Chihuil Sea Catfish Bagre panamensis Viscera as a New Source of Serine Proteases: Semi-purification, Biochemical Characterization and Application for Protein Hydrolysates Production

  • Gissel Daniela Rios-Herrera
  • Idalia Osuna Ruiz
  • Crisantema Hernández
  • Angel Valdez-Ortiz
  • Jorge Manuel Sandoval-Gallardo
  • Emmanuel Martínez-Montaño
  • Jorge Saúl Ramírez-Pérez
  • Jesús Aarón Salazar-LeyvaEmail author
Original Paper


The recovery of proteases from fish viscera could be a strategy to reduce environmental problems caused by inadequate disposal of fish by-products. This study reports the biochemical characterization of proteases isolated from chihuil sea catfish (Bagre panamensis) intestines and the evaluation of their stability to different physical and chemical factors. Protein hydrolysates from chihuil muscle and casein were produced using its semi-purified proteases extract (SPE) and alcalase. Assays with specific protease inhibitors indicate that trypsin and chymotrypsin are the main types of serine proteases in SPE. Semi-purified enzymes exhibited proteolytic activity at alkaline pH (9–12), and high stability at low/mild temperatures (10–40 °C). A 92% of SPE proteolytic activity was retained in the presence of 30% NaCl. The enzyme extract was stable in reducing agents (2-mercaptoethanol and DTT) but lost about 70% of proteolytic activity in anionic detergents like SDS and tween-80. Organic solvents did not affect the enzyme activity of SPE. Finally, maintaining a same E/S ratio for protein hydrolysates elaboration, chihuil serin proteases exhibited a higher hydrolytic efficiency compared to alcalase when casein and proteins from chihuil muscle were hydrolyzed. Thus, the semi-purification of serine proteases from chihuil viscera provided a low-cost source of enzymes with interesting catalytic features.

Graphic Abstract


Protein semi-purification Serine proteases Protein hydrolysis Sea catfish Bagre panamensis 



The authors wish to acknowledge the National Council for Science and Technology (CONACyT) Mexico for financing gran proposal 231525 and the graduate scholarship granted to Gissel Rios. The authors thank PhD. Carmen López Saiz for her editorial work in English.

Compliance with Ethical Standards

Conflict of interest

The authors declare no conflict of interest.


