臺灣博碩士論文加值系統

稀有醣類 (rare sugars)是自然界中含量極為稀少的單醣或其衍生物，稀有糖因含量稀少，限制了其潛在之應用，L-核糖屬於稀有醣類，可用於合成抗病毒及抗癌藥物，但由於產量稀少價格昂貴，造成研究及應用上的限制。本研究將以L-阿拉伯糖利用 L-阿拉伯糖異構酶 (L-arabinose isomerase, L-AI) 轉換成 L-核酮糖，再由 L-核糖異構酶 (L-ribose isomerase, L-RI) 轉換成 L-核糖。酵素來源L-RI是來自 Actinotalea fermentans ATCC 43279 (Af-RI) 之突變型F141Y，L-AI 來自 T. saccharolyticum NTOU1 (Ts-AI) 之突變型 F270N。首先探討先可表現 F141Y Af-RI 及 F270N Ts-AI的大腸桿菌最適培養條件，將小量最適條件至發酵槽放大培養，發現突變型 Af-RI利用發酵槽培養能有效放大菌體量及活性，進而可降低生產成本; 由突變型Ts-AI經由pH-stat控制能有效提升每公升的菌量及活性。將上述酵素使用 HisTrap TM HP 管柱進行純化，並將純化酵素固定於活性碳及離子交換樹脂上，結果顯示F270N Ts-AI 酵素固定在20-40目數活性碳上其結合率較佳，固定在12-20目數活性碳及Q SepharoseTM Fast Flow上其固定化效率較好，且固定在 Q SepharoseTM Fast Flow上其固定化效率高達80%；將F141Y Af-RI 固定在 Q SepharoseTM Fast Flow上其最高的固定化效率為 38.7% 。熱穩定方面，固定於Q Sepharose之突變型RI在50oC下半衰期為167 分鐘；固定於Q Sepharose之突變型AI在60 oC 下半衰期為322分鐘，且固定在Q Sepharos上之酵素，儲藏28天後活性皆保留原有之80%以上，利用F270N Ts-AI 及F141Y Af-RI 的未固定酵素及固定後之酵素進行稀有糖生產，未固定酵素部分，在僅有AI的情況下，核酮糖轉換率有42%，接著加入RI一起反應後， L-核酮糖及 L-核糖轉換率分別為 34% 及 13 %；固定在Q Sepharose上之酵素部分，在僅有固定化AI存在下，核酮糖轉換率有46%，放入固定化RI一起反應後，L-核酮糖及 L-核糖轉換率分別為11%及19%。

Rare sugars are extremely rare in nature, and the rare amounts of these sugars limit their potential applications. L-Ribose is defined as a rare sugar. L-Ribose can be used as a precursor for the synthesis of L-nucleoside analogues, which are constantly used as antiviral drugs. However, the scarcity and high cost of L-ribose limit the pursuit of more applications and research. In this study, L-Arabinose was firstly converted into L-ribulose by L-arabinose isomerase, and then L-ribulose can be converted into L-ribose by L-ribose isomerase. Mutant F141Y L-RI from Actinotalea fermentans ATCC 43279 and mutant F270N L-AI from Thermoanaerobacterium saccharolyticum NTOU1 were used in this study. Flask culturing was used to explore the best feed medium for fermenter culturing. It was found that fermenter culturing can effectively increase cell weights and total activities for mutant Af-RI-containing E. coli, which can greatly reduce the production cost. In addition, the mutant Ts-AI-containing E. coli grown in the fermenter through pH-stat control can effectively increase cell weights and total activities. The purified enzymes were immobilized on activated carbons and ion exchange resins. Mutant Ts-AI turned out that used 20-40 mesh activated carbons as carrier can bind more enzymes, however, used Q Sepharose and 12-20 mesh activated carbons can obtain higher immobilization efficiencies. Q Sepharose has been used to immobilize Af-RI and Ts-AI, the immobilization efficiencies were found to be 38.6% and 80.47%, respectively. In terms of thermal stability, mutant Af-RI immobilized on Q Sepharose have a half-life of 167 mins at 50oC. Ts-AI immobilized on Q Sepharose have a half-life of 322 min at 60oC. The activities of the enzymes immobilized on Q sepharose remained more than 80% after 28 days. At last, using unimmobilized and Q Sepharose-immobilized F270N Ts-AI, respectively, to produce L-ribulose from L-arabinose at 45oC, the reaction reached equilibrium at 48 h with about 42% and 46% conversion yields, respectively. Then the unimmobilized and Q Sepharose-immobilized F141Y Af-RI, respectively, were added to produce L-ribose. For unimmobilized enzymes, the reaction reached equilibrium with about 34% and 13% conversion yields of L-ribose and L-ribulose, respectively. For unimmobilized enzymes, the reaction reached equilibrium with about 11% and 19% conversion yield of L-ribose and L-ribulose, respectively.

