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系統識別號 U0002-0207202013402200
中文論文名稱 液化澱粉芽孢桿菌 TKU050 所生產之果膠酶的純化與定性
英文論文名稱 Purification and characterization of a pectinase from Bacillus amyloliquefaciens TKU050
校院名稱 淡江大學
系所名稱(中) 化學學系碩士班
系所名稱(英) Department of Chemistry
學年度 108
學期 2
出版年 109
研究生中文姓名 陳建霖
研究生英文姓名 Chien-Lin Chen
學號 607160214
學位類別 碩士
語文別 中文
口試日期 2020-06-05
論文頁數 49頁
口試委員 指導教授-王三郎
指導教授-阮文邦
委員-王全祿
委員-呂誌翼
中文關鍵字 液化澱粉芽孢桿菌  果膠酶  麥麩 
英文關鍵字 Bacillus amyloliquefaciens  pectinase  wheat bran 
學科別分類 學科別自然科學化學
中文摘要 在這項研究中,使用了來自香蕉(香蕉皮),大米(米糠),橙子(橙皮),咖啡(廢咖啡渣)以及小麥(麥麩)的各種含農業果膠的副產品,提供碳和氮(C / N)養分。通過篩選自淡水土壤經鑑定為液化澱粉芽孢桿菌的這株果膠酶生產菌TKU050,在含0.5% 麥麩中於37℃連續4天表現出最高的果膠酶生產率(0.76 U / mL)。
B. amyloliquefaciens TKU050於較適培養條件發酵所得上清液,經硫酸銨沉澱、High Q column以及HPLC進行果膠酶之純化分離。純化所得TKU050果膠酶之分子量經SDS-PAGE測得為58 kDa,活性回收率與比活性分別為7.24%與0.91U/mg。TKU050果膠酶之最適反應溫度與pH分別為50℃及pH6,熱穩定性及pH安定性分別為<50℃與pH6~9。TKU050果膠酶活性會受Cu2+和SDS所抑制。
用TKU050果膠酶水解得到的產物對乳酸菌的生長具有增強作用。產物具促進乳酸桿菌生長之效果。
英文摘要 Pectin-containing by products possess potential applications in many fields. In this research, different kinds of agricultural pectin-containing by products from banana (banana peel), rice (rice bran), orange (orange peel), coffee (spent coffee ground), as well as wheat (wheat bran) were used to provide both carbon and nitrogen (C/N) nutrients for the production of a pectinase via Bacillus amyloliquefaciens TKU050, a pectinolytic bacterium isolated from the soils of Tamsui. B. amyloliquefaciens TKU050 expressed the highest pectinase productivity (0.76 U/mL) on 0.5% wheat bran-containing medium at 37 ◦C for 4 days. After purification by ammonium sulfate precipitation, High Q column chromatography, and HPLC, a pectinase (TKU050) was purified from the culture supernatant fermented under optimized culture conditions. The molecular mass of TKU050 pectinase was estimated to be 58 kDa by SDS-PAGE. The recovery ratio and specific activity of TKU050 pectinase were 7.24% and 0.51 U/mg, respectively. The optimum temperature, optimum pH, thermal stability, and pH stability of TKU050 pectinase were 50◦C, pH6, <50◦C, and pH 6-9, respectively. TKU050 pectinase was inhibited Cu2+ and SDS. The reducing sugar obtained by hydrolyzing with TKU050 pectinase showed enhancing effect on the growth of lactobacillus acid bacteria.
論文目次 目錄
中文摘要 Ⅰ
英文摘要 Ⅱ
目錄 Ⅲ
圖目錄 Ⅷ
表目錄 Ⅺ

