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) 可作用在麥芽寡糖 (maltooligosaccharide) 還原末端上的 α-1,4 糖苷鍵，使其轉變為 α-1,1 糖苷鍵，形成麥芽寡糖苷海藻糖，工業上搭配麥芽寡糖苷海藻糖水解酶 (maltooligosyl trehalose trehalohydrolase, MTHase) 水解麥芽寡糖基與海藻糖基之間的鍵結，以此生產海藻糖。而麥芽寡糖苷海藻糖生成酶也會水解麥芽寡糖與生產的麥芽寡糖苷海藻糖，產生葡萄糖與麥芽寡糖，此水解反應則不利於海藻糖的生產。因此本實驗針對 Sulfolobus acidocaldarius 來源之 MTSase 進行定位突變以期能維持轉糖苷活性並減少水解活性。動力學結果可知轉糖苷催化效率並沒有太大變化，突變後 kM 值與 kcat 值皆有提升，因此整體轉糖苷催化速率 kcat/kM 並無提高。水解活性部分，kM 值與 kcat 值皆下降，經突變後水解速率 kcat/kM 由 0.083 s-1∙mM-1 下降至 0.038 s-1∙mM-1，表示突變有成功減少水解速率。
另一方面，酵素於生產上不易回收且不具重複利用性，因此希望藉由固定化酵素改善上述缺點，並進行原生型酵素的最適固定化條件探討。實驗利用海藻酸鈣與 NHS-activated Sepharose 4 Fast Flow 進行酵素的固定化，海藻酸鈣部分固定化後最適作用溫度提升至 65oC，最適作用 pH 為 5，與未固定酵素相同，然而其固定化效率僅有14%，推測原因為酵素從海藻酸鈣孔洞中洩漏導致固定化效率不高。NHS-activated Sepharose 4 Fast Flow 固定化效率有 95%，最適作用溫度為 70oC，較酵素提高 10oC，最適作用 pH 與酵素相同仍為 pH 5，然其成本較高因此再嘗試以海藻酸鈣固定化菌體。固定化菌體相較於固定化酵素其固定化效率提升至 37%，最適作用溫度為 70oC，最適作用 pH 為 5。熱穩定性方面，三種固定化方法固定後之酵素或菌體皆較未固定者差。雖然以 NHS-activated Sepharose 4 Fast Flow 固定化效率較佳，但考量成本後，原生型 MTSase 較適合以菌體進行固定化，期望後續能以此應用於生產。

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. MTSase acts with maltooligosyl trehalose trehalohydrolase (MTHase) which hydrolyzes the linkage between maltooligosaccharide group and trehalose group to produce trehalose. MTSase also hydrolyzes maltooligosaccharides and maltooligosyltrehaloses to liberate maltooligosaccharides and glucose. The hydrolysis reaction is unfavorable to trehalose production. In order to keep the transglycosyl activity and decrease hydrolytic activity of MTSase from Sulfolobus acidocaldarius, the mutant MTSase was constructed by site-directed mutagenesis. The wild-type and mutant MTSases were purified and studied to obtain their kinetic parameters. The kcat/kM value of mutant on transglycosyl reaction is similar to that of wild-type. The kcat/kM value of mutant on hydrolysis reaction reduces from 0.083 s-1∙mM-1 to 0.038 s-1∙mM-1.
On the other hand, enzyme immobilization make enzyme become reusable. Therefore, the wild-type MTSase was immobilized and studied for the optimum immobilization conditions. The alginate and NHS-activated Sepharose 4 Fast Flow were used to immobilize MTSase. The optimum temperature for alginate -immobilized MTSase was 65oC. The optimum pH for alginate -immobilized MTSase was 5. However, the immobilizaion efficiency of alginate -immobilized MTSase was only 14%. The low immobilization efficiency may due to the leakage of MTSase from alginate beads. The immobilizaion efficiency of NHS-activated Sepharose 4 Fast Flow was 95%, the optimum temperature was 65oC, and the optimum pH was 5. Nevertheless, NHS-activated Sepharose 4 Fast Flow was expensive, and then immobilize cells containing MTSase by alginate entraption was also studied. The immobilizaion efficiency of immobilized cells containing MTSase was 37%, the optimum temperature was 70oC, and the optimum pH was 5. Whether immobilized MTSase or MTSase-containing immobilized cells, their thermostabilities are decreased. Although the immobilizaion efficiency using NHS-activated Sepharose 4 Fast Flow is higher than the others, the low cost and medium immobilizaion efficiency of MTSase-containing immobilized cells may be more suitable for trehalose production.

