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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

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• TITLE: Isolation and activation of collagenase from fish processing waste

  AUTHORS: Said M. Daboor, Suzanne M. Budge, Abdel E. Ghaly, Marianne S. Brooks, Deepika Dave

  KEYWORDS: Fish Waste; Collagenase Enzyme; Extraction; Purification; Inhibitors; Activators

  JOURNAL NAME: Advances in Bioscience and Biotechnology, Vol.3 No.3, June 21, 2012

  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.