Applied Microbiology and Biotechnology

, Volume 82, Issue 1, pp 79–86 | Cite as

Isolation and biochemical characterization of a novel leguminous defense peptide with antifungal and antiproliferative potency

  • Shaoyun WangEmail author
  • Pingfan Rao
  • Xiuyun Ye
Biotechnologically Relevant Enzymes and Proteins


Leguminous plants have formed a popular subject of research owing to the abundance of proteins and peptides with important biological activities that they produce. The antifungal proteins and peptides have been purified from a number of leguminous species. However, research continues to discover novel antifungal plant-produced peptides and proteins are being needed, specially those novel ones with both antifungal activity and other significant bioactivities. The objective of this study was to isolate a novel peptide from Phaseolus limensis. A 6.8 kDa peptide designated Limyin, with both antifungal and antiproliferative activity, was isolated from the large lima bean (P. limensis) legumes. The isolation procedure consisted of extraction, precipitation, affinity chromatography on Affi-gel blue gel, ion chromatography on SP-Toyopearl, and gel filtration on Superdex 75. Its N-terminal sequence was determined to be KTCENLATYYRGPCF, showing high homology to defensin and defensin precursors from plants. It potently suppressed mycelial growth in Alternaria alternata, Fusarium solani, and Botrytis cinerea. Its antifungal activity was stable up to 80°C. It showed antiproliferative activity towards tumor cells including human liver hepatoma cells Bel-7402 and neuroblastoma cells SHSY5Y. However, it had no effect on bacteria Staphylococcus aureus and Salmonella. The present findings make a significant addition of the research on leguminous plants.


Large lima bean Peptide Defensin Antifungal Antiproliferative 



This research was supported by the Collaboration Funding of Queen University, United States of Kingdom (number 008200509). The authors are grateful to Dr. Dan Zhu in the College of Agricultural and Life Science at University of Wisconsin, Madison, USA, for valuable assistance.


