SpringerLink
Log in

Purification and properties of a collagenolytic protease produced by marine bacteriumVibrio vulnificus CYK279H

  • Published:
Biotechnology and Bioprocess Engineering Aims and scope Submit manuscript

Abstract

A collagenolytic enzyme, produced byVibrio vulnificus CYK279H, was purified by ultrafiltration, dialysis, Q-Sepharose ion exchange and Superdex-200 gel chromatography. The enzyme from the supernatant was purified 13.2 fold, with a yield of 11.4%. The molecular weight of the purified enzyme was estimated by SDS-PAGE to be approximately 35.0 kDa. The N-terminal sequence of the enzyme was determined as Gly-Asp-Pro-Cys-Met-Pro-Ile-Ile-Asn. The optimum temperature and pH for the enzyme activity were 35°C and 7.5, respectively. The enzyme activity was stable within the pH and temperature ranges 6.8∼8.0 and 20∼35°C, respectively. The purified enzyme was strongly activated by Zn2+, Li2+, and Ca2+, but inhibited by Cu2+. In addition, the enzyme was strongly inhibited by 1, 10-phenanthroline and EDTA. The purified enzyme was suggested to be a neutral metalloprotease.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price includes VAT (France)

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Ravanti, L. and V. M. Kähäri (2000) Matrix metalloproteases in wound repair.Int. J. Mol. Med. 6: 391–407.

    CAS  Google Scholar 

  2. Harrington, D. J. (1996) Bacterial collagenases and collagen-degrading enzymes and their potential role in human disease.Infect. Immun. 64: 1885–1891.

    CAS  Google Scholar 

  3. Van Wart, H. E. and D. E. Steinbrink (1981) A continuous spectrophotometric assay forClostridium histolyticum collagenase.Anal. Biochem. 113: 356–365.

    Article  Google Scholar 

  4. Gross, J. and C. M. Lapiere (1962) Collagenolytic activity in amphibian tissues: A tissue culture assay,Proc. Natl. Acad. Sci. USA 48: 1014–1022.

    Article  CAS  Google Scholar 

  5. Peterkofsky, B. (1982) Bacterial collagenase.Methods Enzymol. 82: 453–471.

    Article  CAS  Google Scholar 

  6. Eisen, A. Z., K. O. Henderson, J. J. Jeffrey, and R. A. Bradshaw (1973) A collagenolytic protease from the hepatopancreas of the fiddler crab,Uca pugilator, Purification and properties.Biochemistry 12: 1814–1822.

    Article  CAS  Google Scholar 

  7. Neurath, H. (1984) Evolution of protelytic enzyme.Science 224: 350–357.

    Article  CAS  Google Scholar 

  8. Watanabe, K. (2004) Collagenolytic proteases from bacteria.Appl. Microbiol. Biotechnol. 63: 520–526.

    Article  CAS  Google Scholar 

  9. Honds, S. (1998) Dietary use of collagen and collagen peptides for cosmetics.Food style. 21: 54–60.

    Google Scholar 

  10. Dreisbach, J. H. and J. R. Merkel (1978) Induction of collagenase production inVibrio B-30.J. Bacteriol. 135: 521–527.

    CAS  Google Scholar 

  11. Matsushita, O., K. Yoshihara, S. Katayama, J. Minami, and A. Okabe (1994) Purification and characterization ofClostridium perfringens 120-kilodalton collagenase and nucleotide sequence of the corresponding gene.J. Bacteriol. 176: 149–156.

    CAS  Google Scholar 

  12. Okamoto, M., Y. Yonejima, Y. Tsujimoto, Y. Suzuki, and K. Watanabe (2001) A thermostable collagenolytic protease with a very large molecular mass produced by thermophilicBacillus sp. strain MO-1.Appl. Microbiol. Biotechnol. 57: 103–108.

    Article  CAS  Google Scholar 

  13. Teo, J. W., L. H. Zhang, and C. L. Poh (2003) Cloning and characterization of a metalloprotease fromVibrio harveyi strain AP6.Gene 303: 147–156.

    Article  CAS  Google Scholar 

  14. Matsushita, O., C. M. Jung, S. Katayama, J. Minami, Y. Takahashi, and A. Okabe (1999) Gene duplication and multiplicity of collagenases inClostridium histolyticum.J. Bacteriol. 181: 923–933.

