臺灣博碩士論文加值系統

麥芽寡糖苷海藻糖生成酶 (maltooligosyl trehalose synthase, MTSase) 可作用於麥芽寡糖 (maltooligosaccharide) 還原末端之α-1,4 糖苷鍵，將其轉為α-1,1 糖苷鍵，形成麥芽寡糖苷海藻糖，研究發現除了轉糖苷作用外，其也具有水解作用，會水解麥芽寡糖苷海藻糖，產生葡萄糖，此水解反應不利於海藻糖之生產，若能改變 MTSase 之基質特異性，降低水解效率同時維持轉糖苷效率，可能增加海藻糖產量。將 Sulfolobus acidocaldarius 來源之 MTSase 進行定位突變，建構 Y225L、I253Y、A438T、T439L、Y225L/A438T/T439L、I253Y/A438T/T439L 及 Y225L/I253Y/A438T/T439L 重組突變型質體，與重組原生型質體分別轉形至 ClearColi BL21 (DE3)-CodonPlus-RIL 中進行蛋白質表現，獲得重組原生型及突變型 MTSase。初步篩選發現 I253Y 及 A438T 之結構裂解，觀察模擬結構得知其與 T439L 三者殘基間會相互影響。轉糖苷動力學結果發現突變型 T439L 、I253Y/A438T/T439L 及 Y225L/I253Y/A438T/T439L 之 kcat 無明顯變化，但 kM 皆下降，轉糖苷催化效率 kcat/kM 皆高於原生型，而水解動力學則僅 I253Y/A438T/T439L 有較原生型低之水解催化效率。計算 selective ratio (相同轉糖苷效率下的水解效率比值)，發現上述突變型皆低於原生型，顯示此四個位置之胺基酸突變能降低 selective ratio。
另一方面，嘗試以 NHS-activated Sepharose 4 Fast Flow 將海藻糖生成相關酵素同時進行固定化並由澱粉生產海藻糖，結果發現固定化後之最高海藻糖產量僅約未固定化酵素產量的一半，且反應速率較慢，於反應 48 小時後仍未達平衡，而隨固定化酵素量增加，產量有提升之趨勢，期望後續能找到最適固定化條件並嘗試大量生產海藻糖。

Maltooligosyl trehalose synthase (MTSase) catalyzes an intramolecular transglycosyl reaction to form maltooligosyltrehalose by converting the α-1,4-glucosidic linkage at the reducing end of maltooligosaccharide to an α-1,1-glucosidic linkage. Except the transglycosyl reaction, the hydrolysis activity also exist causing release of glucose by MTSase. The hydrolysis reaction is unfavorable to trehalose production. If the substrate specificity of MTSase could be altered to decrease the hydrolysis activity while maintaining its transglycosyl activity, an increased trehalose yield would be anticipated. In order to decrease hydrolysis activity of MTSase from Sulfolobus acidocaldarius, the mutations Y225L, I253Y, A438T, T439L, Y225L/A438T/T439L, I253Y/A438T/T439L and Y225L/I253Y/A438T/T439L were constructed by site-directed mutagenesis. The wild-type and mutated recombinant DNA was transformed into ClearColi BL21 (DE3)-CodonPlus-RIL to express wild-type and mutant MTSases, respectively. Then the enzymes were purified and studied. The mutant I253Y and A438T were completely inactive. In the modling structure, the two residues were found interacting with T439L.
The mutantions T439L, I253Y/A438T/T439L and Y225L/I253Y/A438T/T439L have no significantly changes of kcat values for transglycosylation, but have lower kM values. And the kcat/kM values for transglycosylation of these mutations were higher than that of wild-type, the hydrolysis activity of these mutations were higher than that of wild-type, but their selective ratios ([kcat/kM (G3)] / [kcat/kM (G5)] × 100%) lower than that of wild-type.
On the other hand, the wild-type MTSase, along with isoamylase and MTHase, were immobilized on NHS-activated Sepharose 4 Fast Flow at the same time, and were studied for trehalose production. We found that the immobilized enzymes have only about half trehalose yield compared to that of free enzymes, and have lower reaction rate. The reaction still not reached equilibrium after 48 h, and we found that the more amounts of enzymes immobilized the higher trehalose yield obtained. Hope that we can find better immobilization condition and use immobilized enzymes to produce trehalose in the future.