目錄

壹、研究背景與目的 1
一、研究背景 1
二、研究目的 2

貳、文獻整理 3
一、稀有醣類 3
1. 簡介 3
2. 酵素轉換法生成稀有糖例子 3
3. L-核酮糖 (L -ribulose) 3
4. L-核糖 (L-ribose) 3
二、L-核糖 (L-ribose)之生產 4
1. 生產方式 4
2. 生產L-核糖需要的酵素 4
三、重組蛋白在ClearColiTM BL21 (DE3)中之蛋白質表現 5
1. ClearColiTM BL21 (DE3) 5
2. pET 表現系統 6
3. 乳糖操控組 (lactose operon) 6
四、化學伴護子 (chemical chaperone) 6
五、發酵技術 7
1. 發酵簡介 7
2. 發酵槽種類 7
3. 發酵槽培養方式 7
六、固定化 8
1. 固定化方式 8
2. 吸附法材料介紹 9

參、實驗設計與流程 11
以小量培養探討突變型Af-RI 及Ts-AI 的最適培養條件 11
二、以發酵槽進行培養探討饋料方式對於Af-RI 及Ts-AI 菌體收穫量與酵
素活性的影響 11
三、固定化酵素之方法與特性探討 11
四、L-核糖及 L-核酮糖之生產 12

肆、實驗材料與方法 13
一、 材料 13
1. 菌株與載體 13
2. 抗生素 13
3. 標準品 13
4. 市售套組 13
5. 培養基 13
6. 化學藥品 13
7. 實驗設備 15
二、 藥品配置 17
三、 實驗步驟及方法 19
1. 電穿孔轉形作用 19
1.1 宿主 19
1.2 製備 E. coli 電穿孔勝任細胞 19
1.3 電轉形作用 (electrotransformation) 20
1.4 菌種保存 20
2. 小量重組型Af-RI 及 Ts-AI 之表現 20
2.1 以不同條件誘導表現重組型Af-RI 及 Ts-AI 20
2.2 SDS-PAGE 21
3. 發酵槽培養 23
3.1 批式培養 23
3.2 批式饋料培養-定速追加饋料 23
3.3 批式饋料培養-pH-stat 24
4. 重組型 F270N Ts-AI 及 F141Y Af-RI 純化 24
5. 蛋白質定量 25
6. 活性分析與特性探討 25
6.1 酵素活性分析 25
6.2 重組型 F270N Ts-AI 酵素活性測定 25
6.3 重組型 F141Y Af-RI 酵素活性測定 25
6.4 固定化酵素測定 26
7. 活性碳-固定化酵素的製備與特性探討 26
7.1 活性碳-固定化酵素之微觀結構 26
7.2 活性碳-固定化酵素之最適結合溫度 26
7.3 活性碳-固定化酵素之最適作用pH 27
7.4 活性碳-固定化酵素之重複使用性 27
8. 離子交換樹脂-固定化酵素分析與特性探討 27
8.1 Q Sepharose-固定化酵素(Q Sepharose-immobilized enzyme) 27
8.2 Amberlite-固定化酵素 27
8.3 Q Sepharose-固定化酵素之最適溫度 27
8.4 Q Sepharose-固定化酵素之最適pH 27
8.5 Q Sepharose-固定化酵素之半衰期 28
8.6 Q Sepharose-固定化酵素之儲藏性 28
9. 以固定化酵素及純化酵素進行L-核糖的生產 28

伍、結果與討論 30
一、 F141Y Af-RI 表現及純化 30
1. 利用小量培養進行條件探討 30
2. 以發酵槽進行大量生產 30
二、 F270N Ts-AI 表現及純化 31
1. 利用小量培養進行條件探討 31
2. 以發酵槽進行大量生產 31
三、 固定化 32
1. 活性碳 32
2. 離子交換樹脂 33
四、 生產 L-核糖與 L-核酮糖 35

陸、結論 36

柒、參考文獻 37

捌、圖表 46

玖、附錄 86

Ahmd, Z. (2001). Production of natural and rare pentoses using microorganisms and their enzymes. Electronic Journal of Biotechnology, 4(2), 13-14.
