Purification and Characterization of Two Novel Fibrinolytic Proteases from Mushroom, Fomitella fraxinea

  • Lee Jong-Suk (Korea Research Institute of Bioscience and Biotechnology) ;
  • Baik Hyung-Suk (Department of Microbiology, Busan National University) ;
  • Park Sang-Shin (Department of Biotechnology, Dongguk University)
  • Published : 2006.02.01

Abstract

Two fibrinolytic enzymes were purified from the culture supernatant of Fomitella fraxinea mycelia by ion-exchange and gel filtration chromatographies, and were designated as F. fraxenia proteases 1 and 2 (FFP1 and FFP2). The apparent molecular masses of the enzymes were estimated to be 32 kDa and 42 kDa, respectively, by SDS-PAGE and gel filtration chromatography. Both enzymes had the same optimal temperature ($40^{\circ}C$), but different pH optima (10.0 and 5.0 for FFP1 and FFP2, respectively). FFP1 was relatively stable at pH 7.0-9.0 and temperature below $30^{\circ}C$, whereas FFP2 was very stable in the pH range of 4-11 and temperature below $40^{\circ}C$. FFPI activity was completely inhibited by phenylmethylsulfonyl fluoride (PMSF) and aprotinin, indicating that this enzyme is a serine protease. The activity of FFP2 was enhanced by the addition of $CO^{2+}$ and $Zn^{2+}$ and inhibited by $Cu^{2+},\;Ni^{2+}$, and $Hg^{2+}$. Furthermore, FFP2 activity was strongly inhibited by EDTA and 1,10-phenanthroline, implying that the enzyme is a metalloprotease. Both enzymes readily hydrolyzed fibrinogen, preferentially digesting the $A{\alpha}$- and $B{\beta}$-chains of fibrinogen over ${\gamma}$-chain. FFP1 showed broad substrate specificity for synthetic substrates, but FFP2 did not. $K_{m}$ and $V_{max}$ values of FFP1 for a synthetic substrate, N-succinyl-Ala-Ala-Pro-Phe-pNA, were 0.213 mM and 39.68 units/ml, respectively. The first 15 amino acids of the N-terminal sequences of both enzymes were APXXPXGPWGPQRIS and ARPP(G)VDGQ(R,I)SK(L)ETLPE, respectively.

