Skip to main content
SpringerLink
Log in

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

  • Published:
Food Science and Biotechnology Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Anson ML. The estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. J. Gen. Physiol. 22:79–89 (1938)

    Article  CAS  Google Scholar 

  • Arvanitoyannis IS, Kassaveti A. Fish industry waste: treatments, environmental impacts, current and potential uses. Int. J. Food Sci. Technol. 43:726–745 (2008)

    Article  CAS  Google Scholar 

  • 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)

    Article  CAS  Google Scholar 

  • 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)

    Article  CAS  Google Scholar 

  • 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)

    Article  CAS  Google Scholar 

  • 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)

    Article  CAS  Google Scholar 

  • 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 

  • Gildberg A. Aspartic proteinases in fishes and aquatic invertebrates. Comp. Biochem. Physiol. B 91:425–435(1988)

    Article  CAS  Google Scholar 

  • 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)

    Article  CAS  Google Scholar 

  • 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)

    Article  CAS  Google Scholar 

  • Jacob M, Jaros D, Rohm H. Recent advances in milk clotting enzymes. Int. J. Dairy Technol. 64:14–33 (2011)

    Article  CAS  Google Scholar 

  • 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)

    Article  Google Scholar 

  • Kumar D, Bhalla TC. Microbial proteases in peptide synthesis: approaches and applications. Appl. Microbiol. Biotechnol. 68:726–736 (2005)

    Article  CAS  Google Scholar 

  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 (1970)

    Article  CAS  Google Scholar 

  • 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)

    Article  CAS  Google Scholar 

  • 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)

    Article  CAS  Google Scholar 

  • 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)

    Article  CAS  Google Scholar 

  • 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)

    Article  CAS  Google Scholar 

  • 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)

    Article  CAS  Google Scholar 

  • 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)

    Article  CAS  Google Scholar 

  • 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)

    Article  CAS  Google Scholar 

  • 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)

    Article  Google Scholar 

  • 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)

    Article  CAS  Google Scholar 

  • 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)

    Article  CAS  Google Scholar 

  • Ogino H, Ishikawa H. Enzymes which are stable in the presence of organic solvents. J. Biosci. Bioeng. 91(2):109–116 (2001)

    Article  CAS  Google Scholar 

  • 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)

    Article  CAS  Google Scholar 

  • 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)

    Article  CAS  Google Scholar 

  • 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)

    Article  CAS  Google Scholar 

  • 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)

    Article  CAS  Google Scholar 

  • 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)

    Article  Google Scholar 

  • Stauffer C. Enzyme assays for food scientist. New York: Van Nostrand (1989)

    Google Scholar 

  • Squires EJ, Haard F, Feltham LAW. Gastric proteases of the Greenland cod Gadus ogac. II. Structural properties. Biochem. Cell Biol. 64:215–222 (1986)

    Article  CAS  Google Scholar 

  • 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)

    Article  CAS  Google Scholar 

  • 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)

    Article  CAS  Google Scholar 

  • 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)

    Article  Google Scholar 

Download references

Acknowledgements

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)”.

Author information

Authors and Affiliations

  1. Maestría en Ciencias Aplicadas, Unidad Académica de Ingeniería en Biotecnología, Universidad Politécnica de Sinaloa, Mazatlán, Sinaloa, Mexico

    Idalia Osuna-Ruiz, Jesús Aarón Salazar-Leyva & Emmanuel Martínez-Montaño

  2. Ingeniería en Biotecnología. Unidad Académica de Ingeniería en Biotecnología, Universidad Politécnica de Sinaloa, Mazatlán, Sinaloa, Mexico

    María Fernanda Espinoza-Marroquin

  3. Laboratorio de Acuicultura Tropical, División Académica de Ciencias Biológicas, Universidad Juárez Autónoma de Tabasco, Villahermosa, Tabasco, Mexico

    Emyr Peña & Carlos Alfonso Álvarez-González

  4. Cátedras CONACYT, Consejo Nacional de Ciencia y Tecnología, Ciudad de Mexico, Mexico

    Emyr Peña & Emmanuel Martínez-Montaño

  5. Facultad de Ciencias del Mar, Universidad Autónoma de Sinaloa, Mazatlán, Sinaloa, Mexico

    Isaura Bañuelos-Vargas

Authors
  1. Idalia Osuna-Ruiz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. María Fernanda Espinoza-Marroquin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jesús Aarón Salazar-Leyva
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Emyr Peña
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Carlos Alfonso Álvarez-González
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Isaura Bañuelos-Vargas
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Emmanuel Martínez-Montaño
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Emmanuel Martínez-Montaño.

Ethics declarations

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.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Osuna-Ruiz, I., Espinoza-Marroquin, M.F., Salazar-Leyva, J.A. et al. Biochemical characterization of a semi-purified aspartic protease from sea catfish Bagre panamensis with milk-clotting activity. Food Sci Biotechnol 28, 1785–1793 (2019). https://doi.org/10.1007/s10068-019-00614-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10068-019-00614-8

Keywords

Search

Navigation