    CAS  Google Scholar 

  15. Gupta, R., Q. K. Beg, and P. Lorenz (2002) Bacterial alkaline proteases: molecular approaches and industrial applications.Appl. Microbiol. Biotechnol. 59: 15–32.

    Article  CAS  Google Scholar 

  16. Rao, M. B., A. M. Tanksale, M. S. Ghatge, and V. V. Deshpande (1998) Molecular and biotechnological aspects of microbial proteases.Microbiol. Mol. Biol. Rev. 62: 597–635.

    CAS  Google Scholar 

  17. Kang, S. I., Y. M. Kim, Y. B. Jang, D. J. Lim, and J. Y. Kong (2004) The optimal culture condition for the collagenolytic protease production fromVibrio vulnificus CYK279H.Korean J. Biotechnol. Bioeng. 19: 295–300.

    Google Scholar 

  18. Moore, S. and W. H. Stein (1948) Photometric ninhydrin method for use in the chromatography of amino acids.J. Biol. Chem. 176: 367–388.

    CAS  Google Scholar 

  19. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principal of protein-dye binding.Anal. Biochem. 72: 248–254.

    Article  CAS  Google Scholar 

  20. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227: 680–685.

    Article  CAS  Google Scholar 

  21. Edman, P. and G. Begg (1967) A protein sequenator.Eur. J. Biochem. 1: 80–91.

    Article  CAS  Google Scholar 

  22. Batrhomocuf, C., H. Pourrat, and A. Pourrat (1992) Collagenolytic activity of a new semi-alkaline protease fromAspergillus niger.J. Ferment. Bioeng. 73: 233–236.

    Article  Google Scholar 

  23. Roy, P., B. Colas, and P. Durand (1996) Purification, kinetical and molecular characterizations of a serine collagenolytic protease from greenshore crab (Carcinus maenas) digestive gland.Comp. Biochem. Physiol. 115B: 87–95.

    CAS  Google Scholar 

  24. Juarez, Z. E. and M. W. Stinson (1999) An extracellular protease ofStreptococcus gordonii hydrolyzes type IV collagen and collagen analogues.Infect. Immun. 67: 271–278.

    CAS  Google Scholar 

  25. Sasagawa, H., Y. Kamio, Y. Matsubara, Y. Matsubara, K. Suzuki, H. Kojima, and K. Izaki (1993) Purification and properties of collagenase fromCytophaga sp. L43-1 strain.Biosci. Biotechnol. Biochem. 57: 1894–1898.

    Article  CAS  Google Scholar 

  26. Altschul, S. F., T. L. Madden, A. A. Schäffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.Nucleic Acids Res. 25: 3389–3402.

    Article  CAS  Google Scholar 

  27. Aoki, H., M. N. Ahsan, K. Matsuo, T. Hagiwara, and S. Watabe (2003) Purification and characterization of collagenolytic proteases from the hepatopancreas of northern shrimp (Pandalus eous).J. Agric. Food Chem. 51: 777–783.

    Article  CAS  Google Scholar 

  28. Sivakumar, P., P. Sampath, and G. Chandrakasan (1999) Collagenolytic metalloprotease (gelatinase) from the hepatopancreas of the marine crab,Scylla serrata.Comp. Biochem. Physiol. 123B: 273–279.

    CAS  Google Scholar 

  29. Jung, H. J., H. K. Kim, and J. I. Kim (1999) Purification and characterization of Co2+-activated extracellular metallo protease fromBacillus sp. JH108.J. Microbiol. Biotechnol. 9: 861–869.

    CAS  Google Scholar 

  30. Labadie, J. and M. C. Montel (1982) Purification and study of some properties of a collagenase produced byEmpedobacter collagenolyticum.Biochimie 64: 49–53.

    Article  CAS  Google Scholar 

  31. Murai, A., Y. Tsujimoto, H. Matsui, and K. Watanabe (2004) AnAneurinibacillus sp. strain AM-1 produces a praline-specific aminopeptidase useful for collagen degradation.J. Appl. Microbiol. 96: 810–818.

    Article  CAS  Google Scholar 

  32. Häse, C. C. and R. A. Finkelstein (1993) Bacterial extracellular zinc-containing metalloproteases.Microbiol. Rev. 57: 823–837.

