以食品製程之副產物應用於培養基因重組大腸桿菌生產藍藻蛋白之探討

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研究生：	胡維倫 Wei-Lun-Hu
論文名稱：	以食品製程之副產物應用於培養基因重組大腸桿菌生產藍藻蛋白之探討 Utlilize by-products of food production processes to produce cyanophycin with recombinant Escherichia coli
指導教授：	曾文祺 Wen-Chi Tseng
口試委員:	林析右 Shi-Yow Lin 唐建翔 Chien-Hsiang Tang 陳信銘 Hsin-Ming Chen
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：	2020
畢業學年度：	108
語文別：	中文
論文頁數：	103
中文關鍵詞：	藍藻蛋白、食品製程副產物
外文關鍵詞：	cyanophycin
相關次數：	點閱：221 下載：12
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藍藻蛋白(cyanophycin granule polypeptide，CGP)又稱藻青素是一種非核醣體合成的聚合物，於一百多年前就已經被科學家Borzi觀察藍綠藻菌(cyanobacteria)時發現了藍藻蛋白。藍藻蛋白原本是由藍綠藻菌體內所產生的物質，但並不是所有的藍綠藻都能產生藍藻蛋白。天然的藍藻蛋白是由等莫耳比例的精胺酸 (arginine, Arg)跟天門冬胺酸 (aspartic acid, Asp)，以天門冬胺酸為主鏈與精胺酸為側鏈進行胺基酸聚合反應為主鏈，其鍵結為精胺酸的β-羧基以及天門冬胺酸的α-胺基做為連接。
本實驗藉由Plackett-Burman設計12組培養基得到所需的因子，再利用 Response Surface-Central Composite這15組培養基，使用Synechocysis sp.PCC6803 cphA之E. coli BL21 (DE3) CodonPlus-RIL培養於這些培養基，培養溫度為22 oC並用0.01 mM IPTG進行誘導，將培養的菌體經由藍藻蛋白純化步驟，便可得到水溶性藍藻蛋白(sCGP)以及非水溶性藍藻蛋白(inCGP)。
經過計算後，以酵母菌萃取物和胰蛋白腖來培養，改成以糖蜜以及大豆水解液來培養培養基因重組大腸桿菌，在相同的成本下，用大豆水解液以及糖蜜所配成的培養基，能夠得到較多的藍藻蛋白。

Cyanophycin (Cyanophycin Granular Peptide, CGP) is a non-ribosome-synthesized polymer. It has been discovered by scientist Borzi more than a hundred years ago while observing cyanobacteria. The cyanobacterial protein was produced by the Cyanobacteria, but not all Cyanobacteria can produce cyanobacterial protein. The natural cyanobacterial protein is composed of equal molar ratios of arginine (Arg) and aspartic acid (Asp), with aspartic acid as the main chain and arginine as the side chain for connection.
In this experiment, Plackett-Burman designed 12 runs of media to obtain the required factors, and then used Response Surface-Central Composite 15 runs of media, using Synechocissis sp. PCC6803 cphA E. coli BL21 (DE3) CodonPlus-RIL cultured in these media at a culture temperature of 22 oC and induction with 0.01 mM IPTG, the cultured cells undergo a cyanobacteria protein purification step to obtain soluble and insoluble cyanophycin.
After calculation, compare with the medium prepared with yeast extract and trypsin and the medium prepared with molasses and soybean hydrolysate were used to cultivate the genetically recombinant E. coli. At the same cost, the medium prepared with soybean hydrolysate and molasses could get more cyanobacteria protein.

摘要    I
Abstract    II
誌謝    III
目錄    IV
圖目錄    VIII
表目錄    X
附錄圖目錄    XI
第一章  緒論    1
第二章  文獻回顧    2
1 糖蜜    2
1.1糖蜜之來源    2
1.2糖蜜營養價值    2
1.3糖蜜的應用    4
2 大豆水解液    5
2.1大豆水解液之來源    5
3.2大豆水解液的價值    6
3藍藻蛋白    6
2.1藍藻蛋白結構與特性    7
2.2以基因重組菌生產藍藻蛋白    8
2.3藍藻蛋白的應用    9
3 Plackett-Burman 設計    10
4 Surface Response – Central Composite 設計    12
5高效液相層析    15
第三章 實驗材料與方法    16
1藥品與儀器清單    16
2實驗儀器    18
3藥品配置    19
3.1 菌株培養    19
3.1.1二合一抗生素    19
3.1.2 Luria broth medium plate (LB medium plate)    19
3.1.3 Luria broth medium (LB medium)    19
3.1.4 Terrific broth medium (TB medium)    19
3.1.5 Phosphate    20
3.1.6 Molasses solution    20
3.1.7 Vitamin solution    20
3.1.8 Trace element    20
3.1.9 Isopropyl β-D-1-thiogalactopyranoside (IPTG)    21
3.2 SDS-PAGE    21
3.2.1 0.5M Tris-HCl buffer ( pH=6.8 )上層膠    21
3.2.2 1.5M Tris-HCl buffer ( pH=8.8 )下層膠    21
3.2.3 10% SDS (sodium dodecyl sulfate)    21
3.2.4 20% APS (ammonium persulfate)    21
3.2.5 10×protein running buffer    21
3.2.6 protein staining buffer    22
3.2.7 protein de-staining buffer    22
3.2.8 protein preserving buffer    22
3.3 HPLC    22
3.3.1 Pre-derivatization solution    22
3.3.2 Derivatization solution    22
3.3.3 0.14 M 醋酸鈉水溶液 (移動相A)    22
3.3.3 60 %乙腈(移動相B)    22
4實驗步驟    23
4.1 菌株培養    23
4.1.1 培養微生物於LB medium plate    23
4.1.2 培養微生物於2 mL LB medium    23
4.1.3 培養微生物於200 mL LB medium    23
4.1.4 培養微生物於150 mL TB medium    24
4.1.5 以IPTG對微生物進行誘導    24
4.1.6 收集微生物    24
4.2 純化藍藻蛋白    24
4.2.1 水溶性藍藻蛋白純化步驟    25
4.2.2 非水溶性藍藻蛋白純化步驟    25
4.3 SDS-PAGE    25
4.4高效液相層析法    27
4.5 Plackett-Burman設計    28
4.5.1選擇PB因子    28
4.5.2 PB因子的上下限    28
4.5.3 使用Plackett-Burman方法設計出實驗    31
4.6 培養基最佳化-Response Surface    32
第四章  結果與討論    33
1 Plackett-Burman方法    33
1.1 PB因子的預篩選    33
1.2 Plackett-Burman設計    34
1.2.1 Response: Optical density (O.D.)    34
1.2.1 Response: inCGP Yield    38
1.2.1 Response: sCGP Yield    41
1.2.1 Response: CGP Yield    43
1.2.1 Response: inCGP/sCGP yield retio    46
2培養基最適化-Response Surface    49
2.1 Fit Summary    49
2.2變異數分析Analysis of variance (ANOVA)    51
2.3 Residual vs. Run    53
2.4 最佳化    53
2.5 成本計算    54
2.6 使用高效液相層析法分析藍藻蛋白    56
第五章  結論    59
附錄    60
參考文獻    83

                                

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