Skip to main content

Advertisement

SpringerLink
Log in

Characterization of Peptides from Capsicum annuum Hybrid Seeds with Inhibitory Activity Against α-Amylase, Serine Proteinases and Fungi

  • Published:
The Protein Journal Aims and scope Submit manuscript

Abstract

Over the last several years, the activity of antimicrobial peptides (AMPs), isolated from plant species, against different microorganisms has been demonstrated. More recently, some of these AMPs have been described as potent inhibitors of α-amylases and serine proteinases from insects and mammals. The aim of this work was to obtain AMPs from protein extracts of a hybrid Capsicum (Ikeda × UENF 1381) seeds and to evaluate their microbial and enzyme inhibitory activities. Initially, proteins were extracted from the Capsicum hybrid seeds in buffer (sodium phosphate pH 5.4,) and precipitated with ammonium sulfate (90 % saturated). Extract of hybrid seeds was subjected to size exclusion chromatography, and three fractions were obtained: S1, S2 and S3. The amino acid sequence, obtained by mass spectrometry, of the 6 kDa peptide from the S3 fraction, named HyPep, showed 100 % identity with PSI-1.2, a serine protease inhibitor isolated from C. annuum seeds, however the bifunctionality of this inhibitor against two enzymes is being shown for the first time in this work. The S3 fraction showed the highest antifungal activity, inhibiting all the yeast strains tested, and it also exhibited inhibitory activity against human salivary and Callosobruchus maculatus α-amylases as well as serine proteinases.

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

Similar content being viewed by others

Abbreviations

AMPs:

Antimicrobial peptides

HyPep:

6 kDa peptide from the S3 fraction

FITC:

Fluorescein isothiocyanate

BApNA:

N-benzoyl-DL-arginyl-p-nitroanilide

BTpNA:

N-benzoyl-L-tyrosyl-p-nitroanilide

References

  1. Benko-Iseppon AM, Galdino SL, Calsa TJ, Kido EA, Tossi A, Belarmino LC, Crovella S (2010) Overview on plant antimicrobial peptides. Curr Prot Pept Sci 11:181–188

    Article  CAS  Google Scholar 

  2. Peters BM, Shirtliff ME, Jabra-Rizk MA (2010) Antimicrobial peptides: Primeval molecules or future drugs? PLoS Pathog 6:10–16

    Article  Google Scholar 

  3. Baumann T, Kämpfer U, Schürch S, Schaller J, Largiadèr C, Nentwig W, Kuhn-Nentwig L (2010) Ctenidins: antimicrobial glycine-rich peptides from the hemocytes of the spider Cupiennius salei. Cell Mol Life Sci 66:2787–2798

    Article  Google Scholar 

  4. Auvynet C, Rosenstein Y (2009) Multifunctional host defense peptides: antimicrobial peptides, the small yet big players in innate and adaptive immunity. FEBS J 276:6497–6508

    Article  CAS  Google Scholar 

  5. Brandenburg LO, Merres J, Albrecht LJ, Varoga D, Pufe T (2012) Antimicrobial peptides: multifunctional drugs for different applications. Polymers 4:539–560

    Article  Google Scholar 

  6. Kuzina LV, Miller TA, Cooksey DA (2006) In vitro activities of antibiotics and antimicrobial peptides against the plant pathogenic bacterium Xylella fastidiosa. Microbiol Appl Lett 42:514–520

    Article  CAS  Google Scholar 

  7. Diz MSS, Carvalho AO, Ribeiro SFF, Da Cunha M, Beltramini L, Rodrigues R, Nascimento VV, Machado OLT, Gomes VM (2011) Characterisation, immunolocalisation and antifungal activity of a lipid transfer protein from chili pepper (Capsicum annuum) seeds with novel α-amylase inhibitory properties. Physiol Plant 142:233–246

    Article  CAS  Google Scholar 

  8. Pelegrini PB, Lay FT, Murad AM, Anderson MA, Franco OL (2008) Novel insights on the mechanism of action of α-amylase inhibitors from the plant defensin family. Proteins 73:719–729