  1. Broekaert WF, Van Parijs J, Leyns F, Joos H, Peumans WJ (1989) A chitin-binding lectin from stinging nettle rhizomes with antifungal properties. Science 245:1100–1102CrossRefGoogle Scholar
  2. Chilosi G, Caruso C, Caporale C, Leonardi L, Bertini L, Buzi A, Nobile M (2000) Antifungal activity of a Bowman–Birk type trypsin inhibitor from wheat kernel. J Phytopathol 148:477–481CrossRefGoogle Scholar
  3. Chu KT, Ng TB (2003) Isolation of a large thaumatin-like antifungal protein from seeds of the Kweilin chestnut Castanopsis chinensis. Biochem Biophys Res Commun 301:364–370CrossRefGoogle Scholar
  4. Flores T, Alape-Giron A, Flores-Diaz M, Flores HE (2002) Ocatin. A novel tuber storage protein from the Andean tuber crop oca with antibacterial and antifungal activities. Plant Physiol 128:1291–1302CrossRefGoogle Scholar
  5. Gozia O, Ciopraga J, Bentia T, Lungu M, Zamfirescu I, Tuder R, Roseanu A, Nitu F (1995) Antifungal properties of lectin and new chitinase from potato tuber. FEBS Lett 370:245–249CrossRefGoogle Scholar
  6. Grenier J, Potvin C, Asselin A (2000) Some fungi express b-1,3-glucanases similar to thaumatin-like proteins. Mycologia 92:841–848CrossRefGoogle Scholar
  7. Joshi BN, Sainani MN, Bastawade KB, Gupta VS, Ranjekar PK (1998) Cysteine protease inhibitor from pearl millet: a new class of antifungal protein. Biochem Biophys Res Commun 246:382–387CrossRefGoogle Scholar
  8. Laemmli UK, Favre M (1973) Gel electrophoresis of proteins. J Mol Biol 80:575–599CrossRefGoogle Scholar
  9. Lam SK, Ng TB (2001) First simultaneous isolation of a ribosome inactivating protein and an antifungal protein from a mushroom (Lyophyllum shimeji) together with evidence for synergism of their antifungal effects. Arch Biochem Biophys 393:271–280CrossRefGoogle Scholar
  10. Leah R, Tommerup H, Svendsen I, Mundy J (1991) Biochemical and molecular characterization of three barley seed proteins with antifungal properties. J Biol Chem 246:1564–1573Google Scholar
  11. Lichtenstein AK, Ganz T, Selsted ME, Lehrer RI (1986) In vitro tumor cell cytolysis mediated by peptide defensins of human and rabbit granulocytes. Blood 68:1407–1410Google Scholar
  12. Lichtenstein AK, Ganz T, Nguyen TM, Selsted ME, Lehrer RI (1988) Mechanism of target cytolysis by peptide defensins. J Immunol 140:2686–2694Google Scholar
  13. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  14. Mastrolorenzo A, Rusconi S, Scozzafava A, Barbaro G, Supuran CT (2007) Inhibitors of HIV-1 protease: current state of the art 10 years after their introduction. From antiretroviral drugs to antifungal, antibacterial and antitumor agents based on aspartic protease inhibitors. Curr Med Chem 14:2734–2748CrossRefGoogle Scholar
  15. Ngai PHK, Ng TB (2007) A lectin with antifungal and mitogenic activities from red cluster pepper (Capsicum frutescens) seeds. Appl Microbiol Biotechnol 74(2):366–371CrossRefGoogle Scholar
  16. Qureshi A, Colin PL, Faulkner DJ (2000) Microsclerodermins F-I, antitumor and antifungal cyclic peptides from the lithistid sponge Microscleroderma sp. Tetrahedron 56:3679–3685CrossRefGoogle Scholar
  17. Raj PA, Dentino AR (2002) Current status of defensins and their role in innate and adaptive immunity. FEMS Microbiol Lett 266:9–18CrossRefGoogle Scholar
  18. Thevissen K, Ferket KK, Francois IE, Cammue BP (2003a) Interactions of antifungal plant defensins with fungal membrane components. Peptides 24:1705–1712CrossRefGoogle Scholar
  19. Thevissen K, Francois IEJA, Takemoto JY, Ferket KKA, Meert EMK, Cammue BPA (2003b) DmAMP1, an antifungal plant defensin from dahlia (Dahlia merckii), interacts with sphingolipids from Saccharomyces cerevisiae. FEMS Microbiol Lett 226:169–173CrossRefGoogle Scholar
  20. Vogelsang R, Barz W (1993) Purification, characterization and differential hormonal regulation of a b-1,3-glucanase and two chitinases from chickpea (Cicer arietinum L.). Planta 189:60–69CrossRefGoogle Scholar
  21. Vu L, Huyhn QK (1994) Isolation and characterization of a 27-kDa antifungal protein from the fruits of Diospyros texana. Biochem Biophys Res Commun 202:666–672CrossRefGoogle Scholar
  22. Wang HX, Ng TB (2007) Isolation and characterization of an antifungal peptide with antiproliferative activity from seeds of Phaseolus vulgaris cv. ‘Spotted Bean’. Appl Microbiol Biotechnol 74:125–130CrossRefGoogle Scholar
  23. Wang SY, Wu JH, Ng TB, Ye XY, Rao PF (2004a) A non-specific lipid transfer protein with antifungal and antibacterial activities from the mung bean. Peptides 25:1235–1242CrossRefGoogle Scholar
  24. Wang SY, Wu JH, Ye XY, Ng TB, Rao PF (2004b) Crystallization and preliminary X-ray crystallographic analysis of a nonspecific lipid-transfer protein with antipathogenic activity from Phaseolus mungo. Acta Crystallogr Section D Biol Crystallogr 60:2391–2393CrossRefGoogle Scholar
  25. Wang SY, Ng TB, Chen T, Lin DY, Rao PF, Ye XY (2005) First report of a novel plant lysozyme with both antifungal and antibacterial activities from Phaseolus mungo. Biochem Biophys Res Commun 327:820–827CrossRefGoogle Scholar
  26. Wang SY, Lin J, Ye MY, Ng TB, Rao PF, Ye XY (2006) Isolation and characterization of a novel mung bean protease inhibitor with antipathogenic and anti-proliferative activities. Peptides 27:3129–3136CrossRefGoogle Scholar
  27. Wang SY, Shao B, Rao PF, Lee YY, Ye XY (2007) Hypotin, a novel antipathogenic and antiproliferative protein from peanuts with a sequence similar to those of chitinase precursors. J Agric Food Chem 55:9792–9799CrossRefGoogle Scholar
  28. Wang SY, Zhou J, Shao B, Lu YJ, Rao PF (2008) A thermostable chitinase with chitin-binding activity from Phaseolus limensis. J Food Sci 73(6):C452–C457CrossRefGoogle Scholar
  29. Ye XY, Ng TB (2000) Hypogin, a novel antifungal peptide from peanuts with sequence similarity to peanut allergen. J Peptide Res 57:330–336CrossRefGoogle Scholar
  30. Ye XY, Ng TB (2002a) Isolation of a novel peroxidase from French bean legumes and first demonstration of antifungal activity of a non-milk peroxidase. Life Sci 71:1667–1680CrossRefGoogle Scholar
  31. Ye XY, Ng TB (2002b) Isolation of a new cyclophilin-like protein from chickpeas with mitogenic, antifungal and anti-HIV-1 reverse transcriptase activities. Life Sci 70:1129–1138CrossRefGoogle Scholar
  32. Ye XY, Wang HX, Ng TB (2000a) Sativin, a novel antifungal miraculin-like protein isolated from the legumes of the sugar snap Pisum sativum var. macrocarpon. Life Sci 67:775–781CrossRefGoogle Scholar
  33. Ye XY, Wang HX, Ng TB (2000b) Dolichin, a new chitinase-like antifungal protein isolated from field beans (Dolichos lablab). Biochem Biophys Res Commun 269:155–159CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  1. 1.College of Bioscience & BiotechnologyFuzhou UniversityFuzhouChina

Personalised recommendations