摘要 I
Abstract II
目錄 III
表目錄 VI
圖目錄 VII
壹、研究背景與目的 1
貳、文獻整理 2
一、海藻糖 2
1. 簡介 2
2. 特性與應用 2
3. 生產 2
4. 利用澱粉生產海藻糖 4
5. 產量測定 4
二、麥芽寡糖苷海藻糖合成酶 5
1. 簡介 5
2. 結構 5
3. 作用機制 6
四、ClearColi BL21 (DE3) 大腸桿菌表現宿主 7
五、CodonPlus RIL 質體 8
六、固定化 8
1. 簡介 8
2. NHS-activated Sepharose 4 Fast Flow 8
參、實驗設計 9
一、SaMTSase 定位突變 9
二、重組質體於 ClearColi BL21 (DE3) 中表現 9
三、利用 NHS-activated Sepharose 4 Fast Flow 進行酵素固定化 9
肆、實驗材料與方法 11
一、實驗材料 11
1. 實驗用質體與菌株 11
2. 抗生素 11
3. 標準品 11
4. 市售套組 11
5. 酵素 11
6. 化學藥品 12
7. 實驗設備 13
二、藥品配製 15
1. LB 培養液 / 平板培養基 15
2. TB 培養液 15
3. SOB 培養液 15
4. SOC 培養液 16
5. 1% 洋菜糖膠體 16
6. 50X TAE buffer 16
7. Ampicillin (100 mg/mL) 16
8. Chloramphenicol (34 mg/mL) 16
9. 1 M IPTG 17
10. 蛋白質電泳膠體 17
11. 2X SDS sampling buffer 18
12. SDS-PAGE 染色液 18
13. SDS-PAGE 脫色液 18
14. 1 M Tris-HCl (pH 8.5) 18
15. 1 M Benzamidine 18
16. 4X Dye-binding reagent 19
17. BSA (10 mg/mL) 19
18. 0.25 M KHP buffer (pH 5.0) 19
19. DNS reagent 19
20. Glucose oxidase/peroxidase reagent 19
21. I2-KI reagent 19
三、實驗方法 20
1. 定位突變 20
2. DNA 電泳 21
3. 熱休克轉形至 DH5ɑ 勝任細胞 21
4. 以 Colony PCR 篩選轉形株 22
5. 基因定序 22
6. 抽取質體 DNA 22
7. 以電穿孔轉形重組質體至 ClearColi BL21 (DE3) 23
8. 菌種保存 24
9. ClearColi BL21 (DE3) 之蛋白質表現與誘導 24
10. 異澱粉酶、MTSase、MTHase 之大量生產 24
11. 異澱粉酶、MTSase、MTHase 之純化 25
12. 異澱粉酶、MTSase、MTHase 之活性分析 26
13. MTSase 酵素動力學 29
14. 酵素特性探討 30
15. 酵素固定化 30
16. 海藻糖產量試驗 31
伍、結果與討論 32
一、SaMTSase 定位突變 32
1. 突變設計 32
2. 突變方法 32
二、重組質體於 ClearColi BL21 (DE3) 中表現與純化 33
1. 突變之初步篩選 33
2. 表現與純化 33
3. 動力學探討 34
4. 特性分析 34
5. 突變結果分析 35
6. 海藻糖產量試驗 36
三、以固定化酵素由澱粉生產海藻糖 36
1. 利用 NHS-activated Sepharose 4 Fast Flow 固定化酵素 36
2. 由澱粉生產海藻糖 37
陸、 結論 38
柒、 參考文獻 39
捌、 圖表 43
玖、 附錄 73

王美瓔，2005，基質次結合部位殘基突變後對於麥芽糖寡苷海藻糖水解酶之活性與基質選擇性的影響，國立臺灣海洋大學食品科學系碩士學位論文，基隆。
石東原，2004，活性部位殘基突變後對於海藻糖生成酶之活性與基質選擇性的影響，國立臺灣海洋大學食品科學系碩士學位論文，基隆。
余文青，2008，利用 Sulfolobus solfataricus ATCC 35092 之原生型與突變型麥芽寡糖苷海藻糖生成酶及麥芽寡糖苷海藻糖水解酶由澱粉生產海藻糖之研究，國立臺灣海洋大學食品科學系碩士學位論文，基隆。
汪毓屏，2011，增加與減少基質結合部位殘基與基質間氫鍵對於麥芽寡糖苷海藻糖水解酶之活性與基質選擇性的影響，國立臺灣海洋大學食品科學系碩士學位論文，基隆。
黃幸光，2002，嗜高溫海藻糖生成相關酵素之基因選殖以及海藻糖苷糊精生成酶的生產與特性探討，國立臺灣海洋大學食品科學系碩士學位論文，基隆。
黃鈺婷，2016，Sulfolobus acidocaldarius 來源之麥芽寡糖苷海藻糖生成酶突變與固定化探討，國立臺灣海洋大學食品科學系碩士學位論文，基隆。
潘勁行，2004，活性部位苯丙胺酸殘基突變後對於海藻糖苷糊精生成酶之轉糖基與水解作用的影響，國立臺灣海洋大學食品科學系碩士學位論文，隆。
劉英嬋，2014，以枯草桿菌表現重組熱穩定性海藻糖生成相關酵素並由澱粉生產海藻糖，國立臺灣海洋大學食品科學系碩士學位論文，基隆。
蘇子涵，2015，熱穩定重組海藻糖生成相關酵素的固定化之研究，國立臺灣海洋大學食品科學系碩士學位論文，基隆。
Aboulaich N, Chung WK, Thompson JH, Larkin C, Robbins D, & Zhu M. (2014). A novel approach to monitor clearance of host cell proteins associated with monoclonal antibodies. Biotechnology progress, 30(5), 1114-1124.
