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研究生： 謝元傑
YUAN-CHIEH HSIEH
論文名稱： 基因重組大腸桿菌於不同溫度培養條件下所生產的藍藻蛋白經不同分離條件之物性探討
Physical Properties of fractionated cyanophycin prepared with recombinant Escherichia coli at different temperatures
指導教授： 曾文祺
Wen-Chi Tseng
口試委員: 陳秀美
Hsiu-Mei Chen
毛慶豐
C.F. Mao,
學位類別： 碩士
Master
系所名稱： 工程學院 - 化學工程系
Department of Chemical Engineering
論文出版年： 2016
畢業學年度： 104
語文別： 中文
論文頁數： 69
中文關鍵詞： 藍藻蛋白
外文關鍵詞： cyanophycin
相關次數： 點閱：248  下載：0 
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    • 藍藻蛋白為一種非核糖體合成的生物高分子，於一百多年前在藍綠藻菌裡發現天然藍藻蛋白，它結構是以天冬門胺酸為主鏈，側鏈則是接上精胺酸，以精胺酸的α-胺基與天冬門胺酸的β-羧基互相連接，天冬門胺酸再互相接合。將藍藻蛋白酶基因片段並與適當載體作結合，再選殖至其他微生物體內，大量地產生藍藻蛋白，而獲得的藍藻蛋白，其胺基酸組成會由天冬門胺酸、精胺酸、賴胺酸所構成。
    本實驗所使用的菌株為Synechocysis sp.PCC6803 cphA之Escherichia coli BL21 (DE3) CodonPlus-RIL，將藍藻蛋白酶基因片段選殖到大腸桿菌中，於不同溫度培養大腸桿菌，找尋最適合大腸桿菌大量生產藍藻蛋白之最佳溫度。由於產出的藍藻蛋白之分子量分佈廣，故希望改變純化方式將藍藻蛋白更進一步的分離，水溶性藍藻蛋白是藉由非極性的溶液(乙醇) 來改變溶液中極性的程度，來分離水溶性藍藻蛋白。而非水溶性藍藻蛋白是調整溶液中的pH值，來分離非水溶性藍藻蛋白。在進一步分析分離過的藍藻蛋白之間的差異。
    實驗結果顯示在27 oC溫度下培養重組基因大腸桿菌所產出的水溶性與非水溶性藍藻蛋白產量最高。分離過的水溶性藍藻蛋白，在低濃度乙醇分離下之藍藻蛋白其胺基酸比例為Asp：Arg：Lys =52.62±0.55 % : 23.68 ±1.00 % : 23.70±0.76 %;而高%乙醇分離下之藍藻蛋白比例為Asp：Arg：Lys =50.79±0.78 % : 21.21±0.56 % : 28.01±0.41 %;分離過的非水溶性藍藻蛋白，在pH 4分離下其胺基酸比例Asp：Arg：Lys =52.15± 0.82 % : 41.82 ± 0.68 % : 6.03 ± 0.40 %，而在pH 5分離下之藍藻蛋白比例Asp：Arg：Lys =51.21±0.32 % : 40.52±0.09 % : 8.27±0.25 %。因為水溶性與非水溶性藍藻蛋白為熱敏性高分子，可藉由溫度控制粒徑狀態，其個別在不同溫度下會有UCS-T/LCST相變化的形式。

    Cyanophycin is a nonribosomally synthesized biopolymer, and was originally discovered in cyanobacteria about 100 years ago. The structure of natural cyanophycin consists of aspartic acid and arginine, and is arranged as an aspartic acid backbone with an arginine side chain which is linked to the β-carboxyl group of each aspartate by its α-amino group. The gene transformed into microorganisms can be used to produce large amounts of cyanophycin.
    In this study, we used Escherichia coli containing Synechocystis sp. PCC 6803 cphA on BL21-CodonPlus(DE3)-RIL to produce cyanophycin. E. coli was cultivated at different temperatures in order to find an optimal condition for cyanophycin production. The molecular weight distribution of cyanophycin has a wide range. We used solvents with different polarity to separate cyanophycin. Soluble cyanophycin was further separated in a solution containing ethanol. Insoluble cyanophycin are further separated at different pH values. The analysis of physical properties of the fractionated cyanophycin was also carried out.
    The results showed that the yield of soluble/insoluble cyanophycin ratio at 27 oC is higher than at other temperatures. Fractionated cyanophycin by a low polarity solution showed that the amino acid compositions as Asp：Arg：Lys = 52.62±0.55% : 23.68±1.00% : 23.70±0.76%; fractionated cyanophycin by a high polarity solution showed the amino acid compositions as Asp：Arg：Lys = 50.79±0.78% : 21.21±0.56% : 28.01±0.41%. However fractionated cyanophycin by pH 4 solution showed that the amino acid compositions as Asp：Arg：Lys = 52.15±0.82% : 41.82±0.68% : 6.03±0.40%; fractionated cyanophycin by pH 5 solution showed that the amino acid compositions as Asp：Arg：Lys = 51.21±0.32% : 40.52±0.09% : 8.27±0.25%. Both the soluble and insoluble cyanophycin are thermoresponsive polymers. The phase transitions were observed at different temperatures.

