臺灣博碩士論文加值系統

纖溶酶(fibrinolytic enzyme)有破壞和溶解凝血蛋白酵素(thrombin)能力，是身體對抗血液不正常凝固之重要酵素之一。此酵素能裂解纖維蛋白(fibrin)交叉連結網絡而形成穩定聚合體，進而溶解血液凝固恢復血液流通。實驗初步篩選出具有高抗凝血蛋白活性菌株分別選擇兩株細菌(液化澱粉芽胞桿菌、枯草桿菌)及四株真菌(白殭菌、蛹蟲草、黑殭菌、蠟蚧輪枝菌)。其中，蟲生真菌白殭菌菌株具有高潛力生產纖溶酶之能力。白殭菌所分泌發酵液經硫酸銨沉澱、DEAE-Sepharose CL-6B離子交換層析以及Benzamidine sepharose CL 4B親和性層析進行分離純化。經SDS-PAGE電泳和MALDI-TOF MS分析方法得知，分子量約為53、10.7及8 kDa。進一步以caseinolytic, fibrinolytic, amidolytic, fibrinolysis和fibrinogenolysis等分析方法確認抗凝血酵素之存在。在amidolytic activity之測定中發現，N-Suc-Ala-Ala-Pro-Phe-pNA呈現較高基質活性。fibrinolysis activity分析，發現白殭菌發酵液對fibrin之�� subunit有高分解能力，其次為�� subunit，亦可水解��-�� subunit。此外fibrinogenolysis活性方析發現，纖溶酶能快速水解A�� subunit，另外B�狻M�� subunits於一小時後隨之被水解。白殭菌纖溶酶之特性分析得知，此酵素之最適溫度為40�aC，最適pH為 6 (磷酸緩衝液)。酵素活性受到二價金屬離子(10 mM )，如Cu2+, Mg2+, Zn2+, 及 Mn2+抑制，但Fe2+則有助於纖溶酶之活性表現。另外，於EDTA存在下，酵素活性稍微減少，但於PMSF存在下，酵素活性則受到強烈抑制，顯示此酵素應屬於絲胺酸蛋白分解酵素。

Fibrinolytic enzyme, an important enzyme capable of breaking down and dissolving thrombin, can cleave the fibrin network that dissolution the clot to restore blood flow. Two bacteria (Bacillus amyloliquefaciens and B. subtilis) and four fungi (Beauveria bassiana, Cordyceps militari, Metarhizium anisopliae, and Verticllium lecanni) were screening for the ability of fibrinolytic enzyme production. Among, the high potential strain was fond to be entomopathogen Beauveria bassiana, a fungus which causes a disease known as the white muscadine disease in insects. Purification was achieved by means of ammonium sulphate precipitation, DEAE ion-exchange chromatography and subsequent extraction from a Benzamidine sepharose CL4 affinity chromatography. The molecular mass was determined by SDS-PAGE and MALDI-TOF MS to be 53, 10.7, and 8 kDa, respectively. Further verification of fibrinolytic ability of this enzyme through the caseinolytic, fibrinolytic, amidolytic, fibrinolysis, and fibrinogenolysis assays were also investigated. The fibrinolysis activity assay revealed that the enzyme rapidly hydrolyzed the �� subunit of fibrin, followed by the �� subunit. A remarkable hydrolyzed in the ��-�� subunit was also observed. Furthermore, in the fibrinogenolysis activity assay, a rapidly hydrolyzed in the A�� subunit was detected as well, while the B�� and �� subunits were hydrolyzed after 1 h. The purified enzyme displayed an optimum temperature around 40 �aC, exhibited an optimum pH of 6.0 in phosphate buffer. The enzyme activity was inactivated by the addition of a divalent cation, such as Cu2+, Mg2+, Zn2+, and Mn2+ (at 10 mM level). However, the presence of Fe2+ was promoted the enzyme activities. The ionic strength studied revealed that the enzyme retained the similar activity even at high ionic strength. This fibrinolytic enzyme activity decreased slightly in the presence of EDTA, but was strongly inhibited by PMSF. The result with this serine protease inhibitor indicates that the enzyme is indeed a serine protease.

中文摘要…………………………………………………………………I
Abstract ……………………………………………………………III
誌謝………………………………………………………………………V
目錄……………………………………………………………………VII
表目錄…………………………………………………………………XI
圖目錄……………………………………………………………XII

第一章 緒論……………………………………………………………1

第二章 文獻簡介………………………………………………………3

2-1 白殭菌簡介………………………………………………………3
2-2 蛋白質分解酵素的分類及作用機制………………………………4
2-3 纖溶酶簡介…………………………………………………………9
2-4 凝血機制…………………………………………………………11
2-5 血管栓塞症………………………………………………………21
2-5-1 血栓溶解劑……………………………………………………21
2-5-2 抗凝血劑………………………………………………………25
2-6 蛋白質分解酵素在工業與醫療上應用…………………………27
2-7 研究範圍及目的…………………………………………………29

第三章 不同微生物纖溶酶來源之篩選……………………………32

3-1 菌種之來源及培養……………………………………………32
3-2 分析方法………………………………………………………35
3-2-1 酪蛋白分解酵素活性分析(caseinolytic activity)……35
3-2-2 纖維蛋白試驗法(fibrinolytic activity)………………38
3-2-3 纖維醯胺水解之活性方法(amidolytic activity)………40
3-3 結果與討論………………………………………………………43

第四章 纖溶酶之分離純化……………………………………………50

4-1 前言………………………………………………………………50
4-2分離純化…………………………………………………………50
4-3 結果與討論………………………………………………………52

第五章 纖溶酶之分析鑑定……………………………………………58

5-1 纖維蛋白-酶圖(fibrin-zymography)…………………………58
5-2 纖溶酶活性檢驗法(fibrinolysis activity)…………………58
5-3 纖維蛋白原活性檢驗法(fibrinogenolysis)…………………58
5-4 SDS膠體電泳……………………………………………………59
5-5 二維電泳………………………………………………………59
5-6 基質輔助雷射脫附游離飛行時間質譜分析…………………60
5-6-1 前言…………………………………………………………60
5-6-2 實驗方法……………………………………………………61
5-7 結果與討論……………………………………………………62

第六章 纖溶酶之特性探討……………………………………………73

6-1 前言………………………………………………………………73
6-2 實驗方法…………………………………………………………73
6-2-1最適溫度…………………………………………………………74
6-2-2最適pH之測定…………………………………………………74
6-2-3金屬離子對酵素作用之影響…………………………………74
6-2-4離子強度對酵素作用之影響……………………………………75
6-2-5抑制劑特異性……………………………………………………75
6-3 結果與討論………………………………………………………76

第七章 結論與未來展望………………………………………………83

參考文獻………………………………………………………………84

附錄一 …………………………………………………………………94

作者簡歷……………………………………………………………95

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