利用篩選之Bacillus subtilis DYU6菌株生產凝乳酵素及其特性之研究 王曼瑩、吳建一

利用篩選之Bacillus subtilis DYU6菌株生產凝乳酵素及其特性之研究 王曼瑩、吳建一

E-mail: 345441@mail.dyu.edu.tw

摘 要

凝乳酵素(milk-clotting enzyme, MCE)主要源自動物、植物以及微生物。由於動物性凝乳酵素供不應求，導致價格居高不下

，所以，許多學者便開始研究其它替代性的蛋白?，以取代小牛凝乳酵素。本研究的目的是利用篩選之Bacillus subtilis DYU6菌株生產MCE，並探討其特性。利用搖瓶和5-L發酵槽，探討不同培養條件對B. subtilis DYU6菌株發酵生產MCE的 影響。搖瓶部分，發現最佳碳源為澱粉(20 g/ L)，在培養基中添加50mM的NaCl，可得到酵素最佳活性為1,000 SU，最大 的蛋白質水解活性為0.16 U/ mL。在5L發酵槽實驗方面，由實驗結果發現，當培養基pH調控在6、轉速為100 rpm、曝氣 量為0.5 vvm時，可達到最高酵素活性為600 SU。使用(NH4)2SO4沉澱可獲得部分純化的沉澱物。當 (NH4)2SO4飽和度 為50-70%時，由收集到的沉澱物，可測得凝乳酵素活性(milk-clotting activity, MCA)(1,333 SU/ mg)。將收集到的沉澱物再使 用column chromatography (superdexTM grade)進一步地純化，由收集的沖提液中測得MCA為4,176 SU/ mg。研究外在因子(

凝固的溫度、CaCl2濃度、NaCl濃度、pH)對於牛奶凝乳特性的影響。MCA隨著牛奶的pH遞減(從7.5-5.5)而增加。添加如

：Al3+、Zn2+、Fe3+、Fe2+ 金屬離子可以明顯地促進凝乳，而添加Ni2+, Cu2+ 及Na+則會抑制活性。不論粗酵素或是純 化酵素，其酵素活性範圍在pH 5-11，而擁有最佳活性的pH為7。酵素儲存穩定性則是在4℃最穩定，液態或粉末酵素分別 在240天和140天內仍能保留70 %的初始活性。此外，對於B. subtilis DYU6菌體生長可利用Logistic model進行動力學解析，

由結果顯示Logistic model對於菌體生長具有良好的模擬性。

關鍵詞 : 凝乳酵素、純化

目錄

封面內頁 簽名頁 中文摘要 iii 英文摘要 v 誌謝 vi 目錄 viii 圖目錄 xiii 表目錄 xix 1. 緒論 1 1.1 前言 1 1.2 研究動機與目的 6 2.

