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.
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Abbreviations
- PIs:
-
Protease inhibitors
- PR-proteins:
-
Pathogenesis-related proteins
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Acknowledgements
This work was supported by the Thailand Research Fund (TRF) through the Royal Golden Jubilee Ph.D. Program (RGJ-PHD) to Miss Orawan Bunyatang (PHD/0166/2552), the Research Funding through Prince of Songkla University, Thailand (SCI 530063 S), and the Research Funding of the Graduate School Prince of Songkla University. We thank Dr. Brian Hodgson, Prince of Songkla University, for revision of the manuscript and valuable comments.
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Bunyatang, O., Chirapongsatonkul, N. & Churngchow, N. Purification of a Protease Inhibitor from Hevea brasiliensis cell suspension and it’s effect on the growth of Phytophthora palmivora . J. Plant Biochem. Biotechnol. 22, 185–192 (2013). https://doi.org/10.1007/s13562-012-0137-y
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DOI: https://doi.org/10.1007/s13562-012-0137-y