Cejkova J, Cejka C, & Luyckx J. (2012). Trehalose treatment accelerates the healing of UVB-irradiated corneas. Comparative immunohistochemical studies on corneal cryostat sections and corneal impression cytology. Histology and Histopathology, 27(8), 1029-1040.
Chi Z, Wang JM, Chi ZM, & Ye F. (2010). Trehalose accumulation from corn starch by Saccharomycopsis fibuligera A11 during 2-l fermentation and trehalose purification. Journal of Industrial Microbiology and Biotechnology, 37(1), 19-25.
de Pascale D, Sasso MP, Di Lernia I, Di Lazzaro A, Furia A, Farina MC, Rossi M, & De Rosa M. (2001). Recombinant thermophilic enzymes for trehalose and trehalosyl dextrins production. Journal of Molecular Catalysis B: Enzymatic, 11(4), 777-786.
Di Lernia I, Morana A, Ottombrino A, Fusco S, Rossi M, & De Rosa M. (1998). Enzymes from Sulfolobus shibatae for the production of trehalose and glucose from starch. Extremophiles, 2(4), 409-416.
Doran PM, & Bailey JE. (1986). Effects of immobilization on growth, fermentation properties, and macromolecular composition of Saccharomyces cerevisiae attached to gelatin. Biotechnology and Bioengineering, 28(1), 73-87.
El-Bashiti T, Hamamcı H, Öktem HA, & Yücel M. (2005). Biochemical analysis of trehalose and its metabolizing enzymes in wheat under abiotic stress conditions. Plant Science, 169(1), 47-54.
Elbein AD, Pan Y, Pastuszak I, & Carroll D. (2003). New insights on trehalose: A multifunctional molecule. Glycobiology, 13(4), 17R-27R.
Eleutherio EC, Araujo PS, & Panek AD. (1993). Protective role of trehalose during heat stress in Saccharomyces cerevisiae. Cryobiology, 30(6), 591-596.
Fang TY, Hung XG, Shih TY, & Tseng WC. (2004). Characterization of the trehalosyl dextrin-forming enzyme from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092. Extremophiles, 8(4), 335-343.
Gangadharan D, Nampoothiri KM, Sivaramakrishnan S, & Pandey A. (2009). Immobilized bacterial α-amylase for effective hydrolysis of raw and soluble starch. Food research international, 42(4), 436-442.
Gueguen Y, Rolland JL, Schroeck S, Flament D, Defretin S, Saniez MH, & Dietrich J. (2001). Characterization of the maltooligosyl trehalose synthase from the thermophilic archaeon Sulfolobus acidocaldarius. FEMS Microbiology Letters, 194(2), 201-206.
Higashiyama T. (2002). Novel functions and applications of trehalose. Pure and Applied Chemistry, 74(7), 1263-1269.
Hounsa CG, Brandt EV, Thevelein J, Hohmann S, & Prior BA. (1998). Role of trehalose in survival of Saccharomyces cerevisiae under osmotic stress. Microbiology, 144(3), 671-680.
Kammann M, Laufs Jr, Schell J, & Gronenborn B. (1989). Rapid insertional mutagenesis of DNA by polymerase chain reaction (PCR). Nucleic Acids Research, 17(13), 5404.