壹、研究背景與目的 1
貳、文獻整理 2
一、海藻糖 2
1.海藻糖簡介 2
2.海藻糖的應用 2
3.海藻糖的生產 2
二、麥芽寡糖苷海藻糖生成酶 4
1. 麥芽寡糖苷海藻糖生成酶特性 4
2.麥芽寡糖苷海藻糖生成酶反應機制 5
三、定位突變 6
1.QuikChange site-directed mutagenesis 6
2.Megaprimer PCR 6
四、固定化酵素 7
1.固定化酵素簡介 7
2.固定化材料 7
3.固定化方法 8
五、蛋白質表現系統簡介 8
参、實驗設計 9
肆、實驗材料與方法 11
ㄧ、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
8.藥品配製 14
二、實驗方法 20
1.抽取質體DNA 20
2.DNA電泳 20
3.定位突變 20
4.電穿孔轉形至 Clearcoli BL21 (DE3) 勝任細胞 22
5.以Colony PCR 篩選轉形株 23
6.序列分析 23
7.菌種保存 24
8. Clearcoli BL21 (DE3) 添加不同濃度之 IPTG 24
9.蛋白質電泳 24
10. MTSase 之大量生產 25
11.MTSase 之純化 25
12. MTSase之活性測定 26
13. 蛋白質特性分析 27
14. 酵素固定化 28
15. 固定化菌體 32
伍、結果與討論 34
一、 SaMTSase 定位突變探討 34
1. SaMTSase 突變設計 34
2.原生型及突變型 SaMTSase 純化 34
3.動力學探討 35
二、固定化酵素之固定化條件探討 35
1.海藻酸鈣固定化酵素探討 35
2. NHS-activated Sepharose 4 Fast Flow固定化酵素探討 36
三、.固定化菌體之固定化條件探討 37
陸、結論 39
柒、參考文獻 40
捌、圖表 44
玖、附錄 66

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，基隆。
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。
李于昇，2015，源自 Thermoanaerobacterium saccharolyticum 之重組 L-鼠李糖異構酶固定化之探討，國立臺灣海洋大學食品科學系碩士學位論文，基隆。
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。
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。
吳采郿，2009，麥芽寡糖苷海藻糖生成酶之突變與活性篩選，國立臺灣海洋大學食品科學系碩士學位論文，基隆。
吳易皇，2015，Agrobacterium sp. ATCC 31750 D-阿洛酮糖表異構酶之蛋白質工程及利用固定化菌體生產 D-阿洛酮糖，國立臺灣海洋大學食品科學系碩士學位論文，基隆。
魏岑芸，2007，增加基質結合部位殘基與基質間氫鍵對於Sulfolobus solfataricusATCC35092麥芽寡糖苷海藻糖生成酶轉糖苷及水解作用之影響，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。
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。
Aboulaich N, Chung WK, Thompson JH, Larkin C, Robbins D, et al. (2014) A novel approach to monitor clearance of host cell proteins associated with monoclonal antibodies. Biotechnology Progress 30: 1114-1124.
Brady D, andJordaan J (2009) Advances in enzyme immobilisation. Biotechnology Letters 31: 1639-1650.
Cejkova J, Cejka C, andLuyckx 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: 1029-1040.
Cole S, Brosch R, Parkhill J, Garnier T, Churcher C, et al. (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393: 537-544.