Keywords

References

  1. Astrup, T. and S. Mullertz. 1952. The fibrin plate method for estimating fibrinolytic activity. Arch. Biochem. Biophys. 40: 346-351 https://doi.org/10.1016/0003-9861(52)90121-5
  2. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  3. Chihara, G., J. Hamuro, Y. Y. Maeda, Y. Arai, and F. Fukuoka. 1970. Fractionation and purification of the polysaxccharides with marked antitumor activity, especially lentinan, from Lentinus edodes (Berk.) Sing. (an edible mushroom). Cancer Res. 30: 2776-2781
  4. Choi, H. S. and H. H. Shin. 1998. Purification and partial characterization of a fibrinolytic protease in Pleurotus ostreatus. Mycologia 90: 674-679 https://doi.org/10.2307/3761226
  5. Choi, N. S., K. H. Yoo, J. H. Hahm, K. S. Yoon, K. T. Chang, B. H. Hyun, P. J. Maeing, 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. Biothecnol. 15: 72-79
  6. Choi, W. A., J. W. Lee, K. H. Lee, and S. H. Park. 1998. Effects of environmental and nutritional conditions on fibrinolytic enzyme production from Bacillus subtilis BK-17 in flask culture. Kor. J. Biotechnol. Bioeng. 13: 491-496
  7. Collens, D. and H. R. Lijnen.1991. Basic and clinical aspects of fibrinolysis and thrombosis. Blood 78: 3114-3124
  8. Fujita, M., K. Nomura, K. Hong, Y. Ito, A. Asada, and S. Nishimuro. 1993. Purification and characterization of strong fibrinolytic enzyme (nattokinase) in the vegetable cheese natto, a popular soybean fermented food in Japan. Biochem. Biophy. Res. Commun. 197: 1340-1347 https://doi.org/10.1006/bbrc.1993.2624
  9. Harlan, J. M. and L. A. Harker. 1981. Haemostasis, thrombosis and thromboembolic disorder. Med. Clin. North Am. 65: 855-857 https://doi.org/10.1016/S0025-7125(16)31502-4
  10. Jang, J. S., D. O. Kang, M. J. Chun, and S. M. Byun. 1992. Molecular cloning of a subtilisin J gene from Bacillus stearothermophilus and its expression in Bacillus subtilis. Biochem. Biophys. Res. Commun. 184: 277-282 https://doi.org/10.1016/0006-291X(92)91189-W
  11. Jain, S. C., U. Shinde, Y. Li, M. Inouye, and H. M. Berman. 1998. The crystal structure of an autoprocessed Ser221 Cys-subtilisin E propeptide complex at 2.0-A resolution. J. Mol. Biol. 284: 137-144 https://doi.org/10.1006/jmbi.1998.2161
  12. Kim J. H. and Y. S. Kim. 1999. A fibrinolytic metalloprotease from the fruiting bodies of an edible mushroom, Armillariella mellea. Biosci. Biotechnol. Biochem. 63: 2130-2136 https://doi.org/10.1271/bbb.63.2130
  13. Kim J. H. and Y. S. Kim. 2001. Characterization of a metallaloenzyme from a wild mushroom, Tricholoma saponaceum. Biosci. Biotechnol. Biochem. 65: 356-362 https://doi.org/10.1271/bbb.65.356
  14. Kim, W. K., K. H. 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 CK11-4 screened from Chungkook-Jang. Appl. Environ. Microbiol. 62: 2482-2488
  15. Kweon, M. H., H. Jang, W. J. Lim, H. I. Chang, C. W. Kim, H. C. Yang, H. J. Hwang, and H. C. Sung. 1999. Anticomplementary properties of polysaccharides isolated from fruit bodies of mushroom Pleurotus ostreatus. J. Microbiol. Biotechnol. 9: 450-456
  16. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: 680-685 https://doi.org/10.1038/227680a0
  17. Lee, J. H., S. M. Cho, H. M. Kim, N. D. Hong, and I. D. Yoo. 1997. Immunostimulating activity of polysaccharides from mycelia of Phellinus linteus grown under different culture conditions. J. Microbiol. Biotechnol. 7: 52-55
  18. Lee, J. S., H. S. Baik, and S. S. Park. 2002. Optimal production and characterization of fibrinolytic enzymes from Fomitella fraxinea mycelia. Kor. J. Microbiol. Biotechnol. 30: 325- 331
  19. Lee, S. K., D. H. Bae, T. J. Kwon, S. B. Lee, H. H. Lee, J. H. Park, S. Heo, and M. G. Jhonson. 2001. Purification and characterization of a fibrinolytic enzyme from Bacillus sp. KDO-13 isolated from soybean paste. J. Microbiol. Biotechnol. 11: 845-852
  20. Lee, S. Y., J. S. Kim, J. E. Kim, K. Sapkota, M. H. Shen, S. Kim, H. S. Chun, J. C. Yoo, H. S. Choi, M. K. Kim, and S. J. Kim. 2005. Purification and characterization of fibrinolytic enzyme from cultured mycelia of Armillaria mellea. Protein Expr. Purif. 43: 1-7 https://doi.org/10.1016/j.pep.2005.03.016
  21. Liu, K. L., L. X. Du, F. P. Lu, X. Q. Zheng, and J. Xiao. 2005. Purification and characterization of a novel fibrinolytic enzyme from Rhizopus chinensis 12. Appl. Microbiol. Biotechnol. 67: 209-214 https://doi.org/10.1007/s00253-004-1846-5
  22. Nakajima, N., N. Taya, and H. Sumi. 1993. Potent fibrinolytic enzyme from the lysate of Katsuwonus pelamis digestive tract (Shiokara): Purification and characterization. Biosci. Biotechnol. Biochem. 57: 1604-1605 https://doi.org/10.1271/bbb.57.1604
  23. Nakamura, T., Y. Yamafata, and E. Ichishima. 1992. Nucleotide sequence of the subtilisin NAT gene, aprN of Bacillus subtilis(natto). Biosci. Biotechnol. Biochem. 56: 1869-1871 https://doi.org/10.1271/bbb.56.1869
  24. Nonaka, T., H. Ishikawa, Y. Tsumuraya, Y. Hashimoto, and N. Dohmae. 1995. Characterization of a thermostable lysine-specific metallopeptidase from the fruiting bodies of a Basidiomycete Grifola frondosa. J. Biochem. 118: 589-593
  25. Park, S. S., J. S. Lee, K.G. Bae, K. H. Yu, H. C. Han, and T. J. Min. 2001. Antioxidative activity and structural analysis of the steroid compound from Fomitella fraxinea. Kor. J. Mycol. 29: 67-71
  26. 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
  27. Reed, G. L., L. F. Parhami-Seren, and P. Kussie. 1995. Identification of plasminogen binding region in streptokinase that is necessary for the creation of functional streptokinase-plasminogen activator complex. Biochemistry 34: 10266- 10271 https://doi.org/10.1021/bi00032a021
  28. Shin, H. H. and H. S. Choi. 1999. Purification and characterization of metalloenzyme from Pleurotus sajor-caju. J. Microbiol. Biotechnol. 9: 675-678
  29. Sumi, H., H. Hamada, K. Nakashini, and H. Hiratani. 1990. Enhancement of the fibrinolytic activity in plasma by oral administration of nattokinase. Acta Haematol. 84: 139-143 https://doi.org/10.1159/000205051
  30. Sumi, H., H. Hamada, H. Tsushima, H. Mihara, and H. Muraki. 1987. A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese natto; a typical and popular soybean food in the Japanese diet. Experientia 43: 1110-1111 https://doi.org/10.1007/BF01956052
  31. Voet, D. and J. G. Voet. 1990. Biochemistry, pp. 1087-1095, 2nd Ed. John Willey and Sons, New York, U.S.A
  32. Yoshimoto, T., H. Oyama, T. Honda, H. Takeshita, T. Kamiyama, and D. Tsuru. 1988. Cloning and expression of subtilisin amylosacchariticus gene. J. Biochem. 103: 1060- 1065 https://doi.org/10.1093/oxfordjournals.jbchem.a122380
  33. Zaworski, P. G., K. R. Marotti, V. MacKay, C. Yip, and G. S. Gill. 1989. Production and secretion of porcine urokinase in Saccharomyces cerevisiae: Characterization of the secreted gene product. Gene 28: 545-551