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Purification and characterization of three trypsin isoforms from viscera of sardinelle (Sardinella aurita)

Fish Physiology and Biochemistry volume 37, pages 123–133 (2011)Cite this article

Abstract

Three trypsin isoforms A, B and C were purified to homogeneity from the viscera of sardinelle (Sardinella aurita). Purification was achieved by ammonium sulfate precipitation (20–70% (w/v)), Sephadex G-100 gel filtration and Mono Q-Sepharose anion-exchange chromatography. The molecular weights of these purified enzymes were estimated to be 28.8 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Based on the native PAGE and casein-zymography, each purified trypsin appeared as a single band. Trypsins A and C exhibited the maximal activity at 55°C, while trypsin B at 50°C. All isoforms showed the same optimal pH (pH 9.0) using Nα-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as a substrate. The three trypsins were stable at temperatures below 40°C and over a broad pH range (7.0–11.0). The activities of the three isoforms were strongly inhibited by soybean trypsin inhibitor and phenylmethylsulfonyl fluoride, a serine protease inhibitor, and partially inhibited by ethylenediaminetetraacetic acid, a metalloenzyme inhibitor. Kinetic constants of trypsins A, B and C for BAPNA were evaluated at 25°C and pH 9.0. The values of K m and k cat were 0.125, 0.083 and 0.10 mM, and 2.24, 1.21 and 5.76 s−1, respectively. The N-terminal sequences of the first 10 amino acids were “I V G G Y E C Q K Y” for trypsin A and “I V G G Y E A Q S Y” for trypsins B and C. These sequences showed highly homology to other fish trypsins.

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Acknowledgments

This work was funded by the Ministry of Higher Education and Scientific Research-Tunisia.

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  1. Laboratoire de Génie Enzymatique et de Microbiologie, Ecole Nationale d’Ingénieurs de Sfax, Route Soukra Km 3.5, B.P. 1173-3038, Sfax, Tunisia

    Hayet Ben Khaled, Kemel Jellouli, Nabil Souissi, Sofiane Ghorbel, Ahmed Barkia & Moncef Nasri

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  1. Hayet Ben Khaled
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  2. Kemel Jellouli
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  3. Nabil Souissi
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  4. Sofiane Ghorbel
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  5. Ahmed Barkia
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  6. Moncef Nasri
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Correspondence to Moncef Nasri.

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Ben Khaled, H., Jellouli, K., Souissi, N. et al. Purification and characterization of three trypsin isoforms from viscera of sardinelle (Sardinella aurita). Fish Physiol Biochem 37, 123–133 (2011). https://doi.org/10.1007/s10695-010-9424-5

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  • DOI: https://doi.org/10.1007/s10695-010-9424-5

Keywords

  • S. aurita
  • Trypsins
  • Isoforms
  • Viscera
  • Purification
  • N-terminal amino acid sequence