DOI QR코드

DOI QR Code

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

Bacillus amyloliquefaciens HC188이 생산하는 혈전분해 효소의 정제 및 특성

  • 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)
  • 신소희 (연세대학교 생명과학기술학부) ;
  • 홍성욱 (연세대학교 생명과학기술학부) ;
  • 정건섭 (연세대학교 생명과학기술학부)
  • Received : 2012.08.27
  • Accepted : 2012.09.25
  • Published : 2013.03.28

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

References

  1. Agrebi, R., A. Haddar, N. Hmidet, K. Jellouli, L. Manni, and M. Nasri. 2009. BSF1 fibrinolytic enzyme from a marine bacterium Bacillus subtilis A26: Purification, biochemical and molecular characterization. Process Biochem. 44: 1252-1259. https://doi.org/10.1016/j.procbio.2009.06.024
  2. Ahn, Y. S., Y. S. Kim, and D. H. Shin. 2006. Isolation, identification, and fermentation characteristics of Bacillus sp. with high protease activity from traditional Cheonggukjang. Korean. J. Food. Sci. Technol. 38: 82-87.
  3. Anson, M. L. 1939. The Estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin. J. Gen. Physiol. 22: 78-89.
  4. Astrup, T. and S. Müllertz. 1952. The fibrin plate method for estimating fibrinolytic activity. Arch. Biochem. Biophys. 40: 346-347. https://doi.org/10.1016/0003-9861(52)90121-5
  5. Cho, S. J., S. H. Oh., R. D. Pridmore, M. A. Juillerat, and C. H. Lee. 2003. Purification and characterization of proteases from Bacillus amyloliquefaciens isolated from traditional soybean fermentation starter. J. Agric. Food Chem. 51: 7664-7670. https://doi.org/10.1021/jf0259314
  6. Cho, Y. J., W. S. Cha, S. K. Bok, M. U. Kim, S. S. Cheon, and U. K. Choi. 2000. Production and separation of anti-hypertensive peptide during Chunggugjang fermentation with Bacillus subtilis CH-1023. J. Korean Soc. Agric. Chem. Biotechnol. 43: 247-252.
  7. Choi, N. S., K. H. Yoo, J. H. Hahm, K. S. Yoon, K. T. Chang, B. H. Hyun, P. J. Maeng, and S. H. Kim. 2005. Purification and characterization of a new peptidase, bacillopeptidase DJ-2, having fibrinolytic activity produced by Bacillus sp. DJ-2 from Doen- Jang. J. Microbiol. Biotechnol. 15: 72-79.
  8. Collen, D. and H. R. Lijnen. 2009. The tissue-type plasminogen activator story. Arterioscler. Thromb. Vasc. Biol. 29: 1151- 1155. https://doi.org/10.1161/ATVBAHA.108.179655
  9. Craik, C. S., S. Roczniak, C. Largman, and W. J. Rutter. 1987. The catalytic role of the active site aspartic acid in serine protease. Science. 237: 909-913. https://doi.org/10.1126/science.3303334
  10. Dubey, R., J. Kumar, D. Agrawala, T. Char, and P. Pusp. 2011. Isolation, production, purification, assay and characterization of fibrinolytic enzyme (nattokinase, streptokinase and urokinase) from bacterial sources. Afr. J. Biotechnol. 10: 1408-1420.
  11. Edman, P. and G. Begg. 1967. A protein sequenator. European J. Biochem. 1: 80-91. https://doi.org/10.1111/j.1432-1033.1967.tb00047.x
  12. Fujita, M., K. Nomura, K. Hong, Y. Ito, A. Asada, and S. Nishimuro. 1993. Purification and characterization of a strong fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto, a popular soybean fermented food in Japan. Biochem. Biophys. Res. Comm. 197: 1340-1347. https://doi.org/10.1006/bbrc.1993.2624
  13. Hassanein, W. A., E. Kotb, N. M. Awny, and Y. A. El-Zawahry. 2011. Fibrinolysis and anticoagulant potential of a metallo protease produced by Bacillus subtilis K42. J. Biosci. 36: 773- https://doi.org/10.1007/s12038-011-9151-9
  14. Hong, S. W., J. Y. Kim, B. K. Lee, and K. S. Chung. 2006. The bacterial biological response modifier enriched Chungkookjang fermentation. Korean J. Food Sci. Technol. 38: 548-553.