    Article  CAS  Google Scholar 

  9. Santos IS, Carvalho AO, Filho GAS, Nascimento VV, Machado OLT, Gomes VM (2010) Purification of a defensin isolated from Vigna unguiculata seeds, its functional expression in Escherichia coli, and assessment of its insect α-amylase inhibitory activity. Protein Expr Purif 71:8–15

    Article  Google Scholar 

  10. Zottich U, Da Cunha M, Carvalho AO, Dias GB, Silva NCM, Santos IS, Nascimento VV, Miguel EC, Machado OLT, Gomes VM (2011) Purification, biochemical characterization and antifungal activity of a new lipid transfer protein (LTP) from Coffea canephora seeds with α-amylase inhibitor properties. Biochim Biophys Acta 1810:375–383

    Article  CAS  Google Scholar 

  11. Sivakumar S, Mohan M, Franco OL, Thayumanavan B (2006) Inhibition of insect pest α-amylases by little and finger millet inhibitors. Pestic Biochem Phys 85:155–160

    Article  CAS  Google Scholar 

  12. Wang JR, Yan ZH, Wei YM, Nevo E, Baum BR, Zheng YL (2006) Molecular characterization of dimeric alpha-amylase inhibitor genes in wheat and development of genome allele-specific primers for the genes located on chromosome 3BS and 3DS. J Cereal Sci 43:360–368

    Article  CAS  Google Scholar 

  13. Moreira GR, Calimam FRB, Silva DJH, Ribeiro CSC (2006) Espécies e variedades de pimenta. Informe Agropecuário 27:16–29

    Google Scholar 

  14. Argáez-Brito L, Moguel-Salazar F, Zamudio F, González-Estrada T, Islãs-Flores I (2009) Characterization of a Capsicum chinense seed peptide fraction with broad antimicrobial activity. Asian J Biochem 4:77–87

    Article  Google Scholar 

  15. Teixeira FR, Lima MCOP, Almeida HO, Romeiro RS, Silva DJH, Pereira PRG, Fontes EPB, Baracat-Pereira MC (2006) Bioprospection of cationic and anionic antimicrobial peptides from bell pepper leaves for inhibition of Ralstonia solanacearum and Clavibacter michiganensis ssp. michiganensis growth. J Phytopathol 154:418–421

    Article  CAS  Google Scholar 

  16. Cruz LP, Ribeiro SF, Carvalho AO, Vasconcelos IM, Rodrigues R, Da Cunha M, Gomes VM (2010) Isolation and partial characterization of a novel lipid transfer protein (LTP) and antifungal activity of peptides from chilli pepper seeds. Protein Pept Lett 17:311–318

    Article  CAS  Google Scholar 

  17. Diz MSS, Carvalho AO, Rodrigues R, Neves-Ferreira AGC, Da Cunha M, Alves EW, Okorokova-Façanha AL, Oliveira MA, Perales J, Machado OLT, Gomes VM (2006) Antimicrobial peptides from chili pepper seeds causes yeast plasma membrane permeabilization and inhibits the acidification of the medium by yeast cells. Biochem Biophys Acta 1760:1323–1332

    Article  CAS  Google Scholar 

  18. Ribeiro SFF, Carvalho AO, Da Cunha M, Rodrigues R, Melo VMM, Vasconcelos IM, Melo EJT, Cruz LP, Gomes VM (2007) Isolation and characterization of novel peptides from chilli pepper seeds: antimicrobial activities against pathogenic yeasts. Toxicon 50:600–611

    Article  CAS  Google Scholar 

  19. Taveira GB, Mathias LS, Motta OV, Machado OLT, Rodrigues R, Carvalho AO, Teixeira-Ferreira A, Perales J, Vasconcelos IM, Gomes VM (2013) Thionin-like peptides from Capsicum annuum fruits with high activity against human pathogenic bacteria and yeasts. Pept Sci 102:30–39

