Skip to main content
SpringerLink
Log in

Purification and characterization of pepsinogen and pepsin from the stomach of rainbow trout (Oncorhynchus mykiss)

  • Original Paper
  • Published:
European Food Research and Technology Aims and scope Submit manuscript

Abstract

Three pepsinogen isolates (PG-I till III) from the stomach of rainbow trout (Oncorhynchus mykiss) were purified by using ammonium sulfate precipitation, ion exchange chromatography, and two subsequent gel filtrations containing seven pepsinogen isoforms. SDS-PAGE revealed that pepsinogen isolate I contained two isoforms with molecular masses of 45 kDa (PG-I a) and 44 kDa (PG-I b) and the molecular masses of the PG isolates PG-II and PG-III were 42 kDa, respectively. P-I till P-III converted into the corresponding pepsins (P) at pH 2.0 with molecular masses of 37 kDa (P-I a, b, P-III) and 35 kDa (P-II). The isoelectric points were 4.0 (PG-I a, b), 5.9 (PG-II), and pepsinogen isolate III exhibited four isoforms with isoelectric points of 3.73 (PG-III a), 3.78 (PG-III b), 4.0 (PG-III c) and 4.15 (PG-III d). After conversion from pepsinogen, trout pepsins exhibited optimal activity at pH 3.0 and 40 °C (P-I) and pH 2.5 and 30 °C (P-II, P-III). The N-terminal amino acid sequences of the three pepsin isolates (P-I till III) were determined up to 20 amino acids. P-II showed 100 % identity (ID) to pepsin A of Oncorhynchus keta, but P-I and P-III revealed high similarity to chitinases (85 % ID). Catalytic constants KM and kcat for proteolysis of acid-denatured hemoglobin were determined as 2.8 × 10−8 M (P-I), 1.3 × 10−8 M (P-II) and 7.9 × 10−9 M (P-III), and 5.43–19.1 S−1, respectively.

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
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Kageyama T (2002) Pepsinogens, progastricsins, and prochymosins: structure, function, evolution, and development. Cell Mol Life Sci 59(2):288–306

    Article  CAS  Google Scholar 

  2. Bougatef A, Balti R, Zaied SB, Souissi N, Nasri M (2008) Pepsinogen and pepsin from the stomach of smooth hound (Mustelus mustelus): purification, characterization and amino acid terminal sequences. Food Chem 107(2):777–784

    Article  CAS  Google Scholar 

  3. Stanley DW, Emmons DB (1977) Cheddar cheese made with bovine pepsin II. Texture—microstructure—composition relationships. Can Inst Food Sci Technol J 10(2):78–84

    Article  CAS  Google Scholar 

  4. Tavares JF, Baptista JA, Marcone MF (1997) Milk-coagulating enzymes of tuna fish waste as a rennet substitute. Int J Food Sci Nutr 48(3):169–176

    Article  CAS  Google Scholar 

  5. Vlahović B, Popović-Vranješ A, Mugoša I (2014) International cheese market–current state and perspective. Econ Insights Trends Chall 3:35–43

    Google Scholar 

  6. Llarena-Reino M, Pineiro C, Antonio J, Outerino L, Vello C, Gonzalez AF, Pascual S (2013) Optimization of the pepsin digestion method for anisakids inspection in the fishing industry. Vet Parasitol 191(3–4):276–283

    Article  CAS  Google Scholar 

  7. Lassoued I, Jridi M, Nasri R, Dammak A, Hajji M, Nasri M, Barkia A (2014) Characteristics and functional properties of gelatin from thornback ray skin obtained by pepsin-aided process in comparison with commercial halal bovine gelatin. Food Hydrocoll 41:309–318

    Article  CAS  Google Scholar 

  8. Zhao L, Budge SM, Ghaly AE, Brooks MS, Dave D (2011) Extraction, purification and characterization of fish pepsin: a critical review. J Food Process Technol 2(6):2–6

    Article  Google Scholar 

  9. Klomklao Kishimura H, Yabe M, Benjakul S (2007) Purification and characterization of two pepsins from the stomach of pectoral rattail (Coryphaenoides pectoralis). Comp Biochem Physiol B Biochem Mol Biol 147(4):682–689

    Article  Google Scholar 

  10. Gildberg A, Olsen RL, Bjarnason JB (1990) Catalytic properties and chemical composition of pepsins from Atlantic cod (Gadus morhua). Comp Biochem Physiol B Biochem Mol Biol 96(2):323–330