    摘要 I 目錄 VI 圖表目錄 XI 第一章 緒論 1 1.1前言 1 1.2研究動機 1 第二章 文獻回顧 3 2.1藍藻蛋白 3 2.1.1天然之藍藻蛋白 3 2.1.2經基因重組菌株生產之藍藻蛋白 5 2.1.2.1 pET蛋白表現系統 5 2.1.2.2經基因重組大腸桿菌生產之藍藻蛋白 7 2.3熱敏性高分子 9 2.3.1熱敏性高分子的種類 9 2.3.2熱敏性高分子特徵 10 2.3.2.1霧點 10 2.3.2.2熱敏性高分子的線團狀-球狀轉換 10 2.3.2.3滯遲現象 11 第三章 實驗材料與方法 12 3.1 實驗藥品 12 3.2 實驗儀器 14 3.3 藥品配置 15 3.3.1 藍藻蛋白生產與純化 15 3.3.1.1 LB Medium Plate 15 3.3.1.2 LB Medium 15 3.3.1.3 TB Medium 16 3.3.1.4氨苄青黴素/氯黴素二合一抗生素 16 3.3.1.5 Isopropyl β-D-1-thiogalactopyranoside 16 3.3.2 SDS-PAGE 17 3.3.2.1 1.5 M Tris-HCl (pH 8.8) 17 3.3.2.2 1M Tris-HCl (pH 6.8) 17 3.3.2.3 loading buffer 17 3.3.2.4 running buffer(10X) 17 3.3.2.5 staining solution 17 3.3.2.6 de-staining solution 18 3.3.2.7 preserving solution 18 3.3.2.8 4%聚丙烯醯胺膠體 (stacking gel) 18 3.3.2.9 12 %聚丙烯醯胺膠體 (separating gel) 18 3.3.2.10 15 %聚丙烯醯胺膠體 (separating gel) 18 3.3.3 Escherichia coli Protein Extraction 配置 19 3.3.3.1 lysozyme solution 19 3.3.3.2 CaCl2.2H2O solution 19 3.3.3.3 DNase solution 19 3.3.4 高效液相層析法 20 3.3.4.1 前置反應溶液(pre-derivatization solution) 20 3.3.4.2染色反應溶液(derivatization solution) 20 3.3.4.3 0.14 M醋酸鈉水溶液 (移動相A) 20 3.3.4.4 60 %乙腈水溶液 (移動相B) 20 3.4實驗步驟 21 3.4.1 培養基因重組之大腸桿菌 21 3.4.1.1將菌株畫盤於LB medium plate 21 3.4.1.2將菌株轉養至2 mL LB medium 21 3.4.1.3將菌株轉養至60mL LB medium 22 3.4.1.4將菌株轉養至150mL LB medium 22 3.4.1.5 收菌 23 3.4.2純化藍藻蛋白 23 3.4.2.1泡菌和破菌 23 3.4.2.2水溶性藍藻蛋白純化 24 3.4.2.3非水溶性藍藻蛋白純化 25 3.4.2.4水溶性藍藻蛋白分離 26 3.4.2.5非水溶性藍藻蛋白分離 27 3.4.3 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(SDS-PAGE) 27 3.4.3.1鑄膠 28 3.4.3.2跑膠 28 3.4.4 萃取Escherichia coli 所生產的蛋白質 29 3.4.5 膠體滲透層析儀 30 3.4.6 高效液相層析法 31 3.4.6.1標準胺基酸與線下面積之關係 32 3.4.6.2使用高效液相層析法分析藍藻蛋白 33 3.4.7藍藻蛋白溶解度測試 34 3.4.8分析藍藻蛋白對溫度應答之相變化和霧點 34 3.4.9水溶性與非水溶性藍藻蛋白之粒徑分析 35 第四章 結果與討論 37 4.1以不同溫度培養大腸桿菌所生產藍藻蛋白 37 4.1.1 SDS PAGE分析不同溫度下藍藻蛋白合成酶的表現 37 4.1.2純化後不同溫度產率比較 38 4.1.3分離藍藻蛋白 39 4.1.3.1以不同濃度乙醇分離水溶性藍藻蛋白產率 39 4.1.3.2以不同pH值分離非水溶性藍藻蛋白產率 40 4.2不同濃度乙醇分離的水溶性藍藻蛋白分析 41 4.2.1分子量分析 41 4.2.1.1 SDS-PAGE 41 4.2.1.2 GPC 42 4.2.2高效液相層析法分析水溶性藍藻蛋白之胺基酸組成 44 4.2.3不同濃度乙醇分離的水溶性藍藻蛋白溶解度測試 45 4.2.4分析水溶性藍藻蛋白對溫度應答之相變化 46 4.2.5水溶性藍藻蛋白對溫度應答之粒徑變化 50 4.3不同pH值分離非水溶性藍藻蛋白分析 51 4.3.1分子量分析 51 4.3.2高效液相層析法分析非水溶性藍藻蛋白之胺基酸組成 52 4.3.3不同pH值分離的非水溶性藍藻蛋白溶解度測試 53 4.3.4非水溶性藍藻蛋白對溫度應答之相變化 54 4.3.5非水溶性藍藻蛋白對溫度應答之粒徑變化 56 第五章 結論 57 參考文獻 59

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