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KMID : 0613820130230020259
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Journal of Life Science
2013 Volume.23 No. 2 p.259 ~ p.266
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Isolation of Bacteria with Protease Activity from Cheonggukjang and Purification of Fibrinolytic Enzyme
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Choi Yeon-Hee
Lee Jun-Seung
Bae So-Young
Yang Keun-Jae
Yeom Kyu-Won
Jo Dong-Hyeok
Kang Ock-Hwa
Baik Hyung-Suk
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Abstract
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To isolate the fibrinolytic enzyme, 268 strains from 21 samples were morphologically isolated from Cheonggukjang collected from Korea and Japan. Among the 268 strains, protease-producing bacteria were isolated in nutrient agar medium including 1% skimmed milk. As a result of this, 22 strains were isolated. Apiweb site was used to identify these strains based on their biochemical properties. In addition, 16S rRNA sequencing was performed to identify the strain. Most of the identified strains were Bacillus subtilis and B. amyloliquefaciens. Fibrinolytic enzyme activity was measured with the fibrin plate method. Five strains were finally selected: A2-14, A2-20, C1-05, C1-09, and F2-01. Of those five strains, the A2-20 strain, which is close to B. amyloliquefaciens, showed the strongest fibrinolytic activity. The fibrinolytic enzyme produced by the A2-20 strain was partially purified from culture supernatant by gel filtration and ion exchange chromatography. The optimal pH and temperature values of the partially purified enzyme were 7.0 and 35℃, respectively. Purified protein analysis was carried out with SDS-PAGE and zymography. A genetic analysis was also conducted by PCR based on the consensus sequence of fibrinolytic enzyme. Corresponding genes with a partial sequence of the A2-20 strain were identified.
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KEYWORD
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Bacillus amyloliquefaciens, Cheonggukjang, enzyme purification, fibrinolytic enzyme, protease
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