Ahmed, Z., Shimonishi, T., Bhuiyan, S. H., Utamura, M., Takada, G., & Izumori, K. (1999). Biochemical preparation of L-ribose and L-arabinose from ribitol: a new approach. Journal of Bioscience and Bioengineering, 88(4), 444-448.
Akagi, M., Omae, D., Tamura, Y., Ueda, T., Kumashiro, T., & Urata, H. (2002). A practical synthesis of L-ribose. Chemical and Pharmaceutical Bulletin, 50(6), 866-868.
Akdogan, H. A., & Pazarlioglu, N. K. J. P. b. (2011). Fluorene biodegradation by P. osteratus–Part II: Biodegradation by immobilized cells in a recycled packed bed reactor. Process Biochemistry, 46(4), 840-846.
Ali, G., Dulong, V., Gasmi, S. N., Rihouey, C., Picton, L., & Le Cerf, D. (2015). Covalent immobilization of pullulanase on alginate and study of its hydrolysis of pullulan. Biotechnology Progress, 31(4), 883-889.
Alibolandi, M., & Mirzahoseini, H. (2011). Chemical assistance in refolding of bacterial inclusion bodies. Biochemistry Research International, 2011.
Anita, A., Sastry, C., & Hashim, M. (1997). Immobilization of urease using Amberlite MB-1. Bioprocess Engineering, 17(6), 355-359.
Beerens, K., Desmet, T., & Soetaert, W. (2012a). Enzymes for the biocatalytic production of rare sugars. Journal of Industrial Microbiology Biotechnology Progress, 39(6), 823-834.
Beerens, K., Desmet, T., & Soetaert, W. (2012b). Enzymes for the biocatalytic production of rare sugars.Journal of Industrial Microbiology biotechnology, 39(6), 823-834.
Beerens, K., Desmet, T., & Soetaert, W. (2012c). Enzymes for the biocatalytic production of rare sugars. Journal of Industrial Microbiology biotechnology, 39(6), 823-834.
Boland, L. G., Zehner, G. L., & Stevens, R. A. (1988). U.S. Patent No. 4,747,846. Washington, DC: U.S. Patent and Trademark Office.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1-2), 248-254.
Cheetham, P., & Wootton, A. (1993). Bioconversion of D-galactose into D-tagatose. Enzyme and Microbial Technology, 15(2), 105-108.
Chen, S.-Y., Wei, Y.-H., & Chang, J.-S. (2007). Repeated pH-stat fed-batch fermentation for rhamnolipid production with indigenous Pseudomonas aeruginosa S2. Applied Microbiology and Biotechnology, 76(1), 67-74.
Cho, E.-A., Lee, D.-W., Cha, Y.-H., Lee, S.-J., Jung, H.-C., Pan, J.-G., & Pyun, Y.-R. (2007). Characterization of a novel D-lyxose isomerase from Cohnella laevoribosii RI-39 sp. nov. Journal of Bacteriology, 189(5), 1655-1663.
