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

Abstract

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.

Keywords

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

Abbreviations

PIs

Protease inhibitors

PR-proteins

Pathogenesis-related proteins

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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

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