ABB  Vol.3 No.3 , June 2012
Isolation and activation of collagenase from fish processing waste
Abstract: Collagenase was isolated from fish waste (a mixture of haddock, herring, ground fish and flounder) using a Tris-buffer system. The proteins in the crude extract were precipitated using ammonium sulfate (40% - 80%) and purified with gel-filtration chromatography using Sephadex G-100. The results showed that the collagenase enzyme was produced as a latent enzyme and was activated with bovine trypsin and potassium thiocyanate (KSCN). The enzyme activity was affected by pH and temperature. Optimal enzyme activities were found at 35?C and a pH of 7.5 when insoluble collagene type I was used as substrate and the liberated amino acids were measured in relation to L-leucine in the presence of ninhydrin. The enzyme activity was completely inhibited by the action of ethylenediaminetetraacetic acid (EDTA) suggesting that the collagenase enzyme isolated from the fish waste is a metalloproteinase enzyme requiring metal ions for enzyme activity. Dialysis against KSCN decreased the enzyme total activity and increased its specific activity. Sodium dodecyl sulphate polyacryla-mide gel electrophoresis (SDS-PAGE) indicated that the purified procollagenase enzyme have only one band at molecular weight of 50 kilodaltons (kDa). When the enzyme was cleaved with trypsin, it was found to consist of two subunits: a large unit with a molecular weight of 50 kDa and a small unit with a molecular weight of 10 kDa.
Cite this paper: Daboor, S. , Budge, S. , Ghaly, A. , Brooks, M. and Dave, D. (2012) Isolation and activation of collagenase from fish processing waste. Advances in Bioscience and Biotechnology, 3, 191-203. doi: 10.4236/abb.2012.33028.

[1]   Simpson, B.K. and Haard, N.F. (1987) Trypsin and a trypsin-like enzyme from the stomachlesscunner. Catalytic and other physical characteristics. Journal of Agriculture and Food Chemistry, 35, 652-656.

[2]   Liese, A., Seelbach, K. and Wandrey, C. (2006) Advantages of biotransformations over classical chemistry en- zymes are proteins. In: Industrial Biotransformations, Wi-ley-VchverlagGmbh and Co. KGaA, Wein-heim.

[3]   Cronlund, A.L. and Woychik, J.H. (1987) Solubilization of collagen in restructured beef with collagenases and α-Amylase. Journal of Food Science, 52, 857-860. doi:10.1111/j.1365-2621.1987.tb14227.x

[4]   ?gren, M.S., Taplin, C.J., Woessner, J.F. Jr, Eagistein, W.H. and Mertz, P.M. (1992). Collagenase in wound healing: effect of wound age and type. Journal of Investigative Dermatology, 99, 709-714. doi:10.1111/1523-1747.ep12614202

[5]   Ashie, I.N.A. and Lanier, T.C. (2000) Transglutaminases in seafood processing. In: Haard, N.F. and Simpson, B.K. Eds., Sea-food Enzymes Utilization and Influence on Post-harvest Seafood Quality. Marcel Dekker, Inc., New York.

[6]   Püllen. R., Popp, R., Volkers, P. and Füsgen, I. (2002) Prospective randomized double-blind study of the wound-debriding effects of collagenase and fibrinoly-sin/deoxy- ribonuclease in pressure ulcers. Age and Ageing, 31, 126- 130. doi:10.1093/ageing/31.2.126

[7]   Mohorcic, M., Friedrich, J., Renime, I., Andre, P., Mandin, D. and Chaumont, J.P. (2006) Production of melanin bleaching enzyme of fungal origin and its application in cosmetics. Biotechnology and Bioprocess Engineering, 12, 200-206. doi:10.1007/BF02931093

[8]   Kanth, S.V., Venba, R., Madhan, B., Chandrababu, N.K. and Sadulla, S. (2008). Studies on the influence of bacterial collagenase in leather dyeing. Dyes and Pigments, 76, 338-347. doi:10.1016/j.dyepig.2006.08.043

[9]   Raa, J. (1997) New commercial products from waste of the fish processing industry. In: Bremner, A., Davis, C. and Aus-tin, B., Eds., Making the Most of the Catch, Proceedings of the Seafood Symposium, Brisbane.