文獻回顧 7 2.1 酪蛋白之簡介 7 2.2 凝乳酵素之簡介 8 2.2.1 動物性凝乳酵素 9 2.2.2 植物性凝乳酵素 10 2.2.3 微生物性凝乳酵 素 12 2.3 生產凝乳酵素的微生物種類 12 2.3.1 真菌性凝乳酵素 12 2.3.2 細菌性凝乳酵素 14 2.4 環境因子對於生產凝乳酵素 的影響 16 2.4.1 溫度的影響 16 2.4.2 pH的影響 16 2.5 凝乳酵素的生化特性 17 2.5.1 溫度的影響 17 2.5.2 pH的影響 19 2.5.3 鈣離子的影響 20 2.6 凝乳酵素作用機制 21 3. 材料與方法 24 3.1實驗材料 24 3.1.1 材料 24 3.1.2 實驗藥品 25 3.1.3 儀器設備 27 3.2 菌株篩選 29 3.3 凝乳酵素的生產 29 3.3.1 搖瓶生產MCE實驗 29 3.3.2 發酵槽實驗 30 3.4 B. subtilis DYU6凝乳酵素純 化 31 3.4.1 酵素液製備 31 3.4.2 超過濾濃縮裝置 31 3.4.3 硫酸銨沉澱法 33 3.4.4 SuperdexTM grade管柱膠體過濾層析 33 3.5 分析方法 33 3.5.1 凝乳活性分析 33 3.5.2 蛋白質含量分析(Folin-phenol法) 34 3.5.3 蛋白?活性分析 36 3.5.4 凝乳塊強度分析 38 3.5.5 澱粉水解?活性分析 39 3.5.6 澱粉濃度含量分析 40 3.5.7 還原醣定量分析 41 3.6 B. subtilis DYU6凝乳酵素之特性探討 43 3.6.1 pH之影響 43 3.6.2 溫度之影響 43 3.6.3 乳基質濃度之影響 43 3.6.4 NaCl濃度之影響 43 3.6.5 CaCl2濃度之影響 44 3.6.6 金屬離子種類與濃度之影響 44 3.7 B. subtilis DYU6凝乳酵素之穩定性探討 45 3.7.1 酵素pH之影響 45 3.7.2 熱穩定性 45 3.8 儲存穩定性 45 3.9 SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) 46 4. 動力學模式解析 47 4.1 MCE生產動力學解析 47 5. 結果與討論 55 5.1生產凝乳酵素之菌株篩選與鑑定 55 5.2 初始PH對生產凝乳酵素之影響 59 5.3額外添加物對生產之探討 63 5.3.1金屬離子對生產凝乳酵素之影響 63 5.3.2 NaCl濃度對生產凝乳酵素之影響 67 5.4 培養基組成探討 71 5.4.1氮源對生 產凝乳酵素之影響 71 5.4.2 碳源對生產凝乳酵素之影響 75 5.4.3 澱粉種類對生產凝乳酵素之影響 80 5.4.4 澱粉濃度對生產凝 乳酵素之影響 84 5.5發酵槽實驗 90 5.5.1 曝氣量對生產凝乳酵素之影響 90 5.5.2 轉速對生產凝乳酵素之影響 93 5.5.3 調 控pH對生產凝乳酵素之影響 97 5.6 B. subtilis DYU6微生物生產動力學解析 101 5.7 B. subtilis DYU6凝乳酵素純化 107 5.8 不 同乳基質條件對凝乳酵素之特性探討 116 5.8.1 乳基質濃度之探討 118 5.8.2 溫度之探討 122 5.8.3 乳基質pH之探討 125 5.8.4 金屬離子之探討 128 5.8.5 CaCl2濃度之探討 131 5.8.6 NaCl濃度之探討 133 5.9 不同酵素條件對凝乳活性之探討 136 5.9.1 初 始酵素pH對凝乳活性的影響 136 5.10 凝乳塊口感模擬測試 141 5.10.1 乳基質pH對凝乳塊之影響 141 5.10.2 溫度對凝乳塊之 影響 143 5.10.3 酵素量對凝乳塊之影響 146 5.11 凝乳酵素儲存穩定性 148 6. 結論 150 參考文獻 152 圖目錄 Figure 1-1 Schematic of this study procedure. 5 Figure 1-2 Motivation and purpose of this study procedure. 6 Figure 2-1 The coagulation of milk by MCE includes two separate steps: 22 Figure 3-1 Schematic diagram of the fermentor. 31 Figure 3-2 The diafiltration system.

32 Figure 3-3 The Standard Curve of protein concentration. 35 Figure 3-4 The Standard Curve of tyrosine concentration. 37 Figure 3-5 Experimental apparatus for the measurement of curd strength. 38 Figure 3-6 The Standard Curve of starch concentration. 41 Figure 3-7 The Standard Curve of glucose concentration. 42 Figure 5-1 Appearance of cultured with the screened strain of production rennet's bacterial. 56 Figure 5-2 Phylogenetic tree showing species relatedness of Bacillus subtilis DYU6 isolated in

soybean waste residue. 57 Figure 5-3 Scanning electron micrograph (SEM) of B. subtilis DYU6. 58 Figure 5-4 The time course of cell growth and milk-clotting activity of enzyme by B. subtilis DYU6 in various initial pH medium 61 Figure 5-5 Effect of initial pH on MCE and protein content in fermentation broth by B. subtilis 62 Figure 5-6 The time course of cell growth and milk-clotting activity of enzyme by B. subtilis DYU6 in medium containg various metal ions. 65 Figure 5-7 Effect of metal ions on MCE and protein content in fermentation broth by B. subtilis DYU6 after 36h…...66 Figure 5-8 The time course of cell growth and milk-clotting activity of enzyme by B. subtilis DYU6 in medium containg various NaCl concentration. 69 Figure 5-9 Effect of NaCl concentration on MCE and protein content in fermentation broth by B. subtilis DYU6 after 36h 70 Figure 5-10 The time course of cell growth, protein concentration, proteolytic activity and milk-clotting activity of enzyme from different nitrogen source culture by B. subtilis DYU6. 73 Figure 5-11 Effect of different nitrogen source on MCE and proteolytic activity in fermentation broth by B.

