近年來，因生技產業的迅速發展，蛋白酶已廣泛應用在食品及醫療領域，在產業界的機能性食品之開發過程，商用酵素的使用往往會提高產品的製造成本，所以自行生產蛋白酶使用，將會降低產品開發之成本。而本實驗從市售納豆中所篩選之菌株經鑑定後確定為枯草桿菌，也是最常用來研究生產蛋白酶的菌株之一，其培養24小時之菌液粗酵素活性約35.7 U/ml，36小時後活性達47.2 U/ml，其最適培養基成分包括3.6 % (w/v) soluble starch，1.28 % (w/v) yeast extract，0.12 % (w/v) meat dry extract，0.4 % (w/v) KH2PO4，最適培養條件為37℃、150 rpm之轉速、最適起始pH 6與3 % (v/v)之最適接種量。以固態發酵方式大量生產蛋白酶之探討結果，其發酵基質為綠豆片及樹豆片以9：1之比例混合，最適水分含量及接種量為60%及3%，37℃為最適發酵溫度，再額外添加2.5% (w/w)之sucrose與skim milk做為碳氮源，經過60小時發酵後，有最高之酵素生產活性約1196 U/ml。再經過酒精沉澱與薄膜超過濾濃縮之後，其酵素活性可1776 U/ml與8184 U/ml。
蛋白酶之生化特性分析，其最適作用溫度及pH分別為60℃及pH 10，在-80℃下保存一個月可以維持80%以上活性，酵素對鐵離子具有強烈敏感性，不同蛋白酶抑制劑之實驗結果證明，本實驗蛋白酶為絲胺酸蛋白酶，經酵素動力論Lineweaver-Burk雙倒數圖之分析結果，其Km值與Vmax值分別為16.36 mg/ml與714 μg tyrosine/min。以初步純化濃縮酵素液，進行脫脂黃豆粉之水解，其水解條件設定1% (w/v)之脫脂黃豆粉添加1% (v/v)之酵素液(2997 U/ml)，在50℃下水解1小時，其對ACE之抑制活性可達98.1%。

Recently, biotechnology industry is rapidly developed, proteases has been used in food science and medical science popularly. In the development process of new functional foods, using commercial protease usually gets higher costs, so it would reduce the R&D costs by producing protease oneself. In this study, the strain isolated from natto was identified as Bacillus subtilis, and was used to produce protease. The cell-free crude enzyme showed the activity (35.7 U/ml) after 24 hr culture, showed the highest activity (47.2 U/ml) after 36 hr culture. The optimal medium included 3.6% (w/v) soluble starch, 1.28% (w/v) yeast extract, 0.12% (w/v) meat dry extract, 0.6% (w/v) KH2PO4. The optimal culture conditions of the strain were 37℃、150 rpm、pH 6 for initial pH of medium and 3% (v/v) for inocular size. After the study of SSF, the substrate was the mixture of mung bean and tree bean, the optimal moisture content was 60% and 3% (v/w) for inocular size, 5% (w/w) skim milk and sucrose were added, the strain presented the highest activity about 1196 U/ml at 37℃fermented for 60 hours. It showed 1776 U/ml and 8184 U/ml after alcohol precipitate and ultra filtration.
The enzyme exhibited the optimal reaction temperature and pH were 60℃ and pH 10,respectivly. Protease activity kept upon 80% at -80℃ storage for one month, and for iron ion is sensitive. The study of various protease inhibitor proved that the protease belonged to serine type protease. According to the Lineweaver-Burk Double reciprocal plot, the Km value and the Vmax value were 16.36 mg/ml and 714 μg tyrosine/min respectively, initial purification enzyme was used to hydrolyze the defat soy powder. 1% defat soy powder was hydrolyzed with 1% enzyme at 50℃ for 1 hours for hydrolysis condition, the inhibit activity presented about 98.1% to ACE.

中文摘要 I
Abstract III
謝誌 V
目錄 VII
圖表目錄 X
壹、前言 1
貳、文獻回顧 3
一、蛋白酶之介紹 3
(一)蛋白酶之簡介 3
(二)蛋白酶之命名與分類 4
二、蛋白酶之應用 16
(一)皮革工業 16
(二)清潔工業 17
(三)飼料加工 17
(四)食品加工 17
(五)醫藥 19
三、高血壓之介紹 20
(一)高血壓之定義 20
(二)高血壓之分類 22
(三)血管收縮素轉化酶之生化特性 23
四、高血壓之防治與治療 27
(一)降血壓用藥 27
(二)天然ACEI來源 28
參、材料與方法 32
一、實驗架構 32
二、實驗材料 33
(一)菌株來源 33
(二)實驗培養基 33
(三)實驗藥品 35
(四)實驗儀器 35
三、實驗方法 37
(一)菌種篩選 37
(二)菌種特性及最適培養條件探討 38
(三)利用固態培養探討蛋白酶之活性生產 42
(四)蛋白酶之純化與濃縮 44
(五)蛋白酶之生化特性探討 47
(六)蛋白酶水解物對ACE抑制活性探討 49
肆、結果與討論 53
一、菌種分離與篩選 53
二、菌種特性及最適培養條件探討 53
(一)菌種鑑定及介紹 53
(二)選擇基礎培養基 57
(三)起始培養條件之生長曲線 57
(四)最適培養基探討 61
(五)最適培養條件探討 73
(六)最適培養條件之生長曲線 77
三、利用固態培養探討蛋白酶之生產活性 77
(一)最適基質探討 77
(二)最適基質比例探討 80
(三)最適水分含量探討 80
(四)最適接種量探討 84
(五)最適發酵溫度探討 84
(六)最適固態發酵碳氮源探討 84
(七)最適固態發酵生長曲線 88
四、蛋白酶之純化濃縮 88
五、蛋白酶之生化特性探討 91
(一)蛋白酶最適作用溫度 91
(二)蛋白酶之熱穩定性 94
(三)蛋白酶最適作用pH 94
(四)蛋白酶 pH值之穩定性 97
(五)不同溫度之保存性試驗 97
(六)不同蛋白酶抑制劑對蛋白酶活性之影響 101
(七)不同金屬離子添加對蛋白酶活性之影響 101
(八)蛋白酶之酵素動力學研究 101
六、蛋白酶水解物對ACE抑制活性探討 104
(一)最適水解時間 104
(二)最適水解基質濃度 108
(三)最適水解酵素濃度 108
伍、結論 111
參考文獻 113
作者介紹 124

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