[10]   Haard, N.F. (1998). Speciality enzymes from marine organisms. Food Technology, 52, 64-67.

[11]   Venugopal, V., Lakshmanan, R. Doke, S.N. and Bongir- war, D.R. (2000). Enzymes in fish processing, biosensors and quality control: A review. Food Biotechnology, 14, 21-27. doi:10.1080/08905430009549980

[12]   Shahidi, F. and JanakKamil, Y.V.A. (2001) Enzymes from fish and aquatic invertebrates and their application in the food industry. Trends in Food Science and Technology, 12, 435-464. doi:10.1016/S0924-2244(02)00021-3

[13]   DFO (2012) Summary of Canadian commercial catches and values, 2008-2009. Commercial Fisheries, Depart- ment of Fi-sheries and Oceans Canada, Ottawa.

[14]   Roberts, T. (2010) Alternatives are not feasible, producers say, fish waste dumping continues.

[15]   Nagai, Y. (1973) Vertebrate collagenase: Further characterization and the significance of its latent form in vivo. Molecular and Cellular Biochemistry, 1, 137-145. doi:10.1007/BF01659325

[16]   Goshev, I., Gousterova, A., Vasileva-Tonkova, E. and Nedkov, P. (2005) Characterization of the enzyme com- plexes produced by two newly isolated thermophylicac- tinomycete strains during growth on collagen-rich materials. Process Biochemistry, 40, 1627-1631. doi:10.1016/j.procbio.2004.06.016

[17]   Foegeding, E.A. and Larick, D.K. (1986) Tenderization of beef with bac-terial collagenase. Meat Science, 18, 201- 214. doi:10.1016/0309-1740(86)90034-3

[18]   D?az-López, M. and García-Carreńo, F.L. (2000) Applications of fish and shellfish enzymes in food and feed products. In: Haard, N.F. and Simpson, B.K., Eds., Sea- food Enzymes, Utilization and Influence on Postharvest Seafood Quality. Marcel Dekker, New York.

[19]   Gupta, R., Beg, Q.K., Khan, S. and Chauhan, B. (2002) An overview on fermentation, downstream processing and properties of mi-crobial alkaline proteases. Applied Microbiology and Biotechnology, 60, 381-395. doi:10.1007/s00253-002-1142-1

[20]   Shanmugam, S. and Sathishkumar, T. (2009) Enzyme technology. Inter-national Publishing House PVT, New Delhi.

[21]   Chung, L., Dinakarpandian, D., Yoshida, N., Lauer-Fields, J.L., Fields, G.B., Visse, R. and Nagase, H. (2004). Col- lage-nase unwinds triple-helical collagen prior to peptide bond hydrolysis. The European Molecular Biology Or- ganiza-tion Journal, 23, 3020-3030.

[22]   Sp?k, A. (2006) Safety regulations of food enzymes. Food Technology and Biotechnology, 44, 197-209.

[23]   Kristinsson, H.G. and Rasco, B.A. (2000) Fish protein hydrolysates: Production, biochemical and functional prop- erties. Critical Reviews in Food Science and Nutrition, 40, 43-81. doi:10.1080/10408690091189266

[24]   Glidberg, A. (2001) Utilization of male Arctic capelin and Atlantic cod intestines for fish sauce production evaluation of fermentation conditions. Bioresource Technology, 769, 119- 123. doi:10.1016/S0960-8524(00)00095-X

[25]   Shih, I.L., Chenb, L.G., Yu, T.S., Changb, W.T. and Wang, S.L. (2003) Microbial reclamation of fish processing wastes for the production of fish sauce. Enzyme and Microbial Technology, 33, 154-162. doi:10.1016/S0141-0229(03)00083-8

