immobilized Boronic Acid.
Chien, H. C., & Chou, T. C. (2010). Glassy Carbon Paste Electrodes for the Determination of Fructosyl Valine. Electroanalysis, 22(6), 688-693.
Credou, J., & Berthelot, T. (2014). Cellulose: from biocompatible to bioactive material. Journal of Materials Chemistry B, 2(30), 4767. doi:10.1039/c4tb00431k
Ferri, S., Kim, S., Eng, M., Tsugawa, W., & Sode, K. (2009). Review of Fructosyl Amino Acid Oxidase Engineering Research: A Glimpse into the Future of Hemoglobin A1c Biosensing. J Diabetes Sci Technol, 3(3), 585-592.
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
Gabriele, H. B., Katzensteiner, S., Schnedl, W., Pu rstner, P., Pieber, T., & and Martie, W. T. (1997). Comparative evaluation of three assay systems for automated determination of hemoglobin A1c. Clinical Chemistry, 43(3), 511-517.
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:http://dx.doi.org/10.1016/j.bbrc.2003.09.169
Hirokawa, K., Nakamura, K., & Kajiyama, N. (2004). Enzymes used for the determination of HbA1C. FEMS Microbiology Letters, 235(1), 157-162. Retrieved from http://femsle.oxfordjournals.org/content/235/1/157.abstract
http://femsle.oxfordjournals.org/content/femsle/235/1/157.full.pdf
Hirokawa, K., Shimoji, K., & Kajiyama, N. (2005). An enzymatic method for the determination of hemoglobinA1C. Biotechnology Letters, 27, 963-968.
Iberg, N., & Fluckiger, R. (1986). Nonenzymatic Glycosylation of Albumin in Vivo. The Journal of Biological Chemistry, 261(29), 13542-13545.
Iguchi, M., Yamanaka, S., & Budhiono, A. (2000). Bacterial cellulose—a masterpiece of nature's arts. Journal of Materials Science, 35(2), 261-270. doi:10.1023/A:1004775229149
Jeppsson, J. O., Kobold, U., Barr, J., Finke, A., Hoelzel, W., Hoshino, T., . . . Weykamp, C. (2002). Approved IFCC Reference Method for the Measurement of HbA1c in Human Blood. Jeppsson 2002. Clin Chem Lab Med, 40(1), 78–89.
Kauffmann, C., Shoseyov, O., Shpigel, E., Bayer, E. A., Lamed, R., Shoham, Y., & Mandelbaum, R. T. (2000). Novel Methodology for Enzymatic Removal of Atrazine from Water by CBD-Fusion Protein Immobilized on Cellulose. Environmental Science & Technology, 34(7), 1292-1296. doi:10.1021/es990754h
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
Kobold, U., Jeppsson, J. O., Du lffer, T., Finke, A., Hoelzel, W., & Miedema, K. (1997). Candidate reference methods for hemoglobin A1c based on peptide mapping. Clinical Chemistry, 43(10), 1944-1951.
Kuo, C.-H., Chen, J.-H., Liou, B.-K., & Lee, C.-K. (2016). Utilization of acetate buffer to improve bacterial cellulose production by Gluconacetobacter xylinus. Food Hydrocolloids, 53, 98-103. doi:10.1016/j.foodhyd.2014.12.034
Lan, D., Tai, Y., Shen, Y., Wang, F., Yang, B., & Wang, Y. (2012). Efficient purification of native recombinant proteins using proteases immobilized on cellulose. J Biosci Bioeng, 113(4), 542-544. doi:10.1016/j.jbiosc.2011.12.003
Liu, L., Hood, S., Wang, Y., Bezverkov, R., Dou, C., Datta, A., & Yuan, C. (2008). Direct enzymatic assay for %HbA1c in human whole blood samples. Clinical Biochemistry, 41(7–8), 576-583. doi:http://dx.doi.org/10.1016/j.clinbiochem.2008.01.013
Markus Linder, T. T. T. (1997). The roles and function of cellulose-binding domains Journal of Biotechnology, 57(1-3), 14. doi:10.1016/S0168-1656(97)00087-4
Millon, L. E., & Wan, W. K. (2006). The polyvinyl alcohol-bacterial cellulose system as a new nanocomposite for biomedical applications. J Biomed Mater Res B Appl Biomater, 79(2), 245-253. doi:10.1002/jbm.b.30535
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
Nam, J.-M., Fujita, Y., Arai, T., Kondo, A., Morikawa, Y., Okada, H., . . . Tanaka, A. (2002). Construction of engineered yeast with the ability of binding to cellulose. Journal of Molecular Catalysis B: Enzymatic, 17(3), 197-202.
