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Recovery and Characterization of a Serine Collagenolytic Extract from Snow Crab (Chionoecetes opilio) By-products

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Abstract

Sequential acidic precipitation followed by a single chromatographic step (gel filtration) allowed the recovery of a collagenolytic fraction containing several proteases from by-products of snow crab (Chionoecetes opilio). The partial purification was particularly efficient to recover tryptic (purification fold = 1,352.5; yield = 110%) but also chymotryptic, elastolytic, and collagenolytic activities. A temperature of 40 °C and pH 8.0–8.5 were optimal for enzyme activity, which was stable for 2 h under these conditions. Calcium was not required for stability and thus activity. The isoelectric points of the protein components ranged from 3.7 to 4.6. Zymography revealed 29 and 48 kDa major components and others from 22 to 56 kDa. Enzymes were inhibited by PMSF and TLCK but were insensitive to TPCK. In view of these properties, the proteases likely belong to the serine collagenase group. Inhibition by EDTA could be due to a mechanism other than Ca2+ chelation. Using a food system (ground fish), the fraction was more proteolytic than a commercial bacterial protease, suggesting potential applications in enzymatic hydrolysis processes.

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References

  1. Québec Ministry of Agriculture, Fisheries and Food (2007). Innovation and Technologies Directorate. Banque de données: pêches maritimes 2007

  2. Beaulieu, L., Thibodeau, J., Bryl, P., & Carbonneau, M. E. (2009). Characterization of enzymatic hydrolyzed snow crab (Chionoecetes opilio) by-product fractions: A source of high-valued biomolecules. Bioresource Technology, 100, 3332–3342.

    Article  CAS  Google Scholar 

  3. Rustad, T., & Falch, E. (2002). Making the most of fish catches. Food Science and Technology, 16, 36–39.

    Google Scholar 

  4. Rudenskaya, G. N. (2003). Brachyurins, serine collagenolytic enzymes from crabs. Russian Journal of Bioorganic Chemistry, 29, 101–111.

    Article  CAS  Google Scholar 

  5. Barret, A. J., Rawlings, N. D., & Woessner, J. F. (1998). Handbook of Proteolytic Enzymes. New York: Academic.

    Google Scholar 

  6. Tsu, C. A., Perona, J. J., Fletterick, R. J., & Craik, C. S. (1997). Structural basis for the broad substrate specificity of fiddler crab collagenolytic serine protease 1. Biochemistry, 36, 5393–5401.

    Article  CAS  Google Scholar 

  7. Grant, G. A., Eisen, A. Z., & Bradshaw, R. A. (1981). Collagenolytic protease from fiddler crab (Uca pugilator). Methods in Enzymology, 80, 722–734.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  9. Semenova, S. A., Rudenskaya, G. N., Lyutova, L. V., & Nikitina, O. A. (2008). Isolation and properties of collagenolytic serine proteinase isoenzyme from king crab Paralithodes camtschatica. Biochemistry (Moscow), 73, 1125–1133.

    Article  CAS  Google Scholar 

  10. Sakharov, I. Y., Glyanzev, S. P., Litvin, F. E., & Savvina, T. V. (1993). Potent debriding ability of collagenolytic protease isolated from the hepatopancreas of the king crab Paralithodes camtschatica. Archives for Dermatological Research, 285, 32–35.

    Article  CAS  Google Scholar 

  11. Sakharov, I. Y., Litvin, F. E., & Artyukov, A. A. (1994). Purification and characterization of two serine collagenolytic proteases from crab Paralithodes camtschatica. Comparative Biochemistry and Physiology, 108B, 561–568.

    CAS  Google Scholar 

  12. Roy, P., Colas, B., & Durand, P. (1996). Purification, kinetical and molecular characterizations of a serine collagenolytic protease from greenshore crab (Carcinus maenas) digestive gland. Comparative Biochemistry and Physiology, 115B, 87–95.

    CAS  Google Scholar 

  13. Klimova, O. A., Vedishcheva, I., & Strongin, A. Y. (1991). Isolation and characteristics of collagenolytic enzymes from the hepatopancreas of crab Chionoecetes opilio. Doklady Akademii nauk UzSSR, 317, 482–484.

    CAS  Google Scholar 

  14. Aoki, H., Ahsan, M. N., Matsuo, K., Hagiwara, T., & Watabe, S. (2003). Purification and characterization of collagenolytic proteases from the hepatopancreas of northern shrimp (Pandalus eous). Journal of Agricultural and Food Chemistry, 51, 777–783.

    Article  CAS  Google Scholar 

  15. Kristjánsson, M. M., Gudmundsdόttir, S., Fox, J. W., & Bjarnason, J. B. (1995). Characterization of a collagenolytic serine proteinase from the Atlantic cod (Gadus morhua). Comparative Biochemistry and Physiology, 110B, 707–717.

    Google Scholar 

  16. Simpson, B. K. (2000). In Seafood Enzymes, chap. In N. F. Haard & B. K. Simpson (Eds.), 8: Digestive proteinases from marine animals (pp. 191–21). New York: Marcel Dekker.

