Skip to main content

Controlled Enzymatic Hydrolysis: A New Strategy for the Discovery of Antimicrobial Peptides

Abstract

The use of antimicrobial peptides (AMPs) is an alternative to traditional antibiotics. AMPs are obtained using different methods such as bacterial synthesis, chemical synthesis and controlled enzymatic hydrolysis. The later is an interesting approach that deserves our attention because of the yields gathered and peptides engineered. Usually, activities of AMPs obtained in such a way are tightly dependent on the hydrolysis mechanism used. This paper deals with the hydrolysis of hemoglobin mechanism as a potential source of AMPs. Production of AMPs from hemoglobin using enzymatic controlled system is linked to hemoglobin structure. Further, we show that bovine hemoglobin, which is sensitive to peptic hydrolysis, results upon enzymatic digestion as a great source of AMPs. The hemoglobin in native and denatured states was hydrolyzed by “one-by-one” and “zipper” mechanisms, respectively. Nevertheless, a new mechanism named “semi-zipper” mechanism is obtained when protein is in molten globule structural state, constituting an original strategy for AMPs production. Seventy seven percentage of the peptides obtained by this new strategy showed antibacterial activity against nine strains.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. 1.

    Adje YE, Balti R, Kouach M, Dhulster P, Guillochon D, Nedjar-Arroume N (2011) Obtaining antimicrobial peptides by controlled peptic hydrolysis of bovine hemoglobin. Int J Biol Macromol 49:143–153

    Article  CAS  Google Scholar 

  2. 2.

    Adje YE, Balti R, Kouach M, Guillochon D, Nedjar-Arroume N (2011) α 67–106 of bovine hemoglobin: a new family of antimicrobial and angiotensin I-converting enzyme inhibitory peptides. Eur Food Res Technol 232:637–646

    Article  CAS  Google Scholar 

  3. 3.

    Barkhudaryan N, Kellermann J, Galoyan A, Lottspeich F (1993) High molecular weight aspartic endopeptidase generates a coronaro-constrictory peptide from the beta-chain of hemoglobin. FEBS Lett 329:215–218

    Article  CAS  Google Scholar 

  4. 4.

    Barkhudaryan N, Oberthuer W, Lottspeich F, Galoyan AA (1992) Structure of hypothalamic coronaro-constrictory peptide factor. Neurochem Res 17:1217–1221

    Article  CAS  Google Scholar 

  5. 5.

    Bhakuni V (1998) Alcohol-induced molten globule intermediates of proteins: are they real unfolding intermediates or off pathway products? Arch Biochem Biophys 357:274–284

    Article  CAS  Google Scholar 

  6. 6.

    Brian BL, Konermann L (2007) Folding and assembly of haemoglobin monitored by electrospray Mass spectrometry using an on-line dialysis system. J Am Soc Mass Spectrom 18:8–16

    Article  Google Scholar 

  7. 7.

    Brian LB, Mark C, Konermann KL (2007) Symmetric behavior of hemoglobin α- and β- subunits during acid-induced denaturation observed by electrospray mass spectrometry. Biochemistry 46:10675–10684

    Article  Google Scholar 

  8. 8.

    Buck M, Radford SE, Dobson CM (1993) A partially folded state of hen egg white lysozyme in trifluoroethanol: structural characterization and implications for protein folding. Biochemistry 32:669–678

    Article  CAS  Google Scholar 

  9. 9.

    Buck M, Schwalbe H, Dobson CM (1995) Characterization of conformational preferences in a partly folded protein by heteronuclear NMR spectroscopy: assignment and secondary structure analysis of hen egg white lysozyme in trifluoroethanol. Biochemistry 34:13219–13232

    Article  CAS  Google Scholar 

  10. 10.

    Dalgalarrondo M, Dufour E, Chobert JM, Bertrand-Harb C, Haertlk T (1993) Proteolysis of β-Lactoglobulin and β-Casein by Pepsin in Ethanolic Media. Int Dairy J 5:1–14

    Article  Google Scholar 

  11. 11.

    Daoud R, Dubois V, Bors-Dodita L, Nedjar-Arroume N, Krier F, Chihib NE, Mary P, Kouach M, Briand G, Guillochon D (2005) New antibacterial peptide derived from bovine hemoglobin. Peptides 26:713–719

    Article  CAS  Google Scholar 

  12. 12.

    Swati De, Girigoswami A (2006) A fluorimetric and circular dichroism study of hemoglobin–effect of pH and anionic amphiphiles. J Colloid Interface Sci 296:324–331

    Article  Google Scholar 

  13. 13.