Kato M, Miura Y, Kettoku M, Shindo K, Iwamatsu A, & Kobayashi K. (1996). Purification and characterization of new trehalose-producing enzymes isolated from the hyperthermophilic archae, Sulfolobus solfataricus KM1. Bioscience, Biotechnology, and Biochemistry, 60(3), 546-550.
Kato M, Takehara K, Kettoku M, Kobayashi K, & Shimizu T. (2000). Subsite structure and catalytic mechanism of a new glycosyltrehalose-producing enzyme isolated from the hyperthermophilic archaeum, Sulfolobus solfataricus KM1. Bioscience, Biotechnology, and Biochemistry, 64(2), 319-326.
Kobayashi K, Kato M, Miura Y, Kettoku M, Komeda T, & Iwamatsu A. (1996). Gene cloning and expression of new trehalose-producing enzymes from the hyperthermophilic archaeum Sulfolobus solfataricus KM1. Bioscience, Biotechnology, and Biochemistry, 60(11), 1882-1885.
Kobayashi K, Komeda T, Miura Y, Kettoku M, & Kato M. (1997). Production of trehalose from starch by novel trehalose-producing enzymes from Sulfolobus solfataricus KM1. Journal of fermentation and bioengineering, 83(3), 296-298.
Kobayashi M, Kubota M, & Matsuura Y. (2003). Refined structure and functional implications of trehalose synthase from Sulfolobus acidocaldarius. Journal of Applied Glycoscience, 50(1), 1-8.
Kubota M, Maruta K, Fukuda S, Kurimoto M, Tsujisaka Y, Kobayashil M, & Matsuura Y. (2001). Structure and function analysis of malto-oligosyltrehalose synthase. Journal of Applied Glycoscience, 48(2), 153-161.
Lama L, Nicolaus B, Trincone A, Morzillo P, De Rosa M, & Gambacorta A. (1990). Starch conversion with immobilized thermophilic archaebacterium Sulfolobus solfataricus. Biotechnology Letters, 12(6), 431-432.
Ling MM, & Robinson BH. (1997). Approaches to DNA mutagenesis: an overview. Analytical biochemistry, 254(2), 157-178.
Mamat U, Wilke K, Bramhill D, Schromm AB, Lindner B, Kohl TA, Corchero JL, Villaverde A, Schaffer L, & Head SR. (2015). Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins. Microbial cell factories, 14(1), 1.
Mamat U, Woodard RW, Wilke K, Souvignier C, Mead D, Steinmetz E, Terry K, Kovacich C, Zegers A, & Knox C. (2013). Endotoxin-free protein production-ClearColi (TM) technology. In): Nature publishing group, macmillan building, 4 crinan st, London N1 9XW, England.
Maruta K, Hattori K, Nakada T, Kubota M, Sugimoto T, & Kurimoto M. (1996). Cloning and sequencing of trehalose biosynthesis genes from Rhizobium sp. M-ll. Bioscience, Biotechnology, and Biochemistry, 60(4), 717-720.
Matsuo T. (2001). Trehalose protects corneal epithelial cells from death by drying. British Journal of Ophthalmology, 85(5), 610-612.
Nakada T, Ikegami S, Chaen H, Kubota M, Fukuda S, Sugimoto T, Kurimoto M, & Tsujisaka Y. (1996). Purification and characterization of thermostable maltooligosyl trehalose trehalohydrolase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Bioscience, biotechnology, and biochemistry, 60(2), 263-266.
Richards A, Krakowka S, Dexter L, Schmid H, Wolterbeek A, Waalkens Berendsen D, Shigoyuki A, & Kurimoto M. (2002). Trehalose: a review of properties, history of use and human tolerance, and results of multiple safety studies. Food and Chemical Toxicology, 40(7), 871-898.
Rosano GL, & Ceccarelli EA. (2009). Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain. Microbial cell factories, 8(1), 41.
Schiraldi C, Di Lernia I, & De Rosa M. (2002). Trehalose production: exploiting novel approaches. Trends in Biotechnology, 20(10), 420-425.
Teramoto N, Sachinvala ND, & Shibata M. (2008). Trehalose and trehalose-based polymers for environmentally benign, biocompatible and bioactive materials. Molecules, 13(8), 1773-1816.
Tseng WC, Lin JW, Wei TY, & Fang TY. (2008). A novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method. Analytical Biochemistry, 375(2), 376-378.
Zhang Y, Zhang T, Chi Z, Wang JM, Liu GL, & Chi ZM. (2010). Conversion of cassava starch to trehalose by Saccharomycopsis fibuligera A11 and purification of trehalose. Carbohydrate Polymers, 80(1), 13-18.