Biochemical characterization of a semi-purified aspartic protease from sea catfish Bagre panamensis with milk-clotting activity

  • Idalia Osuna-Ruiz
  • María Fernanda Espinoza-Marroquin
  • Jesús Aarón Salazar-Leyva
  • Emyr Peña
  • Carlos Alfonso Álvarez-González
  • Isaura Bañuelos-Vargas
  • Emmanuel Martínez-MontañoEmail author


Pepsin from stomach of Bagre panamensis was semi-purified and biochemically characterized. The acid proteolytic activity and purification fold were 3875 U/mg protein and 91.85, respectively, after purification process. The optimum pH and temperature for semi-purified protease were 2–3 and 65 °C, respectively. The enzyme activity was stable after heating proteases at 50 °C for 120 min, but only 30% residual activity was detected after heating at 65 °C for 30 min. SDS-PAGE analysis showed two proteins bands after dialysis (26.1 and 38.6 kDa). Only the band of 38.6 kDa had proteolytic activity, which was inhibited using pepstatin A. Organic solvents, surfactants and reducing agents affect the proteolytic activity at different extent; however, metal ions or EDTA have no impact on protease activity. The semi-purified protease exhibited milk coagulant activity, with a maximum activity at 45 °C. The obtained results highlight the potential biotechnological use of B. panamensis pepsin.


Proteases Milk clotting activity Catfish Pepsin Protein purification By-products 



The authors would like to thank to MC Gissel Rios and Reyna Tiznado for her laboratory assistance and MC Jesús Martín Moreno Hernandez for his advices for milk-clotting assays. Also, the authors thank María Elena Sánchez Salazar for her editorial work in English. This work has been elaborated as an activity from thematic research network of CONACYT: “RED 12.4, Para reducir y valorizar las pérdidas y desperdicios de alimentos: Hacia sistemas alimentarios sostenibles (No. 294768)”.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Human and animal rights statement

This article does not contain studies with animals subjects performed by the any of the authors.


