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研究生:張哲源
研究生(外文):Chang, Che-Yuan
論文名稱:源自 Thermoanaerobacterium saccharolyticum L-鼠李糖異構酶之突變、固定化條件探討及 D-阿洛糖的生產
論文名稱(外文):Mutation and Immobilization of RecombinantL-Rhamnose Isomerase from Thermoanaerobacterium saccharolyticum and Production of D-Allose
指導教授:方翠筠
指導教授(外文):Fang, Tsuei-Yun
口試委員:方翠筠曾文祺蔡國珍
口試委員(外文):Fang, Tsuei-YunTseng, Wen-ChiTsai,Guo-Jane
口試日期:2016-07-13
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:65
中文關鍵詞:L-鼠李糖異構酶固定化定位突變D-阿洛糖
外文關鍵詞:L-rhamnose isomeraseimmobilizationsite-directed mutagenesisD-allose
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D-阿洛糖 (D-allose) 為六碳醛糖,為一種稀有醣類,有研究利用價格低廉之 D-果糖,以D-阿洛酮糖表異構酶 (D-psicose-3-epimerase, DPE) 和 L-鼠李糖異構酶 (L-rhamnose isomerse, L-RhI) 進行異構化得到 D-阿洛糖。本研究先培養只含有 pET-21b-Tsrhi 或同時帶有 pET-21b-Tsrhi 及 CodonPlus-RIL 質體的 ClearColi BL21 (DE3),經過誘導後,利用 SDS-PAGE 分析 Thermoanaerobacterium saccharolyticum L-RhI (TsRhI) 的表現量,發現含有兩個質體的 ClearColi BL21 (DE3) 有最好的TsRhI 表現量,且經 37oC、32 小時誘導後具最高的菌液活性。
接著以最適誘導條件培養帶有兩個質體的 ClearColi BL21 (DE3),並用海藻酸鈣固定菌體後,以反應曲面法 (Response surface methodology, RSM) 探討固定化條件,結果得知,菌體濃度對固定化菌體之酵素活性具有最大的影響,經 Design-Expert 軟體預測,以 100 g/L 的菌體濃度、0.2 M 氯化鈣溶液及 0.05% 十六烷基三甲基溴化銨 (cetyltrimethylammonium bromide, CTAB) 進行菌體固定化後具有最高的酵素活性,以此條件進行固定化菌體可得 404 U/g-cells。
另外,本研究利用定位突變將 pET-21b-L59M/S60G-Tsrhi 雙突變質體突變為 pET-21b-L59M/S60G/I102Q-Tsrhi 三突變質體,並將其與 CodonPlus-RIL 同時轉形至 E. coli 與 ClearColi BL21 (DE3),以表現目標蛋白質,經純化後得到酵素比活性分別為 140 與 151 U/mg,接著進行酵素動力學分析,發現三突變酵素對 L-鼠李糖為基質的催化效率 (kcat/ KM) 提昇,對 D-阿洛糖為基質時的催化效率下降。
最後,將 30% (w/v) D-果糖、50 mM Tris-HCl (pH 7) 緩衝溶液及 0.25 mM 鈷離子先利用含有 DPE 活性之固定化菌體反應且經酵母菌去除果糖,即可獲得 D-阿洛酮糖反應液,利用含有 TsRhI 之固定化菌體進行重複利用性試驗,反應三次皆於 4 – 7 分鐘後達平衡,平衡後 D-阿洛酮糖與 D-阿洛糖的比例為 7 : 3。

D-Allose is an aldohexose, and it belongs to rare sugars. Obtaining D-allose from inexpensive D-fructose has been studied using D-psicose-3-epimerase (DPE) and L-rhamnose isomerase (L-RhI). In this study, the expression of Thermoanaerobacterium saccharolyticum L-RhI (TsRhI) in ClearColi BL21 (DE3) carrying pET-21b-Tsrhi only or both pET-21b-Tsrhi and CodonPlus-RIL was analysed by SDS-PAGE. ClearColi BL21 (DE3) carrying two vectors has the better expression of TsRhI and induced at 37oC for 32 h has the highest activity.
Response surface methodology (RSM) was then used to optimize the TsRhI activity of immobilized ClearColi BL21 (DE3) carrying two vectors by entrapping cells with calcium alginate. The results had shown that cell concentration significantly affects the TsRhI activity of immobilized cells. The optimized conditions to obtain the highest TsRhI activity of immobilized cells can be predicted by the Design-Expert software. The optimized conditions are 100 g/L cell concentration, 0.2 M calcium chloride, and 0.05% cetyltrimethylammonium bromide (CTAB). By using predicted optimized conditions to immobilize cells, the activity of TsRhI of immobilized cells can obtain 404 U/g-cells.