    Article  Google Scholar 

  20. Maokam C, Ruangviriyachai C, Chanthai S (2014) Determination of major and minor capsaicinoids in different varieties of the Capsicum fruits using GC-MS and their inhibition effect of the chilli extract on α-amylase activity. Int Food Res J 21:2237–2243

    CAS  Google Scholar 

  21. Oboh G, Adedayo O, Ademiluyi FYM (2011) Effect of Combination on the antioxidant and inhibitory properties of tropical pepper varieties against α-amylase and α-glucosidase activities in vitro. J Med Food 14:1152–1158

    Article  CAS  Google Scholar 

  22. Dias GB, Gomes VM, Pereira UZ, Ribeiro SFF, Carvalho AO, Rodrigues R, Machado OLT, Fernandes KVS, Ferreira AT, Perales J, Da Cunha M (2013) Isolation, characterization and antifungal activity of proteinase inhibitors from Capsicum chinense Jacq. seeds. Protein J 32:15–26

    Article  CAS  Google Scholar 

  23. Ribeiro SF, Fernandes KVS, Santos IS, Taveira GB, Carvalho AO, Lopes JLS, Beltramini LM, Rodrigues R, Vasconcelos IM, Da Cunha M, Souza-Filho GA, Gomes VM (2013) New small proteinase inhibitors from Capsicum annuum seeds: characterization, stability, spectroscopic analysis and a cDNA cloning. Pept Sci 100:132–140

    Article  CAS  Google Scholar 

  24. Terras FRG, Torrekens S, Van Leuven F, Osborn RW, Vandeleyden J, Cammue BPA, Broekaert WF (1993) A new family of basic cysteine-rich plant antifungal proteins from Brassicaceae species. FEBS Lett 316:233–240

    Article  CAS  Google Scholar 

  25. Schagger H, Jagow Von (1987) Tricine-sodium dodecylsulfate polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379

    Article  CAS  Google Scholar 

  26. León IR, Neves-Ferreira AG, Valente RH, Mota EM, Lenzi HL, Perales J (2007) Improved protein identification efficiency by mass spectrometry using N-terminal chemical derivatization of peptides from Angiostrongylus costaricensis, a nematode with unknown genome. J Mass Spectrom 42:781–792

    Article  Google Scholar 

  27. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Article  CAS  Google Scholar 

  28. Broekaert WF, Terras FRG, Cammue BPA, Vanderleyden J (1990) An automated quantitative assay for fungal growth inhibition. FEMS Microbiol Lett 69:55–60

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  30. Bernfeld P (1995) Amylase α and β. Methods Enzymol 1:149–154

    Article  Google Scholar 

  31. Macedo MLR, Garcia VA, Freire MGM, Richardson M (2007) Characterization of a Kunitz trypsin inhibitor with a single disulfide bridge from seeds of Inga laurina (SW.) Willd. Phytochemistry 68:1104–1111

    Article  CAS  Google Scholar 

  32. Antcheva N, Pintar A, Patthy A, Simoncsits A, Barta E, Tchorbanov B, Pongor S (2001) Proteins of circularly permuted sequence present within the same organism: the major serine proteinase inhibitor from Capsicum annuum seeds. Protein Sci 10:2280–2290

    Article  CAS  Google Scholar 

  33. Bryant J, Green TR, Gurusaddaiah T, Ryan CA (1976) Proteinase inhibitor II from potatoes: isolation and characterization of its protomer components. Biochemistry 15:3418–3424

    Article  CAS  Google Scholar 

  34. Plunkett G, Senear DF, Zuroske G, Ryan CA (1982) Proteinase inhibitors I and II from leaves of wounded tomato plants: purification and properties. Arch Biochem Biophys 213:463–472

    Article  CAS  Google Scholar 

  35. Games PD, Mello EO, Santos IS, Diz MSS, Carvalho AO, Souza Filho GA, Da Cunha M, Vasconcelos IM, Ferreira BS, Gomes VM (2008) Isolation, characterization and cloning of a cDNA encoding a new antifungal defensin from Phaseolus vulgaris L. seeds. Peptides 29:2090–2100

