臺灣博碩士論文加值系統

本實驗主要利用羧酸化磁珠固定C端帶有 7 個離胺酸標籤之 L-鼠李糖異構酶 (L-RhI-7K)，進行固定化之條件探討以及特性分析。
L-鼠李糖異構酶 (L-RhI) 可催化 L-rhamnose 及 L-rhamnulose 之間的轉換外，還可催化多種醛糖與酮糖間的轉換。Thermoanaerobacterium saccharolyticum NTOU1 來源之 L-RhI 為目前酵素活性產量最高之 L-RhI，比其他來源之 L-RhI 高三倍以上，故有潛力運用在固定化上。本實驗之羧酸化磁珠利用 Fe2O3 與 FeO 之共同沉澱反應 (co-precipitation) 形成 Fe3O4，再經己二酸修飾後產生。而重組鼠李糖異構酶則是源自 Thermoanaerobacterium saccharolyticum NTOU1，其蛋白質序列 C 端經修飾上 7 個離胺酸，成為離胺酸標籤 (Lysine tag)。利用此胺基酸之正電荷與羧酸化磁珠進行結合。以 DNA 重組技術建構 pET-21b-rhi-7k 後，送入 E. coli BL21-CodonPlus(DE3)-RIL (簡稱為 BL21C) 表現宿主中，以 IPTG 誘導其大量表現，經過修飾之 L-RhI-7K於 BL21C 中最佳 IPTG 誘導濃度為 0.05 mM，其分子量與原生型之 L-RhI 相近，為 48 kDa，為了讓 L-RhI-7K 的純度提高，以利於固定化條件之測試，先將粗酵素液以 80 ℃ 熱處理 15 分鐘後去除掉大部份雜蛋白，再以離子交換進行純化。
L-RhI-7K 之最適作用溫度為 75 ℃，最適作用 pH為 7，有良好之熱穩定性及 pH 穩定性 (pH 5-9)，但置於 65 ℃， pH 5-9 中 24小時，活性下降約 50% ，酵素反應時需金屬離子才有活性，與原生型之 L-RhI 相比並無明顯改變。將 L-RhI-7K 進行固定化之條件探討，當酵素存在於 pH 8 及 80 mM之緩衝液的條件下，經固定 10 分鐘後，可達到最大吸附率，故本研究之後續實驗皆採用 pH 8 及 80 mM 緩衝液之條件下進行固定化。使用反應曲面法預測固定化酵素之最佳反應溫度及 pH 值為 73.55 ℃ 及 pH 7.6，熱穩定性及 25 ℃ 之 pH 穩定性無明顯改變，而 65 ℃ 之 pH 穩定性的探討中，固定化酵素之活性大幅下降 90%。固定化酵素在儲藏 30 天後，下降 49.5% 之活性。經過重複反應 7 次後，與第一次反應相比，剩餘 20% 之相對活性。

In this study, we immobilized C-terminally lysine-tagged L-rhamnose isomerase (L-RhI-7K) by carboxylated magnetic particles, and optimised the immobilization conditions and characterized of the immobilized L-RhI-7K.
L-rhamnose isomerase (L-RhI) not only catalyzes the aldose-ketose isomerization between L-rhamnose and L-rhammulose, but also between many aldose and ketoses. The activity of L-RhI from T. saccharolyticum is three times higher than L-rhamnose isomerases from other sources, so it has the potential to be immobilized. In this study, the carboxylated magnetic particles were prepared by the co-precipitation of Fe2O3 and FeO in aqueous medium and then modified with adipic acid. The gene of the recombinant L-RhI is obtained from T. saccharolyticum, and the recombinant L-RhI is tagged C-terminally with hepta-lysines (lysine tag). The positive charge of lysine residues allows the emzyme to bind to the negatively charged magnetic particles. The pET-21b-rhi-7k was constructed, and transformed into E. coli BL21-CodonPlus(DE3)-RIL (BL21C), and expressed under the presence of IPTG. The molecular weights of monomer of L-RhI-7K, which is close to that of wild-type L-RhI is 48kDa. For raising the purifity of L-RhI-7K, the crude enzyme is heated at 80 ℃ in 15 min to denature other proteins, and then purified by ion-exchange chromatography.
