白殭菌纖溶酶之特性分析與其在動物血栓溶解作用之探討

A Study on Purification of Fibrinolytic Enzyme from Beauveria bassiana and its in vivo Thrombolytic Activity

陳耀霖(Yao-Lin Chen)

指導教授 ： 劉炳嵐

朝陽科技大學/理工學院/應用化學系/碩士(2009年)

摘要

纖溶酶(Fibrinolytic enzyme)在血液的抗凝血系統中扮演一個重要的角色，它能溶解在凝血終了所形成使血栓更加穩固的纖維蛋白(fibrin)網狀聚合物及破壞纖維蛋白原(fibrinogen)，進而使血栓溶解，恢復血液流通。本論文的主要研究是利用五公升發酵槽培養白殭菌(Beauveria bassians)發酵液，經由硫酸銨沉澱、陰離子交換層析(DEAE-Sepharose FF)及親合性層析(Benzamidine Sepharose 4 FF)等一系列的純化步驟，分離出一個具有纖溶酶活性的蛋白酶。經由SDS-PAGE、活性電泳與二維電泳分析得知，白殭菌纖溶酶(BBFE)之分子量為47 kDa，等電點(pI)為5.3。近一步利用纖維蛋白活性分析證實，BBFE具有溶解Fibrin活性。從特性分析發現，此酵素之最適反應溫度與pH為50°C與pH 7，在30~50°C可維持90%以上的活性，同時在pH 8有較佳的穩定性。另外，酵素活性會受到Cu2+抑制，但添加10 mM的Ba2+、Ca2+及Mg2+則能增加活性。而PMSF、leupeptin、aprotinin會抑制BBFE活性，顯示BBFE應屬於絲胺酸蛋白酶。
最後經由動物實驗證實，以膠原蛋白(collagen)及腎上腺素(epinephrine)誘導小鼠肺血栓的模式中，給予t-PA (0.29×106 IU/kg)、BBFE低劑量(1200 U/kg)與高劑量(2400 U/kg)後分析血液凝固時間，結果顯示t-PA及低劑量組能延長APTT時間及降低fibrinogen含量，而高劑量卻沒有較佳的效果。另外，以肺組織栓塞面積量化分析發現，t-PA、BBFE低劑量可以分解血栓，降低因血栓栓塞所引起的併發症。

關鍵字

纖溶酶 ； 組織型纖溶酶原活化劑 ； 血栓 ； 分離純化 ； 白殭菌

並列摘要

The fibrinolytic enzyme acts as an important role in the blood anticoagulant system. It can dissolve the thrombus by disrupt the insoluble fibrin polymers and cleave fibrinogen formed during the coagulation. As a result, the blood flow was restored. In this study, a 5-L stirred-tank bioreactor was employed to produce the fibrinolytic enzyme from Beauveria bassians. Purification was achieved by means of ammonium sulphate precipitation, DEAE-Sepharose FF ion-exchange chromatography and subsequent extraction from a Benzamidine Sepharose 4 FF affinity chromatography. The molecular weight of Beauveria bassians fibrinolytic enzyme (BBFE) was estimated to be 47 kDa as judged by SDS-PAGE and fibrin-zymography. The pI of 5.3 was found as determined from 2-Dimensional Electrophoresis (2DE). The specific activity was determined to be 41.2 U/mg using the fibrinolytic assay with a chromogenic substrate Suc-Ala-Ala-Pro-Phe-pNA. The purified enzyme displayed optimal activity at 50°C and pH 7. It was stable between 30-50°C, over 90% of residual activity was retained. The enzyme also exhibited a better stability at pH 8. The BBFE activity was inhibited by the addition of a Cu2+. However, the presence of Ba2+, Ca2+ and Mg2+ was stimulated the enzyme activity. The fibrinolytic activity was strongly inhibited by PMSF, Leupeptin, Aprotinin. These results indicated that the BBFE is a serine protease. In vivo study was investigated using a collagen and epinephrine induced pulmonary thrombosis model in male mice. Three groups were established: t-PA (0.29×106 IU/kg), BBFE low-dose (1200 U/kg), high dose (2400 U/kg) and a saline control group. After treatment with various samples, the plasma coagulation times were analysis. The results showed that the t-PA and BBFE low-dose group extended the APTT (activated partial thromboplastin time) and reduced the fibrinogen content, while the higher BBEF dose did not show the better results. In addition, lung tissue biopsy and quantitative analysis of embolism area suggested that the t-PA and low dose BBFE can dissolve thrombin and subsequently reduce the thromboembolic complications.

並列關鍵字

tissue plasminogen activator ； thrombus ； purification ； fibrinolytic enzyme ； Beauveria bassians

參考文獻

15.高穗生、蔡勇勝(1995)蟲生病原真菌在蟲害防治上之利用(下)。藥試所專題報導，台中。

31.Ahn, M. Y., Hahn, B. S., Ryu, K. S., Kim, J. W., Kim, I. and Kim Y. S. (2003) Purification and characterization of a serine protein with fibrinolytic activity from the dung beetles, Catharsius molossus. Thrombosis Research, 112, 339-347.

32.Ballabeni, V., Calcina, F., Tognolini, M., Bruno, O., Manotti, C., Barocelli, E. (2004) Effects of subacute treatment with benzopyranopyrimidines in hemostasis and experimental thrombosis in mice. Life Sciences, 74, 1851-1859.

33.Bello, C. A., Hermogenes, A. L. N., Magalhaes, A., Veiga, S. S., Gremski, L. H., Richardson, M. and Sanchez, E. F. (2006) Isolation and biochemical characterization of a fibrinolytic proteinase from Bothrops leucurus (white-tailed jararaca) snake venom. Biochimie, 88, 189-200.

34.Bidochka, M. J. and Khachatourians, G. G. (1987) Purification and properties of the entomopathogenic fungus Beauveria bassiana. Applied and Environmental Microbiology, 53, 1679-1684.