  1. 1.
    FAO. The State of World Fisheries and Aquaculture - Meeting the sustainable development goals. Rome. pp. 2–193. (2018)
  2. 2.
    Olsen, R.L., 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(2), 144–151 (2014). CrossRefGoogle Scholar
  3. 3.
    Bougatef, A.: Trypsins from fish processing waste: characteristics and biotechnological applications—Comprehensive review. J. Clean Prod. 57, 552–567 (2013). CrossRefGoogle Scholar
  4. 4.
    Shen, X., Zhang, M., Bhandari, B., Gao, Z.: Novel technologies in utilization of byproducts of animal food processing: a review. Crit. Rev. Food Sci. Nutr. (2018). CrossRefGoogle Scholar
  5. 5.
    Klein, M.D., Oleskowicz-Popiel, P., Simmons, B.A., Blanch, H.W.: The challenge of enzyme cost in the production of ligno cellulosic biofuels. Biotechnol. Bioeng. 109, 1083–1087 (2012). CrossRefGoogle Scholar
  6. 6.
    Bezerra, R., Lins, E., Alencar, R., Paiva, P., Chaves, M., Coelho, L.: Alkaline proteinase form intestine of Nile tilapia (Oreochromis niloticus). Process Biochem. 40, 1829–1834 (2005). CrossRefGoogle Scholar
  7. 7.
    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
  8. 8.
    Neklyudov, A.D., Ivankin, A.N., Berdutina, A.V.: Properties and uses of protein hydrolysates. Appl. Biochem. Microbiol. 36, 452–459 (2000). CrossRefGoogle Scholar
  9. 9.
    Hou, Y., Wu, Z., Dai, Z., Wang, G., Wu, G.: Protein hydrolysates in animal nutrition: industrial production, bioactive peptides, and functional significance. J. Anim. Sci. Biotechnol. 8(1), 24 (2017). CrossRefGoogle Scholar
  10. 10.
    Blanco, M., Sotelo, C.G., Pérez-Martín, R.I.: Hydrolysis as a valorization strategy for unused marine food biomass: boarfish and small-spotted catshark discards and by-products. J. Food Biochem. 39(4), 368–376 (2015). CrossRefGoogle Scholar
  11. 11.
    Murthy, L.N., Phadke, G.G., Unnikrishnan, P., Annamalai, J., Joshy, C.G., Zynudheen, A.A., Ravishankar, C.N.: Valorization of fish viscera for crude proteases production and its use in bioactive protein hydrolysate preparation. Waste Biomass Valoriz. 9(10), 1735–1746 (2017). CrossRefGoogle Scholar
  12. 12.
    Raghavan, S., Kristinsson, H.G.: Antioxidative efficacy of alkali-treated Tilapia protein hydrolysates: a comparative study of five enzymes. J. Agric. Food Chem. 56, 1434–1441 (2008). CrossRefGoogle Scholar
  13. 13.
    Chalamaiah, M., Dinesh-Kumar, B., Hemalatha, R., Jyothirmayi, T.: Fish protein hydrolysates: proximate composition, amino acid composition, antioxidant activities and applications: a review. Food Chem. 135(4), 3020–3038 (2012). CrossRefGoogle Scholar
  14. 14.
    Zamani, A., Benjakul, S.: Trypsin from unicorn leatherjacket (Aluterus monoceros) pyloric caeca: purification and its use for preparation of fish protein hydrolysate with antioxidative activity. J. Sci. Food Agric. 6(96), 962–969 (2016). CrossRefGoogle Scholar
  15. 15.
    Muro-Torres, V.M., Amezcua, F., Lara-Mendoza, R.E., Buszkiewicz, J.T., Amezcua-Linares., F.: Trophic ecology of the chihuil sea catfish (Bagre panamensis) in the south-east Gulf of California, México. J. Mar. Biol. Assoc. UK 98(4), 885–893 (2017). CrossRefGoogle Scholar
  16. 16.
    Castillo-Yáñez, F., Pacheco-Aguilar, R., García-Carreño, F., Toro, M., López, M.: Purification and biochemical characterization of chymotrypsin from the viscera of Monterey sardine (Sardinops sagax caerulea). Food Chem. 99(2), 252–259 (2006). CrossRefGoogle Scholar
  17. 17.
    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
  18. 18.
