Browse > Article

Purification and Properties of a Collagenolytic Protease Produced by Marine Bacterium Vibrio vulnificus CYK279H  

Kang, Sung-Il (Department of Biotechnology & Bioengineering, Pukyong National University)
Jang, Young-Boo (Department of Biotechnology & Bioengineering, Pukyong National University)
Choi, Yeung-Joon (Division of Marine Bioscience/Institute of Marine Industry, Gyeongsang National University)
Kong, Jai-Yul (Department of Biotechnology & Bioengineering, Pukyong National University)
Publication Information
Biotechnology and Bioprocess Engineering:BBE / v.10, no.6, 2005 , pp. 593-598 More about this Journal
Abstract
A collagenolytic enzyme, produced by Vibrio 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 35.0kDa. The N-terminal sequence of the enzyme was determined as Gly-Asp-Pro-Cys-Met-Pro-Ile-Ile-Ser-Asn. The optimum temperature and pH for the enzyme activity were $35^{\circ}C$ and 7.5, respectively. The enzyme activity was stable within the pH and temperature ranges 6.8-8.0 and $20{\sim}35^{\circ}C$, respectively. The purified enzyme was strongly activated by $Zn^{2+},\;Li^{2+},\;and\;Ca^{2+}$, but inhibited by $Cu^{2+}$. 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;
Citations & Related Records