  1. Anson ML. The estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. J. Gen. Physiol. 22:79–89 (1938)CrossRefGoogle Scholar
  2. Arvanitoyannis IS, Kassaveti A. Fish industry waste: treatments, environmental impacts, current and potential uses. Int. J. Food Sci. Technol. 43:726–745 (2008)CrossRefGoogle Scholar
  3. Barberis S, Quiroga E, Morcelle S, Priolo N, Luco JM. Study of phytoproteases stability in aqueous-organic biphasic systems using linear free energy relationships. J. Mol. Catal. B Enzym. 38:95–103 (2006)CrossRefGoogle Scholar
  4. Bkhairia I, Mhamdi S, Jridi M, Nasri M. New acidic proteases from Liza aurata viscera: Characterization and application in gelatin production. Int. J. Biol. Macromol. 92:533–542 (2016)CrossRefGoogle Scholar
  5. Bougatef A, Balti B, Zaied SB, Soussi N, Nasri M. Pepsinogen and pepsin from the stomach of smooth hound (Mustelus mustelus): Purification, characterization and amino acid terminal sequences. Food Chem. 107:777–784 (2008)CrossRefGoogle Scholar
  6. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of dye binding. Anal. Biochem. 72:248–254 (1976)CrossRefGoogle Scholar
  7. Burgess RR. Protein precipitation techniques. Chapter 20, pp. 331–342. In: Methods in Enzymology. Volume 463. Guide to protein purification. Burgess RR, Deutscher MP (eds). Academic Press, San Diego, CA, USA (2009)Google Scholar
  8. Gildberg A. Aspartic proteinases in fishes and aquatic invertebrates. Comp. Biochem. Physiol. B 91:425–435(1988)CrossRefGoogle Scholar
  9. Harpaz S, Eshel A, Lindner P. Effect of 1-propanol on the activity of intestinal proteolytic enzymes of the European sea bass Dicentrarchus labrax. J. Agric. Food Chem. 42:49–52 (1994)CrossRefGoogle Scholar
  10. Homaei A, Lavajoo F, Sariri R. Development of marine biotechnology as a resource for novel proteases and their role in modern biotechnology. Int. J. Biol. Macromol. 88:542–552 (2016)CrossRefGoogle Scholar
  11. Jacob M, Jaros D, Rohm H. Recent advances in milk clotting enzymes. Int. J. Dairy Technol. 64:14–33 (2011)CrossRefGoogle Scholar
  12. Klomklao S, Kishimura H, Yabe M, Benjakul S. Purification and characterization of two pepsins from the stomach of pectoral rattail (Coryphaenoides pectoralis). Comp. Biochem. Physiol. B 147:682–689 (2007)CrossRefGoogle Scholar
  13. Kumar D, Bhalla TC. Microbial proteases in peptide synthesis: approaches and applications. Appl. Microbiol. Biotechnol. 68:726–736 (2005)CrossRefGoogle Scholar
  14. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 (1970)CrossRefGoogle Scholar
  15. Mazorra-Manzano MA, Moreno-Hernández JM, Ramírez Suárez JC, Torres-Llanez MJ, González-Córdova AF, Vallejo-Córdoba B. Sour orange Citrus aurantium L. flowers: a new vegetable source of milk-clotting proteases. LWT Food Sci. Technol. 54:325–330 (2013)CrossRefGoogle Scholar
  16. Miura Y, Kageyama T, Moriyama A. Pepsinogens and pepsins from largemouth bass, Micropterus salmoides: Purifications and characterization with special reference to high proteolytic activities of bass enzymes. Comp. Biochem. Physiol. B 183:42–48 (2015)CrossRefGoogle Scholar
  17. Nalinanon S, Benjakul S, Kishimura H, Shahidi F. Functionalities and antioxidant properties of protein hydrolysates from the muscle of ornate threadfin bream treated with pepsin from skipjack tuna. Food Chem. 124:1354–1362 (2011)CrossRefGoogle Scholar
  18. Nalinanon S, Benjakul S, Kishimura H. Biochemical properties of pepsinogen and pepsin from the stomach of albacore tuna (Thunnus alalunga). Food Chem. 121:49–55 (2010)CrossRefGoogle Scholar
  19. Nalinanon S, Benjakul S, Visessanguan W, Kishimura, H. Use of pepsin for collagen extraction from the skin of bigeye snapper (Priacanthus tayenus). Food Chem. 104:593–601 (2007)CrossRefGoogle Scholar
  20. Nalinanon S, Benjakul S, Visessanguan W, Kishimura H. Improvement of gelatin extraction from bigeye snapper skin using pepsin-aided process in combination with protease inhibitor. Food Hydrocoll. 22:615–622 (2008a)CrossRefGoogle Scholar