  15. Hwang, H. A., N. K. Lee, I. J. Cho, Y. T. Hahm, K. O. Kwon, and B. Y. Kim. 2008. Selection of microorganisms and optimization of manufacture process for Cheonggukjang. Korean J. Food Sci. Technol. 40: 406-411.
  16. Hwang, J. S., H. J. Yoo, S. J. Kim, and H. B. Kim. 2008. Charactrization of $\beta$-1,4-glucanase activity of Bacillus licheniformis B1 in Chungkookjang. Korean J. Microbiol. 44: 69-73.
  17. Hwang, J. S., S. J. Kim, and H. B. Kim. 2009. Antioxidant and blood-pressure reduction effects of fermented soybean, Chungkookjang. Korean J. Microbiol. 45: 54-57.
  18. Hwang, K. J., K. H. Choi, M. J. Kim, C. S. Park, and J. H. Cha. 2007. Purification and charaterization of a new fibrinolytic enzyme of Bacillus licheniformis KJ-31, isolated from Korean traditional Jeot-gal. J. Microbiol. Biotechnol. 17: 1469-1476.
  19. Jaouadi, B., S. Ellouz-Chaabouni, M. Rhimi, and S. Bejar. 2008. Biochemical and molecular characterization of a detergentstable serine alkaline protease from Bacillus pumilus CBS with high catalytic efficiency. Biochimie 90: 1291-1305. https://doi.org/10.1016/j.biochi.2008.03.004
  20. Jo, H. D., H. A. Lee, S. J. Jeong, and J. H. Kim. 2011. Purification and characterization of a major fibrinolytic enzyme from Bacillus amyloliquefaciens MJ5-41 isolated from Meju. J. Microbiol. Biotechnol. 21: 1166-1173. https://doi.org/10.4014/jmb.1106.06008
  21. Kim, A. R., J. J. Lee, H. Lee, H. C. Chang, and M. Y. Lee. 2010. Body-weight-loss and cholesterol-lowering effects of Cheonggukjang (a fermented soybean paste) given to rats fed a high-fat/high-cholesterol diet. Korean J. Food Preserv. 17: 688-697.
  22. Kim, G. M., A. R. Lee, K. W. Lee, J. Y. Park, J. Y. Chun, J. H. Cha, Y. S. Song, and J. H. Kim. 2009. Characterzation of a 27kDa fibrinolytic enzyme from Bacillus amyloliquefaciens CH51 isolated from Cheonggukjang. J. Microbiol. Biotechnol. 19: 997-1004. https://doi.org/10.4014/jmb.0811.600
  23. Kim, H. K., G. T. Kim, D. K. Kim, W. A. Choi, S. H. Park, Y. K. Jeong, and I. S. Kong. 1997. Purification and characterization of a novel fibrinolytic enzyme from Bacillus sp. KA38 originated from fermented fish. J. Ferment. Bioeng. 84: 307-312. https://doi.org/10.1016/S0922-338X(97)89249-5
  24. Kim, S. H., N. S. Choi, W. Y. Lee, J. W. Lee, and D. H. Kim. 1998. Isolation of Bacillus strains secreting fibrinolytic enzymes from Doenjang. Korean J. Microbiol. 34: 87-90.
  25. Kim, Y. M., M. A. Kim, and Y. W. Kim. 1999. The changes of lipoxygenase-2, -3 and urease activities and of trypsin inhibitor, tannin and phytic acid contents in process of Chunggukjang. Korean Soybean Digest. 16: 56-62.
  26. Kim, W. K., K. Y. Choi, Y. T. Kim, H. H. Park, J. Y. Choi, Y. S. Lee, H. I. Oh, I. B. Kwon, and S. Y. Lee. 1996. Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from Chungkook-jang. Appl. Environ. Microbiol. 62: 2482-2488.
  27. Korea National Statistical Office. A study on causes of death for 2011 (2012).
  28. Kunamneni, A., B. D. Ravuri, P. Ellaiah, T. Prabhakhar, and V. Saisha. 2008. Urokinase - a strong plasmingen activiator. Biotechnol. Mol. Biol. Rev. 3: 58-70.
  29. Kwon, H. Y., Y. S. Kim, G. S. Kwon, C. S. Kwon, and H. Y. Sohn. 2004. Isolation of immuno-stimulation strain Bacillus pumilus JB-1 from Chungkook-jang and fermentational characteristics of JB-1. Korean J. Microbiol. Biotechnol. 32: 291-296.
  30. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227: 680-685. https://doi.org/10.1038/227680a0
  31. Lee, D. G., N. Y. Kim, M. K. Jang, B. H. Yoo, K. Y. Kim, S. G. Kim, Y. K. Jeong, and S. H. Lee. 2006. Isolation of a fibrinolytic bacterium from Cheongkukjang and characterization of its bioactivity. Korean J. Microbiol. Biotechonol. 34: 299-305.
