The Purification and Characterization of Fibrinolytic Enzyme frome Beauveria bassiana

• Main Authors: Ching-Ju Chang, 張靜如
• Other Authors: Bing-Lan Liu
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/13303978871456053350

碩士 === 朝陽科技大學 === 生物技術研究所 === 94 === 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.