  21. Nalinanon S, Benjakul S, Visessanguan W, Kishimura H. Tuna pepsin: Characteristics and its use for collagen extraction from the skin of threadfin bream (Nemipterus spp.). J. Food Sci. 73:C413–C419 (2008b)CrossRefGoogle Scholar
  22. Nasri RN, Younes I, Lassoued I, Ghorbel S, Ghorbel-Bellaaj O, Nasri M. Digestive alkaline proteases from Zosterisessor ophiocephalus, Raja clavata, and Scorpaena scrofa: Characteristics and application in chitin extraction. J. Amino Acids 2011:913616 (2011)CrossRefGoogle Scholar
  23. Noda M, Murakami K. Studies on proteinases from the digestive organs of sardine. II. Purification and characterization of two acid proteinases from the stomach. Biochim. Biophys. Acta 658:27–34 (1981)CrossRefGoogle Scholar
  24. Olsen RL, Toppe J, Karunasagar I. Challenges and realistic opportunities in the use of by-products from processing of fish and shellfish. Trends Food Sci. Technol. 36:144–151 (2014)CrossRefGoogle Scholar
  25. Ogino H, Ishikawa H. Enzymes which are stable in the presence of organic solvents. J. Biosci. Bioeng. 91(2):109–116 (2001)CrossRefGoogle Scholar
  26. Pereira NA, Fernández-Gimenez AV. Exogenous enzymes in dairy technology: Acidic proteases from processing discards of shrimp Pleoticus muelleri and their use as milk-clotting enzymes for cheese manufacture. Int. J. Food Sci. Technol. 52:341–347 (2017)CrossRefGoogle Scholar
  27. Saborowski R, Sahling G, Navarrete del Toro MA, Walter I, García-Carreño FL. Stability and effects of organic solvents on endopeptidases from the gastric fluid of the marine crab Cancer pagurus. J. Mol. Catal. B Enzym. 30:109–118 (2004)CrossRefGoogle Scholar
  28. Sathivel S, Yin H, Prinyawiwatkul W, King JM. Comparisons of chemical and physical properties of catfish oils prepared from different extracting processes. J. Food Sci. 74(2):E70–E76 (2009)CrossRefGoogle Scholar
  29. Shankar S, Laxman RS. Biophysicochemical characterization of an alkaline protease from Beauveria sp. MTCC 5184 with multiple applications. Appl. Biochem. Biotechnol. 175:589-602 (2015)CrossRefGoogle Scholar
  30. Simmons M, Ru G, Casalone C, Iulini B, Cassar C, Seuberlich T. Discontools: identifying gaps in controlling bovine spongiform encephalopathy. Transbound. Emerg. Dis. 65:9–21 (2018)CrossRefGoogle Scholar
  31. Stauffer C. Enzyme assays for food scientist. New York: Van Nostrand (1989)Google Scholar
  32. Squires EJ, Haard F, Feltham LAW. Gastric proteases of the Greenland cod Gadus ogac. II. Structural properties. Biochem. Cell Biol. 64:215–222 (1986)CrossRefGoogle Scholar
  33. Tavares JFP, Baptista JAB, Marcone MF. Milk-coagulating enzymes of tuna fish waste as a rennet substitute. Int. J. Food Sci. Nutr. 48:169–176 (1997)CrossRefGoogle Scholar
  34. Wu T, Sun LC, Du CH, Cai QF, Zhang QB, Su WJ, Cao MJ. Identification of pepsinogens and pepsins from the stomach of European eel (Anguilla Anguilla). Food Chem. 115:137–142 (2009)CrossRefGoogle Scholar
  35. Zhao L, Budge SM, Ghaly AE, Brooks MS, Dave D. Extraction, purification and characterization of fish pepsins: a critical review. J. Food Process. Technol. 2:1–14 (2011)CrossRefGoogle Scholar

Copyright information

© The Korean Society of Food Science and Technology 2019

Authors and Affiliations

  • Idalia Osuna-Ruiz
    • 1
  • María Fernanda Espinoza-Marroquin
    • 2
  • Jesús Aarón Salazar-Leyva
    • 1
  • Emyr Peña
    • 3
    • 4
  • Carlos Alfonso Álvarez-González
    • 3
  • Isaura Bañuelos-Vargas
    • 5
  • Emmanuel Martínez-Montaño
    • 1
    • 4
    Email author
  1. 1.Maestría en Ciencias Aplicadas, Unidad Académica de Ingeniería en BiotecnologíaUniversidad Politécnica de SinaloaMazatlánMexico
  2. 2.Ingeniería en Biotecnología. Unidad Académica de Ingeniería en BiotecnologíaUniversidad Politécnica de SinaloaMazatlánMexico
  3. 3.Laboratorio de Acuicultura Tropical, División Académica de Ciencias BiológicasUniversidad Juárez Autónoma de TabascoVillahermosaMexico
  4. 4.Cátedras CONACYTConsejo Nacional de Ciencia y TecnologíaCiudad de MexicoMexico
  5. 5.Facultad de Ciencias del MarUniversidad Autónoma de SinaloaMazatlánMexico

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