[26]   Tao, H., Zhao, M.M., Wang, J.S., Yang, B., Cui, C., Zhao, Q.Z. and Wu, H. (2008) Activity distribution of digestive proteases from Nemipterusvirgatus and their responses to pH value and temperature. Journal of Food Processing Engineering, 31, 21-33. doi:10.1111/j.1745-4530.2007.00141.x

[27]   Kl?ck, G., Kowalski, M.B., Hering, B.J., Eiden, M.E., Weidemann, A., Langer, S., Zimmermann, U., Federlin, K. and Bretzel, R.G. (1996) Fractions from commercial collagenase preparations: Use in enzymic isolation of the islets of Langerhans from porcine pancreas. Cell Transplantation, 5, 543-551. doi:10.1016/0963-6897(96)00023-1

[28]   Mookhtiar, K., Steinbrink, S.D. and Van Wart, H.E. (1985) Mode of hy-drolysis of collagen-like peptidase by class I and class II Clostridium histolyticum collagenases: Evidence for both indopeptidase and tripeptidylcarboxypep- tidase activities. Biochemistry, 24, 6527-6533. doi:10.1021/bi00344a033

[29]   Goldberg, G.I., Wilhelm, S.M., Kronberger, A., Bauer, E.A., Grant, G.A. and Eisen, A.Z. (1986) Human fibro-blast collagenase. Complete primary structure and homology to an oncogene transformation-induced rat protein. The Journal of Biological Chemistry, 261, 6600-6605.

[30]   Endo, A., Murakawa, S., Shimizu, H. and Shiraishi, Y. (1987) Purification and properties of collagenase from a Streptomyces species. The Journal of Biochemistry, 102, 163-170.

[31]   Lim, D.V., Jackson, R.J. and Pull-VonGruenigen, C.M. (1993) Purification and assay of bacterial collagenase. Journal of Microbiological Methods, 18, 241-253. doi:10.1016/0167-7012(93)90039-K

[32]   Matsushita, O., Yoshihara, K., Katayama, S.I., Minami, J. and Okabe, A. (1994) Purification and characterization of a Clostridium perfringes 120-Kilodalton collagenase and nucleotide sequence of the corresponding gene. Journal of Bacteri-ology, 176, 149-156.

[33]   Wu, Q., Li, C., Li, C., Chen, H. and Shuliang, L. (2009). Purification and characterization of a novel collagenase from Bacillus pumilus Col-J. Applied Journal of Bio-chemistry and Biotechnology, 160, 129-139.

[34]   Garcia-Carreno, F.L., Hernandez-Cortes, M.P. and Haard, N.F. (1994) Enzymes with peptidase and proteinase ac- tivity from the digestive system of a freshwater and ma- rine decapod. Journal of Agricultural and Food Chemistry, 42, 1456-1461. doi:10.1021/jf00043a013

[35]   Kristjánsson, M.M., Gudmundsdóttir, S., Fox, J.W. and Bjarnason, J.B. (1995) Characterization of collagenolytic serine proreinase from the Atlantic cod (Gadusmorhua). Comparative Biochemistry and Physiology, Part B, 110, 707-717.

[36]   Vaithilingam, I.S., McDonald, W., Malott, D.W. and Del Maestro, R.F. (1995) An extracellular pro-teasome-like structure from C6 astrocytoma cells with serine collagenase IV activity and metallo-dependent activity on α- casein and β-insuline. The Journal of Biological Chemistry, 270, 4588-4593.

[37]   Sriket, C., Ben-jakul, S., Visessanguan, W. and Kishimura, H. (2011) Collagenolytic serine protease in fresh water prawn (Ma-crobrachiumrosenbergii): Characteristics and its impact on muscle during iced storage. Food Chemistry, 124, 29-35. doi:10.1016/j.foodchem.2010.05.098

[38]   Murado, M.A., González, M.P. and Vázquez, J.A. (2009). Recovery of proteolytic and collagenolytic activities from viscera by-products of Rayfish (Raja clavata). Marine Drugs, 7, 803-815. doi:10.3390/md7040803

[39]   Birkedal-Hansen H., Cobb, C.M., Taylor, R.E. and Fullmer, H.M. (1975). Trypsin activation of latent collagenase from several mammalian sources. European Journal of Oral Sciences, 83, 302-305. doi:10.1111/j.1600-0722.1975.tb00442.x

[40]   Sellers, A. and Murphy, G. (1981). Collagenolytic enzymes and their naturally occurring inhibitors. Interna- tional Review of Connective Tissue Research, 9, 151-190.