Choi, Keum, K. C., & Lee, S. Y. (2006). Production of recombinant proteins by high cell density culture of Escherichia coli. Chemical Engineering Science, 61(3), 876-885.
Choi, J. M., Lee, Y.-J., Cao, T.-P., Shin, S.-M., Park, M.-K., Lee, H.-S., Di Luccio, E., Kim, S.-B., Lee, S.-J., & Lee, S. J. (2016). Structure of the thermophilic L-Arabinose isomerase from Geobacillus kaustophilus reveals metal-mediated intersubunit interactions for activity and thermostability. Archives of Biochemistry and Biophysics, 596, 51-62.
Cárdenas‐Fernández, M., Neto, W., López, C., Álvaro, G., Tufvesson, P., & Woodley, J. M. (2012). Immobilization of Escherichia coli containing ω‐transaminase activity in LentiKats®. Biotechnology Progress, 28(3), 693-698.
Daoud, F. B.-O., Kaddour, S., & Sadoun, T. (2010). Adsorption of cellulase Aspergillus niger on a commercial activated carbon: kinetics and equilibrium studies. Colloids Surfaces B: Biointerfaces, 75(1), 93-99.
Das, N., Kayastha, A. M., & Malhotra, O. (1998). Immobilization of urease from pigeonpea (Cajanus cajan L.) in polyacrylamide gels and calcium alginate beads. Biotechnology and Applied Biochemistry, 27(1), 25-29.
Datta, S., Christena, L. R., & Rajaram, Y. R. S. (2013). Enzyme immobilization: an overview on techniques and support materials. 3 Biotech, 3(1), 1-9.
de Lima, J. S., Cabrera, M. P., Casazza, A. A., da Silva, M. F., Perego, P., de Carvalho Jr, L. B., & Converti, A. (2018). Immobilization of Aspergillus ficuum tannase in calcium alginate beads and its application in the treatment of boldo (Peumus boldus) tea. International Journal of Biological Macromolecules, 118, 1989-1994.
de Taxis, d. P. P., Arcand, Y., Bernier Jr, R., Barbotin, J., & Thomas, D. (1987). Plasmid stability in immobilized and free recombinant Escherichia coli JM105 (pKK223-200): importance of oxygen diffusion, growth rate, and plasmid copy number. Applied and Environmental Microbiology, 53(7), 1548.
De Vuyst, L., & Vandamme, E. J. (1991). Microbial manipulation of misin biosynthesis and. Nisin Novel lantibiotics, 397.
Dhanoa, T. S., & Housner, J. A. (2007). Ribose: More than a simple sugar? Current Sports Medicine Reports, 6(4), 254-257.
Fazlena, H., Kamaruddin, A., & Zulkali, M. (2006). Dynamic kinetic resolution: alternative approach in optimizing S-ibuprofen production. Bioprocess and Biosystems Engineering, 28(4), 227-233.
Fu, G., Zhang, S., Dong, H., Chen, J., Tu, R., & Zhang, D. (2019). Enhanced production of D‐psicose 3‐epimerase in Bacillus subtilis by regulation of segmented fermentation. Biotechnology and Applied Biochemistry.
Glick, B. R. (1995). Metabolic load and heterologous gene expression. Biotechnology Advances, 13(2), 247-261.
Groboillot, A., Boadi, D., Poncelet, D., & Neufeld, R. (1994). Immobilization of cells for application in the food industry. Critical Reviews in Biotechnology, 14(2), 75-107.
Gupta, K., Jana, A. K., Kumar, S., & Maiti, M. (2013). Immobilization of α-amylase and amyloglucosidase onto ion-exchange resin beads and hydrolysis of natural starch at high concentration. Bioprocess and Biosystems Engineering, 36(11), 1715-1724.
Helanto, M., Kiviharju, K., Granström, T., Leisola, M., & Nyyssölä, A. (2009a). Biotechnological production of L-ribose from L-arabinose. Applied Microbiology and Biotechnology, 83(1), 77-83.