    Deng L, Pan X, Wang Y, Wang L, Zhou XE, Li M, Feng Y, Wu Q, Wang B, Huang N (2009) Hemoglobin and its derived peptides may play a role in the antibacterial mechanism of the vagina. Hum Reprod 24:211–218

    Article  CAS  Google Scholar 

  14. 14.

    Fogaça AC, Da Silva PI, Miranda TMM, Bianchi AG, Mirandai A, Ribolla PEM, Daffre S (1999) Antimicrobial activity of a bovine hemoglobin fragment in the tick Boophilus microplus. J Biol Chem 274:25330–25334

    Article  Google Scholar 

  15. 15.

    Froidevaux R, Krier F, Nedjar-Arroume N, Vercaigne-Marko D, Kosciarz E, Ruckebusch C, Dhulster P, Guillochon D (2001) Antibacterial activity of a pepsin-derived bovine hemoglobin fragment. FEBS Lett 491:159–163

    Article  CAS  Google Scholar 

  16. 16.

    González-Jiménez J, Cortijo M (2002) Urea-induced denaturation of human serum albumin labeled with acrylodan. J Protein Chem 21(2):75–79

    Article  Google Scholar 

  17. 17.

    Griffith WP, Kaltashov IA (2003) Highly asymmetric interactions between globin chains during hemoglobin assembly revealed by electrospray ionization mass spectrometry. Biochemistry 42:10024–10033

    Article  CAS  Google Scholar 

  18. 18.

    Hancock RE, Falla T, Brown M (1995) Cationic bactericidal peptides. Adv Microb Physiol 37:135–175

    Article  CAS  Google Scholar 

  19. 19.

    Hancock RE, Lehrer R (1998) Cationic peptides: a new source of antibiotics. Trends Biotechnol 16:82–88

    Article  CAS  Google Scholar 

  20. 20.

    Herskovits TT, Gadegbeku B, Jaillet H (1970) On the structural stability and solvent denaturation of proteins: I denaturation by the alcohols and glycols. J Biol Chem 245:588–2598

    Google Scholar 

  21. 21.

    Hobson D, Hirsch JG (1958) The antibacterial activity of hemoglobin. J Exp Med 107:167–183

    Article  Google Scholar 

  22. 22.

    Jansson H, Bergman R, Swenson J (2005) Relation between solvent and protein dynamics as studied by dielectric spectroscopy. J Phys Chem B 109:24134–24141

    Article  CAS  Google Scholar 

  23. 23.

    Lebrun F, Bazus A, Dhulster P, Guillochon D (1998) Solubility of heme in heme-iron enriched bovine haemoglobin hydrolysates. J Agric Food Chem 46:5017–5025

    Article  CAS  Google Scholar 

  24. 24.

    Linderstrom-Lang K (1953) The initial phases of the enzymatic degradation of proteins. Bull Soc Chim Biol 35:100–116

    CAS  Google Scholar 

  25. 25.

    Liu Y, Bolen DW (1995) The peptide backbone plays a dominant role in protein stabilization by naturally occurring osmolytes. Biochemistry 34:12884–12891

    Article  CAS  Google Scholar 

  26. 26.

    Mahmoud MI, Malone WT, Cordle CT (1992) Enzymatic hydrolysis of casein: effect of degree of hydrolysis on antigenicity and physical properties. J Food Sci 57:1223–1229

    Article  CAS  Google Scholar 

  27. 27.

    Mak P, Wójcik K, Silberring J, Dubin A (2000) Antimicrobial peptides derived from heme-containing proteins: hemocidins. Antonie Van Leeuwenhoek 77:197–207

    Article  CAS  Google Scholar 

  28. 28.

    Mak P, Wójcik K, Wicherek L, Suder P, Dubin A (2004) Antibacterial hemoglobin peptides in human menstrual blood. Peptides 25:1839–1847

    Article  CAS  Google Scholar 

  29. 29.

    Mito K, Fujii M, Kuwahara M, Matsumura N, Shimizu T, Sugano S, Karaki H (1996) Antihypertensive effect of angiotensin I-converting enzyme inhibitory peptides derived from hemoglobin. Eur J Pharmacol 304:93–98

    Article  CAS  Google Scholar 

  30. 30.

    Nedjar-Arroume N, Dubois-Delval V, Adje EY, Traisnel J, Krier F, Mary P, Kouach M, Briand G, Guillochon D (2008) Bovine hemoglobin: an attractive source of antibacterial peptides. Peptides 29:969–977

    Article  CAS  Google Scholar 

  31. 31.