    Article  CAS  Google Scholar 

  36. Ng TB (2004) Antifungal proteins and peptides of leguminous and non-leguminous origins. Peptides 25:1215–1222

    Article  CAS  Google Scholar 

  37. Ye XY, Wang HX, Ng TB (1999) First chromatographic isolation of an antifungal thaumatin-like protein from French bean legumes and demonstration of its antifungal activity. Biochem Biophys Res Commun 263:130–134

    Article  CAS  Google Scholar 

  38. Wong JH, Ng TB (2003) Gymnin, a potent defensin-like antifungal peptide from the Yunnan bean (Gymnocladus chinensis Baill). Peptides 24:963–968

    Article  CAS  Google Scholar 

  39. Moguel-Salazar F, Brito-Ágarez L, Diaz-Brito M, Islãs-Flores I (2011) A review of a promising therapeutic and agronomical alternative: antimicrobial peptides from Capsicum sp. Afr J Biotechnol 10:19918–19928

    CAS  Google Scholar 

  40. Tailor RH, Acland DP, Attenborough S, Cammue BP, Evans IJ, Osborn RW, Ray JA, Rees SB, Broekaert WF (1997) A novel family of small cysteine-rich antimicrobial peptides from seed of Impatiens balsamina is derived from a single precursor protein. J Biol Chem 272:24480–24487

    Article  CAS  Google Scholar 

  41. Roberts W, Selitrennikoff CP (1990) Zeamatin, an antifungal protein from maize with membrane-permeabilizing activity. J Gen Microbiol 136:1771–1778

    Article  CAS  Google Scholar 

  42. Schimoler-O’Rourke R, Richardson M, Selitrennikoff CP (2001) Zeamatin inhibits trypsin and α-amylase activities. Appl Environ Microbiol 67:2365–2366

    Article  Google Scholar 

  43. Campos FAP, Richardson M (1983) The complete amino acid sequence of the bifunctional α-amylase/trypsin inhibitor from seeds of ragi (Indian finger millet, Eleusine coracana Gaertn.). FEBS Lett 152:300–304

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This study forms part of the M.Sc. degree thesis of GCVB, carried out at the Universidade Estadual do Norte Fluminense Darcy Ribeiro. We acknowledge the financial support of the Brazilian agencies CNPq, FAPERJ and CAPES through the CAPES/Toxinology project (No. 063/2010). We are also thankful for the financial support to Drª. Viviane Veiga do Nascimento through fellowship CAPES/PNPD. We are grateful to L.C.D.

Author information

Authors and Affiliations

  1. Laboratório de Fisiologia e Bioquímica de Microrganismos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, RJ, 28013-602, Brazil

    Gabriela C. Vieira Bard, Viviane V. Nascimento, Suzanna F. F. Ribeiro, André O. Carvalho, Katia Valevski S. Fernandes & Valdirene M. Gomes

  2. Centro de Ciências e Tecnologias Agropecuárias, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil

    Rosana Rodrigues

  3. Laboratório de Toxinologia, Fundação Osvaldo Cruz (FIOCRUZ), Rio de Janeiro, RJ, Brazil

    Jonas Perales & André Teixeira-Ferreira

Authors
  1. Gabriela C. Vieira Bard
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Viviane V. Nascimento
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Suzanna F. F. Ribeiro
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rosana Rodrigues
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jonas Perales
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. André Teixeira-Ferreira
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. André O. Carvalho
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Katia Valevski S. Fernandes
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Valdirene M. Gomes
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Valdirene M. Gomes.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Vieira Bard, G.C., Nascimento, V.V., Ribeiro, S.F.F. et al. Characterization of Peptides from Capsicum annuum Hybrid Seeds with Inhibitory Activity Against α-Amylase, Serine Proteinases and Fungi. Protein J 34, 122–129 (2015). https://doi.org/10.1007/s10930-015-9604-3

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10930-015-9604-3

Keywords

Search

Navigation