Purification of a Protease Inhibitor from Hevea brasiliensis cell suspension and it’s effect on the growth of Phytophthora palmivora

  • Orawan Bunyatang
  • Nion Chirapongsatonkul
  • Nunta Churngchow
Original Article


Protease inhibitors (PIs) are one family of pathogenesis-related proteins (PR-proteins) that play essential roles in defense mechanisms against an attack by a pathogenic microorganism or insect. Cell suspension derived from a seed integument of rubber tree (Hevea brasiliensis) treated for 48 h with 20 μM copper sulphate, an abiotic elicitor, had an increased production of PIs. The intracellular PIs were detected in an extract of treated cells; however, much higher levels of PIs were found in the medium (extracellular). Using azocasein as substrate, these PIs possessed strong inhibitory activity against subtilisin A but not against trypsin, chymotrysin and papain. These extracellular PIs were purified by anion exchange chromatography, DEAE-Sepharose (CL-6B), eluted with 0.06 M NaCl in 20 mM Tris-HCl (pH 7.0). The active fractions were then subjected to native and SDS preparative gel electrophoresis, respectively. A single band of a purified PI with a molecular weight of 25 kDa was revealed after a tricine SDS-PAGE and stained with silver nitrate. The yield of this purified protein was 3.14 ng.g−1. The activity of the purified PI was stable up to 70 °C, and its activity was retained in the buffer pH values of 2–10. The biological activity of the obtained PI was investigated. It was found that the PI at 5 μg.mL−1 (0.2 μM) inhibited the mycelium growth of Phytophthora palmivora, a rubber tree pathogen.


Defense responses Hevea brasiliensis Pathogenesis-related proteins Phytophthora palmivora Protease inhibitors 