subtilis DYU6 after 36h. 74 Figure 5-12 The time course of cell growth, proteolytic activity and milk-clotting activity of crude enzyme from different carbon sources culture by B. subtilis DYU6. 77 Figure 5-13 The time course of cell growth, protease activity and milk-clotting activity of crude enzyme from different sugar cane molasses culture by B. subtilis DYU6. 78 Figure 5-14 Effect of different carbon source on MCE and protein content in fermentation broth by B. subtilis DYU6 after 36h. 79 Figure 5-15 The time course of cell growth, milk-clotting activity, starch concentration, starch hydrolysis enzyme activity and production of reducing sugars of enzyme from different starch of carbon source culture by B. subtilis DYU6. 82 Figure 5-16 Effect of starch culture on MCE and protein content in fermentation broth by B. subtilis DYU6 after 48h. 83 Figure 5-17 The time course of cell growth and

milk-clotting activity of enzyme in medium containing different concentration of starch (Reagent Grade) by B. subtilis DYU6. 86 Figure 5-18 Effect of starch concentration on MCE and protein content in fermentation broth by B. subtilis DYU6 after 36h 87 Figure 5-19 The time course of cell growth and milk-clotting activity of enzyme in medium containing different concentration of starch (sensibly tapioca) by B. subtilis DYU6. 88 Figure 5-20 Effect of different Sensibly tapioca starch concentration on MCE and protein content in fermentation broth by B. subtilis DYU6 after 36h. 89 Figure 5-21 Time course of MCE production by B. subtilis DYU6 at various aeration rate in a 3L fermentor. 91 Figure 5-22 Effect of various aeration rate on MCE and protein content in fermentation broth by B. subtilis DYU6 after 36h 92 Figure 5-23 Time course of MCE production by B. subtilis DYU6 at various agitation rate in a 3L fermentor (aeration rate: 0.5vvm). 95 Figure 5-24 Effect of various agitation rate on MCE and protein content in fermentation broth by B. subtilis DYU6 after 36h 96 Figure 5-25 Time course of MCE production by B. subtilis DYU6 at various pH medium in a 3L fermentor 99 Figure 5-26 Effect of various pH on MCE and protein content in fermentation broth by B. subtilis DYU6 after 24h 100 Figure 5-27 Comparison of experimental data and kinetic model predicitions of the growth at various aeration rate by using Eq. (4-2). 102 Figure 5-28 Comparison of experimental data and kinetic model predicitions of the growth at various agitation rate by using Eq. (4-2). 104 Figure 5-29 Comparison of experimental data and kinetic model predicitions of the growth at various pH culture by using Eq. (4-2). 106 Figure 5-30 Elution diagram of MCE by using superdexTM grade column

chromatography. 110 Figure 5-31 SDS-PAGE of milk-clotting enzyme purification steps. 112 Figure 5-32 Effect of milk source on milk-clotting activity of enzyme from different carbon source in fermentation broth by B. subtilis DYU6. 117 Figure 5-33 Effect of skim milk concentration on milk-clotting activity of enzyme from fermentation liquid produced by B. subtilis DYU6. 120 Figure 5-34 Lineweaver-Burk plot for velocity versus substrate concnetration for milk-clotting enzyme produced by B. subtilis DYU6. 121 Figure 5-35 Effect of different temperature on milk-clotting activity of enzyme from fermentation liquid produced by B. subtilis DYU6. 124 Figure 5-36 Effect of various pH of skim milk on milk-clotting activity of enzyme from fermentation liquid produced by B. subtilis DYU6. 127 Figure 5-37 Effect of different metal ions on milk-clotting activity from fermentation liquid produced by B. subtilis DYU6. 128 Figure 5-38 Effect of CaCl2 concentration on Milk-clotting activity of enzyme from fermentation liquid produced by B.

subtilis DYU6. 132 Figure 5-39 Effect of NaCl concentration on milk-clotting activity of enzyme from fermentation liquid produced by B. subtilis DYU6. 135 Figure 5-40 Effect of various pH of enzyme on milk-clotting activity by Bacillus subtilis DYU6. 138 Figure 5-41 Effect of heating time and temperature of MCE from B. subtilis DYU6. 140 Figure 5-42 Comparison of the strength of rennet gels made from MCE by different pH. 142 Figure 5-43 Comparison of the strength of rennet gels made from MCE by different temperature. 145 Figure 5-44 Comparison of the strength of rennet gels made from MCE concentration by B. subtilis DYU6. 147 Figure 5-45 Effect of storage time on milk-clotting activity of enzyme concentration and enzyme powder from fermentation broth produced by B. subtilis DYU6. 149 表目錄 Table 2-1 List of fungal reported to produce milk clotting enzyme. 14 Table 2-2 List of bacteria reported to produce milk clotting enzyme. 15 Table 3-1 Anchor dry skim milk powder standard analysis per 100 grams of powder. 24 Table 3-2 Dry skim milk powder from Fluka company standard analysis. 26 Table 5-1 Milk-clotting activity of precipitation fraction of the B. subtilis DYU6. 109 Table 5-2 Purification of rennet from B. subtilis DYU6. 111 Table 5-3 Milk-clotting enzymes produced by various microorganisms. 113

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