    Google Scholar 

  17. Masui, Y., Takemoto, T., Sakakibara, S., Hori, H., & Nagai, Y. (1977). Synthetic substrates for vertebrate collagenase. Biochemical Medicine, 17, 215–221.

    Article  CAS  Google Scholar 

  18. Rudenskaya, G. N., Isaev, V. A., Shmoylov, M. A., Karabasova, M. A., Shvets, S. V., Miroshnikov, A. I., et al. (2000). Preparation of proteolytic enzymes from kamchatka crab Paralithodes camtschatica hepatopancreas and their application. Applied Biochemistry and Biotechnology, 88, 175–183.

    Article  CAS  Google Scholar 

  19. Klimova, O. A., & Chebotarev, V. Y. (2000). Collagenolytic protease preparations from invertebrates: Biochemical aspects of medical and cosmetological applications. Bulletin of Experimental Biology and Medicine, 130, 671–675.

    Article  CAS  Google Scholar 

  20. Díaz-López, M., & Garcia-Carreño, F. L. (2000). Applications of fish and shellfish enzymes in food and feed products. In N. F. Haard & B. K. Simpson (Eds.), Seafood Enzymes (pp. 571–618). New York: Marcel Dekker.

    Google Scholar 

  21. Gildberg, A., Simpson, B. K., & Haard, N. F. (2000). Uses of enzymes from marine organisms. In N. F. Haard & B. K. Simpson (Eds.), Seafood Enzymes (pp. 619–639). New York: Marcel Dekker.

    Google Scholar 

  22. Linder, M., Fanni, J., & Parmentier, M. (2005). Proteolytic extraction of salmon oil and PUFA concentration by lipases. Marine Biotechnology, 15, 70–76.

    Article  Google Scholar 

  23. Liaset, B., Lied, E., & Espe, M. (2000). Enzymatic hydrolysis of by-products from the fish-filleting industry; chemical characterization and nutritional evaluation. Journal of the Science of Food and Agriculture, 80, 581–589.

    Article  CAS  Google Scholar 

  24. Haard, N. F. (1992). A review of proteolytic enzymes from marine organisms and their application in the food industry. Journal of Aquatic Food Product Technology, 1, 17–35.

    Article  CAS  Google Scholar 

  25. Asgeirsson, B., Fox, J. W., & Bjarnason, J. B. (1989). Purification and characterization of trypsin from the poikilotherm Gadus morhua. European Journal of Biochemistry, 180, 85–94.

    Article  CAS  Google Scholar 

  26. Burgos-Hernández, A., Farias, S. I., Torres-Arreola, W., & Ezquerra-Brauer, J. M. (2005). In vitro studies of the effects of aflatoxin B1 and fumonisin B1 on trypsin-like and collagenase-like activity from the hepatopancreas of white shrimp (Litopeanus vannamei). Aquaculture, 250, 399–410.

    Article  Google Scholar 

  27. Aoki, H., Ahsan, M. N., Matsuo, K., Hagiwara, T., & Watabe, S. (2004). Partial purification of proteases that are generated by processing of the northern shrimp Pandalus borealis and which can tenderize beef. International Journal of Food Science & Technology, 29, 471–480.

    Article  Google Scholar 

  28. Byun, H. G., Park, P. J., Sung, N. J., & Kim, S. K. (2003). Purification and characterization of a serine proteinase from the tuna pyloric caeca. Journal of Food Biochemistry, 26, 479–494.

    Article  CAS  Google Scholar 

  29. Park, P. J., Lee, S. H., Byun, H. G., Kim, S. H., & Kim, S. K. (2002). Purification and characterization of a collagenase from the mackerel Scomber japonicus. Journal of Biochemistry and Molecular Biology, 35, 576–582.

    CAS  Google Scholar 

  30. Tsu, C. A., Perona, J. J., Schellenberger, V., Turck, C. W., & Craik, C. S. (1994). The substrate specificity of Uca pugilator collagenolytic serine protease 1 correlates with the bovine type 1 collagen cleavage sites. The Journal of Biological Chemistry, 269, 19565–19572.

    CAS  Google Scholar 

  31. Grant, G. A., Sacchettini, J. C., & Welgus, H. G. (1983). A collagenolytic serine protease with trypsin-like specificity from the fiddler crab Uca pugilator. Biochemistry, 22, 354–358.

    Article  CAS  Google Scholar 

  32. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.

    Article  CAS  Google Scholar 

  33. Moore, S., & Stein, W. H. (1954). A modified ninhydrin reagent for the photometric determination of amino acids and related compounds. The Journal of Biological Chemistry, 211, 907–913.

    CAS  Google Scholar 

  34. Stocknes, I. (1994). Proteases in fish, purification and characterization of alkaline muscle proteases. Trondheim, Norway: PhD thesis, Norwegian Institute of Technology.

    Google Scholar 

  35. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M., et al. (1985). Measurement of protein using bicinchoninic acid. Analytical Biochemistry, 150, 76–85.