    Google Scholar 

  33. Chakraborty, R. and A. L. Chandra (1986) Purification and characterization of a streptomycete collagenase.J. Appl. Bacteriol. 61: 331–337.

    CAS  Google Scholar 

  34. Jackson, R. J., D. V. Lim, and M. L. Dao (1997) Identification and analysis of a collagenolytic activity inStreptococcus mutans.Curr. Microbiol. 34: 49–54.

    Article  CAS  Google Scholar 

  35. Chae, Y. R. and K. G. Ryu (2004) Partial purification and characterization of an extracellular protease fromXenorhabdus nematophilus, a symbiotic bacterium isolated from anEntomopathogenic nematode, Steinernema glaseri.Biotechnol. Bioprocess Eng. 9: 379–382.

    Article  CAS  Google Scholar 

  36. Lee, Y. J., J. H. Kim, H. K. Kim, and J. S. Lee (2004) Production and characterization of keratinase fromParacoccus sp. WJ-98.Biotechnol. Bioprocess Eng. 9: 17–22.

    Article  CAS  Google Scholar 

  37. Nimmi, M. E. (2000)Collagen Metabolism, pp. 25–31.Collagen. CRC Press, Inc., Boca Raton, Florida, USA.

    Google Scholar 

  38. Bae, M. and P. Y. Park (1989) Purification and characterization of thermotolerable alkaline protease by alkalophilicBacillus sp. No. 8-16.Kor. J. Appl. Microbiol. Biotechnol. 17: 545–551.

    CAS  Google Scholar 

  39. Takami, H., T. Akiba, and K. Horikoshi (1990) Characterization of an alkaline protease fromBacillus sp. No. AH-101.Appl. Microbiol. Biotechnol. 33: 519–523.

    Article  CAS  Google Scholar 

  40. Yu, M. S. and C. Y. Lee (1999) Expression and characterization of the prtV gene encoding a collagenase fromVibrio parahaemolyticus inEscherichia coli.Microbiology 145: 143–150.

    CAS  Google Scholar 

  41. Takeuchi, H., Y. Shibano, K. Morihara, J. Fukushima, S. Inami, B. Keil, A. M. Gilles, S. Kawamoto, and K. Okada (1992) Structure gene and complete amino acid sequence ofVibrio alginolyticus collagenase.Biochem. J. 281: 705–708.

    Google Scholar 

  42. Lee, J. H., G. T. Kim, J. Y. Lee, H. K. Jun, J. H. Yu, and I. S. Kong (1998) Isolation and sequence analysis of metalloprotease gene fromVibrio mimicus.Biochim. Biophys. Acta. 1384: 1–6.

    CAS  Google Scholar 

  43. Kim, B. J., H. J. Kim, S. H. Hwang, S. K. Bae, S. D. Ha, J. D. Kim, and J. Y. Kong (1998) Cloning and expression of a collagenase gene from the marine bacteriumVibrio vulnificus CYK279H.J. Microbiol. Biotechnol. 8: 245–250.

    Google Scholar 

  44. Sasagawa, Y., K. Izaki, Y. Matsubara, K. Suzuki, H. Kojima, and Y. Kamio (1995) Molecular cloning and sequence analysis of the gene encoding the collagenase fromCytophaga sp. L43-1 strain.Biosci. Biotechnol. Biochem. 59: 2068–73.

    CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biotechnology & Bioengineering, Pukyong National University, 608-737, Pusan, Korea

    Sung-Il Kang, Young-Boo Jang & Jai-Yul Kong

  2. Division of Marine Bioscience/Institute of Marine Industry, Gyeongsang National University, 650-160, Gyeongnam, Korea

    Yeung-Joon Choi

Authors
  1. Sung-Il Kang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Young-Boo Jang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yeung-Joon Choi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jai-Yul Kong
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jai-Yul Kong.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kang, SI., Jang, YB., Choi, YJ. et al. Purification and properties of a collagenolytic protease produced by marine bacteriumVibrio vulnificus CYK279H. Biotechnol. Bioprocess Eng. 10, 593–598 (2005). https://doi.org/10.1007/BF02932300

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02932300

Keywords

Search

Navigation