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研究生:吳昱昇
研究生(外文):Yu-Sheng Wu
論文名稱:納豆醱酵分離菌株之鑑定與其蛋白質分解酵素之分析
論文名稱(外文):Identification of Bacterial Isolates from Natto Fermentative Broth and the Analyses of their Proteases
指導教授:呂誌翼
指導教授(外文):Jyh-Yih Leu
學位類別:碩士
校院名稱:輔仁大學
系所名稱:生命科學系碩士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:86
中文關鍵詞:納豆蛋白質分解酵素醱酵基質
外文關鍵詞:NattoProtease
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納豆在日本是一種具有悠久歷史也是近年來最受矚目的傳統醱酵食品,是由枯草桿菌 (Bacillus subtilis natto) 將煮熟的大豆醱酵而來。本研究目的在探討納豆菌株之菌種鑑定及其蛋白質分解酵素 (pretease) 之分析。自西元1987年須見博士在納豆食品中發現能夠溶解血栓纖維的蛋白質分解酵素—納豆激酶 (nattokinase) 以來。從不同來源的Bacillus菌屬或其他微生物中,已發現許多菌株能夠產生功能類似的凝血纖維蛋白分解酵素。本研究從醱酵製程中篩選出高生長速率的菌株FZ - 01、FZ - 02與FZ - 03,以菌種鑑定系統進行型態特徵,酸鹼度、生長溫度、耐鹽度等生理測試,碳源利用與酵素活性等生化測試及16S rDNA片段序列分析的遺傳鑑定,確認實驗菌株的分類地位;另外,本研究挑選菌株FZ - 01,針對培養基組成,分別以脫脂奶粉、黃豆粉、小麥胚芽與酵母菌萃取物作為醱酵基質並收取發酵產物,探討不同醱酵基質對菌株產生蛋白質分解酵素的影響。結果顯示,在分類鑑定上FZ - 01、FZ - 02、FZ - 03與標準菌株BCRC - 14718在分類上皆屬於Bacillus subtilis,且菌株FZ - 01、FZ - 02與FZ - 03有極為相近的親緣關係。在培養基組成不同的情況下,菌株FZ - 01所產生的蛋白質產物在NATIVE - PAGE的分子量、種類與表現量上皆有差異;從蛋白質分解酵素對血栓纖維蛋白與酪蛋白的蛋白質分解活性測試中可以發現活性表現的區域亦有所不同。從本研究結果可以推論單一菌株在不同培養條件下,可能產生不同種類的蛋白質分解酵素與血栓纖維分解酵素。
In Japan natto has a long history of being a premium traditional fermentive food, and been received much attention for health today. Natto is made of Bacillus subtilis natto in steamed soy bean, and was found containing with nattokinase, a fibrinolytic protease discovered by Dr. Sumi in 1987. According to many scientific publications, many similar proteases functioning in digesting fibrin were generated from a variety of bacillus strains and other microorganisms by using various substrates. Strains FZ-01, FZ-02 and FZ-03 were isolated from natto fermentation broth with high growth rates. The three strains were identified by analyzing following characterizations: pH, temperature, salt adaptation, carbon source and various enzyme activity. Furthermore, phylogenetic analysis was carried out by 16S rDNA fragment sequencing and showed that FZ-01, FZ-02 and FZ-03 are closely related and clustered with bacillus subtilis. In this study, strain FZ-01 was selected for protease production and analyses. Strain FZ-01was cultured with four different media: milk, soybean, wheat acrospire, and yeast extract-based media. Fermentative broths of strain FZ-01 were analyzed with protease using NATIVE-PAGE, which revealed that the produced proteins under four different media showed differences in molecular weight. The activities of these protease varied on fibrin- and casein-containing plates. From these results, we deducted that strain FZ-01 can produce different protease using different nitrogen sources.
中文摘要 I

英文摘要 II

第一章 前言 1
1.1 納豆 1
1.2 菌種分類鑑定系統 2
1.3 Bacillus的分類 4
1.4 納豆菌 5
1.5 蛋白質分解酵素 6
1.6 納豆激酶 8
1.7 納豆激酶之功能性 9
1.8 研究目的 11
第二章 材料與方法 12
2.1 菌株來源、培養與保存 12
2.2 本實驗使用之培養基 12
2.3 蛋白質分析實驗所使用之膠體配方、緩衝液與染劑 14
2.4 型態特徵與格蘭氏染色 17
2.5 基本生理特性-酸鹼度、生長溫度、鹽度的測試 17
2.5.1 酸鹼度生長範圍測試 17
2.5.2 溫度生長範圍測試 18
2.5.3 耐鹽度測試 18
2.5.4 生長曲線測定 18
2.6 基本生化特性-碳源利用與酵素活性測試 19
2.6.1 單一碳源利用能力 19

2.6.2 澱粉水解能力測試 19
2.6.3 Modified酪蛋白 (Casein) 水解測試 19
2.6.4 白明膠 (Gelatin) 水解測試 20
2.6.5 解脂酶 (Lipase) 活性測試 20
2.6.6 API-ZYM酵素活性檢測 20
2.7 菌種遺傳鑑定-16S rDNA片段序列分析 20
2.7.1 細菌genomic DNA抽取 20
2.7.2 PCR引子設計 21
2.7.3 PCR反應 21
2.7.4 16S rDNA片段序列分析 22
2.8 醱酵培養與回收 23
2.8.1 醱酵前培養 23
2.8.2 醱酵實驗 23
2.8.3 醱酵液回收 23
2.9 醱酵產物分析 24
2.9.1 置備式電泳 24
2.9.2 膠體染色 24
2.9.3 血栓溶解活性測試 24
2.9.4 酪蛋白分解活性測試 25
第三章 結果 26
3.1 型態特徵與格蘭氏染色 26
3.2 基本生理特性-酸鹼度、生長溫度、鹽度的測試 26
3.2.1 酸鹼度生長範圍測試 26

3.2.2 溫度生長範圍測試 26
3.2.3 耐鹽度測試 26
3.2.4 生長曲線測試 27
3.3 基本生化特性-碳源利用與酵素活性測試 27
3.3.1 單一碳源的利用能力 27
3.3.2 澱粉水解能力測試 27
3.3.3 Modified酪蛋白 (Casein) 水解測試 28
3.3.4 白明膠 (Gelatin) 水解測試 28
3.3.5 解脂酶 (Lipase) 活性測試 28
3.3.6 APIZYM酵素活性檢測 28
3.4 菌種遺傳鑑定-16S rDNA片段序列分析 28
3.5 醱酵培養與回收 29
3.6 醱酵產物分析 29
3.6.1 置備式電泳與膠體染色 29
3.6.2 血栓溶解活性測試 30
3.6.3 酪蛋白分解活性測試 30
第四章 討論 31
4.1 菌株FZ-01、FZ-02與FZ-03之菌種鑑定 31
4.2 不同醱酵基質對菌株FZ-01產生蛋白質產物的影響 32
4.3 不同醱酵基質對菌株FZ-01產生蛋白質分解酵素的影響 33
4.4 未來展望 34
參考文獻 36
圖表 44
附錄
附錄一、實驗菌株FZ-01之16S rDNA序列 72
附錄二、實驗菌株FZ-02之16S rDNA序列 73
附錄三、實驗菌株FZ-03之16S rDNA序列 74
附錄四、標準菌株BCRC-14718之16S rDNA 75
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