Helanto, M., Kiviharju, K., Granström, T., Leisola, M., & Nyyssölä, A. (2009b). Biotechnological production of L-ribose from L-arabinose. Applied Microbiology and Biotechnology, 83(1), 77-83.
Helanto, M., Kiviharju, K., Leisola, M., & Nyyssölä, A. (2007). Metabolic engineering of Lactobacillus plantarum for production of L-ribulose. Applied and Environmental Microbiology, 73(21), 7083-7091.
Hung, X.-G., Tseng, W.-C., Liu, S.-M., Tzou, W.-S., & Fang, T.-Y. (2014). Characterization of a thermophilic L-arabinose isomerase from Thermoanaerobacterium saccharolyticum NTOU1. Biochemical Engineering Journal, 83, 121-128.
Hung, X.-G., Yu, M.-Y., Chen, Y.-C., & Fang, T.-Y. (2015). Characterization of a Recombinant L-ribose isomerase from Geodermatophilus obscurus DSM 43160 and pplication of this enzyme to the production of L-ribose from L-arabinose. Journal of Marine Science and Technology-Taiwan, 23(4), 558-566.
Hunt, J. P., Zhao, E. L., Soltani, M., Frei, M., Nelson, J. A. D., & Bundy, B. C. (2019). Streamlining the preparation of “endotoxin-free” ClearColi cell extract with autoinduction media for cell-free protein synthesis of the therapeutic protein crisantaspase. Synthetic and Systems Biotechnology, 4(4), 220-224.
Jesionowski, T., Zdarta, J., & Krajewska, B. (2014a). Enzyme immobilization by adsorption: A review. Adsorption, 20(5-6), 801-821.
Kharrat, N., Ali, Y. B., Marzouk, S., Gargouri, Y.-T., & Karra-Châabouni, M. (2011). Immobilization of Rhizopus oryzae lipase on silica aerogels by adsorption: Comparison with the free enzyme. Process Biochemistry, 46(5), 1083-1089.
Kim, Hong, S.-H., Shin, K.-C., Jo, Y.-S., & Oh, D.-K. (2014). Characterization of a F280N variant of L-arabinose isomerase from Geobacillus thermodenitrificans identified as a D-galactose isomerase. Applied Microbiology and Biotechnology, 98(22), 9271-9281.
Kim, K. R., Seo, E. S., & Oh, D. K. (2014). L-Ribose production from L-arabinose by immobilized recombinant Escherichia coli co-expressing the L-arabinose isomerase and mannose-6-phosphate isomerase genes from Geobacillus thermodenitrificans. Applied Biochemistry and Biotechnology, 172(1), 275-288.
Kim, P.-S., Shin, H.-D., Park, J.-K., & Lee, Y.-H. (2000). Immobilization of cyclodextrin glucanotransferase on amberlite IRA-900 for biosynthesis of transglycosylated xylitol. Biotechnology and Bioprocess Engineering, 5(3), 174.
Kiviharju, K., Helanto, M., & Nyyssölä, A. (2007). L-Ribulose production using resting cells of genetically modified Lactobacillus plantarum. Journal of Biotechnology, 2(131), S98-S99.
Kumar, A. G., Swarnalatha, S., Kamatchi, P., Kirubagaran, R., Perinmbam, K., & Sekaran, G. (2009). Immobilization of proteolytic enzyme on highly porous activated carbon derived from rice bran. Journal of Porous Materials, 16(4), 439-445.
Kweon, D.-H., Kim, S.-G., Han, N. S., Lee, J. H., Chung, K. M., & Seo, J.-H. (2005). Immobilization of Bacillus macerans cyclodextrin glycosyltransferase fused with poly-lysine using cation exchanger. Enzyme and Microbial Technology, 36(4), 571-578.
Leandro, P., & Gomes, C. M. (2008). Protein misfolding in conformational disorders: rescue of folding defects and chemical chaperoning. Mini Reviews in Medicinal Chemistry, 8(9), 901-911.
Lee, S. Y. (1996). High cell-density culture of Escherichia coli. Trends in Biotechnology, 14(3), 98-105.
