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

  • Sung-Il Kang
  • Young-Boo Jang
  • Yeung-Joon Choi
  • Jai-Yul Kong
Article

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.

Keywords

collagenase gelatin metalloprotease purification Vibrio vulnificus 

References

  1. [1]
    Ravanti, L. and V. M. Kähäri (2000) Matrix metalloproteases in wound repair.Int. J. Mol. Med. 6: 391–407.Google Scholar
  2. [2]
    Harrington, D. J. (1996) Bacterial collagenases and collagen-degrading enzymes and their potential role in human disease.Infect. Immun. 64: 1885–1891.Google Scholar
  3. [3]
    Van Wart, H. E. and D. E. Steinbrink (1981) A continuous spectrophotometric assay forClostridium histolyticum collagenase.Anal. Biochem. 113: 356–365.CrossRefGoogle Scholar
  4. [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.CrossRefGoogle Scholar
  5. [5]
    Peterkofsky, B. (1982) Bacterial collagenase.Methods Enzymol. 82: 453–471.CrossRefGoogle Scholar
  6. [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.CrossRefGoogle Scholar
  7. [7]
    Neurath, H. (1984) Evolution of protelytic enzyme.Science 224: 350–357.CrossRefGoogle Scholar
  8. [8]
    Watanabe, K. (2004) Collagenolytic proteases from bacteria.Appl. Microbiol. Biotechnol. 63: 520–526.CrossRefGoogle Scholar
  9. [9]
    Honds, S. (1998) Dietary use of collagen and collagen peptides for cosmetics.Food style. 21: 54–60.Google Scholar
  10. [10]
    Dreisbach, J. H. and J. R. Merkel (1978) Induction of collagenase production inVibrio B-30.J. Bacteriol. 135: 521–527.Google Scholar
  11. [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.Google Scholar
  12. [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.CrossRefGoogle Scholar
  13. [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.CrossRefGoogle Scholar
  14. [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.Google Scholar
  15. [15]
    Gupta, R., Q. K. Beg, and P. Lorenz (2002) Bacterial alkaline proteases: molecular approaches and industrial applications.Appl. Microbiol. Biotechnol. 59: 15–32.CrossRefGoogle Scholar
  16. [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.Google Scholar
  17. [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. [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.Google Scholar
  19. [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.CrossRefGoogle Scholar
  20. [20]
    Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227: 680–685.CrossRefGoogle Scholar
  21. [21]
    Edman, P. and G. Begg (1967) A protein sequenator.Eur. J. Biochem. 1: 80–91.CrossRefGoogle Scholar
  22. [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.CrossRefGoogle Scholar
  23. [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.Google Scholar
  24. [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.Google Scholar
  25. [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.CrossRefGoogle Scholar
  26. [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.CrossRefGoogle Scholar
  27. [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.CrossRefGoogle Scholar
  28. [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.Google Scholar
  29. [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.Google Scholar
  30. [30]
    Labadie, J. and M. C. Montel (1982) Purification and study of some properties of a collagenase produced byEmpedobacter collagenolyticum.Biochimie 64: 49–53.CrossRefGoogle Scholar
  31. [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.CrossRefGoogle Scholar
  32. [32]
    Häse, C. C. and R. A. Finkelstein (1993) Bacterial extracellular zinc-containing metalloproteases.Microbiol. Rev. 57: 823–837.Google Scholar
  33. [33]
    Chakraborty, R. and A. L. Chandra (1986) Purification and characterization of a streptomycete collagenase.J. Appl. Bacteriol. 61: 331–337.Google Scholar
  34. [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.CrossRefGoogle Scholar
  35. [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.CrossRefGoogle Scholar
  36. [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.CrossRefGoogle Scholar
  37. [37]
    Nimmi, M. E. (2000)Collagen Metabolism, pp. 25–31.Collagen. CRC Press, Inc., Boca Raton, Florida, USA.Google Scholar
  38. [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.Google Scholar
  39. [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.CrossRefGoogle Scholar
  40. [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.Google Scholar
  41. [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. [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.Google Scholar
  43. [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. [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.Google Scholar

Copyright information

© The Korean Society for Biotechnology and Bioengineering 2005

Authors and Affiliations

  • Sung-Il Kang
    • 1
  • Young-Boo Jang
    • 1
  • Yeung-Joon Choi
    • 2
  • Jai-Yul Kong
    • 1
  1. 1.Department of Biotechnology & BioengineeringPukyong National UniversityPusanKorea
  2. 2.Division of Marine Bioscience/Institute of Marine IndustryGyeongsang National UniversityGyeongnamKorea

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