In addition, pET-21b-L59M/S60G/I102Q-Tsrhi was obtained from pET-21b-L59M/S60G-Tsrhi by site-directed mutagenesis. To express target protein, this study transformed both pET-21b-L59M/S60G/I102Q-Tsrhi and CodonPlus-RIL into E. coli BL21 (DE3) and ClearColi BL21 (DE3), respectively. Afer purification, the specific activities of L59M/S60G/I102Q TsRhIs expressed from E. coli BL21 (DE3) and ClearColi BL21 (DE3) are 140 and 151 U/mg, respectively. L59M/S60G/I102Q TsRhI has higher catalytic efficiency (kcat/ KM) against L-rhamnose and lower kcat/ KM against D-allose.
At last, immobilized cells containing DPE were used to produce D-psicose from 30% (w/v) D-fructose in 50 mM Tris-HCl buffer (pH 7.0) plus 0.25 mM Co2+. After epimerization, D-fructose was removed from reaction mixture by baker’s yeast treatment, and the D-psicose was obtained. To investigate the repeatability of immobilized cells containing TsRhI, the immobilized cells were used three times to produce D-allose from D-psicose. The reaction can reach equilibrium at the ratio of D-psicose : D-allsoe = 7 : 3 after 4 - 7 minutes in all three cycles of usage.

摘要 I
Abstract II
目錄 III
圖目錄 V
表目錄 VI
附錄 VII
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 (rare sugar) 3
二、L-鼠李糖異構酶 (L-rhamnose isomerase, L-RhI) 5
三、ClearColi BL21 (DE3) 6
四、E. coli BL21(DE3)-CodonPlus-RIL 7
五、固定化 7
六、反應曲面法 (Response surface methodology, RSM) 10
參、實驗設計與流程 12
一、單獨含有 pET-21b-Tsrhi 或同時含有 pET-21b-Tsrhi 及 CodonPlus-RIL 質體的 ClearColi BL21 (DE3) 之 L-鼠李糖異構酶表現 12
二、菌體固定化最適條件探討 12
三、TsRhI 活性區域基質結合相關胺基酸之定位突變 12
四、以海藻酸鈣固定化菌體生產 D-阿洛糖 13
肆、實驗材料與方法 14
一、實驗材料 14
二、實驗方法 19
伍、結果與討論 33
一、單獨含有 pET-21b-Tsrhi 或同時含有 pET-21b-Tsrhi 及 CodonPlus-RIL 質體的 ClearColi BL21 (DE3) 之 L-鼠李糖異構酶表現 33
二、菌體固定化最適條件探討 34
三、 TsRhI 活性區域基質結合相關胺基酸之定位突變 36
四、以海藻酸鈣固定化菌體生產 D-阿洛糖 37
陸、結論 38
柒、參考文獻 39
捌、圖表 44
玖、附錄 61


王蘭欣,2002,紅麴菌 Monascus pilosus 產孢與其天然色素生產之研究,朝陽科技大學應用化學系碩士學位論文,臺中。
方弘懿,2016,以蛋白質工程及固定化提昇 Agrobacterium sp. ATCC 31750 來源重組 D-阿洛酮糖表異構酶之熱穩定性,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。
吳文騰,2003,生物產業技術概論,國立清華大學出版社,台北。
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。
陳于君,2014,利用蛋白質工程改變 Thermoanaerobacterium saccharolyticum NTOU1 L-鼠李糖異構酶的基質特異性,國立臺灣海洋大學食品科學系碩士學位論文,基隆。
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,國立臺灣海洋大學食品科學系碩士學位論文,基隆。
彭詩婷,2014,利用 D-阿洛酮糖表異構酶及 L-鼠李糖異構酶生產 D-阿洛酮糖及 D-阿洛糖之最適化探討,國立臺灣海洋大學食品科學系碩士學位論文,基隆。
Baker DC, Horton D, Tindall CG (1972) Large-scale preparation of D-allose: observations on the stereoselectivity of the reduction of 1, 2: 5, 6-di-O-isopropylidene-α-D-ribo-hexofuranos-3-ulose hydrate. Carbohydrate research 24: 192-197.
Baysal Z, Bulut Y, Yavuz M, Aytekin Ç (2014) Immobilization of α‐amylase via adsorption onto bentonite/chitosan composite: Determination of equilibrium, kinetics, and thermodynamic parameters. Starch‐Stärke 66: 484-490.
Chibata I, Tosa T (1977) Transformations of organic compounds by immobilized microbial cells. Advances in Applied Microbiology 22: 27.