Protease inhibitors


Pathogenesis-related proteins


  1. Angelova L, Dalgalarrondo M, Minkov I, Danova S, Kirilov N, Serkedjieva J et al (2006) Purification and characterisation of a protease inhibitor from Streptomyces chromofuscus 34-1 with an antiviral activity. Biochim Biophys Acta 1760:1210–1216PubMedCrossRefGoogle Scholar
  2. Antao CM, Maclcata FX (2005) Plant serine protease biochemical and physiological and molecular feature. Plant Physiol Biochem 43:637–650PubMedCrossRefGoogle Scholar
  3. Bangrak P, Chotigeat W (2011) Molecular cloning and biochemical characterization of a novel cystatin from Hevea rubber latex. Plant Physiol Biochem 49:244–250PubMedCrossRefGoogle Scholar
  4. Bode W, Huber R (1992) Natural protein proteinase inhibitors and their interaction with proteinases. Eur J Biochem 204:433–451PubMedCrossRefGoogle Scholar
  5. Bowles DJ (1990) Defense-related proteins in higher plants. Annu Rev Biochem 59:873–907PubMedCrossRefGoogle Scholar
  6. Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principal of protein dye-binding. Anal Biochem 72:246–254CrossRefGoogle Scholar
  7. Brady RL (2003) Plant protease inhibitors: significance in nutrition, plant protection, cancer prevention and genetic engineering. Phytochemistry 64:1419CrossRefGoogle Scholar
  8. Brian TM, Dawson TE (2002) Differential induction of trichomes by three herbivores of black mustard. Oecologia 13:526–532Google Scholar
  9. Carlini CR, Grossi-de-Sá MF (2002) Plant toxic proteins with insecticidal properties, A review on their potentialities as bioinsecticides. Toxicon 40:1515–1539PubMedCrossRefGoogle Scholar
  10. Carrillo L, Herrero I, Cambra I, Sánchez-Monge R, Diaz I, Martinez M (2011) Differential in vitro and in vivo effect of barley cysteine and serine protease inhibitors on phytopathogenic microorganisms. Plant Physiol Biochem 49:1191–1200PubMedCrossRefGoogle Scholar
  11. De Leo F, Volpicella M, Licciulli F, Liuni S, Gallerani R, Ceci LR (2002) PLANT-PIs: a database for plant protease inhibitors and their genes. Nucleic Acids Res 30:347–348PubMedCrossRefGoogle Scholar
  12. Erwin DC, Ribeiro OK (1996) The American Phytopathological Society, in: Phytophthora disease worldwide. APS Press, St Paul, 408Google Scholar
  13. Giudici AM, Regente MC, De la Canal L (2000) A potent antifungal protein from Helianthus annuus flowers is a trypsin inhibitor. Plant Physiol Biochem 38:881–888CrossRefGoogle Scholar
  14. Hartl M, Giri AP, Kaur H, Baldwin IT (2010) Serine protease inhibitors specifically defend Solanum nigrum against generalist herbivores but do not influence plant growth and development. Plant Cell 22:4158–4175PubMedCrossRefGoogle Scholar
  15. Hermosa MR, Turra D, Fogliano V, Monte E, Lorito M (2006) Identification and characterization of potato protease inhibitors able to inhibit pathogenicity and growth of Botrytis cinerea. Physiol Mol Plant Pathol 68:138–148CrossRefGoogle Scholar
  16. Jouili H, Ferjani EE (2003) Changes in antioxidant and lignifying enzyme activities in sunflower roots (Helianthus annuus L.) stressed with copper excess. C R Biol 326:639–644PubMedCrossRefGoogle Scholar
  17. Khan H, Subhan M, Durrani MF, Abbas S, Khan S (2008) Purification and characterization of serine protease from seeds of Holarrhena antidysenterica. Biotechnology 7:94–99CrossRefGoogle Scholar
  18. Kim JY, Park SC, Kim MH, Lim HT, Park Y, Hahm KS (2005) Antimicrobial activity studies on a trypsin–chymotrypsin protease inhibitor obtained from potato. Biochem Bioph Res Co 330:921–927CrossRefGoogle Scholar
  19. Koch E, Slusarenko A (1990) Arabidopsis 1 s susceptible to infection by a Downy Mildew fungus. Plant Cell 2:437–445PubMedGoogle Scholar
  20. Musor A, Chotigeat W, Phongdara A (2008) Cloning and characterization of protease inhibitor from latex of rubber tree (Hevea brasiliensis). KKU Res J (GS) 8:12–17Google Scholar
  21. Seidl DS, Abreu H, Jaffe WG (1978) Purification of a subtilisin inhibitor from black bean seeds. FEBS J 92:245–250CrossRefGoogle Scholar
  22. Somssich I, Hahlbrock K (1998) Pathogen defense in plants–A paradigm of biological complexity. Trends Plant Sci 3:86–90CrossRefGoogle Scholar
  23. Sritanyarat W, Pearce G, Siems WF, Ryan CA, Wititsuwannakul R, Wititsuwannakul D (2006) Isolation and characterization of isoinhibitors of the potato protease inhibitor I family from the latex of the rubber trees Hevea brasiliensis. Phytochemistry 67:1644–1650PubMedCrossRefGoogle Scholar
  24. Techato S, Niyagij C, Suranilpong P (2002) Callus formation from protoplasts derived from cell suspension culture of rubber tree (Hevea brasiliensis Muell. Arg.). Thai J Agric Sci 35:165–173Google Scholar
  25. Van der Hoorn RAL (2008) Plant proteases: From phenotypes to molecular mechanisms. Annu Rev Plant Biol 59:191–223PubMedCrossRefGoogle Scholar
  26. Van Loon LC, Van Strien EA (1999) The families of patho-genesis-related proteins, their activities, and comparative analysis of PR-1 type proteins. Physiol Mol Plant Pathol 3:85–97CrossRefGoogle Scholar
  27. Wang S, Lin J, Ye M, Bun NT, Rao P, Ye X (2006) Isolation and characterization of a novel mung bean protease inhibitor with antipathogenic and anti-proliferative activities. Peptides 27:3129–3136PubMedCrossRefGoogle Scholar

Copyright information

© Society for Plant Biochemistry and Biotechnology 2012

Authors and Affiliations

  • Orawan Bunyatang
    • 1
  • Nion Chirapongsatonkul
    • 1
  • Nunta Churngchow
    • 1
  1. 1.Department of Biochemistry, Faculty of SciencePrince of Songkla UniversityHat-YaiThailand

Personalised recommendations