    Article  CAS  Google Scholar 

  11. Brier S, Maria G, Carginale V, Capasso A, Wu Y, Taylor RM, Borotto NB, Capasso C, Engen JR (2007) Purification and characterization of pepsins A1 and A2 from the Antarctic rock cod Trematomus bernacchii. FEBS J 274(23):6152–6166

    Article  CAS  Google Scholar 

  12. Nalinanon S, Benjakul S, Kishimura H (2010) Biochemical properties of pepsinogen and pepsin from the stomach of albacore tuna (Thunnus alalunga). Food Chem 121(1):49–55

    Article  CAS  Google Scholar 

  13. Wu T, Sun L-C, Du C-H, Cai Q-F, Zhang Q-B, Su W-J, Cao M-J (2009) Identification of pepsinogens and pepsins from the stomach of European eel (Anguilla anguilla). Food Chem 115(1):137–142

    Article  CAS  Google Scholar 

  14. Cao MJ, Chen WQ, Du CH, Yoshida A, Lan WG, Liu GM, Su WJ (2011) Pepsinogens and pepsins from Japanese seabass (Lateolabrax japonicus). Comp Biochem Physiol B 158(4):259–265

    Article  Google Scholar 

  15. Vannabun A, Ketnawa S, Phongthai S, Benjakul S, Rawdkuen S (2014) Characterization of acid and alkaline proteases from viscera of farmed giant catfish. Food Biosci 6:9–16

    Article  CAS  Google Scholar 

  16. Zhou Q, Liu GM, Huang YY, Weng L, Hara K, Su WJ, Cao MJ (2008) Pepsinogens and pepsins from mandarin fish (Siniperca chuatsi). J Agric Food Chem 56(13):5401–5406

    Article  CAS  Google Scholar 

  17. Liaset B, Lied E, Espe M (2000) Enzymatic hydrolysis of by-products from the fish-filleting industry; chemical characterisation and nutritional evaluation. J Sci Food Agric 80(5):581–589

    Article  CAS  Google Scholar 

  18. FEAP (2015) European aquaculture production report 2005–2014. Federation of European Aquaculture Producers. http://www.feap.info/Default.asp?SHORTCUT=582. Accessed 20 Aug 2015

  19. Chen YC, Jaczynski J (2007) Protein recovery from rainbow trout (Oncorhynchus mykiss) processing byproducts via isoelectric solubilization/precipitation and its gelation properties as affected by functional additives. J Agric Food Chem 55(22):9079–9088

    Article  CAS  Google Scholar 

  20. Twining SS, Alexander PA, Huibregtse K, Glick DM (1983) A pepsinogen from rainbow trout. Comp Biochem Physiol B 75(1):109–112

    CAS  Google Scholar 

  21. Tanji M, Yakabe E, Kageyama T, Yokobori S, Ichinose M, Miki K, Ito H, Takahashi K (2007) Purification and characterization of pepsinogens from the gastric mucosa of African coelacanth, Latimeria chalumnae, and properties of the major pepsins. Comp Biochem Physiol B Biochem Mol Biol 146(3):412–420

    Article  Google Scholar 

  22. Zhou Q, Fu X-P, Zhang L-J, Su W-J, Cao M-J (2007) Purification and characterization of sea bream (Sparus latus houttuyn) pepsinogens and pepsins. Food Chem 103(3):795–801

    Article  CAS  Google Scholar 

  23. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1):248–254

    Article  CAS  Google Scholar 

  24. Xu RA, Wong RJ, Rogers ML, Fletcher GC (1996) Purification and characterization of acidic proteases from the stomach of the deepwater finfish orange roughy (Hoplostethus atlanticus). J Food Biochem 20(6):31–48

    Article  CAS  Google Scholar 

  25. Ryle AF (1984) Pepsins, gastricsins and their zymogens. In: Bergmeyer HU (ed) Methods of enzymatic analysis. Verlag Chemie, Weinheim, pp 223–238

    Google Scholar 

  26. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685

    Article  CAS  Google Scholar 

  27. Altinelataman C, Kündiger R, Cakli S, Rehbein H (2009) Comparison of IEF patterns of sarcoplasmic proteins of fish from North Atlantic and Aegean Sea. Food Control 20(11):980–985

    Article  CAS  Google Scholar 

  28. Heukeshoven J, Dernick R (1985) Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis 6(3):103–112

    Article  CAS  Google Scholar 

  29. Lineweaver H, Burk D (1934) The determination of enzyme dissociation constants. J Am Chem Soc 56(3):658–666

    Article  CAS  Google Scholar 

  30. Edman P (1949) A method for the determination of amino acid sequence in peptides. Arch Biochem Biophys 22(3):475

    CAS  Google Scholar 

  31. Gildberg A, Raa J (1983) Purification and characterization of pepsins from the arctic fish capelin (Mallotus villosus). Comp Biochem Physiol A Comp Physiol 75(3):337–342