Nanjo, Y., Hayashi, R., & Yao, T. (2007). An enzymatic method for the rapid measurement of the hemoglobin A1c by a flow-injection system comprised of an electrochemical detector with a specific enzyme-reactor and a spectrophotometer. Analytica Chimica Acta, 583, 45-54.
Nathan, D. M., Kuenen, J., Borg, R., Zheng, H., Schoenfeld, D., Heine, R. J., & Group, A. c.-D. A. G. S. (2008). Translating the A1C assay into estimated average glucose values. Diabetes Care, 31(8), 1473-1478. doi:10.2337/dc08-0545
Ong, E. (1992). Enzyme immobilization using the cellulose-binding domain of the Cellulomonas Fimi exoglucanase. (Text). Retrieved from https://open.library.ubc.ca/collections/831/items/1.0098964
Prashant R. Chawla, I. B. B., Shrikant A. Survase and Rekha S. Singhal*. (2009). Microbial Cellulose: Fermentative Production and Applications. Food Technology and Biotechnology, 47(2).
Sakurabayashi, Watano, T., Yonehara, S., Ishimaru, K., Hirai, K., Komori, T., & Yagi, M. (2003). New Enzymatic Assay for Glycohemoglobin. Clinical Chemistry, 49(2), 269-274.
Schnedl, W. J., Krause, R., Gabriele, H. B., Trinker, M., LIPP, R. W., & Krejs, G. J. (2000). Evaluation of HbA1c Determination Methods in Patients With Hemoglobinopathies. Diabetes Care, 23, 339-344.
Shapiro, R., McManus, J. M., Zalut, C., & Bunn, H. F. (1980). . Biological Chemistry, 255(7), 3120-3127.
Sheikholeslam, S., Pritzker, M. D., & Chen, P. (2011). Electrochemical Biosensor for Glycated Hemoglobin (HbA1c). Biosensors for Health, Environment and Biosecurity, Pier Andrea Serra (Ed.).
Shi, Z., Zhang, Y., Phillips, G. O., & Yang, G. (2014). Utilization of bacterial cellulose in food. Food Hydrocolloids, 35, 539-545. doi:10.1016/j.foodhyd.2013.07.012
Shih, T.-Y., & Tsai, S.-L. (2014). Simultaneous silver recovery and bactericidal bionanocomposite formation via engineered biomolecules. RSC Advances, 4(77), 40994-40998.
Shpigel, E., Goldlust, A., Efroni, G., Avraham, A., Eshel, A., Dekel, M., & Shoseyov, O. (1999). Immobilization of recombinant heparinase I fused to cellulose-binding domain. Biotechnol Bioeng, 65(1), 17-23. doi:10.1002/(SICI)1097-0290(19991005)65:1<17::AID-BIT3>3.0.CO;2-Y
Song, S. Y., & Yoon, H. C. (2009). Boronic acid-modified thin film interface for specific binding of glycated hemoglobin (HbA1c) and electrochemical biosensing. Sensors and Actuators B: Chemical, 140(1), 233-239. doi:10.1016/j.snb.2009.04.057
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
Van Tilbeurgh, H., Tomme, P., Claeyssens, M., Bhikhabhai, R., & Pettersson, G. (1986). Limited proteolysis of the cellobiohydrolase I from Trichoderma reesei. FEBS letters, 204(2), 223-227.
Weets, Gorus, F. K., & Gerlo, E. (1996). Evaluation of an Immunoturbidimetric Assay for Haemoglobin Alc on a Cobas® Mira S Analyser. Ilse Weets 1996. Eur J Clin Chem Clin Biochem, 34, 449-453.
歐陽嘉, 李鑫, 王向明, 嚴明, & 徐琳. (2008). 纖維素結合域的研究進展. 生物加工過程(02).