Times Cited By Web Of Science : 6  (Related Records In Web of Science)
Times Cited By SCOPUS : 7
연도 인용수 순위
1 Harrington, D. J. (1996) Bacterial collagenases and collagen- degrading enzymes and their potential role in human disease. Infect. Immun. 64: 1885-1891
2 Van Wart, H. E. and D. E. Steinbrink (1981) A continuous spectrophotometric assay for Clostridium histolyticum collagenase. Anal. Biochem. 113: 356-365   DOI   ScienceOn
3 Gross, J. and C. M. Lapiere (1962) Collagenolytic activity in amphibian tissues : A tissue culture assay. Proc. Natl. Acad. Sci. USA 48: 1014-1022
4 Watanabe, K. (2004) Collagenolytic proteases from bacteria. Appl. Microbiol. Biotechnol. 63: 520-526   DOI   PUBMED   ScienceOn
5 Dreisbach, J. H. and J. R. Merkel (1978) Induction of collagenase production in Vibrio B-30. J. Bacteriol. 135: 521-527
6 Moore, S. and W. H. Stein (1948) Photometric ninhydrin method for use in the chromatography of amino acids. J. Biol. Chem. 176: 367-388
7 Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685   DOI   PUBMED   ScienceOn
8 Juarez, Z. E. and M. W. Stinson (1999) An extracellular protease of Streptococcus gordonii hydrolyzes type IV collagen and collagen analogues. Infect. Immun. 67: 271-278
9 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   DOI   ScienceOn
10 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   DOI   ScienceOn
11 Chae, Y. R. and K. G. Ryu (2004) Partial purification and characterization of an extracellular protease from Xenorhabdus nematophilus, a symbiotic bacterium isolated from an Entomopathogenic nematode, Steinernema glaseri. Biotechnol. Bioprocess Eng. 9: 379-382   DOI   ScienceOn
12 Nimmi, M. E. (2000) Collagen Metabolism. pp. 25-31. Collagen. CRC Press, Inc., Boca Raton, Florida, USA
13 Bae, M. and P. Y. Park (1989) Purification and characterization of thermotolerable alkaline protease by alkalophilic Bacillus sp. No. 8-16. Kor. J. Appl. Microbiol. Biotechnol. 17: 545-551
14 Matsushita, O., C. M. Jung, S. Katayama, J. Minami, Y. Takahashi, and A. Okabe (1999) Gene duplication and multiplicity of collagenases in Clostridium histolyticum. J. Bacteriol. 181: 923-933
15 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   DOI   ScienceOn
16 Ravanti, L. and V. M. Kähäri (2000) Matrix metalloproteases in wound repair. Int. J. Mol. Med. 6: 391-407
17 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 from Cytophaga sp. L43-1 strain. Biosci. Biotechnol. Biochem. 59: 2068-73   DOI   ScienceOn
18 Peterkofsky, B. (1982) Bacterial collagenase. Methods Enzymol. 82: 453-471
19 Matsushita, O., K. Yoshihara, S. Katayama, J. Minami, and A. Okabe (1994) Purificaton and characterization of Clostridium perfringens 120-kilodalton collagenase and nucleotide sequence of the corresponding gene. J. Bacteriol. 176: 149-156   DOI
20 Hase, C. C. and R. A. Finkelstein (1993) Bacterial extracellular zinc-containing metalloproteases. Microbiol. Rev. 57: 823-837
21 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   DOI   PUBMED   ScienceOn
22 Batrhomoeuf, C., H. Pourrat, and A. Pourrat (1992) Collagenolytic activity of a new semi-alkaline protease from Aspergillus niger. J. Ferment. Bioeng. 73: 233-236   DOI   ScienceOn
23 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 from Vibrio vulnificus CYK279H. Korean J. Biotechnol. Bioeng. 19: 295-300
24 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
25 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 from Vibrio mimicus. Biochim. Biophys. Acta 1384: 1-6   DOI   ScienceOn
26 Jackson, R. J., D. V. Lim, and M. L. Dao (1997) Identification and analysis of a collagenolytic activity in Streptococcus mutans. Curr. Microbiol. 34: 49-54   DOI   ScienceOn
27 Neurath, H. (1984) Evolution of protelytic enzyme. Science 224: 350-357   DOI   PUBMED
28 Edman, P. and G. Begg (1967) A protein sequenator. Eur. J. Biochem. 1: 80-91   DOI   ScienceOn
29 Takeuchi, H., Y. Shibano, K. Morihara, J. Fukushima, S. Inami, B. Keil, A. M. Gilles, S, Kawamoto, and K. Okuda (1992) Structure gene and complete amino acid sequence of Vibrio alginolyticus collagenase. Biochem. J. 281: 703- 708   DOI
30 Yu, M. S. and C. Y. Lee (1999) Expression and characterization of the prtV gene encoding a collagenase from Vibrio parahaemolyticus in Escherichia coli. Microbiology 145: 143-150   DOI   ScienceOn
31 Labadie, J. and M. C. Montel (1982) Purification and study of some properties of a collagenase produced by Empedobacter collagenolyticum. Biochimie 64: 49-53   DOI   ScienceOn
32 Lee, Y. J., J. H. Kim, H. K. Kim, and J. S. Lee (2004) Production and characterization of keratinase from Paracoccus sp. WJ-98. Biotechnol.Bioprocess Eng. 9: 17-22   DOI   ScienceOn
33 Honds, S. (1998) Dietary use of collagen and collagen peptides for cosmetics. Food style. 21: 54-60
34 Teo, J. W., L. H. Zhang, and C. L. Poh (2003) Cloning and characterization of a metalloprotease from Vibrio harveyi strain AP6. Gene 303: 147-156   DOI   PUBMED   ScienceOn
35 Jung, H. J., H. K. Kim, and J. I. Kim (1999) Purification and characterization of Co2+-activated extracellular metallo protease from Bacillus sp. JH108. J. Microbiol. Biotechnol. 9: 861-869
36 Murai, A., Y. Tsujimoto, H. Matsui, and K. Watanabe (2004) An Aneurinibacillus sp. strain AM-1 produces a praline-specific aminopeptidase useful for collagen degradation. J. Appl. Microbiol. 96: 810-818   DOI   ScienceOn
37 Okamoto, M., Y. Yonejima, Y. Tsujimoto, Y. Suzuki, and K. Watanabe (2001) A thermostable collagenolytic protease with a very large molecular mass produced by thermophilic Bacillus sp. strain MO-1. Appl. Microbiol. Biotechnol. 57: 103-108   DOI   ScienceOn
38 Sasagawa, Y., Y. Kamio, Y. Matsubara, Y. Matsubara, K. Suzuki, H. Kojima, and K. Izaki (1993) Purification and properties of collagenase from Cytophaga sp. L43-1 strain. Biosci. Biotechnol. Biochem. 57: 1894-1898   DOI   ScienceOn
39 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 bacterium Vibrio vulnificus CYK279H. J. Microbiol. Biotechnol. 8: 245-250
40 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   DOI   ScienceOn
41 Takami, H., T. Akiba, and K. Horikoshi (1990) Characterization of an alkaline protease from Bacillus sp. No. AH-101. Appl. Microbiol. Biotechnol. 33: 519-523
42 Gupta, R., Q. K. Beg, and P. Lorenz (2002) Bacterial alkaline proteases: molecular approaches and industrial applications. Appl. Microbiol. Biotechnol. 59: 15-32   DOI   ScienceOn
43 Chakraborty, R. and A. L. Chandra (1986) Purification and characterization of a streptomycete collagenase. J. Appl. Bacteriol. 61: 331-337   DOI
44 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   DOI   ScienceOn