    Article  CAS  Google Scholar 

  32. Marciniszyn J Jr, Huang JS, Hartsuck JA, Tang J (1976) Mechanism of intramolecular activation of pepsinogen. Evidence for an intermediate delta and the involvement of the active site of pepsin in the intramolecular activation of pepsinogen. J Biol Chem 251(22):7095–7102

    CAS  Google Scholar 

  33. Sanchez-Chiang L, Cisternas E, Ponce O (1987) Partial purification of pepsins from adult and juvenile salmon fish Oncorhynchus keta. Effect of NaCl on proteolytic activities. Comp Biochem Physiol B Biochem Mol Biol 87(4):793–797

    Article  CAS  Google Scholar 

  34. Arunchalam K, Haard NF (1985) Isolation and characterization of pepsin from polar cod (Boreogadus saida). Comp Biochem Physiol B Biochem Mol Biol 80(3):467–473

    Article  Google Scholar 

  35. Tanji M, Yakabe E, Kubota K, Kageyama T, Ichinose M, Miki K, Ito H, Takahashi K (2009) Structural and phylogenetic comparison of three pepsinogens from Pacific bluefin tuna: molecular evolution of fish pepsinogens. Comp Biochem Physiol B Biochem Mol Biol 152(1):9–19

    Article  Google Scholar 

  36. Tanji M, Kageyama T, Takahashi K (1988) Tuna pepsinogens and pepsins. Purification, characterization and amino-terminal sequences. Eur J Biochem 177(2):251–259

    Article  CAS  Google Scholar 

  37. Sugiura SH, Roy PK, Ferraris RP (2006) Dietary acidification enhances phosphorus digestibility but decreases H+/K+−ATPase expression in rainbow trout. J Exp Biol 209(19):3719–3728. doi:10.1242/jeb.02436

    Article  CAS  Google Scholar 

  38. Gildberg A (1988) Aspartic proteinases in fishes and aquatic invertebrates. Comp Biochem Physiol B Comp Biochem 91(3):425–435

    Article  CAS  Google Scholar 

  39. Bucking C, Wood CM (2006) Gastrointestinal processing of Na+, Cl−, and K+ during digestion: implications for homeostatic balance in freshwater rainbow trout. Am J Physiol Regul Integr Comp Physiol 291(6):R1764–R1772. doi:10.1152/ajpregu.00224.2006

    Article  CAS  Google Scholar 

  40. Brier S, Maria G, Carginale V, Capasso A, Wu Y, Taylor RM, Borotto NB, Capasso C, Engen JR (2007) Purification and characterization of pepsins A1 and A2 from the Antarctic rock cod Trematomus bernacchii. FEBS J 274(23):6152–6166

    Article  CAS  Google Scholar 

  41. Benhabiles MS, Abdi N, Drouiche N, Lounici H, Pauss A, Goosen MFA, Mameri N (2012) Fish protein hydrolysate production from sardine solid waste by crude pepsin enzymatic hydrolysis in a bioreactor coupled to an ultrafiltration unit. Mater Sci Eng C 32(4):922–928

    Article  CAS  Google Scholar 

  42. Hartsuck JA, Koelsch G, Remington SJ (1992) The high-resolution crystal structure of porcine pepsinogen. Proteins 13(1):1–25

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Hessen’s state government under the program “research for practice”.

Author information

Authors and Affiliations

  1. Department of Food Technology, Institute of Human Nutrition and Food Science, University of Kiel, Heinrich-Hecht Platz 10, 24118, Kiel, Germany

    Maleen Wald & Karin Schwarz

  2. Faculty of Food Technology, Fulda University of Applied Sciences, Leipziger Straße 123, 36037, Fulda, Germany

    Maleen Wald, Christopher Beermann & Bettina Bußmann

  3. Department of Safety and Quality of Milk and Fish, Max Rubner-Institute, Palmaille 9, 22767, Hamburg, Germany

    Hartmut Rehbein

Authors
  1. Maleen Wald
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hartmut Rehbein
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christopher Beermann
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bettina Bußmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Karin Schwarz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Maleen Wald.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 93 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wald, M., Rehbein, H., Beermann, C. et al. Purification and characterization of pepsinogen and pepsin from the stomach of rainbow trout (Oncorhynchus mykiss). Eur Food Res Technol 242, 1925–1935 (2016). https://doi.org/10.1007/s00217-016-2692-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00217-016-2692-2

Keywords

Search

Navigation