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http://dx.doi.org/10.4014/kjmb.1208.08010

Purification and Characterization of a Fibrinolytic Enzyme Produced by Bacillus amyloliquefaciens HC188  

Shin, So Hee (Division of Biological Science and Technology, Yonsei University)
Hong, Sung Wook (Division of Biological Science and Technology, Yonsei University)
Chung, Kun Sub (Division of Biological Science and Technology, Yonsei University)
Publication Information
Microbiology and Biotechnology Letters / v.41, no.1, 2013 , pp. 33-43 More about this Journal
Abstract
A bacterium producing a fibrinolytic enzyme was isolated from Cheonggukjang. The bacterium was identified as a strain of Bacillus amyloliquefaciens by 16S rDNA analysis and designated as B. amyloliquefaciens HC188. The optimum culture medium appeared to be one containing 0.5% (w/v) maltose and 0.5% (w/v) soytone. Bacterial growth in the optimal medium at $37^{\circ}C$ reached the stationary phase after 27 h of incubation and the fibrinolytic enzyme showed optimum activity at 24 h. The enzyme was purified by 20-80% ammonium sulfate precipitation, CM Sepharose fast flow ion exchange chromatography, and Sephacryl S-200HR column chromatography. Its specific activity was 38359.3 units/mg protein and the yield was 5.5% of the total activity of the crude extracts. The molecular weight was 24.7 kDa and the amino acids of the N-terminal sequence were AQSVPYGVSQIKAPA. The fibrinolytic enzyme activity had an optimum temperature of $40^{\circ}C$ and an optimum pH of 8.0, and the enzyme was stable in the ranges $20-40^{\circ}C$ and pH 6.0-8.0. Enzyme activity was increased by $Ca^{2+}$ and $Co^{2+}$ but inhibited by $Cu^{2+}$, EDTA, and PMSF. It is suggested that the purified enzyme is a metallo-serine protease.
Keywords
Purification; fibrinolytic enzyme; Bacillus amyloliquefaciens; Cheonggukjang;
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