第一章 緒論 1
第二章文獻回顧 2
2.1 液化澱粉芽孢桿菌(Bacillus amyloliquefaciens) 2
2.2 果膠質 3
2.3 果膠酶 5
2.4 果膠製備 7
2.5 香蕉 8
2.6 麥麩 9
第三章材料與方法 11
3.1 實驗菌株 11
3.2 實驗材料 11
3.3 實驗儀器 12
3.4 實驗菌株之篩選 13
3.4.1 革蘭氏染色(Gram staining) 13
3.4.2 16S rDNA之定序分析 13
3.4.3 API(Analytical Profile Index)之鑑定 14
3.5 實驗菌株之探討條件 14
3.5.1 不同碳源的影響 14
3.5.2 碳源濃度之影響 14
3.5.3 培養液pH 14
3.5.4 培養溫度 15
3.5.5 培養速率 15
3.5.6果膠酶活性之測定 15
3.5.7 DNS試劑的配製 15
3.6 果膠酶之分離純化 16
3.6.1 粗酵素液之製備 16
3.6.2 陰離子交換層析(一) 16
3.6.3 HPLC 16
3.7 蛋白質電泳分析 16
3.8 酵素之特性分析 17
3.8.1 酵素最適反應溫度 17
3.8.2 酵素熱安定性 17
3.8.3 酵素最適反應 pH 17
3.8.4 酵素 pH 安定性 18
3.8.5 金屬離子及化學藥品對酵素活性之影響 18
3.8.6 界面活性劑對酵素活性之影響 18
3.8.7 酵素之基質特異性 18
3.9 果膠酶水解基質及寡醣分析 19
3.9.1 基質水解 19
3.9.2 還原糖量之測定 19
3.10 促進乳酸菌之生長 19
第四章結果與討論 20
4.1 果膠酶生產菌之篩選與鑑定 20
4.2 酵素較適生產條件探討 24
4.2.1不同碳源的影響 24
4.2.2 碳源濃度之影響 25
4.2.3 培養液pH 26
4.2.4 培養溫度 27
4.2.5 培養速率 28
4.2.6 適培養條件結果與比較 29
4.3 果膠酶之分離純化 30
4.3.1粗酵素液之製備 31
4.3.2離子交換樹脂層析(一) 31
4.3.3HPLC 32
4.3.4 分離純化之綜合結果 33
4.4 幾丁聚醣酶分子量之測定與鑑定 34
4.4.1 SDS-PAGE 34
4.5果膠酶之特性分析 35
4.5.1最適反應溫度及熱安定性 35
4.5.2最適 pH 及 pH 安定性 36
4.5.3金屬離子對果膠醣酶活性之影響 37
4.5.4 基質特異性之比較 38
4.5.5 果膠酶產生之比較 39
4.6水解基質之探討 40
4.7促進乳酸菌之生長 40
第五章結論 42
第六章參考文獻 43

圖目錄

圖2.1 果膠質之結構(A)原果膠質及果膠(B)果膠酸 4
圖2.2 果膠酶種類及作用(A)果膠酶酯(B)聚辦乳醣醛酸酶(C)果膠裂解酶 6
圖 4.1 Bacillus amyloliquefaciens TKU050之顯微鏡照片 20
圖 4.2 B. amyloliquefaciens TKU050之16SDNA部分簡基序列 21
圖 4.3 B. amyloliquefaciens TKU050系統演化樹 22
圖 4.4 不同碳源對果膠酶生產之影響 24
圖 4.5 不同麥麩粉濃度對果膠酶生產之影響 25
圖 4.6 不同pH對果膠酶生產之影響 26
圖 4.7 不同溫度對果膠酶生產之影響 27
圖 4.8 不同轉速對果膠酶生產之影響 28
圖 4.9 果膠酶之純化流程 30
圖 4.10 TKU050果膠酶之High Q 層析圖譜 32
圖 4.11 TKU050果膠酶之純化SDS-PAGE之分子量 34
圖 4.12 果膠酶之最適反應溫度熱安定性 35
圖 4.13 果膠酶之最適反應pH及pH熱安定性 36
圖 4.14 TKU050生產之果膠酶基質特異性 38
圖 4.15 TKU050生產之粗酵素液在不同基質下不同反應間之還原糖 40
圖 4.16 TKU050生產之之水解產物對L. paracasei subsp. paracasei BCRC14023 (A), Lactobacillus rhamnosus (BCRC16000) (B), L. rhamnosus BCRC10494 (C), 和 L. lactis subsp (BCRC10791) (D)生長影響 41

表目錄
表 4.1 API鑑定分析結果 23
表 4.2 B. amyloliquefaciens TKU050 所生產果膠酶活性之較適條件 29
表 4.3 B. amyloliquefaciens TKU050果膠酶之純化總表 33
表4.4 金屬離子和表面活性劑對TKU050果膠酶活性的影響 37
表4.5 果膠酶產生之比較
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