L-RhI-7K shows the optimal temperature at 75 ℃ and the optimal pH at pH 7, and presents wide pH stability (pH 5-9) and good thermostability. However the residual activity is decreased to about 50% at 65 ℃ and pH 5-9 for 24 h. When L-RhI-7K is incubated in the condition of pH 8 and 80 mM Tris-HCl and immobilized 10 min, the best immobilized conditionc was obtained and allowed the adsorption efficiency to reach maximum, so we use this condition to immobilize L-RhI-7K. The optimal temperature and pH of immobilized L-RhI-7K estimated by using response surface methodology are 73.55 ℃ and pH 7.6. The thermostability and pH stability at 25 ℃ are not changed obviously, but the residual activity is decreased about 90% at 65 ℃ under various pH values. After the storage of 30 days, the immobilized L-RhI-7K has decreased 49.5% activity. After 7 times of usage, the immobilized L-RhI-7K remains only 20% relative activity of the first reaction.

圖目錄 V
表目錄 VI
壹、研究背景與目的 1
貮、文獻整理 2
一、L-鼠李糖異構酶 2
1. 鼠李糖異構酶背景 2
2. 源自 T. saccharolyticum NTOU1之 L-RhI 2
二、稀有糖類 (RARE SUGARS) 2
1. 簡介 2
2. D-阿洛糖 (D-allose) 2
三、固定化酵素 3
1. 簡介 3
2. 固定化酵素之方法 3
3. 磁珠 (magnetic particles) 4
4. 羧酸化磁珠 (carboxylated magnetic particles) 4
叁、實驗設計與流程 5
一、C 端修飾離胺酸標籤 L-RHI 之基因重組、表現及純化及特性分析 5
1. T. saccharolyticum NTOU1 之 L-RhI 之胺基酸序列 C 端修飾離胺酸標籤 5
2. L-RhI-7K 表現、生產與純化 5
3. L-RhI-7K特性分析 6
二、利用羧酸化磁珠固定 L-RHI-7K 之條件探討 6
三、固定化 L-RHI-7K特性分析 6
肆、實驗材料與方法 7
一、實驗材料與設備 7
1. 實驗菌株、基因 7
2. 酵素 7
3. 實驗材料清單 7
3. 固定化載體材料 8
4. 儀器設備 8
5. 電腦軟體 9
二、實驗方法 10
1. C 端修飾離胺酸標籤 L-RhI 基因重組、表現、純化及特性分析 10
2. 利用羧酸化磁珠固定 L-RhI-7K 之條件探討 15
3. 固定化 L-RhI-7K 之特性探討 16
伍、結果與討論 18
一、C 端修飾離胺酸標籤 L-RHI 之基因重組、表現及純化及特性分析 18
二、利用羧酸化磁珠固定 L-RHI-7K 之條件探討 18
三、固定化 L-RHI-7K 之特性探討 19
陸、結論 20
柒、參考文獻 21

王鼎中，2012，由攜帶藍藻蛋白合成酶基因的重組乳酸菌生產藍藻蛋
白與利用反應曲面法進行培養基最適化之探討，國立臺灣海洋大學食品科學系碩士學位論文，基隆。
林嘉銳，2012，熱穩定鼠李糖異構酶之表現、純化、特性探討及蛋白質工程，國立臺灣海洋大學食品科學系博士學位論文，基隆。
吳文騰，2003，生物產業技術概論，國立清華大學出版社，台北。
范逸軒，1998，脂肪分解酵素 Pseudomonas cepacia 固定於奈米纖維膜並應用在生質柴油製程之研究，國立成功大學化學工程學系碩士學位論文，台南。
陳國誠，1992，微生物酵素工程學，藝軒圖書出版社，台北。
陳雅嵐，2012，源自 T. saccharolyticum NTOU1 之 L-鼠李糖異構酶的固定化探討，國立臺灣海洋大學食品科學系碩士學位論文，基隆
黃偉成，2003，以反應曲面法探討鱈魚魚漿製品最適化之研究，屏東科技大學食品科學系碩士學位論文，屏東。
Abudiab T (1998) Preparation of magnetic immobilized metal affinity separation media and its use in the isolation of proteins. J Chromatogr A 795: 211-217.
Bertelsen H, Jensen BB, Buemann B (1999) D-tagatose a novel low-calorie bulk sweetener with prebiotic properties. World Rev Nutr Diet 85: 98-109.
Bhuiyan SH, Itami Y, Takada G, Izumori K (1999) Preparation of L-talose and D-gulose form L-tagatose and D-sorbose,respectively,using immobilized L-rhamnose isomerase. Biosci Bioeng 88: 567-570.
Baker DC, Horton D, Tindall CG (1972) Large-scale preparation of allose: Obervations on the stereoselectivity of the reduction of 1,2:5,6-di-O-isopropyl-idene-α-D-ribo-hexofuranos-3-ulose hydrate. Carbohydr Res 24: 192-197.