Levin, Y. (2002). Electrostatic correlations: from plasma to biology. Reports on Progress in Physics, 65(11), 1577.
Li, Z., Gao, Y., Nakanishi, H., Gao, X., & Cai, L. (2013). Biosynthesis of rare hexoses using microorganisms and related enzymes. Beilstein Journal of Organic Chemistry, 9(1), 2434-2445.
Mamat, U., Woodard, R. W., Wilke, K., Souvignier, C., Mead, D., Steinmetz, E., Terry, K., Kovacich, C., Zegers, A., & Knox, C. (2013a). Endotoxin-free protein production—ClearColi™ technology. Nature Methods, 10(9), 916.
Mateus, D., Alves, S., & Da Fonseca, M. (1999). Diffusion in cell‐free and cell immobilising κ‐carrageenan gel beads with and without chemical reaction. Biotechnology and Bioengineering, 63(5), 625-631.
Min, L., & Changqing, C. (2000). Progress Studies of High Cell-density Culture of Recombinant Escherichia coli. Progress in Biotechnology-Peking, 20(2), 26-31.
Mitraki, A., & King, J. (1989). Protein folding intermediates and inclusion body formation. Biotechnology, 7(7), 690-697.
Morimoto, Terami, Y., Maeda, Y.-i., Yoshihara, A., Takata, G., & Izumori, K. (2013a). Cloning and characterization of the L-ribose isomerase gene from Cellulomonas parahominis MB426. Journal of Bioscience and Bioengineering, 115(4), 377-381.
Naranjo, J. C., Córdoba, A., Giraldo, L., García, V. S., & Moreno-Piraján, J. C. (2010). Lipase supported on granular activated carbon and activated carbon cloth as a catalyst in the synthesis of biodiesel fuel. Journal of Molecular Catalysis B:Enzymatic, 66(1-2), 166-171.
Nguyen, L. N., Hai, F. I., Dosseto, A., Richardson, C., Price, W. E., & Nghiem, L. D. (2016). Continuous adsorption and biotransformation of micropollutants by granular activated carbon-bound laccase in a packed-bed enzyme reactor. Bioresource Technology, 210, 108-116.
Okano, K. (2009). Synthesis and pharmaceutical application of L-ribose. Tetrahedron, 10(65), 1937-1949.
Önal, S., & Telefoncu, A. (2003). Comparison of chitin and amberlite IRA‐938 for α‐galactosidase immobilization. Artificial Cells, Blood Substitutes, and Biotechnology, 31(1), 19-33.
Osman, A., Symeou, S., Trisse, V., Watson, K. A., Tzortzis, G., & Charalampopoulos, D. (2014a). Synthesis of prebiotic galactooligosaccharides from lactose using bifidobacterial β-galactosidase (BbgIV) immobilised on DEAE-Cellulose, Q-Sepharose and amino-ethyl agarose. Biochemical Engineering Journal, 82, 188-199.
Ozes, O. N., Mayo, L. D., Gustin, J. A., Pfeffer, S. R., Pfeffer, L. M., & Donner, D. B. (1999). NF-κB activation by tumour necrosis factor requires the Akt serine–threonine kinase. Nature, 401(6748), 82-85.
Papp, E., & Csermely, P. (2006). Chemical chaperones: mechanisms of action and potential use. In Molecular Chaperones in Health and Disease, (pp. 405-416): Springer.
Pastinen, O., Visuri, K., Schoemaker, H. E., & Leisola, M. (1999). Novel reactions of xylose isomerase from Streptomyces Rubiginosus. Enzyme and Microbial Technology, 25(8), 695-700.
Ramana, S., Sharma-Natu, P., & Ghildiyal, M. C. (2002). Cytoplasmic effects on photosynthesis and ribulose-1, 5-bisphosphate carboxylase activity in Brassica species. Euphytica, 123(3), 361-365.
