臺灣博碩士論文加值系統

本研究從中篩選具有分解澱粉能力之放線菌，並進行生產澱粉酶之條件優化。實驗利用高氏選擇性培養基並添加制真菌素 (Nystatin) 作為篩選平台，以塗抹培養法培養放線菌，挑選外觀不同之菌落進行分離純化。菌種鑑定以聚合酶連鎖反應擴增放線菌之16S rDNA片段，經定序後將定序結果與NCBI基因資料庫進行BLAST比對，以得知菌種。本實驗從黃水中篩出六種放線菌，分別由第一道黃水篩出Streptomyces lienomycini (菌株編號1-1)，由第二道黃水篩出Cellulosimicrobium funkei (菌株編號2-3)、Gordonia bronchialis (菌株編號2-4)、Streptomyces graminisoli (菌株編號2-5)、Streptomyces griseus (菌株編號2-8)、Oerskovia paurometabola (菌株編號2-9)。將各菌株之培養液進行離心後取上清液作為粗酵素液，測定其澱粉酶活性，結果發現編號1-1菌株產粗酵素液之酵素活性為0.23 ± 0.07 U/ml，編號2-3菌株為0.60 ± 0.05 U/ml，編號2-4菌株為0.16 ± 0.05 U/ml，編號2-5菌株為0.24 ± 0.03 U/ml，編號2-8菌株為1.79 ± 0.07 U/ml，編號2-9菌株為0.32 ± 0.03 U/ml ，其中編號2-8菌株 (Streptomyces griseus) 為最高者，故選該菌株以反應曲面法 (Response surface methodology, RSM) 為模型，選擇溫度、pH值與MgSO4濃度進行三因子三級距之最適化實驗，以得最大量澱粉酶。研究結果顯示於培養溫度29.6oC、pH值8.0、MgSO4濃度0.77 g/L，發酵60小時可得最高澱粉酶產量2.22 U/ml。

The objective of this research is to screen actinobacteria which is able to produce amylase from yellow water and to enhance the production of amylase by optimization of nutritional constituents using Response Surface Methodology (RSM). In this research, we use Gauze selective medium, with Nystain, to screen actinobacteria. Identification of the strains can be conducted by 16S rDNA gene sequencing after amplification by Polymerase Chain Reaction (PCR) and electrophoresis. The results of sequencing were compared with NCBI gene database for BLAST. We screened 6 strains from yellow water, which are Streptomyces lienomycini, Cellulosimicrobium funkei, Gordonia bronchialis, Streptomyces graminisoli, Streptomyces griseus, and Oerskovia paurometabola. Collected supernatant liquid after centrifuging the broth culture to get crude enzyme solution which can act on starch to produce reducing sugar that can be detected by DNS method to know their amylase activity, and found that the amylase activity of the crude enzyme solution produced by strain 1-1 is 0.23±0.07 U/ml, 2-3 is 0.60±0.05 U/ml, 2-4 is 0.16±0.05 U/ml ,2-5 is 0.24±0.03 U/ml, 2-8 is 1.79±0.07 U/ml, and 2-9 is 0.32±0.03 U/ml. Strain 2-8 showed the highest amylase activity among the strains. Strain 2-8 was chosen for optimization of culture conditions to enhance production of amylase. Optimum fermentation condition showed, at 29.6oC, pH 8.0, adding 0.77 g/L MgSO4, and cultured for 60 hours, the strain produced 2.22 U/ml amylase.