    Article  CAS  Google Scholar 

  36. Britton, H.T.S., & Robinson, R.A. (1931). Universal buffer solutions and the dissociation constant of Veronal. Journal of Chemical Society, 1456–1462

  37. Klimova, O. A., Borukhov, S. I., Solovyeva, N. I., Balaevskaya, T. O., & Strongin, A. Y. (1990). The isolation and properties of collagenolytic proteases from crab hepatopancreas. Biochemical and Biophysical Research Communications, 166, 1411–1420.

    Article  CAS  Google Scholar 

  38. Tsu, C. A., & Craik, C. S. (1996). Substrate recognition by recombinant serine collagenase 1 from Uca pugilator. The Journal of Biological Chemistry, 271, 11563–11570.

    Article  CAS  Google Scholar 

  39. Klimova, O. A., & Chebotarev, Y. V. (1999). Collagenic complex of proteases from hepatopancreas of kamtchatka crab. Bulletin of Experimental Biology and Medicine, 128, 308–313.

    CAS  Google Scholar 

  40. Rudenskaya, G. N., Isaev, V. A., Stepanov, V. M., Dunaevsky, Y. E., Baratova, L. A., Kalebina, T. S., et al. (1996). Isolation and properties of serine proteinase PC of the kamchatka crab Paralithodes camtschatica, a proteolytic enzyme of broad specificity. Biochemistry, 61, 804–814.

    Google Scholar 

  41. Tsai, I. H., Lu, P. J., & Chuang, J. L. (1991). The midgut chymotrypsins of shrimps (Penaeus monodon, Penaeus japonicus and Penaeus penicillatus). Biochimica et Biophysica Acta, 1080, 59–67.

    Article  CAS  Google Scholar 

  42. Salamanca, M. H., Barría, C., Asenjo, J. A., & Andrews, B. A. (2002). Isolation, purification and preliminary characterization of cryophilic proteases of marine origin. Bioseparation, 10, 237–241.

    Article  Google Scholar 

  43. Rudenskaya, G. N., Kislitsin, Y. A., & Rebrikov, D. V. (2004). Collagenolytic serine protease PC and trypsin PC from king crab Paralithodes camtschaticus: cDNA cloning and primary structure of the enzymes. BMC Structural Biology, 4, 1–9.

    Article  Google Scholar 

  44. Dendinger, J. E. (1990). Purification and characterization of a trypsin-like enzyme from the midgut gland of the Atlantic blue crab, Callinectes sapidus. Comparative Biochemistry and Physiology, 95, 525–530.

    Article  Google Scholar 

  45. García-Carreño, F. L., Hernández-Cortés, M. P., & Haard, N. F. (1994). Enzymes with peptidase and proteinase activity from the digestive system of a freshwater and a marine decapod. Journal of Agricultural and Food Chemistry, 42, 1456–1461.

    Article  Google Scholar 

  46. Grant, G. A., & Eisen, A. Z. (1980). Substrate specificity of the collagenolytic serine protease from Uca pugilator: Studies with non collagenous substrates. Biochemistry, 19, 6089–6095.

    Article  CAS  Google Scholar 

  47. Simpson, B. K., & Haard, N. F. (1987). Trypsin and trypsin-like enzymes from the stomachless cunner. Journal of Agricultural and Food Chemistry, 35, 652–656.

    Article  CAS  Google Scholar 

  48. Simpson, B. K., Simpson, M. V., & Haard, N. F. (1989). On the mechanism of enzymes action: digestive proteases from selected marine organisms. Biotechnology and Applied Biochemistry, 11, 226–234.

    CAS  Google Scholar 

  49. Raa, J. (1990). Biotechnology in aquaculture and the fish processing industry: A success story in Norway. In M. N. Voigt & J. R. Botta (Eds.), Advances in Fisheries Technology and Biotechnology for Increased Profitability (pp. 509–524). Lancaster, PA: Technomic Publishing.

    Google Scholar 

  50. Beaulieu, L., Thibodeau, J., Bryl, P., & Carbonneau, M. E. (2009). Proteolytic processing of herring (Clupea harengus): biochemical and nutritional characterization of hydrolysates. International Journal of Food Science & Technology, 44, 2113–2119.

    Article  CAS  Google Scholar 

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Acknowledgments

The authors wish to thank the Innovation and Technologies Directorate (DIT-MAPAQ) for their financial support and the Aquatic Products Technology Centre (CTPA) for helpful technical assistance.

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  1. Department of Biology, Chemistry and Geography, Université du Québec à Rimouski (UQAR), Rimouski, Québec, Canada, G5L 3A1

    Nathalie Souchet & Serge Laplante

  2. Aquatic Products Technology Centre (CTPA), Québec Ministry of Agriculture Fisheries and Food (MAPAQ), Gaspé, QC, Canada, G4X 2V6

    Nathalie Souchet & Serge Laplante

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  1. Nathalie Souchet
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Correspondence to Serge Laplante.

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Souchet, N., Laplante, S. Recovery and Characterization of a Serine Collagenolytic Extract from Snow Crab (Chionoecetes opilio) By-products. Appl Biochem Biotechnol 163, 765–779 (2011). https://doi.org/10.1007/s12010-010-9081-2

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