Restaino, O. F., Cimini, D., De Rosa, M., Catapano, A., & Schiraldi, C. (2011). High cell density cultivation of Escherichia coli K4 in a microfiltration bioreactor: a step towards improvement of chondroitin precursor production. Microbial Cell Factories, 10(1), 10.
Ringe, D., & Petsko, G. A. (2009). Q&A: What are pharmacological chaperones and why are they interesting? Journal of Biology, 8(9), 80.
Rodrigues, D. S., Cavalcante, G. P., Ferreira, A. L., & Gonçalves, L. R. (2008). Immobilization of Candida antarctica lipase type B by adsorption on activated carbon. Chemical and Biochemical Engineering Quarterly, 22(1), 125-133.
Sadana, A., & Henley, J. P. (1987). Single‐step unimolecular non‐first‐order enzyme deactivation kinetics. Biotechnology and Bioengineering, 30(6), 717-723.
Schein, C. H., & Noteborn, M. H. (1988). Formation of soluble recombinant proteins in Escherichia coli is favored by lower growth temperature. Biotechnology, 6(3), 291-294.
Schlieker, M., & Vorlop, K. D. (2006). A novel immobilization method for entrapment: LentiKats®. In Immobilization of enzymes and cells (pp. 333-343).
Shih, T. L., & Tseng, J. H. (2004). An efficient synthesis of L-allono-1, 4-lactone from 2, 3: 5, 6-di-O-isopropylidene-D-mannono-1, 4-lactone. Tetrahedron Letters, 45(8), 1789-1791.
Shimonishi, T., & Izumori, K. (1996). A new enzyme, L-ribose isomerase from Acinetobacter sp. strain DL-28. Journal of Fermentation and Bioengineering, 81(6), 493-497.
Spinelli, D., Fatarella, E., Di Michele, A., & Pogni, R. (2013). Immobilization of fungal (Trametes versicolor) laccase onto Amberlite IR-120 H beads: optimization and characterization. Process Biochemistry, 48(2), 218-223.
Tejayadi, S., & Cheryan, M. (1995). Lactic acid from cheese whey permeate. Productivity and economics of a continuous membrane bioreactor. Applied Microbiology and Biotechnology, 43(2), 242-248.
Thiry, M., & Cingolani, D. (2002). Optimizing scale-up fermentation processes. Trends in Biotechnology, 20(3), 103-105.
Tsumoto, K., Umetsu, M., Kumagai, I., Ejima, D., Philo, J. S., & Arakawa, T. (2004). Role of arginine in protein refolding, solubilization, and purification. Biotechnology Progress, 20(5), 1301-1308.
Ueda, T., Akuta, T., Kikuchi-Ueda, T., Imaizumi, K., & Ono, Y. (2016). Improving the soluble expression and purification of recombinant human stem cell factor (SCF) in endotoxin-free Escherichia coli by disulfide shuffling with persulfide. Protein Expression and Purification, 120, 99-105.
Vaz, R. P., & Ferreira Filho, E. X. (2019). Ion exchange chromatography for enzyme immobilization. In Applications of Ion Exchange Materials in Biomedical Industries, (pp. 13-27): Springer.
Vera, A., González‐Montalbán, N., Arís, A., & Villaverde, A. (2007). The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures. Biotechnology and Bioengineering, 96(6), 1101-1106.
Verdich, C., Madsen, J. L., Toubro, S., Buemann, B., Holst, J., & Astrup, A. (2000). Effect of obesity and major weight reduction on gastric emptying. International Journal of Obesity, 24(7), 899-905.
Vieira, I. d. C., & Fatibello-Filho, O. (1997). Amperometric biosensor for the determination of phenols using a crude extract of sweet potato (Ipomoea Batatas (L.) Lam.). Analytical Letters, 30(5), 895-907.
Wilding, K. M., Hunt, J. P., Wilkerson, J. W., Funk, P. J., Swensen, R. L., Carver, W. C., Christian, M. L., & Bundy, B. C. (2019). Endotoxin‐Free E. coli Based Cell Free Protein Synthesis: Pre‐Expression Endotoxin Removal Approaches for on Demand Cancer Therapeutic Production. Biotechnology Journal, 14(3), 1800271.