    Sarath, G., De La Motte, R., Wagner, F., 1989. In R. Beynon (Ed.). Proteolytic enzymes a practical approach, Vol. 3: protease assay methods, 25-55, IRL Press, OxfordGoogle Scholar
  19. 19.
    Erlanger, B.F., Kokowski, N., Cohen, W.: The preparation and properties of two new chromogenic substrates of trypsin. Arch. Biochem. Biophys. 95(2), 271–278 (1961). CrossRefGoogle Scholar
  20. 20.
    Laemmli, U.K.: Cleavage of structural proteins during assembly of the head bacteriophage T4. Nature 227, 680–685 (1970). CrossRefGoogle Scholar
  21. 21.
    García-Carreño, F.L., Haard, N.F.: Characterization of proteinase classes in langostilla (Pleuroncodes punipes) and crayfish (Paczfastacus astacus) extracts. J. Food Biochem. 17, 97–113 (1993). CrossRefGoogle Scholar
  22. 22.
    Villalba-Villalba, A.G., Ramírez-Suárez, J.C., Valenzuela-Soto, E.M., García-Sánchez, G., Carvallo, R.G., Pacheco-Aguilar, R.: Trypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991: its purification and characterization. Food Chem. 141, 940–945 (2013). CrossRefGoogle Scholar
  23. 23.
    García-Carreño, F.L.: Proteinase inhibitors. Trends Food Sci. Technol. 7, 197–203 (1996). CrossRefGoogle Scholar
  24. 24.
    Klomklao, S., Benjakul, S., Visessanguan, W.: Comparative studies on proteolytic activity of spleen extracts from three tuna species commonly used in Thailand. J. Food Biochem. 28, 355–372 (2004). CrossRefGoogle Scholar
  25. 25.
    Simkhada, J.R., Cho, S.S., Park, S.J., Mander, P., Choi, Y.H., Lee, H.J., Yoo, J.C.: An oxidant and organic solvent-resistant alkaline metalloprotease from Streptomyces olivochromogenes. Appl. Biochem. Biotechnol. 162, 1457–1470 (2010). CrossRefGoogle Scholar
  26. 26.
    Rahman, R.N.Z.R.A., Geok, L.P., Basri, M., Salleh, A.B.: An organic solvent-stable alkaline protease from Pseudomonas aeruginosa strain K: enzyme purification and characterization. Enzyme Microb. Technol. 39, 1484–1491 (2006). CrossRefGoogle Scholar
  27. 27.
    Moreno-Hernández, J.M., Hernández-Mancillas, X.D., Coss, N.E., Mazorra-Manzano, M.A., Osuna-Ruiz, I., Rodríguez-Tirado, V.A., Salazar-Leyva, J.A.: Partial characterization of the proteolytic properties of an enzymatic extract from “aguama” Bromelia pinguin L. fruit grown in Mexico. Appl. Biochem. Biotechnol. 182, 181–196 (2017). CrossRefGoogle Scholar
  28. 28.
    Adler-Nissen, J.: A review of food hydrolysis specific areas. In: Adler-Nissen, J. (ed.) Enzymatic hydrolysis of food proteins, pp. 57–109. Elsevier, Copenhagen (1986)Google Scholar
  29. 29.
    Navarrete-del-Toro, M.A., García-Carreño, F.L.: Evaluation of the progress of protein hydrolysis. In: Wrolstad, R.E., Decker, E.A., Schwartz, S.J., Sporns, P. (eds.) Handbook of Food Analytical Chemistry. Water, Proteins, Enzymes, Lipids, and Carbohydrates, Vol. 1, pp. B2.2.1–B2.2.14. Wiley, Hoboken (2002)Google Scholar
  30. 30.
    Silva, J., Espósito, T., Marcuschi, M., Ribeiro, K., Cavalli, R., Oliveira, V.: Purification and partial characterization of a trypsin from the processing waste of the silver mojarra (Diapterus rhombeus). Food Chem. 129, 777–782 (2011). CrossRefGoogle Scholar
  31. 31.
    Sila, A., Nasri, R., Jridi, M., Balti, R., Nasri, M., Bougatef, A.: Characterisation of trypsin purified from the viscera of Tunisian barbel (Barbus callensis) and its application for recovery of carotenoproteins from shrimp wastes. Food Chem. 132, 1287–1295 (2012). CrossRefGoogle Scholar
  32. 32.
    El-Hadj, A.N., Hmidet, N., Ghorbel-Bellaaj, O., Fakhfakh-Zouari, N., Bougatef, A., Nasri, M.: Solvent-stable digestive alkaline proteinases from striped seabream (Lithognathus mormyrus) viscera: characteristics, application in the deproteinization of shrimp waste, and evaluation in laundry. Appl. Biochem. Biotechnol. 164, 1096–1110 (2011). CrossRefGoogle Scholar