    Nedjar-Arroume N, Dubois-Delval V, Miloudi K, Daoud R, Krier F, Kouach M, Briand G, Guillochon D (2006) Isolation and characterization of four antibacterial peptides from bovine hemoglobin. Peptides 27:2082–2089

    Article  CAS  Google Scholar 

  32. 32.

    Nielsen PM, Peterson D, Dambmann C (2001) Improved method for determining food protein degree of hydrolysis. J Food Sci 66:642–646

    Article  CAS  Google Scholar 

  33. 33.

    Pande VS, Grosberg AY, Tanaka T, Rokhsar DS (1998) Pathways for protein folding: is a new view needed? Curr Opin Struct Biol 8:68–79

    Article  CAS  Google Scholar 

  34. 34.

    Parish CA, Jiang H, Tokiwa Y, Berova N, Nakanishi K, Mc Cabe D, Zuckerman W, Xia MM, Gabay JE (2001) Broad-Spectrum antimicrobial activity of hemoglobin. Bioorg Med Chem 9:377–382

    Article  CAS  Google Scholar 

  35. 35.

    Piot JM, Zhao QY, Guillochon D, Ricart G, Thomas D (1992) Isolation and characterization of two opioid peptides from a bovine hemoglobin peptic hydrolysate. Biochem Biophys Res Commun 189:101–110

    Article  CAS  Google Scholar 

  36. 36.

    Povey JF, Smales CM, Hassard SJ, Howard MJ (2007) Comparison of the effects of 2,2,2-trifluoroethanol on peptide and protein structure and function. J Struct Biol 157:329–338

    Article  CAS  Google Scholar 

  37. 37.

    Rezaei-Ghaleh N, Ebrahim-Habibi A, Moosavi-Movahedi AA, Nemat-Gorgani M (2007) Effect of polyamines on the structure, thermal stability and 2,2,2-trifluoroethanol-induced aggregation of alpha-chymotrypsin. Int J Biol Macromol 41:597–604

    Article  CAS  Google Scholar 

  38. 38.

    Roccatano D, Colombo G, Fioroni M, Mark AE (2002) Mechanism by which 2,2,2-trifluoroethanol/water mixtures stabilize secondary-structure formation in peptides: a molecular dynamics study. PNAS Biophys 99:12179–12184

    Article  CAS  Google Scholar 

  39. 39.

    Rupley JA (1967) Susceptibility to attack by proteolytic enzymes. Methods Enzymol 11:905–917

    Article  CAS  Google Scholar 

  40. 40.

    Schally AV, Nair RMG, Tommie WR, Arimura A (1971) Isolation of the luteinizing hormone and follicle-stimulating hormone-releasing hormone from porcine hypothalami. J Biol Chem 246:7230–7239

    CAS  Google Scholar 

  41. 41.

    Tsuzuki W, Ue A, Nagao A (2003) Polar organic solvent added to an aqueous solution changes hydrolytic properties of lipase. Biosci Biotechnol Biochem 67:1660–1666

    Article  CAS  Google Scholar 

  42. 42.

    Van’t Hof W, Veerman EC, Helmerhorst EJ, Amerongen AV (2001) Antimicrobial peptides: properties and applicability. Biol Chem 382:597–619

    Google Scholar 

  43. 43.

    Zhao Q, Piot JM (1997) Investigation of inhibition angiotensin-converting enzyme (ECA) activity and opioid activity of two hemorphins, LVV-hemorphin-5 and VV-hemorphin-5, isolated from a defined peptic hydrolysate of bovine hemoglobin. Neuropeptides 2:147–153

    Article  Google Scholar 

  44. 44.

    Zhao QY, Piot JM, Gautier V, Cottenceau G (1996) Peptic peptide mapping by HPLC, on line with photodiode array detection, of a haemoglobin hydrolysate produced at pilot-plant scale from an ultrafiltration process. Appl Microbiol Biotechnol 45:778–784

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors thank Christine Vanuxem from University of Lille I for assisting in the review of this manuscript and Dr. Gilbert Briand “Laboratoire d’Application de Spectrométrie de Masse, Service Commun de Physicochimie, Faculté des Sciences Pharmaceutiques et Biologiques Lille II, France” for analysis of peptide sequences. Estelle Yaba Adjé has a fellowship from Ivorian government.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Rafik Balti.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Adje, E.Y., Balti, R., Lecouturier, D. et al. Controlled Enzymatic Hydrolysis: A New Strategy for the Discovery of Antimicrobial Peptides. Probiotics & Antimicro. Prot. 5, 176–186 (2013). https://doi.org/10.1007/s12602-013-9138-y

Download citation

Keywords

  • Antimicrobial peptides
  • Hydrolysis mechanism
  • Bovine hemoglobin
  • Protein structure