Woodward, J. (1985). Immobilised Cells and Enzymes: IRL press.
Woodyer, R. D., Wymer, N. J., Racine, F. M., Khan, S. N., & Saha, B. C. (2008). Efficient production of L-ribose with a recombinant Escherichia coli biocatalyst. Applied and Environmental Microbiology, 74(10), 2967-2975.
Xin, F., Dong, W., Dai, Z., Jiang, Y., Yan, W., Lv, Z., Fang, Y., & Jiang, M. (2019). Biosynthetic Technology and Bioprocess Engineering. In Current Developments in Biotechnology and Bioengineering, (pp. 207-232): Elsevier.
Xu, Z., Li, S., Feng, X., Liang, J., & Xu, H. (2014). L-Arabinose isomerase and its use for biotechnological production of rare sugars. Applied Microbiology and Biotechnology, 98(21), 8869-8878.
Yeom, S.-J., Kim, N.-H., Park, C.-S., & Oh, D.-K. (2009). L-ribose production from L-arabinose by using purified L-arabinose isomerase and mannose-6-phosphate isomerase from Geobacillus thermodenitrificans. Applied and Environmental Microbiology, 75(21), 6941-6943.
Yeom, S.-J., Seo, E.-S., Kim, B.-N., Kim, Y.-S., & Oh, D.-K. (2011). Characterization of a mannose-6-phosphate isomerase from Thermus thermophilus and increased L-ribose production by its R142N mutant. Applied and Environmental Microbiology, 77(3), 762-767.
Yun, M., Moon, H. R., Kim, H. O., Choi, W. J., Kim, Y.-C., Park, C.-S., & Jeong, L. S. (2005). A highly efficient synthesis of unnatural L-sugars from D-ribose. Tetrahedron Letters, 46(35), 5903-5905.
Zhang, J., Suflita, M., Li, G., Zhong, W., Li, L., Dordick, J. S., Linhardt, R. J., & Zhang, F. (2015). High cell density cultivation of recombinant Escherichia coli strains expressing 2-O-sulfotransferase and C5-epimerase for the production of bioengineered heparin. Applied Biochemistry and Biotechnology, 175(6), 2986-2995.
Zhou, Y., Ma, X., Hou, Z., Xue, X., Meng, J., Li, M., Jia, M., & Luo, X. (2012). High cell density cultivation of recombinant Escherichia coli for prodrug of recombinant human GLPs production. Protein Expression and Purification, 85(1), 38-43.
吳泰徵. (2017). Actinotalea fermentans ATCC 43279 來源重組 L-核糖異構酶之特性與固定化並以蛋白質工程改變其熱穩定性. 國立臺灣海洋大學, 基隆市.
許仲霆. (2014). 利用蛋白質工程提昇 D-阿洛酮糖表異構酶之活性回收及熱穩定性. 國立臺灣海洋大學, 基隆市.
游銘遠. (2012). Geodermatophilus obscurus DSM 43160 來源之 L-核糖異構酶之基因選殖、表現、純化及特性探討. 國立臺灣海洋大學, 基隆市.
黃幸光. (2014). 熱穩定性阿拉伯糖異構酶之表現、特性探討、稀有糖類生產及蛋白質工程. 國立臺灣海洋大學, 基隆市.
劉俞均. (2018). 以蛋白質工程及固定化菌體改變 Actinotalea fermentans ATCC 43279 來源重組 L-核糖異構酶之熱穩定性. 國立臺灣海洋大學, 基隆市.
蔡佳樺. (2017). Thermoanaerobacterium saccharolyticum NTOU1 L-阿拉伯糖異構酶之固定化與利用定位點突變提昇其反應速率. 國立臺灣海洋大學, 基隆市.
羅珮芸. (2019). 利用固定化的 L-阿拉伯糖異構酶及 L-核糖異構酶生產稀有醣類. 國立臺灣海洋大學, 基隆市.