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Purification and biochemical properties of pepsins from the stomach of skipjack tuna (Katsuwonus pelamis)

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Abstract

Pepsins 1 and 2 from the stomach of skipjack tuna (Katsuwonus pelamis) were purified to homogeneity by using a series of chromatographic purification involving DEAE-cellulose, Sephadex G-50 and Sephadex G-75 with increase in purity of 246-fold and 213-fold, respectively. Molecular weights of pepsins 1 and 2 were estimated by SDS–PAGE to be 33.9 and 33.7 kDa, respectively. The N-terminal amino acid sequences of the first 20 amino acids of both isoenzymes were YQDGTEPMTNDADLSYYGVI. The optimal pH and temperature for pepsin 1 were 2.5 and 50 °C, respectively, while pepsin 2 showed optimal activity at pH 2.0 and 45 °C. The activity of two pepsins was stable in the pH range of 2–5 and at temperatures up to 50 °C. The activity of purified pepsins was strongly inhibited by pepstatin A in a dose-dependent manner. SDS and cysteine showed inhibitory effects toward both pepsins. Activity of pepsin 2 was slightly activated by NaCl, but NaCl had no effect on pepsin 1. Pepsins 1 and 2 had high affinity and hydrolytic activity toward hemoglobin with K m of 54 and 71 μM, respectively. k cat of pepsins 1 and 2 were 38.1 and 44.3 s−1, respectively. Both pepsins effectively hydrolyzed bovine serum albumin, egg white, natural actomyosin from brownstripe red snapper muscle and acid-solubilized collagen from arabesque greenling skin. Nevertheless, the hydrolytic activity was slightly less than that of pepsin from porcine stomach.

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Acknowledgments

This work was supported by the Thailand Research Fund under the Royal Golden Jubilee Ph.D. Program to Sitthipong Nalinanon (PHD/0171/2549) and TRF Senior Research Scholar program.

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Authors and Affiliations

  1. Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla, 90112, Thailand

    Sitthipong Nalinanon & Soottawat Benjakul

  2. Laboratory of Marine Products and Food Science, Research Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido, 041-8611, Japan

    Hideki Kishimura

Authors
  1. Sitthipong Nalinanon
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  2. Soottawat Benjakul
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  3. Hideki Kishimura
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Correspondence to Soottawat Benjakul.

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Nalinanon, S., Benjakul, S. & Kishimura, H. Purification and biochemical properties of pepsins from the stomach of skipjack tuna (Katsuwonus pelamis). Eur Food Res Technol 231, 259–269 (2010). https://doi.org/10.1007/s00217-010-1275-x

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  • DOI: https://doi.org/10.1007/s00217-010-1275-x

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