本研究係以TKU001這株真菌細胞壁分解菌發酵蝦殼粉（shrimp shell powder；簡稱SSP）所得，探討所生產TKU001蛋白酶之純化分離及定性。TKU001生產蛋白酶之較適培養基為0.5%SSP、0.1%K2HPO4、0.05%MgSO4‧7H2O之50 mL液態培養基（pH 6）。於含50 mL較適培養基的250 mL三角錐形瓶中，於37℃振盪（150 rpm）培養3天後，可得較佳之蛋白酶活性（0.11 U/mL）。
    所得發酵液經DEAE Sepharose CL-6B、Sephacryl S-200等層析分離步驟，可純化出二種蛋白酶F1及F2。利用SDS-PAGE測得F1及F2蛋白酶的分子量分別為41 kDa及75 kDa。F1之最適反應溫度為60℃、最適反應pH 8、熱安定性＜60℃、pH安定性6~9；F2蛋白酶最適反應溫度為60℃、最適反應pH 7、熱安定性＜50℃、pH安定性7~9。蛋白酶抑制劑及金屬離子對酵素活性的影響，分別受到EDTA和Fe2+、Cu2+ 、Mn2+的抑制，但F2蛋白酶不受Mn2+離子的抑制。界面活性劑對酵素的影響方面，F1及F2蛋白酶皆受到2 mM SDS作用而抑制。有機溶劑影響對F1蛋白酶之活性，在25%乙醚、甲苯、乙晴等有機溶劑存在下蛋白酶仍有70%以上之相對活性；F2蛋白酶之活性，在乙醚、甲苯、乙晴、丙酮等有機溶劑存在下蛋白酶仍有70%以上之相對活性。

The protease producing bacterium, TKU001 strain, was isolated from the cell wall screened via hydrolysising the fungi, and an experiment is with the fermented shrimp shell powder（Abbreviated as SSP）. TKU001 optimized culture was composed of 0.5% SSP, 0.1% K2HPO4, 0.05% MgSO4‧7H2O condition at pH 7. The bacterium was incubated in 250 mL Erlenmeyer flask containing 50 mL of above liquid medium and kept shaking at 37℃ for three day（150 rpm）.
    The protease of Bacteria strain TKU001 was produced under the optimized culture condition. The supernatant further purification and separation procedures of protease were by DEAE-Sepharose and Sephacryl S-200 gel chromatography. The purification two kinds of proteases（F1&F2）. The apparent molecular mass based on SDS-PAGE was estimated 41 kDa from F1 protease；the estimate 75 kDa from F2 protease. The optimum temperature and pH of the F1 protease were 60℃ and 8 respectively, and the F1 protease was stable at＜60℃ and pH 6~9. The optimum temperature and pH of the F2 protease were 60 ℃ and 7 respectively, and the F2 protease was stable at＜50℃ and pH 7~9. The inhibitor and metal ions, separately receives was inactivated by EDTA and Fe2+, Cu2+, Mn2+ suppression, but F2 protease not Mn2+ suppression. Effect of surfactants on the activities of protease, was inactivated by SDS. In the presence of organic solvent such as Ethyl Ether, Toluene, Acetontitrile, the F1 protease retained more than 70% of its activity. In the presence of organic solvent such as Ethyl ether, toluene, aacetontitrile, acetone, the F2 protease retained more than 70% of its activity.

封面內頁
簽名頁
授權書
中文摘要…………………………………………………………....I
英文摘要…………………………………………………………...II
誌謝………………………………………………………………...III
目錄………………………………………………………………...IV
圖目錄…………………………………………………………….VIII
表目錄……………………………………………………………....X