[41]   Armour, P.C., Levi, S., Golds, E.E., Poole, A.R., Mort, J.S. and Roughley, P.J. (1984). Activation of latent collagenase by serum proteinases that interact with immobilized immunoglobulin G. Rheumatology International, 4, 151-155. doi:10.1007/BF00541205

[42]   He, C., Wilhelm, S.M., Pentland, A.P., Marmer, B.L., Grant, G.A., Eisen, A.Z. and Goldberg, G.I. (1989) Tis- sue cooperation in a proteolytic cascade activating human interstitial collagenase. Proceeding of the National Academy of Science, 86, 2632-2636.

[43]   Roy, P., Bernard, C. and Patrick, D. (1996) Purification, kinetical and molecular characterizations of a serine collagenolytic protease from green shore Crab (Carcinus-maenas) digestive gland. Comparative Biochemistry and Physiology, Part B, 115, 87-95.

[44]   Zefirova, O.N., Mamaeva, A.V., Chupov, V.V., Valuev, L.I. and Plate, N.A. (1996) Synthesis and properties of immobilized collagenolytic protease from hepatopancreas of the king crab Paralithoidescamtschatica. Applied Bio- chemistry and Microbiology, 32, 461-464.

[45]   Freije, J.P., Diez-Itza, I., Balbin, M., Sanchez, L.M., Blasco, R., Tolivia, J. and Lopez-Otin, C. (1994) Mo- lecular cloning and expression of collage-nase-3, a novel human matrix metalloproteinase produced by breast car- cinomas. The Journal of Biological Chemistry, 269, 16766- 16773.

[46]   Sivakumar, P., Sampath, P. and Chandrakasan, G. (1999). Collagenolytic metalloprotease (gelatinase) from the hepato- pancreas of the marine crab Scylla serrata. Comparative Biochemistry and Physiology, Part B, 123, 273-279.

[47]   Park, P.J., Lee, S.H., Byun, H.G., Kim, S.H. and Kim, S.K. (2002). Purification and characterization of a colla- genase from the Mackerel, Scomberjaponicus. Journal of Biochemistry and Molecular Biology, 35, 576-582. doi:10.5483/BMBRep.2002.35.6.576

[48]   Sellers, A, and Murphy, G. (1981). Collagenolytic enzymes and their naturally occurring inhibitors. International Review of Connective Tissue Research, 9, 151-190.

[49]   Harris, E.D. and Vatar, C.A. (1982). Vertebrate colla-genases. Methods Enzymology, Part A, 82, 423-452.

[50]   McCroskery, P.A., Richards, J.F. and Harris, E.D.J. (1975) Purification and characterization of a collagenase extracted from rabbit tumours. Biochemical Journal, 152, 131-142.

[51]   Teruel, S.R.L. and Simpson, B.K. (1995) Characterization of the collagenolytic en-zyme fraction from winter flounder (Pseudopleuronectes americanus). Compendium of Biochemistry and Physiology, Part B, 112, 131-136.

[52]   Hernández-Herrero, M.M., Duflos, G., Malle, P. and Bou- quelet, S. (2003) Collagenase activity and protein hy-drolysis as related to spoilage of iced cod (Gadusmorhua). Food Research In-ternational, 36, 141-147. doi:10.1016/S0963-9969(02)00129-1

[53]   Indra, D., Ramalingam, K. and Babu, M. (2005) Isolation, purifica-tion and characterization of collagenase from heap- topan-creas of the land snail Achatinafulica. Comparative Bio-chemistry and Physiology, Part B, 142, 1-7.