Cao L (2005) Carrier-bound immobilized enzymes : principles, application and design. Chapters 2: 53-168.
Chibata I (1978) Applications of immobilized enzyme and microbial cells. New york: Halsted press.
Chen WP, Anderson AW (1979) Purification, Immobilization, and Some Properties of Glucose Isomerase from Streptomyces flavogriseus. Appl Environ Microbiol 38: 1111-1119.
Cowan DA, Fernandez-Lafuente R (2011) Enhancing the functional properties of thermophilic enzymes by chemical modification and immobilization. Enzyme Microb Technol 49: 326-346.
Fang Y, Huang XJ, Chen PC, Xu ZK (2011) Polymer materials for enzyme immobilization and their application in bioreactors. BMB Rep 44: 87-95.
Fernandes P (2010) Enzymes in food processing: a condensed overview on strategies for better biocatalysts. Enzyme Res 2010: 862537.
Granström TB, Takata G, Tokuda M, Izumori K (2004) Izumoring: a novel and complete strategy for bioproduction of rare sugars. J Biosci Bioeng 97: 89-94.
Garcia J E, Shen GJ, Alajarin R, Wong CH (1995) Cloning and overexpression of rhamnose isomerase and fucose isomerase. Bioorg Med Chem 3: 1349-1355.
Hirata Y, Saito M, Tsukamoto I, Yamaguchi F, Sui L, Kamitori K, Dong Y, Uehara E, Konishi R, Janjua N, Tokuda M (2009) Analysis of the inhibitory mechanism of D-allose on MOLT-4F leukemia cell proliferation. J Biosci Bioeng 107: 562-568.
Hornby WE, Lilly MD, Crook EM (1966) The preparation and properties of ficin chemically attached to carboxymethylcellulose. Biochem J 98: 420-425.
Hossain MA, Izuishi K, Tokuda M, Izumori K, Maeta H. (2004) D-Allose has a strong suppressive effect against ischemia-reperfusion injury: a comparative study with allopurinol and superoxide dismutase. J Hepatobiliary Pancreat Surg 11: 181–9.
Hoshikawa H, Mori T, Mori N (2010) In vitro and in vivo effects of D-allose: up-regulation of thioredoxin-interacting protein in head and neck cancer cells. Ann Otol Rhinol Laryngol 119: 567-571.
Jeong LS, Yoo SJ (1998) Synthesis and antiviral activity of novel isodideoxy nucleosides with exocyclic methylene. Bioorg Med Chem Lett 8: 847-852.
Kim HJ, Oh DK (2005) Purification and characterization of an L-arabinose isomerase from an isolated strain of Geobacillus thermodenitrificans producing D-tagatose. J Biotechnol 120: 162-173.
Korndorfer IP, Fessner WD, Matthews BW (2000) The structure of rhamnose isomerase from Escherichia coli and its relation with xylose isomerase illustrates a change between inter and intra-subunit complementation during evolution. J Mol Biol 300: 917-933.
Kuo CH, Liu YC, Chang CM, Chen JH, Chang C,Shieh CJ (2012) Optimum conditions for lipase immobilization on chitosan-coated Fe3O4 nanoparticles. Carbohydrate Polymers 87: 2538-2545.
Leang K, Takada G, Ishimura A, Okita M, Izumori K (2004) Cloning, nucleotide sequence, and overexpression of the L-rhamnose isomerase gene from Pseudomonas stutzeri in Escherichia coli. Appl Environ Microbiol 70: 3298-3304.
Levin GV (2002) Tagatose, the new GRAS sweetener and health product. J. Med. Food 5: 1-19.
Li CK, Wen AY, Shen BC, Lu J, Huang Y, Chang YC (2011) FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method. BMC Biotechnology 11: 92.
Lin LL, Chen YH, Chi MC, Wang TF, Chen JC (2012) Preparation of Magnetic Nanoparticles and Their Use for Immobilization of C-Terminally Lysine-Tagged Bacillus sp. TS-23 α-Amylase. Appl Biochem Biotechnol 166: 1711–1722
Lin LL, Huang CL, Cheng WC, Yang JC, Chi MC, Chen JH, Lin HP (2010) Preparation of carboxylated magnetic particles for the efficient immobilization of C-terminally lysine-tagged Bacillus stearothermophilus aminopeptidase II. J Ind Microbiol Biotechnol 37: 717–725.
Menavuvu BT, Poonperm w, Leang k, Noguchi N, Okada H, Morimoto k, Granstrom TB, Takasa G, Izumori K (2006) Efficient biosynthesis of D-allose from D-psicose by cross-linked recombinant L-rhamnose isomerase:separation of product by ethanol crystallization. Biosci bioeng 101: 340-345.