  32. Lee, S. K., D. H. Bae, T. J. Kwon, S. B. Lee, H. H. Lee, J. H. Park, S. Heo, and Michael G. Johnson. 2001. Purification and characterization of a fibrinolytic enzyme from Bacillus sp. KOD-13 isolated from soybean paste. J. Microbiol. Biotechnol. 11: 845-852.
  33. Mahajan, P. M., S. Nayak, and S. S. Lele. 2012. Fibrinolytic enzyme from newly isolated marine bacterium Bacillus subtilis ICTF-1: media optimization, purification and characterization. J. Biosci. Bioeng. 113: 307-314.
  34. Markland, F. S., B. R. Dumas, and E. L. Smith. 1967. Subtilisin BPN' VI. Isolation and sequence of peptic peptides. J. Biol. Chem. 242: 5174-5197.
  35. Mine, Y., A. H. K. Wong, and B. Jiang. 2005. Fibrinolytic enzymes in asian traditional fermented foods. Food Res. Inter. 38: 243-250. https://doi.org/10.1016/j.foodres.2004.04.008
  36. Noh, K. A. D. H. Kim, N. S. Choi, and S. H. Kim. 1999. Isolation of fibrinolytic enzyme producing strains from kimchi. Korean J. Food Sci. Technol. 31: 219-223.
  37. Ohnishi, T. S. and J. K. Barr. 1978. A simplified method of quantitationg protein using the biuret and phenol reagents. Anal. Biochem. 86: 193-200. https://doi.org/10.1016/0003-2697(78)90334-2
  38. Paik, H. D., S. K. Lee, S. Heo, S. Y. Kim, H. H. Lee, and T. J. Kwon. 2004. Purification and characterization of the fibrinolytic enzyme produced by Bacillus subtilis KCK-7 from Chungkookjang. J. Microbiol. Biotechnol. 14: 829-835.
  39. Rhee, J. H., K. H. Park, K. R. Yoon, C. B. Yim, and I. H. Lee. 2004. Isolation of Bacillus subtilis producing the Cheongkukjang with reduced off-flavor by suppressing the growth of bacteria causing off-flavor. Food Sci. Biotechonol. 13: 801-805.
  40. Saitou, N. and M. Nei. 1987. The neighbor-joining methods: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406-425.
  41. Shon, M. Y., K. I. Seo, S. K. Park, Y. S. Cho, and N. J. Sung. 2001. Some biological activities and isoflavone content of Chungkuhjang prepared with black beans and Bacillus strain. J. Korean Soc. Food Sci. Nutr. 30: 662-667.
  42. Sikri, N. and A. Bardia. 2007. A history of streptokinase use in acute myocardial infarction. Tex. Heart Inst. J. 34: 318-327.
  43. Sneath, P. H. A. 1986. Endospore forming Gram-positive rods and cocci, pp. 1104-1139. In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (eds.), Bergey's Manual of Systematic Bacteriology, vol. 2. Williams & Wilkins, Baltimore, MD. USA.
  44. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25: 4876-4882. https://doi.org/10.1093/nar/25.24.4876
  45. Wang, C. T., B. P. Ji, B. Li, R. Nout, P. L. Li, H. Ji, and L. F. Chen. 2006. Purification and characterization of a fibrinolytic enzyme of Bacillus subtillis DC33, isolated from Chinese traditional Douchi. J. Ind. Microbiol. Biotechnol. 33: 750-758. https://doi.org/10.1007/s10295-006-0111-6
  46. Yang, J. L., H. S. Kim, J. H. Hong, and Y. S. Song. 2006. Purification and characteristics of fibrinolytic enzyme from Chungkukjang. J. Food Sci. Nutr. 11: 127-132. https://doi.org/10.3746/jfn.2006.11.2.127
  47. Yeo, W. S., M. J. Seo, M. J. Kim, H. H. Lee, B. W. Kang, J. U. Park, Y. H. Choi, and Y. K. Jeong. 2011. Biochemical analysis of a fibrinolytic enzyme purification from Bacillus subtilis strain A1. J. Microbiol. 49: 376-380. https://doi.org/10.1007/s12275-011-1165-3
  48. Yin. L. J., H. H. L, and S. T. Jiang. 2010. Bioproperties of potent nattokinase from Bacillus subtilis YJ1. J. Agric. Food Chem. 58: 5737-5742. https://doi.org/10.1021/jf100290h
  49. Yoon, K. D., D. J. Kwon, S. S. Hong, S. I. Kim and K. S. Chung. 1996. Inhibitory effect of soybean and fermented soybean products on the chemically induced mutagenesis. Korean J. Appl. Microbiol. Biotechnol. 24: 525-528.