[54]   Shinkai, H. and Nagai, Y. (1977) A latent collagenase from embryonic human skin explants. The Journal of Bio- che-mistry, 81, 1261-1268.

[55]   Jr. Woessner, J.F. (1977). A latent form of collagenase in the involuting rat uterus and its activation by a serine proteinase. Biochemical Journal, 161, 535-542.

[56]   Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quan-tities of protein utilizing the principle of protein-dye binding. Analytical Bio-chemistry, 72, 248-254. doi:10.1016/0003-2697(76)90527-3

[57]   Mandl, I., MacLennan, J.D., Howes, E.L., DeBellis, R.H. and Sohler, A. (1953). Isolation and characterization of proteinase and collagenase from C. histolyticum. Journal of Clinical Investigation, 32, 1323-1329. doi:10.1172/JCI102861

[58]   Moor, S. and Stein, W. (1954) A modified ninhydrin reagent for the photometric determination of amino acids and related compounds. Journal of Biochemistry, 211, 907- 913.

[59]   Sigma-Aldrich (1996) Enzymatic assay of collagenase1 (EC digestion assay.

[60]   Laemmli, U.K. (1970) Cleavage of structural proteins dur- ing the assembly of the head of bacteriophage T4. Nature, 227, 680-685. doi:10.1038/227680a0

[61]   Wray, W., Boulikas, T., Wray, V.P. and Hancock, R. (1981) Sliver staining of proteins in polyacrylamide gels. Ana- lytical Biochemistry, 118, 197-203. doi:10.1016/0003-2697(81)90179-2

[62]   Eisen, A.Z., Henderson, K.O., Jeffrey, J.J. and Bradshaw, R.A. (1973). A collagenolytic protease from the hepato- pancreas of the fiddler crab, Ucapugilator. Purification and properties. Biochemistry, 12, 1814-1822. doi:10.1021/bi00733a024

[63]   Bauer, E.A., Stricklm, G.P., Jeffrey, J.J. and Eisen, A.Z. (1975) Collagenase production by human skin fibroblasts. Biochemical and Biophysical Research Communications, 64, 232-240. doi:10.1016/0006-291X(75)90243-0

[64]   Vaes, G. and Eeckhout, Y. (1975) In dynamics of connective tissue macromolecules. North-Holland Publishing Company, Amsterdam-Oxford.

[65]   Abe, S. and Nagai, Y. (1972) Interaction between tadpole collagenase and human α2-macroglobluin. Biochimicaet- Biophysica Acta, 278, 125-132.

[66]   Ohyama, H. and Hashimoto, K. (1977) Collagenase of human skin basal cell epithelioma. Bio-chemistry, 82, 175- 183.

[67]   Vaes, G. (1972) The re-lease of collagenase as an inactive proenzyme by bone ex-plants in culture. Biochemical Journal, 126, 275-289.

[68]   Shinkai, H., Kawamoto, T., Hori, H. and Nagai, Y. (1977) A complex of collagenase with low molecular weight inhibitors in the culture medium of em-bryonic chick skin explants. Journal of Biochemistry, 81, 261-263.

[69]   Sakamoto, S., Goldhaber, P. and Glimcher, M.J. (1972) The further purification and characterization of mouse bone collagenase. Calcified Tissiue Research, 10, 142-151. doi:10.1007/BF02012544

[70]   Woessner, J.F. Jr. (1991) Matrix metalloproteinases and their inhibitors in connective tissue remodeling. The FASEB Journal, 5, 2145-2154.

[71]   Grant, G.A., Henderson, K.O., Eisen, A.Z. and Bradshaw, R.A. (1980) Amino acid sequence of a collagenolytic pro- tease from the hepatopancreas of the fiddler crab, Ucapu- gilator. Biochemistry, 19, 4653-4659. doi:10.1021/bi00561a018

[72]   Yoshinaka, R., Sato, M., Itoko, M., Yamashita, M. and Ikeda, S. (1986) Purification and characterization of a collagenolytic serine proteinase from the catfish pancreas. Journal of Biochemistry, 99, 459-467.