  33. 33.
    Younes, I., Nasri, R., Bkhairia, I., Jellouli, K., Nasri, M.: New proteases extracted from red scorpionfish (Scorpaena scrofa) viscera: characterization and application as a detergent additive and for shrimp waste deproteinization. Food Bioprod. Process. 94, 453–462 (2015). CrossRefGoogle Scholar
  34. 34.
    Villalba-Villalba, A.G., Pacheco-Aguilar, R., Ramírez-Suárez, J.C., Valenzuela-Soto, E.M., Castillo-Yáñez, F.J., Márquez-Ríos, E.: Partial characterization of alkaline proteases from viscera of vermiculated sailfin catfish Pterygoplichthys disjunctivus Weber, 1991. Food Sci. Tech. 77, 697–705 (2011). CrossRefGoogle Scholar
  35. 35.
    Castillo-Yáñez, F.J., Aguilar, R.P., Lugo-Sanchez, M.E., Sanchez, G.G., Reyes, Q.E.: Biochemical characterization of an isoform of chymotrypsin from the viscera of Monterey sardine (Sardinops sagax caerulea), and comparison with bovine chymotrypsin. Food Chem. (2009). CrossRefGoogle Scholar
  36. 36.
    Jellouli, K., Bougatef, A., Daasi, D., Balti, R., Barkia, A., Nasri, M.: New alkaline trypsin from the intestine of grey triggerfish (Balistes capriscus) with high activity at low temperature: purification and characterization. Food Chem. 116, 644–650 (2009). CrossRefGoogle Scholar
  37. 37.
    Kudre, T., Thongraung, C.: Organic solvent and laundry detergent stable crude protease from Nile tilapia (Oreochromis niloticus) viscera. J. Aquat. Food Prod. Technol. 23(1), 87–100 (2014). CrossRefGoogle Scholar
  38. 38.
    Klomklao, S.: Digestive proteinases from marine organisms and their applications. J. Sci. Technol. 30(1), 37–46 (2008)Google Scholar
  39. 39.
    Valdez-Melchor, R.G., Ezquerra-Brauer, J.M., Castillo-Yáñez, F.J., Cárdenas-López, J.L.: Purification and partial characterization of trypsin from the viscera of tropical sierra (Scomberomorus sierra) from the Gulf of California. J. Food Biochem. (2012). CrossRefGoogle Scholar
  40. 40.
    Aranishi, F., Hara, K., Osatomi, K., Ishihara, T.: Purification and characterization of cathepsin B from hepatopancreas of carp Cyprinus carpio. Comp. Biochem. Physiol. B 117, 579–587 (1997). CrossRefGoogle Scholar
  41. 41.
    Hernández-Sámano, A.C., Guzmán-García, X., García-Barrientos, R., Ascencio-Valle, F., Sierra-Beltrán, A., Vallejo-Córdoba, B., González-Córdova, A.F., Torres-Llanez, M.J., Guerrero-Legarreta, I.: Extracción y caracterización de proteasas de pepino de mar Isostichopus fuscus recolectado en el golfo de California, México. Rev. Mex. Ing. Quim. 14(1), 35–47 (2015)Google Scholar
  42. 42.
    Zhu, B.W., Zhao, L., Sun, L., Li, D., Murata, Y., Yu, L., Zhang, L.: Purification and characterization of a cathepsin L-like enzyme from the body wall of the sea cucumber Stichopus japonicus. Biosci. Biotechnol. Biochem. 72(6), 1430–1437 (2008). CrossRefGoogle Scholar
  43. 43.
    Prado, B.A., Hernández, O.A., Sánchez, E.O., Hernández, M.R.: Criterios de selección de cepas fúngicas para la producción de proteasas termoestables por cultivo en medio sólido. Rev Iberoamericana Cien 1(6), 61 (2014)Google Scholar
  44. 44.
    Lu, C.-H., Lin, Y.-F., Lin, J.-J., Yu, C.-S.: Prediction of metal ion–binding sites in proteins using the fragment transformation method. PLoS ONE 7(6), 1–12 (2012). CrossRefGoogle Scholar
  45. 45.
    Flores-Fernández, M.L., Zavaleta, A.I., Chávez-Hidalgo, E.L.: Halotolerant bacteria with lipolytic activity isolated from Pilluana salterns - San Martin. Cienc. Invest 13(2), 87–91 (2010)Google Scholar
