Bacillus amyloliquefaciens V656係株分離自台灣中部土壤之蛋白酶生產菌。本研究以B. amyloliquefaciens V656發酵全脂奶粉生產蛋白酶。B. amyloliquefaciens V656生產蛋白酶之較適培養條件為0.5% milk powder，0.05% MgSO4．7H2O，0.1% K2HPO4 ,於pH 7，100mL，30℃,所得蛋白酶活性為55.15 U/mL。B. amyloliquefaciens V656發酵全脂奶粉所得上清液經硫酸銨沉澱、DEAE-Sepharos陰離子交換層析法及Sephacryl-S100膠體過濾層析，可純化出一種蛋白酶。此蛋白酶之最適反應pH、最適反應溫度、pH安定性及熱安定性分別為7、50℃、6-9及50℃。所得蛋白酶經SDS-PAGE電泳分析測得分子量為29 KDa，EDTA及馬麥特鐵對其具抑制效果。

A protease producing microorganism, Bacillus amyloliquefaciens V656, capable of utilizing whole milk powder as nutrition source was screened out from the soils in central Taiwan. In this research, whole milk powder was fermented by B. amyloliquefaciens V656 to produce protease. An optimal protease activity55.15 U/mL was obtained with a medium containing 0.5% milk powder，0.05% MgSO4．7H2O，0.1% K2HPO4  at pH 7,100 mL, 30℃.A protease was purified from the culture supernatant with ammonium sulfate precipitation followed by DEAE-Sepharose column chromatogrephy and  Sephacryl-S100 gel chromatography. The purified protease was stable within pH 6-9. The optimal temperature and pH for the protease were 50℃ and 7 respectively. The molecular weight of the protease was 29 KDa assayed by SDS-PAGE. EDTA and marmite irons showed inhibitory effect on this protease.

論文口試委源審議通過委員簽名表、授權書
中文論文提要                                         I
英文論文提要                                         II

第一章　研究目的                                     1
第二章　文獻回顧                                     2
2.1 發酵作用                                    2
2.1.1發酵作用的起源                             5
2.1.2結論                                   14
2.1.3主要研究者                                14
2.1.4參考資料                                15
2.2 酵素之應用                                   21 
2.3 蛋白酶                                      27       2.3.1 發現與簡介                               27   
2.3.2 命名與其分類依據                       28
2.4 一般性質                                     30
2.4.1熱變性                                  30
2.4.2表面變性                                30
2.4.3壓力變性                                30
2.4.4 pH變性                                 31
2.4.5有機溶劑變性                            31
2.5附錄 蛋白質研究歷史                          32
第三章 研究材料與方法                                 36
3.1材料及藥品                                   36
3.2研究設備與器材                               37    
3.3實驗菌株                                     38
    3.4實驗方法                                     38
      3.4.1 蛋白酶活性之測定                          38
      3.4.2蛋白質定量分析                             39
3.5蛋白酶最適生長條件探討                         39
      3.5.1碳源濃度對酵素的影響                       39
      3.5.2 基礎培養基起始之pH對酵素的影響           39
      3.5.3溶氧量對酵素的影響                       39
      3.5.4 培養時間對酵素的影響                    40
      3.5.5 微量金屬對酵素的影響                    40
      3.5.6 培養溫度                                40
3.6 蛋白酶之分離純化                            40
      3.6.1 大量培養                                40
      3.6.2 上清液的製備                            40
      3.6.3硫酸銨沉澱                                 41
3.6.4陰離子交換樹脂（Anion exchange chromatography）41
3.6.5膠體過濾層析（Gel filtration chromatography）41
3.7 蛋白酶之生化性質探討                         41
      3.7.1酵素之最適反應pH測定                    41
      3.7.2 酵素之pH安定性測定                     42
      3.7.3 酵素之最適反應溫度測定                    42
      3.7.4 酵素之熱安定性測定                        42
      3.7.5 蛋白質電泳分子量的測定                    43
      3.7.6 等電點測定                                45
 3.7.7 金屬離子及蛋白酶抑制劑對酵素活性的影響  46
 3.7.8 有機溶劑對酵素活性之影響                  46
 3.7.9 CaCl2存在下之熱安定性                      47
3.7.10 測何種金屬離子為酵素之輔酶          47
第四章 研究結果與討論                              49
4.1 Bacillus amyloliquefaciens V656生產蛋白酶最適培養條件之探討                                 49
4.1.1 氮源                                     49
4.1.2基礎起始培養基之pH值                     49
4.1.3培養液充填體積                            50
4.1.4培養時間對酵素活性的影響                  50
4.1.5金屬離子及EDTA對酵素活性的影響           50
4.1.6培養溫度對酵素活性的影響                  51
4.1.7綜合結果                                  51
4.2 Bacillus amyloliquefaciens V656之酵素純化     56
4.2.1 Diethylaminoethyl（DEAE）Sepharose CL-6B 陰離子層析法                                    57
4.2.2 Sephacryl S-100 gel filtration            57
4.2.3酵素之純化概要表                          57
4.3 Bacillus amyloliquefaciens V656酵素生化性質的分析 61
      4.3.1 酵素之最適反應pH值測定                   61
      4.3.2 酵素之pH安定性測定                       61
      4.3.3 酵素之最適反應溫度測定                    61
      4.3.4 酵素之溫度穩定性測定                      61
      4.3.5 酵素之等電點測定                          61
      4.3.6 酵素之Km及Vmax值測定                    62
      4.3.7 酵素之分子量測定                          68
      4.3.8金屬離子及抑制試劑對酵素活性的影響         69
      4.3.9 有機溶劑對酵素活性之影響                  71   
      4.3.10 CaCl2存在下之熱安定性                     72
      4.3.11 測何種金屬離子為酵素之輔酶          73
第五章 結論                                           78
第六章 參考文獻                                       79

圖目錄
  
圖 4.1.1氮源和培養天數對Bacillus amyloliquefaciensV656蛋白酶生產能之影響                              52
圖 4.1.2 起始pH值對於Bacillus amyloliquefaciensV656蛋白酶生產能之影響                                53
圖 4.1.3 溶氧量對Bacillus amyloliquefaciensV656蛋白酶生產能之影響                                      54
圖 4.1.6 培養溫度對Bacillus amyloliquefaciensV656蛋白酶生產能之影響                                    55
圖4.2.1 DEAE Sepharose CL-6B之蛋白酶陰離子層析法      58
圖4.2.2 Sephacryl S-100之蛋白酶過濾層析法             59
圖4.3.1蛋白酶之最適反應(pH 7)                          63
圖4.3.2蛋白酶之PH安定性( pH 7)                         64
圖4.3.3蛋白酶之最適反應溫度 (30℃)                    65
圖4.3.4蛋白酶之熱安定性 (30℃)                        66
圖4.3.5蛋白酶之等電點層析圖譜                         67
圖4.3.7蛋白酶之SDS-PAGE                               68
圖4.3.8金屬離子及抑制試劑對酵素活性的影響             74
圖4.3.9 有機溶劑對酵素活性之影響                       75
圖4.3.10 CaCl2存在下之熱安定性                          76
圖 4.3.11 測何種金屬離子為酵素之輔酶                   77

表目錄

表2.5附錄 蛋白質研究歷史                              32
表3.1電泳片之配置組成                                 44
表4.2.1 Bacillus amyloliquefaciensV656蛋白酶之純化表   60
表4.1.5 各種濃度之金屬離子及EDTA對蛋白酶活性的影響    70

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