Messing RA (1975) Immobilized Enzymes for industrial reactors. Academic Press 1-10.
Milgrom P, Ly KA, Roberts MC, Rothen M, Mueller G, Yamaguchi DK (2006) Mutans Streptococci dose response to xylitol chewing gum. J Dent Res. 85: 177-181.
Miyazaki M, Maeda H (2006) Microchannel enzyme reactors and their applications for processing. Trends Biotechnol 24: 463-470.
Morimoto K, Park CS, Ozaki M, Takeshita K, Shimonishi T, Granström TB, Takata G, Tokuda M, Izumori K (2006) Large scale production of D-allose from D-psicose using continuous bioreactor and separation system. Enzyme Microb Technol 38: 855-859.
Nakamura T, Tanaka S, Hirooka K, Toyoshima T, Kawai N, Tamiya T, Shiraga F, Tokuda M, Keep RF, Itano T, Miyamoto O (2011) Anti-oxidative effects of D-allose, a rare sugar, on ischemia-reperfusion damage following focal cerebral ischemia in rat. Neurosci Lett 487: 103-106.
Nelson JM and Griffin EG (1916) Adsorption of invertase. J Am Chem Soc 38: 1109-15.
Panesar PS, Kumari S, Panesar R (2010) Potential Applications of Immobilized beta-Galactosidase in Food Processing Industries. Enzyme Res 2010: 473137.
PetroviT J, Clark RA (2005) Impact of surface immobilization and solution ionic strength on the formal potential of immobilized cytochrome. Langmuir 21: 6308–6316.
Poonperm W, Takata G, Okada H, Morimoto K, Granström TB, Izumori K (2007) Cloning, sequencing, overexpression and characterization of L-rhamnose isomerase from Bacillus pallidus Y25 for rare sugar production. Appl Microbiol Biotechnol 76: 1297-1307.
Prabhu P, Doan TT, Jeya M, Kang LW, Lee JK (2010) Cloning and characterization of a rhamnose isomerase from Bacillus halodurans. Appl Microbiol Biotechnol 89: 635–644.
Shaw SY, Chen YJ, Ou JJ, Ho L (2006) Preparation and characterization of Pseudomonas putida esterase immobilized on magnetic nanoparticles. Enzyme and Microbial Technol 39: 1089-1095.
Sultana I, Rahman Mizanur MD, Takeshita K, Takada G, Izumori K (2003) Direct production of D-arabinose from D -xylose by a coupling reaction using D -xylose isomerase, D -tagatose 3-epimerase and D -arabinose isomerase. J Biosci Bioeng 95: 342-347.
Sui L, Dong Y, Watanabe Y, Yamaguchi F, Hatano N, Izumori K, Tokuda M (2005) Growth inhibitory effect of D-allose on human ovarian carcinoma cells in vitro. Anticancer Res 25: 2639-2644.
Sui L, Nomura R, Dong Y, Yamaguchi F, Izumori K, Tokuda M (2007) Cryoprotective effects of D-allose on mammalian cekks. Cryobiology 55: 87-92
Tischer W, Wedekind F (1991) Immobilized Enzymes: Methods and Applications. Top curr chem 200: 95-126.
Tsai TL, Liu SM, Lee SC, Chen WJ, Chou SH, Hsu TC, Guo GL, Hwang WS, Wiegel J (2011) Ethanol production efficiency of an anaerobic hemicellulolytic thermophilic bacterium, strain NTOU1, isolated from a marine shallow hydrothermal vent in Taiwan. Microbes Environ 26: 317-324.
Wilson DM, Aji S (1957) Metabolism of L-rhamnose by Escherichia coli. Ι. L-rhamnose isomerase. J Bacteriol 73: 410-414.
Yasin Y (2011) Optimization of immobilization conditions of Thermomyces lanuginosus lipase on olive pomace powder using response surface methodology. Biocatal Agric Biotechnol 1: 39-44.
Yamaguchi F, Kamitori K, Sanada K, Horii M, Dong Y, Sui L, Tokuda M (2008) Rare sugar D-allose enhances anti-tumor effect of 5-fluorouracil on the human hepatocellular carcinoma cell line HuH-7. J Biosci Bioeng 106: 248-252.
Yoshida H, Yamada M, Ohyama Y, Takada G, Izumori K, Kamitori S (2007) The structures of L-rhamnose isomerase from Pseudomonas stutzeri in complexes with L-rhamnose and D-allose provide insights into broad substrate specificity. J Mol Biol 365: 1505-1516.