Purification of a Fibrinolytic Enzyme from Bacillus Sp. Isolated from Budu
DOI:
https://doi.org/10.11113/jt.v59.1586Keywords:
Bacillus sp., Fibrinolytic enzyme, fermented food, chromatographyAbstract
Bacillus sp. strain B1 producing wild type fibrinolytic enzyme was isolated from Budu. The fibrinolytic enzyme was collected from the supernatant of Bacillus sp. strain B1 culture broth and purified to electrophoretic homogeneity through a combination of various purification schemes, which include ammonium sulphate precipitation, followed by anion exchange chromatography using DEAE–Sepharose Fast Flow and gel filtration chromatography on Sephadex G–75 column. During ammonium sulphate precipitation screening, it was observed that the crude enzyme from Bacillus sp. strain B1 precipitated at 40% and 50% of ammonium sulphate saturation respectively. The fibrinolytic enzyme was purified 58.5–fold with a final yield of 0.51%. The specific activity was determined to be 1.17 Units/mg using plasmin as standard and the final total protein content was 8.58 mg/ml. After the successive purification steps, the estimated molecular mass of fibrinolytic enzymes from strain B1 was estimated via sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE). SDS–PAGE analysis showed a single band at 45 kDa corresponding to the purified fraction with fibrinolytic activity.References
Astrup, T. & Mullertz, S. 1952. The Fibrin Plate Method for Estimating
Fibrinolytic Activity. Arch. Biochem. Biophys. 40: 346–351.
Balaraman, K. & Prakabaran, G. 2007. Production and Purification of a
Fibrinolytic Enzyme (Thrombinase) from Bacillus Sphaericus. Indian J
Med Res. 126: 459–464.
Deng, Z., Wong, S., Li, Q., Ji, X., Zhang, L., & Hong, M. 2009.
Purification and Characterization of a Novel Fibrinolytic Enzyme from
the Polychaete, Neanthes Japonica (Iznka). Bioresource Technology.
: 1954–1960.
Hua, Y., Jiang, B., Mine, Y., & Mu, W. 2008. Purification and
Characterization of a Novel Fibrinolytic Enzyme from Bacillus sp. nov.
SK006 Isolated from an Asian Traditional Fermented Shrimp Paste.
Journal of Agricultural Food Chemistry. 56: 1451–1457.
Hwang, Ju, K., Choi, K. H., Kim, M. J., Park, C. S., & Cha, J. 2007.
Purification and Characterization of a New Fibrinolytic Enzyme of
Bacillus licheniformis KJ-31, Isolated from Korean Traditional Jeot-gal,
Journal of Microbiol Biotechnol. Vol 17(9): 1469-1476.
Jeong, Y. K., Park, J. U., Baek, H., Park, S. H., Kong, I. S., Kim, D. W.,
& Joo, W. H. 2001. Purification and Biochemical Characterization of a
Fibrinolytic Enzyme from Bacillus subtilis BK-17. World Journal of
Microbiology & Biotechnology. 17: 89–92.
Jo, Deok, H., Lee, H. A., Jeong, S. J., & Kim, J. H. 2011. Purification
and Characterization of a Major Fibrinolytic Enzyme from Bacillus
amyloliquefaciens MJ5-41 Isolated from Meju. J. Microbiol. Biotechnol.
(11): 1166–1173.
Kim, W., Choi, K., Kim, Y., Park, H., Choi, J., Lee, Y., Oh, H., Kwon, I.,
& Lee, S. 1996. Purification and Characterization of a Fibrinolytic
Enzyme Produced from Bacillus sp. strain CK11-4 Screened from
Changkook-Jang. Applied and Environmental Microbiology. 62(7):
–2488.
Mine, Y., Wong, A. H. K., & Jiang, B. 2005. Fibrinolytic Enzymes in
Asian Traditional Fermented Foods. Food Research International. 38:
–250.
Nordenhem, A. 2006. The fibrinolytic Enzyme System: New Markers of
Potential Interest in Cardiovascular Disease. Department of Molecular
Medicin and Surgery, Division of clinical Chemistry and Blood
Coagulation. 1–41.
Peng, Y., Huang, Q., Zhang, R., & Zhang, Y. 2003. Purification and
Characterization of a Fibrinolytic Enzyme Produced by Bacillus
Amyloliquefaciens DC-4 Screened from Douchi, A Traditional Chinese
Soybean Food. Comparative Biochemistry and Physiology, Part B. 134:
–52.
Peng, Y., Yang, X., & Zhang, Y. 2005. Microbial Finrinolytic Enzymes:
An Overview of Source, Production, Properties, and Thrombolytic
Activity in Vivo. Applicaton Microbiol Biotechnology. 69: 126–132.
Scopes, R. K. 1994. Protein Purification: Principles and Practice. 3
rd
Edition. New York: Springer-Verlag.
Sumi, H., Hamada, H., Tsushima, H., Mihara, H., & Muraki, H. 1987. A
Novel Fibrinolytic Enzyme (Nattokinase) in the Vegetable Cheese Natto;
aTypical and Popular Soybean Food in The Japanese Diet. Experientia.
(10): 1110–1111.
Vengadaramana, A., Balakumar, S., & Arasaratnam, V. 2011.
Purification and Comparison Properties of Crude Enzyme with Purified
Α-Amylase from Bacillus licheniformis ATCC 6346. European Journal
of Experimental Biology. 1(3): 58–69.
Wang, D. S., Koo, T. Y., Lin, I. P., Liu, H. R., & Chou, C. Y. 2006.
Determination of Nattokinase Production Condition Using Taguchi
Parameter Design. Food Science and Technology International. 12(3):
–220.
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