Chu CK, Ma T, Shanmuganathan K, Wang C, Xiang Y, Pai SB, Yao G-Q, Sommadossi J-P, Cheng Y-C (1995) Use of 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil as a novel antiviral agent for hepatitis B virus and Epstein-Barr virus. Antimicrobial Agents and Chemotherapy 39: 979-981.
DiCosimo R, McAuliffe J, Poulose AJ, Bohlmann G (2013) Industrial use of immobilized enzymes. Chemical Society Reviews 42: 6437-6474.
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: 73-87.
Fukada K, Ishii T, Tanaka K, Yamaji M, Yamaoka Y, Kobashi K-i, Izumori K (2010) Crystal structure, solubility, and mutarotation of the rare monosaccharide D-psicose. Bulletin of the Chemical Society of Japan 83: 1193-1197.
Fukumoto T, Kano A, Ohtani K, Yamasaki-Kokudo Y, Kim B-G, Hosotani K, Saito M, Shirakawa C, Tajima S, Izumori K (2011) Rare sugar D-allose suppresses gibberellin signaling through hexokinase-dependent pathway in Oryza sativa L. Planta 234: 1083-1095.
Groboillot A, Boadi D, Poncelet D, Neufeld R (1994) Immobilization of cells for application in the food industry. Critical Reviews in Biotechnology 14: 75-107.
Gumina G, Song G-Y, Chu CK (2001) L-Nucleosides as chemotherapeutic agents. FEMS Microbiology Letters 202: 9-15.
Hoshikawa H, Indo K, Mori T, Mori N (2011) Enhancement of the radiation effects by D-allose in head and neck cancer cells. Cancer Letters 306: 60-66.
Hossain M, Wakabayashi H, Goda F, Kobayashi S, Maeba T, Maeta H. Effect of the immunosuppressants FK506 and D-allose on allogenic orthotopic liver transplantation in rats; 2000. Elsevier. pp. 2021-2023.
Hossain MA, Izuishi K, Maeta H (2003) Protective effects of D‐allose against ischemia reperfusion injury of the rat liver. Journal of Hepato-Biliary-Pancreatic Surgery 10: 218-225.
Hossain MA, Kitagaki S, Nakano D, Nishiyama A, Funamoto Y, Matsunaga T, Tsukamoto I, Yamaguchi F, Kamitori K, Dong Y (2011) Rare sugar D-psicose improves insulin sensitivity and glucose tolerance in type 2 diabetes Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Biochemical and Biophysical Research Communications 405: 7-12.
Iga Y, Nakamichi K, Shirai Y, Matsuo T (2010) Acute and sub-chronic toxicity of D-allose in rats. Bioscience, Biotechnology, and Biochemistry 74: 1476-1478.
Kim SB, Kim HJ, Kim CJ. Enhancement of the enzymatic digestibility of waste newspaper using Tween; 2006. Springer. pp. 486-495.
Kim Y-S, Shin K-C, Lim Y-R, Oh D-K (2013) Characterization of a recombinant L-rhamnose isomerase from Dictyoglomus turgidum and its application for L-rhamnulose production. Biotechnology Letters 35: 259-264.
Kimura S, Zhang G-X, Nishiyama A, Nagai Y, Nakagawa T, Miyanaka H, Fujisawa Y, Miyatake A, Nagai T, Tokuda M (2005) D-allose, an all-cis aldo-hexose, suppresses development of salt-induced hypertension in Dahl rats. Journal of Hypertension 23: 1887-1894.
Korndörfer IP, Fessner W-D, 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. Journal of Molecular Biology 300: 917-933.
López A, Llinares F, Cortell C, Herraez M (2000) Comparative enhancer effects of Span® 20 with Tween® 20 and Azone® on the in vitro percutaneous penetration of compounds with different lipophilicities. International Journal of Pharmaceutics 202: 133-140.
Levin GV (2002) Tagatose, the new GRAS sweetener and health product. Journal of Medicinal Food 5: 23-36.
Lim Y-R, Oh D-K (2011) Microbial metabolism and biotechnological production of D-allose. Applied microbiology and biotechnology 91: 229-235.
Lin C-J, Tseng W-C, Lin T-H, Liu S-M, Tzou W-S, Fang T-Y (2010) Characterization of a thermophilic L-rhamnose isomerase from Thermoanaerobacterium saccharolyticum NTOU1. Journal of Agricultural and Food Chemistry 58: 10431-10436.
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 ™ technology. Nature Methods 10.
Matsuo T (2006) Inhibitory effects of D-psicose on glycemic responses after oral carbohydrate tolerance test in rats. Journal of Japanese Society of Nutrition and Food Science (Japan).