  46. 46.
    Castillo-Rivera, M., Ortiz-Burgos, S., Zárate-Hernández, R.: Estructura de la comunidad de peces estuarinos en un hábitat con vegetación sumergida: variación estacional y nictémera. Hidrobiológica 21(3), 311–321 (2011)Google Scholar
  47. 47.
    Le, C.M., Donnay-Moreno, C., Bruzac, S., Baron, R., Thi My Nguyen, H., Pascal Bergé, J.: Proteolysis of sardine (Sardina pilchardus) and anchovy (Stolephorus commersonii) by commercial enzymes in saline solutions. Food Technol. Biotechnol. 53, 87–90 (2015). CrossRefGoogle Scholar
  48. 48.
    Saborowski, R., Sahling, G., Navarrete del Toro, M.A., Walter, I., García-Carreño, F.L.: Stability and effects of organic solvents on endopeptidases from the gastric fluid of the marine crab Cancer pagurus. J. Mol. Catal. B 30, 109–118 (2004). CrossRefGoogle Scholar
  49. 49.
    Doukyu, N., Ogino, H.: Review: organic solvent-tolerant enzymes. Biochem. Eng. J. 48(3), 270–282 (2010). CrossRefGoogle Scholar
  50. 50.
    Barberis, S., Quiroga, E., Morcelle, S., Priolo, N., Luco, J.M.: Study of phytoproteases stability in aqueous-organic biphasic systems using linear free energy relationships. J. Mol. Catal. B 38, 95–103 (2006). CrossRefGoogle Scholar
  51. 51.
    Osuna-Ruiz, I., Espinoza-Marroquin, M.F., Salazar-Leyva, J.A., Peña, E., Álvarez-González, C.A., Bañuelos-Vargas, I., Martínez-Montaño, E.: Biochemical characterization of a semi-purified aspartic protease from sea catfish Bagre panamensis with milk-clotting activity. Food Sci. Biotechnol. (2019). CrossRefGoogle Scholar
  52. 52.
    Carrera, G., Riva, S.: Organic synthesis with enzymes in non-aqueous media. In: Adlercreutz, P. (ed.) Fundamentals of Biocatalysis in Neat Organic Solvents, pp. 1–24. Wiley, Weinheim (2008). CrossRefGoogle Scholar
  53. 53.
    Ogino, H., Ishikawa, H.: Enzymes which are stable in the presence of organic solvents. J. Biosci. Bioeng. 91, 109–116 (2001). CrossRefGoogle Scholar
  54. 54.
    Adamson, N.J., Reynolds, E.C.: Characterization of casein phosphopeptides prepared using alcalase: determination of enzyme specificity. Enzyme Microb. Technol. 19, 202–207 (1996). CrossRefGoogle Scholar
  55. 55.
    Doucet, D., Otter, D.E., Gauthier, S.F., Allen, F.E.: Enzyme-induced gelation of extensively hydrolyzed whey proteins by alcalase: peptide identification and determination of enzyme specificity. J. Agric. Food Chem. 51(21), 6300–6308 (2003). CrossRefGoogle Scholar
  56. 56.
    Li, Z.Y., Youravong, W., Youravong, H.K.: Protein hydrolysis by protease isolated from tuna spleen by membrane filtration: a comparative study with commercial proteases. Food Sci. Technol. 43, 166–172 (2010). CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Gissel Daniela Rios-Herrera
    • 1
  • Idalia Osuna Ruiz
    • 2
  • Crisantema Hernández
    • 3
  • Angel Valdez-Ortiz
    • 4
  • Jorge Manuel Sandoval-Gallardo
    • 1
  • Emmanuel Martínez-Montaño
    • 2
    • 5
  • Jorge Saúl Ramírez-Pérez
    • 1
  • Jesús Aarón Salazar-Leyva
    • 2
    Email author
  1. 1.Doctorado en Ciencias en Recursos Acuáticos. Facultad de Ciencias del MarUniversidad Autónoma de SinaloaMazatlánMexico
  2. 2.Maestría en Ciencias Aplicadas, Unidad Académica de Ingeniería en BiotecnologíaUniversidad Politécnica de Sinaloa (UPSIN)MazatlánMexico
  3. 3.Centro de Investigación en Alimentación y Desarrollo, A. C. MazatlánMazatlánMexico
  4. 4.Facultad de Ciencias Químico-BiológicasUniversidad Autónoma de SinaloaCuliacánMexico
  5. 5.Cátedras CONACyT, Consejo Nacional de Ciencia y TecnologíaMexico CityMexico

Personalised recommendations