第一章	緒論……………………………………………………….1
  1.1水產廢棄物…………………………………………………....1
    1.1.1 水產廢棄物之來源……………………………………...1
    1.1.2 水產廢棄物之微生物利用……………………………...1
  1.2 蛋白酶………………………………………………………...2
    1.2.1 蛋白酶的發現與簡介…………………………………...2
    1.2.2 微生物蛋白酶之來源…………………………………...3
    1.2.3 蛋白酶的分類…………………………………………...4
    1.2.4 蛋白酶之生化特性……………………………………...7
    1.2.5 蛋白酶的應用…………………………………………...9
  1.3 酵素純化……………………………………………………...9
    1.3.1 硫酸銨沉澱法…………………………………………..10
    1.3.2 離子交換層析法………………………………………..10
    1.3.3 膠體過濾層析法........................................................11
第二章 材料與方法...........................................................14
  2.1 實驗菌株……………………………………………………14
  2.2 實驗材料……………………………………………………14
    2.2.1 培養基..............................................................14
    2.2.2 化學材料...........................................................14
    2.2.3 膠體材料……………………………………………….15
  2.3 儀器設備……………………………………………………16
  2.4 菌株生物活性及其成份之探討……………………………17
  2.5 蛋白酶活性之測定…………………………………………17
  2.6 碳源的選擇…………………………………………………18
  2.7 蛋白質酶較適生產條件探討………………………………18
    2.7.1 培養時間……………………………………………….18
    2.7.2 碳源濃度……………………………………………….18
    2.7.3 培養溫度……………………………………………….18
    2.7.4 培養基酸鹼值………………………………………….19
    2.7.5 培養通氣量…………………………………………….19
    2.7.6 培養時間對於細菌TKU001生長曲線、pH值及活性的影
         響……………………………………………………….19
  2.8 蛋白酶之分離純化…………………………………………19
    2.8.1 酵素液之製備………………………………………….20
    2.8.2 離子交換樹脂層析法………………………………….20
    2.8.3 硫酸銨沉澱…………………………………………….20
    2.8.4 膠體過濾層析法……………………………………….21
  2.9 蛋白質電泳分析……………………………………………21
  2.10 蛋白質定量分析…………………………………………..22
  2.11 酵素生化特性分析………………………………………..22
    2.11.1 酵素最適溫度…………………………………...........22
    2.11.2 酵素熱安定…………………………………...............22
    2.11.3 酵素最適pH………………………………………….22
    2.11.4 酵素pH安定性………………………………………23
    2.11.5 蛋白酶抑制劑及金屬離子對酵素的影響…………...23
    2.11.6 有機溶劑對酵素活性及安定性之影響……………...26
    2.11.7 界面活性劑對酵素活性之影響……………………...26
  2.12 蛋白酶之基質特異性………………………………..........26
第三章 結果與討論……………………………………………….27
  3.1 細菌TKU001生物活性及其成份之探討…………………27
  3.2 碳源的選擇…………………………………………………32
  3.3 細菌TKU001所生產蛋白酶最適培養條件探討…………32
    3.3.1 培養時間……………………………………………….32
    3.3.2 主要碳源蝦殼粉之含量……………………………….33
    3.3.3 培養溫度……………………………………………….33
    3.3.4 初始培養基酸鹼值…………………………………...33
    3.3.5 培養通氣量…………………………………………….33
    3.3.6 綜合結果……………………………………………….34
  3.4 蛋白酶之分離純化…………………………………………34
    3.4.1 酵素液的製備………………………………………….34
    3.4.2 離子交換樹脂層析法………………………………….42
    3.4.3 膠體過濾層析法……………………………………….42
    3.4.4 綜合結果……………………………………………….42
  3.5 蛋白質電泳分析……………………………………………47
  3.6 TKU001所生產蛋白酶之生化特性分析…………………..47
    3.6.1 酵素最適反應溫度…………………………………….47
    3.6.2 酵素熱安定性………………………………………….48
    3.6.3 酵素最適反應pH……………………………………...48
    3.6.4 酵素pH安定性………………………………………..48
    3.6.5 蛋白酶抑制劑及金屬離子對酵素的影響…………….60
    3.6.6 有機溶劑對酵素活性及安定性之影響……………….60
    3.6.7 界面活性劑對酵素活性之影響……………………….61
  3.7 蛋白酶之基質特異性………………………………………61
第四章 結論……………………………………………………68
參考文獻………………………………………………………70
圖 目 錄
圖2.1 細菌TKU001 生產蛋白酶之純化分離流程圖……………24
圗3.1 H-NMR 圖譜………………………………………………...30
圗3.2 C-NMR 圖譜………………………………………………...30
圗3.3 EI-Mass 圖譜………………………………………………...31
圗3.4 不同碳源對於蛋白酶生產之影響…………………………35
圖3.5 不同培養時間對蛋白酶生產之影響………………….…...36
圖3.6 蝦殼粉含量對蛋白酶生產之影響…………………………37
圖3.7 培養溫度對蛋白酶生產之影響……………………………38
圖3.8 不同pH 值對蛋白酶生產之影響……………………….…39
圖3.9 不同培養體積對蛋白酶生產之影響………………………40
圖3.10 細菌TKU001 在不同培養時間對蛋白酶活性、培養基菌體
濃度與pH 值的變化……………………………………...41
圖3.11 DEAE-Sepharose CL-6B 之蛋白酶層析圖譜…………….43
圖3.12 Sephacryl S-200 之F1 蛋白酶層析圖譜……………….…44
圖3.13 Sephacryl S-200 之F2 蛋白酶層析圖譜……………….....45
圖3.14 蛋白酶於12.5%之SDS-PAGE 之分子量分析…………..49
圖3.15 F1 蛋白酶之最適反應溫度………………………………..52
圖3.16 F2 蛋白酶之最適反應溫度………………………………..53
圖3.17 F1 蛋白酶之熱安定性……………………………………..54
圖3.18 F2 蛋白酶之熱安定性……………………………………..55
圖3.19 F1 蛋白酶之最適反應pH…………………………………56
圖3.20 F2 蛋白酶之最適反應pH…………………………………57
圖3.21 F1 蛋白酶之pH 安定性…………………………………...58
圖3.22 F2 蛋白酶之pH 安定性…………………………………...59
圖3.23 有機溶劑對蛋白酶活性之影響…………………………..62
圖3.24 有機溶劑對F1 蛋白酶安定性之影響……………………63
圖3.25 有機溶劑對F2 蛋白酶安定性之影響……………………64
表 目 錄
表1.1 水產加工廢棄物資源化之實施例…………………………05
表1.2 蛋白酶在食品及其他工業上的應用………………………12
表1.3 依照不同蛋白質性質所使用之分離方法…………………13
表2.1 蛋白質標準品組成之分子量………………………….…...25
表3.1 細菌TKU001 有機溶劑萃取物之細胞毒殺試驗…………28
表3.2 細菌TKU001 有機溶劑萃取物之抗氧化試驗……………29
表3.3 細菌TKU001 所生產蛋白酶之純化.……………….……..46
表3.4 本研究酵素與其它蛋白酶的生化特性之比較……………50
表3.5 金屬離子與蛋白酶抑制劑對細菌TKU001 蛋白酶活性的影
響…………………………………………………….……...65
表3.6 界面活性劑對細菌TKU001 蛋白酶活性的影響…………66
表3.7 蛋白酶之基質特異性………………………………………67

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