臺灣博碩士論文加值系統

菊糖為一種線性 β-(2-1)-連接的果糖聚合物，由n個果糖和末端一個葡萄糖線性組合成的GFn長鏈，醣類組成聚合度 (DP) 在 2 到 60 之間，可從菊芋、菊苣、牛蒡和蒜頭等菊科植物的根和塊莖中獲得。經由不同微生物的水解可產生寡糖(oligofructose)，此種寡糖DP在3~7之間，可作為甜味劑添加在食品中，有助於改善消化系統及降低血糖等功效。本研究利用菊芋當作碳源，使黑麴黴Aspergillus niger CCU分泌出的兩種酵素，菊粉酶(inulinase)和轉化酶(invertase)。
本研究的實驗分為兩部份：第一部份，透過測量菊糖(I)和蔗糖(S)兩種基質中釋放還原糖的濃度來分析菊粉酶與轉化酶活性，將每12小時取的上清液加入菊粉和蔗糖基質進行反應，接著利用3,5-二硝基水楊酸(DNS)與還原糖進行反應可測得I及S活性，再利用I/S比值區分酵素，當I/S比值>10-2為菊粉酶，當I/S比值<10-2為蔗糖酶。然後利用不同基質濃度(20 g/L、50 g/L或100 g/L)、培養基體積(50 ml或100 ml)和pH值(5、6.5或9)找出最佳的菊粉酶生產條件，結果發現在50 g/L、100 ml及pH6.5的搖瓶培養之下可產出最大菊粉酶活性為29.98±1.54 U/ml，轉化酶活性為29.50±1.76 U/ml ，I/S比值為0.94±0.12。最後利用十二烷基硫酸鈉聚丙烯醯胺凝膠電泳，將A. niger CCU所分泌的菊粉酶分離成內切菊粉酶及外切菊粉酶。從膠片中可得到三個蛋白質條帶，將條帶切下進行酵素反應發現其中二個條帶具有菊粉酶活性。接著經蛋白質鑑定後發現，外切菊粉酶蛋白質分子量為63kDa，內切菊粉酶蛋白質分子量為35kDa。因A. niger CCU會分泌較多的外切菊粉酶，不利於利用菊粉生產寡糖，會導致產物中含有大量果糖，本研究第二部份利用基因重組技術將分泌內切菊粉酶基因片段合成後接至質體pET15b(+)上，得到的重組菌可表現內切菊粉酶。

Inulin is a linear β-(2-1)-linked fructose polymer composed of n fructose units with a terminal glucose unit, forming a GFn chain. The degree of polymerization (DP) of carbohydrates in this chain ranges from 2 to 60. Inulin can be obtained from the roots and tubers of plants in the Asteraceae family, such as Jerusalem artichoke, chicory, burdock, and garlic. Oligofructose, a shorter chain variant of inulin with a DP of 3 to 7, can be produced through enzymatic hydrolysis by various microorganisms. Oligofructose serves as a sweetener in foods and offers benefits such as improving the digestive system and lowering blood sugar levels.

This study utilizes Jerusalem artichoke as a carbon source to induce the secretion of two enzymes, inulinase and invertase, from the filamentous fungus Aspergillus niger CCU. The experiment is divided into two parts. In the first part, the activity of inulinase and invertase is analyzed by measuring the concentration of released reducing sugars from two substrates: inulin (I) and sucrose (S). The clear supernatant obtained every 12 hours is added to the inulin and sucrose substrates for reaction. The reaction mixture is then assayed using 3,5-dinitrosalicylic acid (DNS) with reducing sugars to determine the activity of I and S. The I/S ratio is used to differentiate the enzymes: an I/S ratio >10^-2 indicates inulinase, while an I/S ratio <10^-2 indicates invertase.

Various conditions are tested, including substrate concentrations (20 g/L, 50 g/L, or 100 g/L), culture volumes (50 ml or 100 ml), and pH values (5, 6.5, or 9), to identify the optimal conditions for inulinase production. The results show that under conditions of 50 g/L substrate concentration, 100 ml culture volume, and pH 6.5, the highest inulinase activity is 29.98 ± 1.54 U/ml, invertase activity is 29.50 ± 1.76 U/ml, and I/S ratio is 0.94 ± 0.12. Gel electrophoresis using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is used to separate the inulinase secreted by A. niger CCU into endoinulinase and exoinulinase. Two of the three protein bands on the gel exhibit inulinase activity upon enzyme reaction. Protein identification reveals that the exoinulinase has a molecular weight of 63 kDa, while the endoinulinase has a molecular weight of 35 kDa.