謝誌...................................................................................................................................I
摘要..................................................................................................................................II
Abstract........................................................................................................................... III
目錄................................................................................................................................ IV
圖目錄 ..........................................................................................................................VIII
表目錄 ............................................................................................................................ IX
壹、 前言 ........................................................................................................................ 1
貳、 文獻回顧 ................................................................................................................ 3
1.1 放線菌 .............................................................................................. 3
1.1.1 放線菌簡介.............................................................................. 3
1.1.2 放線菌的分佈 .......................................................................... 5
1.1.3 放線菌的分類 .......................................................................... 5
1.1.4 放線菌對人類的貢獻.............................................................. 6
1.1.5 放線菌的分離純化 .................................................................11
1.2 高粱酒 ............................................................................................ 12
1.2.1 金門高粱酒............................................................................ 12
1.2.2 高粱酒製程............................................................................ 12
1.2.2.1 製麴 ........................................................................ 14
1.2.2.2 原料處理 ................................................................ 14
1.2.2.3 蒸煮、冷卻 ............................................................ 15
1.2.2.4 拌麴 ........................................................................ 15
1.2.2.5 固態發酵、翻醪 .................................................... 15
1.2.2.6 固態蒸餾 ................................................................ 15
1.2.3 高粱酒製作之相關微生物 .................................................... 16
1.2.4 高粱酒製作過程中酵素反應作用 ........................................ 16
1.2.5 黃水........................................................................................ 19
1.3 澱粉酶 ............................................................................................ 21
1.3.1 澱粉酶簡介 ........................................................................ 21
1.3.2 澱粉分解酵素在工業上的應用 ........................................ 22
1.3.3 澱粉分解酵素的來源 ........................................................ 24
1.4 反應曲面法 (Response surface methodology, RSM) ................... 24
1.4.1 RSM 簡介............................................................................... 24
1.4.2 RSM 步驟............................................................................... 25
參、 實驗目的與架構 .................................................................................................. 28
3.1 實驗目的 ........................................................................................ 28
3.2 實驗架構 ........................................................................................ 29
肆、 材料與方法 .......................................................................................................... 30
4.1 實驗材料 ........................................................................................ 30
4.1.1 黃水樣品................................................................................ 30
4.1.2 培養基材料............................................................................ 30
4.1.3 菌株鑑定材料 ........................................................................ 31
4.1.4 酵素活性測試材料 ................................................................ 32
4.1.5 儀器設備................................................................................ 32
4.2 實驗方法 ........................................................................................ 33
4.2.1 培養基配製方法 .................................................................... 33
4.2.2 菌種篩選、純化培養 ............................................................ 39
4.2.2.1 菌株篩選 ................................................................ 39
4.2.2.2 菌種分離純化 ........................................................ 39
4.2.2.3 菌種液態培養 ........................................................ 39
4.2.2.4 菌株之冷凍保存 .................................................... 39
4.2.3 鑑定放線菌............................................................................ 40
4.2.3.1 萃取及純化放線菌之基因體 DNA....................... 40
4.2.3.2 以聚合酶連鎖反應擴增 16S rDNA 片段............. 41
4.2.3.3 DNA 定序及 BLAST 比對.................................... 43
4.2.4 .................................................................... 43
4.2.4.1 碳源利用性試驗 .................................................... 43
4.2.4.2 氮源利用性試驗 .................................................... 43
4.2.5 澱粉酶活性試驗 .................................................................... 44
4.2.5.1 粗酵素液製備 ........................................................ 44
4.2.5.2 澱粉酶活性測試 .................................................... 44
4.2.5.2.1 葡萄糖標準溶液配製 ......................................... 44
4.2.5.2.2 DNS 法測定還原醣 ............................................. 45
4.2.5.2.3 DNS 試劑配製 ..................................................... 45
4.2.6 培養條件最適化 .................................................................... 47
4.2.6.1 種菌培養 ................................................................ 47
4.2.6.2 溫度對澱粉酶活性影響之實驗 ............................ 47
4.2.6.1 pH 值對澱粉酶活性影響之實驗 .......................... 47
4.2.6.2 MgSO4濃度對澱粉酶活性影響之實驗................ 48
4.2.6.3 反應曲面法實驗 .................................................... 48
伍、 結果與討論 .......................................................................................................... 49
5.1 樣本採集、菌株培養與分離 ........................................................ 49
5.2 菌株鑑定 ........................................................................................ 49
5.3 後續實驗菌株選擇 ........................................................................ 55
5.4 澱粉酶產量最適化 ........................................................................ 59
5.4.1 各實驗因子範圍評估試驗 .................................................... 59
5.4.2 澱粉酶產量最適化結果 ........................................................ 64
陸、 結論 ...................................................................................................................... 69
柒、 參考文獻 .............................................................................................................. 70
捌、 附錄 ...................................................................................................................... 84

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