Datta S, Christena LR, andRajaram YRS (2013) Enzyme immobilization: an overview on techniques and support materials. Biotech 3: 1-9.
DiCosimo R, McAuliffe J, Poulose AJ, andBohlmann G (2013) Industrial use of immobilized enzymes. Chemical Society Reviews 42: 6437-6474.
Doran PM, andBailey JE (1986) Effects of immobilization on growth, fermentation properties, and macromolecular composition of Saccharomyces cerevisiae attached to gelatin. Biotechnology and Bioengineering 28: 73-87.
Draget KI, Skjåk-Bræk G, andSmidsrød O (1997) Alginate based new materials. International Journal of Biological Macromolecules 21: 47-55.
El-Bashiti T, Hamamcı H, Öktem HA, andYücel M (2005) Biochemical analysis of trehalose and its metabolizing enzymes in wheat under abiotic stress conditions. Plant Science 169: 47-54.
Elbein AD, Pan Y, Pastuszak I, andCarroll D (2003) New insights on trehalose: a multifunctional molecule. Glycobiology 13: 17R-27R.
Fang T-Y, Hung X-G, Shih T-Y, andTseng W-C (2004) Characterization of the trehalosyl dextrin-forming enzyme from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092. Extremophiles 8: 335-343.
Fersht AR, Shi J, Knill J, Jones D, Lowe A, et al. (1985) Hydrogen bonding and biological specificity analysed by protein engineering. Nature 314: 235-238.
Fundueanu G, Nastruzzi C, Carpov A, Desbrieres J, andRinaudo M (1999) Physico-chemical characterization of Ca-alginate microparticles produced with different methods. Biomaterials 20: 1427-1435.
Giannou V, andTzia C (2008) Cryoprotective role of exogenous trehalose in frozen dough products. Food and Bioprocess Technology 1: 276-284.
Groboillot A, Boadi D, Poncelet D, andNeufeld R (1994) Immobilization of cells for application in the food industry. Critical Reviews in Biotechnology 14: 75-107.
Gueguen Y, Rolland J-L, Schroeck S, Flament D, Defretin S, et al. (2001) Characterization of the maltooligosyl trehalose synthase from the thermophilic archaeon Sulfolobus acidocaldarius. FEMS Microbiology Letters 194: 201-206.
Higashiyama T (2002) Novel functions and applications of trehalose. Pure and Applied Chemistry 74: 1263-1269.
Kammann M, Laufs Jr, Schell J, andGronenborn B (1989) Rapid insertional mutagenesis of DNA by polymerase chain reaction (PCR). Nucleic Acids Research 17: 5404.
Kato M, Takehara K, Kettoku M, Kobayashi K, andShimizu 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: 319-326.
Ke S-H, andMadison EL (1997) Rapid and efficient site-directed mutagenesis by single-tube ‘megaprimer’PCR method. Nucleic Acids Research 25: 3371-3372.
Kobayashi K, Kato M, Miura Y, Kettoku M, Komeda T, et al. (1996) Gene cloning and expression of new trehalose-producing enzymes from the hyperthermophilic archaeum Sulfolobus solfataricus KM1. Bioscience, Biotechnology, and Biochemistry 60: 1882-1885.
Kobayashi K, Komeda T, Miura Y, Kettoku M, andKato M (1997) Production of trehalose from starch by novel trehalose-producing enzymes from Sulfolobus solfataricus KM1. Journal of Fermentation and Bioengineering 83: 296-298.
Kobayashi M, Kubota M, andMatsuura Y (2003) Refined structure and functional implications of trehalose synthase from Sulfolobus acidocaldarius. Journal of Applied Glycoscience 50: 1-8.
Krajewska B (2004) Application of chitin-and chitosan-based materials for enzyme immobilizations: a review. Enzyme and Microbial Technology 35: 126-139.
Kubota M, Maruta K, Fukuda S, Kurimoto M, Tsujisaka Y, et al. (2001) Structure and function analysis of malto-oligosyltrehalose synthase. Journal of Applied Glycoscience 48: 153-161.