[73]   Turkiewicz, M., Galas, E. and Kalinowska, H. (1991) Collagenolytic serine proteinase from Euphasiasuper- badana (Antarctic krill). Comparative Biochemistry and Physiology, Part B, 99, 359-371.

[74]   Tsai, I.H., Lu, P.J. and Chuang, J.L. (1991). The mid- gutchymotrypsins of shrimps (Penaeus-monodon, Penaeus- japonicusand Penaeuspenicillatus). Biochimicaet Biophysica Acta, 1080, 59-67. doi:10.1016/0167-4838(91)90112-D

[75]   Werner, M.H. and Wemmer, D.E. (1991). 1H assign- ments and sec-ondary structure determination of the soy- bean tryp-sin/chymotrypsin Bowman-Birk inhibitor. Bio- chemistry, 30, 3356-3364. doi:10.1021/bi00228a002

[76]   Fiedler-Nagy, C., Coffey, J.W. and Salvador, R.A. (1977) Factors influencing the apparent molecular weight of collagenase produced by human-skin explants. European Journal of Biochemistry, 76, 291-297. doi:10.1111/j.1432-1033.1977.tb11595.x

[77]   Ozaki, I., Zhao, G., Mizuta, T., Ogawa, Y., Hara, T., Kajihara, S. Hisatomi, A., Sakai, T. and Yamamoto, K. (2002) Hepa-tocyte growth factor induces collagenase (matrix metal-loproteinase-1) via the transcription factor Ets-1 in human hepatic stellate cell line. Journal of Hepa- tology, 36, 169-178. doi:10.1016/S0168-8278(01)00245-8

[78]   Murphy, G., Reynolds, J.J., Bretz, U. and Baggiolini, M. (1982) Partial purification of collagenase and gelatinase from human polymorphonuclear leucocytes. Biochemical Journal, 203, 209-221.

[79]   Vojdani, F. (1996) Solubility. In: Hall, G.M. Ed., Meth- ods of Testing Protein Functionality, Chapman and Hall, New York. doi:10.1007/978-1-4613-1219-2_2

[80]   Omondi, J.G. and Stark, J.R. (2001) Studies on digestive proteases from midgut glands of a shrimp, Penaeusindicus, and a lobster, Nephropsnorvegicus: Part 1. Proteolytic activity. Applied Biochemistry and Biotechnology, 90, 137-153. doi:10.1385/ABAB:90:2:137

[81]   ?sgeirsson, B., Fox, J.W. and Bjarnason J.B. (1989) Purification and charac-terization of trypsin from the poi- kilotherm Gadusmorhua. European Journal of Biochem- istry, 180, 85-94. doi:10.1111/j.1432-1033.1989.tb14618.x

[82]   ?sgeirsson, B. and Bjarnason, J.B. (1991) Structural and kinetic properties of chymotrypsin from Atlantic cod (Gadus-morhua). Comparison with bovine chymotrypsin. Compartive Biochemistry and Physiology, 99, 327-335.

[83]   ?sgeirsson, B. and Bjarnason, J.B. (1993). Properties of elastase from Atlantic cod a cold-adapted proteinase. Bio-chimicaet Biophysica Acta—Protein Structure and Molecular Enzymology, 1164, 91-100.

[84]   Kristjánsson, M.M. (1991). Purification and characterization of trypsin from the pyloric caeca of rainbow trout (Oncorhynchusmykiss). Journal of Agriculture and Food Chemistry, 39, 1738-1742. doi:10.1021/jf00010a009

[85]   Kristjánsson, M.M. and Nielsen, H.H. (1992) Purification and characterization of two chymotrypsin-like proteases from the pyloric caeca of rainbow trout (Oncorhynchus-mykiss). Comparative Biochemistry and Physiology, 101, 247-253.