Matsuo T, Izumori K (2009) D-Psicose inhibits intestinal alpha-glucosidase and suppresses the glycemic response after ingestion of carbohydrates in rats. Journal of Clinical Biochemistry and Nutrition 45: 202.
Matsuo T, Suzuki H, Hashiguchi M, Izumori K (2002) D-psicose is a rare sugar that provides no energy to growing rats. Journal of Nutritional Science and Vitaminology 48: 77-80.
Mu W, Zhang W, Feng Y, Jiang B, Zhou L (2012) Recent advances on applications and biotechnological production of D-psicose. Applied Microbiology and Biotechnology 94: 1461-1467.
Murata A, Sekiya K, Watanabe Y, Yamaguchi F, Hatano N, Izumori K, Tokuda M (2003) A novel inhibitory effect of D-allose on production of reactive oxygen species from neutrophils. Journal of Bioscience and Bioengineering 96: 89-91.
Myers RH, Montgomery DC, Anderson-Cook CM (2009) Response surface methodology: process and product optimization using designed experiments: John Wiley & Sons.
Najafpour G, Younesi H, Ismail KSK (2004) Ethanol fermentation in an immobilized cell reactor using Saccharomyces cerevisiae. Bioresource Technology 92: 251-260.
Nakamura T, Tanaka S, Hirooka K, Toyoshima T, Kawai N, Tamiya T, Shiraga F, Tokuda M, Keep RF, Itano T (2011) Anti-oxidative effects of D-allose, a rare sugar, on ischemia-reperfusion damage following focal cerebral ischemia in rat. Neuroscience Letters 487: 103-106.
Nokhodchi A, Shokri J, Dashbolaghi A, Hassan-Zadeh D, Ghafourian T, Barzegar-Jalali M (2003) The enhancement effect of surfactants on the penetration of lorazepam through rat skin. International Journal of Pharmaceutics 250: 359-369.
Oshima H, Kimura I, IZUMORI K (2006) Psicose contents in various food products and its origin. Food Science and Technology Research 12: 137-143.
Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Recombinant Protein Expression in Microbial Systems: 7.
Salem-Bekhit MM, Jamous Y, Al-Anazi F, Bayomi M, Alsarrah I, Fathallah M (2012) Quality control of optimized hepatitis B plasmid DNA vaccine. African Journal of Biotechnology 11: 1231-1239.
Smidsrød O, Skja G (1990) Alginate as immobilization matrix for cells. Trends in Biotechnology 8: 71-78.
Sui L, Dong Y, Watanabe Y, Yamaguchi F, Hatano N, Tsukamoto I, Izumori K, Tokuda M (2005) The inhibitory effect and possible mechanisms of D-allose on cancer cell proliferation. International Journal of Oncology 27: 907-912.
Sun Y, Hayakawa S, Izumori K (2004) Modification of ovalbumin with a rare ketohexose through the Maillard reaction: effect on protein structure and gel properties. Journal of Agricultural and Food Chemistry 52: 1293-1299.
Sun Y, Hayakawa S, Ogawa M, Izumori K (2007) Antioxidant properties of custard pudding dessert containing rare hexose, D-psicose. Food Control 18: 220-227.
Sun Y, Hayakawa S, Puangmanee S, Izumori K (2006) Chemical properties and antioxidative activity of glycated α-lactalbumin with a rare sugar, D-allose, by Maillard reaction. Food Chemistry 95: 509-517.
Takata MK, Yamaguchi F, Nakanose K, Watanabe Y, Hatano N, Tsukamoto I, Nagata M, Izumori K, Tokuda M (2005) Neuroprotective effect of D-psicose on 6-hydroxydopamine-induced apoptosis in rat pheochromocytoma (PC12) cells. Journal of Bioscience and Bioengineering 100: 511-516.
Tang SY (2012) Rare Sugars: Applications and Enzymatic Production. Journal of Biocatalysis & Biotransformation 2: 363-368.
Tischer W, Wedekind F (1999) Immobilized enzymes: methods and applications. Biocatalysis-from discovery to application: Springer. pp. 95-126.
Tseng W-C, Lin J-W, Wei T-Y, Fang T-Y (2008) A novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method. Analytical Biochemistry 375: 376-378.
Yokohira M, Hosokawa K, Yamakawa K, Saoo K, Matsuda Y, Zeng Y, Kuno T, Imaida K (2008) Potential inhibitory effects of D-allose, a rare sugar, on liver preneoplastic lesion development in F344 rat medium-term bioassay. Journal of Bioscience and Bioengineering 105: 545-553.
Zhang L, Jiang B, Mu W, Zhang T (2009) Bioproduction of D-psicose using permeabilized cells of newly isolated Rhodobacter sphaeroides SK011. Frontiers of Chemical Engineering in China 3: 393-398.


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