Since A. niger CCU predominantly secretes exoinulinase, which is less favorable for oligofructose production from inulin due to higher levels of fructose in the resulting product, the second part of the study employs recombinant gene technology to synthesize the gene fragment of endoinulinase and express it in a vector (pET15b(+)) to obtain a recombinant strain capable of producing endoinulinase.

致謝 i
中文摘要 ii
Abstract iv
目錄 vi
圖目錄 xi
表目錄 xiv
1 第一章研究動機及目的 16
2 第二章文獻回顧 17
2.1 菊糖 (lnulin) 17
2.1.1 菊芋介紹 17
2.1.2 菊芋粉處理 18
2.2 Fructooligosaccharide (FOS) 20
2.3 Inulo-oligosaccharides (IOS) 21
2.4 黑麴黴(Aspergillus niger) 22
2.5 菊粉酶(inulinase) 23
2.6 轉化酶(Invertase) 26
2.7 生產內切菊粉酶 27
2.7.1 內切菊粉酶(Inu)的重因重組 27
2.7.2 蛋白質純化 28
2.7.3 SDS-PAGE分析 30
3 第三章 實驗藥品及器材 32
3.1 實驗藥品 32
3.1.1 菌種來源 32
3.1.2 基因重組 33
3.1.3 SDS-PAGE所需藥品 34
3.2 實驗設備 35
4 第四章 實驗步驟 37
4.1.1 A. niger CCU生產酵素方法 37
4.1.2 菊芋處理 37
4.1.3 A. niger CCU培養及醱酵 37
4.1.4 酵素活性測定 38
4.1.5 蛋白質定量 41
4.1.6 利用A. niger生產之酵素生產菊寡糖 43
4.1.7 組成分析 43
4.1.8 菊粉酶的蛋白質SDS-PAGE分析 43
(1)膠片製作 44
(2)膠體電泳 45
(3)膠片後續處理 46
(4)膠片蛋白質的活性分析 46
4.2 建構表達菊粉內切酶基因的重組菌 46
4.2.1 重組菌的建構 46
4.2.2 熱休克轉型法Heat-shock 47
4.2.3 菌落PCR (Colony PCR) 48
4.2.4 驗證: 49
(1)pET15b(+)-InuA染色體萃取 49
(2)DNA 純化 50
(3)PCR 51
(4)瓊脂膠體電泳分析 53
4.3 培養和誘導E. coli DH5α-inuA以及酵素活性分析 54
4.3.1 E. coli DH5α-inuA培養 54
4.3.2 酵素活性測定 56
4.3.3 酵素反應生產菊寡糖 57
5 第五章 結果與討論 58
5.1 不同反應條件下，菊粉酶活性和菊寡糖產量的影響 58
5.1.1 不同培養基體積下，菊粉酶活性和菊寡糖產量的影響 58
5.1.2 不同菊芋粉濃度下，菊粉酶活性和菊寡糖產量的影響 62
5.1.3 不同pH值對菊粉酶活性和菊寡糖產量的影響 65
5.1.4 不同菊芋粉處理方式對菊粉酶活性和菊寡糖產量的影響 69
5.1.5 利用A. niger生產之酵素生產菊寡糖 74
5.1.6 A. niger 分泌蛋白質的SDS-PAGE分析 76
5.1.7 內切菊粉酶基因的DNA合成 79
5.1.8 熱休克轉型法(heat shock method) 80
5.1.9 染色體萃取和菌落 PCR (Colony PCR) 80
5.1.10 重組菌E. coli DH5α-pET15b(+)-inuA蛋白質SDS-PAGE分析 82
5.1.11 重組菌的目標基因DNA定序 83
5.1.12 E. coli DH5α-pET15b(+)-inuA之測定 88
(1)E. coli DH5α-pET15b(+)-inuA酵素活性、蛋白質濃度和比活性測定 88
(2) 利用E. coli DH5α-pET15b(+)-inuA生產之酵素生產菊寡糖 89
5.1.13 E. coli BL21(DE3)-pET15b(+)-inuA測定 90
(1) E. coli BL21(DE3)-pET15b(+)-inuA分泌蛋白質的SDS-PAGE分析 90
E. coli BL21(DE3)-pET15b(+)-inuA酵素活性、蛋白質濃度和比活性測定 91
(3)利用E. coli BL21(DE3)-pET15b(+)-inuA生產之酵素生產菊寡糖 92
6 結論 94
7 參考文獻 96
8 附錄A A. niger CCU 的內切菊粉酶DNA序列定序結果及其與文獻比對 102
9 附錄B A. niger CCU的內切菊粉酶胺基酸序列及其與文獻比對 104
10 附錄C 不同比例DNS檢量線 105
11 附錄D HPLC個成份檢量線(粗體數字為滯留時間(min)) 106

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