Lee KY, andMooney DJ (2012) Alginate: properties and biomedical applications. Progress in Polymer Science 37: 106-126.
Mamat U, Wilke K, Bramhill D, Schromm AB, Lindner B, et al. (2015) Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins. Microbial Cell Factories 14: 57.
Maruta K, Hattori K, Nakada T, Kubota M, Sugimoto T, et al. (1996) Cloning and sequencing of trehalose biosynthesis genes from Rhizobium sp. M-ll. Bioscience, Biotechnology, and Biochemistry 60: 717-720.
Mateo C, Palomo JM, Fernandez-Lorente G, Guisan JM, andFernandez-Lafuente R (2007) Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme and Microbial Technology 40: 1451-1463.
Migneault I, Dartiguenave C, Bertrand MJ, andWaldron KC (2004) Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking. Biotechniques 37: 790-806.
Nakada T, Ikegami S, Chaen H, Kubota M, Fukuda S, et al. (1996) Purification and characterization of thermostable maltooligosyl trehalose trehalohydrolase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Bioscience, Biotechnology, and Biochemistry 60: 263-266.
Nishizaki Y, Yoshizane C, Toshimori Y, Arai N, Akamatsu S, et al. (2000) Disaccharide-trehalose inhibits bone resorption in ovariectomized mice. Nutrition Research 20: 653-664.
Ohtake S, andWang YJ (2011) Trehalose: current use and future applications. Journal of Pharmaceutical Sciences 100: 2020-2053.
Panesar PS, Kumari S, andPanesar R (2010) Potential applications of immobilized β-galactosidase in food processing industries. Enzyme Research 2010.
Reignault P, Cogan A, Muchembled J, Lounes‐Hadj Sahraoui A, Durand R, et al. (2001) Trehalose induces resistance to powdery mildew in wheat. New Phytologist 149: 519-529.
Richards A, Krakowka S, Dexter L, Schmid H, Wolterbeek A, et al. (2002) Trehalose: a review of properties, history of use and human tolerance, and results of multiple safety studies. Food and Chemical Toxicology 40: 871-898.
Rodrigues RC, Ortiz C, Berenguer-Murcia A, Torres R, andFernández-Lafuente R (2013) Modifying enzyme activity and selectivity by immobilization. Chemical Society Reviews 42: 6290-6307.
Ross CA, andPoirier MA (2004) Protein aggregation and neurodegenerative disease. Nature Medicine 10: S10–S17.
Séraphin B, andKandels-Lewis S (1996) An efficient PCR mutagenesis strategy without gel purificiation step that is amenable to automation. Nucleic Acids Research 24: 3276-3277.
Saitoh Si, Akashi S, Yamada T, Tanimura N, Kobayashi M, et al. (2004) Lipid A antagonist, lipid IVa, is distinct from lipid A in interaction with Toll‐like receptor 4 (TLR4)‐MD‐2 and ligand‐induced TLR4 oligomerization. International Immunology 16: 961-969.
Schiraldi C, Di Lernia I, andDe Rosa M (2002) Trehalose production: exploiting novel approaches. TRENDS in Biotechnology 20: 420-425.
Sriamornsak P (1998) Preliminary investigation of some polysaccharides as a carrier for cell entrapment. European Journal of Pharmaceutics and Biopharmaceutics 46: 233-236.
Taqieddin E, andAmiji M (2004) Enzyme immobilization in novel alginate–chitosan core-shell microcapsules. Biomaterials 25: 1937-1945.
Teramoto N, Sachinvala ND, andShibata M (2008) Trehalose and trehalose-based polymers for environmentally benign, biocompatible and bioactive materials. Molecules 13: 1773-1816.
Tyagi R, Lai R, andDuggleby RG (2004) A new approach to'megaprimer'polymerase chain reaction mutagenesis without an intermediate gel purification step. BMC Biotechnology 4: 1.
White O, Eisen JA, Heidelberg JF, Hickey EK, Peterson JD, et al. (1999) Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1. Science 286: 1571-1577.