Purification and characterization of a novel antifungal protein from Bacillus subtilis strain B29


An antifungal protein was isolated from a culture of Bacillus subtilis strain B29. The isolation procedure comprised ion exchange chromatography on diethylaminoethyl (DEAE)-52 cellulose and gel filtration chromatography on Bio-Gel® P-100. The protein was absorbed on DEAE-cellulose and Bio-Gel® P-100. The purified antifungal fraction was designated as B29I, with a molecular mass of 42.3 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), pI value 5.69 by isoelectric focusing (IEF)-PAGE, and 97.81% purity by high performance liquid chromatography (HPLC). B29I exhibited inhibitory activity on mycelial growth in Fusarium oxysporum, Rhizoctonia solani, Fusarium moniliforme, and Sclerotinia sclerotiorum. The 50% inhibitory concentrations (IC50) of its antifungal activity toward Fusarium oxysporum and Rhizoctonia solani were 45 and 112 μmol/L, respectively. B29I also demonstrated an inhibitory effect on conidial spore germination of Fusarium oxysporum and suppression of germ-tube elongation, and induced distortion, tumescence, and rupture of a portion of the germinated spores.

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  1. Asaka, O., Shoda, M., 1996. Biocontrol of Rhizoctonia solani damping-off of tomato with Bacillus subtilis RB14. Appl. Environ. Microbiol., 62(11):4081–4085.

  2. Benhamou, N., Broglie, K., Broglie, R., Chet, I., 1993. Antifungal effect of bean endochitinase on Rhizoctonia solani: ultrastructural changes and cytochemical aspect of chitin breakdown. Can. J. Microbiol., 39(3):318–328.

  3. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of milligram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem., 72(1–2):248–254. [doi:10.1016/0003-2697(76)90527-3]

  4. Broekaert, W.F., van Parijs, J., Leyns, F., Joos, H., Peumans, W.J., 1989. A chitin-binding lectin from stinging nettle rhizomes with antifungal properties. Science, 245(4922): 1100–1102. [doi:10.1126/science.245.4922.1100]

  5. Carrillo, C., Teruel, J.A., Aranda, F.J., Ortiz, A., 2003. Molecular mechanism of membrane permeabilization by the peptide antibiotic surfactin. Biochim. Biophys. Acta, 1611(1–2):91–97. [doi:10.1016/S0005-2736(03)00029-4]

  6. Delcambe, L., Peypoux, F., Besson, F., Guinand, M., Michel, G., 1977. Structure of iturin and iturin-like substance. Biochem. Society Transac., 5(4):1122–1124.

  7. Hwang, S.F., Chakravarty, P., 1992. Potential for the integrated control of Rhizoctonia root-rot of Pisum sativum using Bacillus subtilis and a fungicide. Z. PflKrankh. PflSchutz., 99:626–636.

  8. Iijima, R., Kurata, S., Natori, S., 1993. Purification, characterization and cDNA cloning of an antifungal protein from the hemolymph of Sarcophaga peregrine (flesh fly). J. Biol. Chem., 268(16):12055–12062.

  9. Joshi, B.N., Sainani, M.N., Bastawade, K.B., Gupta, V.S., Ranjekar, P.K., 1998. Cysteine protease inhibitor from pearl millet: a new class of antifungal protein. Biochem. Biophys. Res. Commun., 246(2):382–387. [doi:10.1006/bbrc.1998.8625]

  10. Kim, P.I., Chung, K.C., 2004. Production of an antifungal protein for control of Colletotrichum lagenarium by Bacillus amyloliquefaciens MET0908. FEMS. Microbiol. Lett., 234(1):177–183. [doi:10.1016/j.femsle.2004.03.032]

  11. Laemmli, U.K., Favre, M., 1973. Maturation of the head of bacteriophage T4. I. DNA packaging events. J. Mol. Biol., 80(4):575–599. [doi:10.1016/0022-2836(73)90198-8]

  12. Lam, S.K., Ng, T.B., 2001a. 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(2):271–280. [doi:10.1006/abbi.2001.2506]

  13. Lam, S.K., Ng, T.B., 2001b. Isolation of a novel thermolabile heterodimeric ribonuclease with antifungal and antiproliferative activities from roots of the sanchi ginseng Panax notoginseng. Biochem. Biophys. Res. Commun., 285(2):419–423. [doi:10.1006/bbrc.2001.5193]

  14. Lam, S.K., Ng, T.B., 2001c. Isolation of a small chitinase-like antifungal protein from Panax notoginseng (sanchi ginseng) roots. Int. J. Biochem. Cell Biol., 33(3):287–292. [doi:10.1016/S1357-2725(01)00002-4]

  15. Lam, Y.W., Wang, H.X., Ng, T.B., 2000. A robust cysteine-deficient chitinase-like antifungal protein from inner shoots of the edible chive Allium tuberosum. Biochem. Biophys. Res. Commun., 279(1):74–80. [doi:10.1006/bbrc.2000.3821]

  16. Leah, R., Tommerup, H., Svendsen, I., Mundy, J., 1991. Biochemical and molecular characterization of three barley seed proteins with antifungal properties. J. Biol. Chem., 266(3):1564–1573.

  17. Li, J., Yang, Q., 2005. Biological Control of Fusarisum Wilt with an Antagonistic Strain of Bacillus subtilis. In: Yang, Q., Yu, Z.N. (Eds.), Study on Plant Pest and Diseases Biological Control and Bio-technology. The 3rd International Symposium on Bio-Control and Biotechnology, Heilongjiang Science Technology Press, Harbin, China, p.229–236.

  18. Li, J., Yang, Q., Zhao, L., Wang, Y., 2008. Antifungal substance from biocontrol Bacillus subtilis B29 strain. China Biotechnology, 28(2):59–65 (in Chinese).

  19. Liu, Y., Chen, Z., Ng, T.B., Zhang, J., Zhou, M., Song, F., Lu, F., Liu, Y., 2007. Bacisubin, an antifungal protein with ribonuclease and hemagglutinating activities from Bacillus subtilis strain B-916. Peptides, 28(3):553–559. [doi:10.1016/j.peptides.2006.10.009]

  20. Loeffler, W., Tschen, J.S.M., Vanittanakom, N., Kulger, M., Knorpp, E., Hsieh, T.F., Wu, T.G., 1986. Antifungal effects of bacilysin and fengycin from Bacillus subtilis F29-3. A comparison with activities of other Bacillus antibiotics. J. Phytopathol., 115(3):204–213. [doi:10.1111/j. 1439-0434.1986.tb00878.x]

  21. Majumdar, S.K., Bose, S.K., 1960. Isolation and homogeneity of mycobacillin. Arch. Biochem. Biophy., 90(1):154–158. [doi:10.1016/0003-9861(60)90626-3]

  22. Molina, A., Segura, A., Garia-Olmedo, F., 1993. Lipid transfer proteins (nsLTPS) from barley and maize leaves are potent inhibitors of bacterial and fungal plant pathogens. FEBS Lett., 316(2):119–122. [doi:10.1016/0014-5793(93)81198-9]

  23. Moyne, A.L., Cleveland, T.E., Tuzun, S., 2004. Molecular characterization and analysis of operon encoding the antifungal lipopeptide bacillomycin D. FEMS Microbiol. Lett., 234(1):43–49. [doi:10.1111/j.1574-6968.2004.tb09511.x]

  24. Ng, T.B., Wang, H.X., 2001. Panaxagin, a new protein from Chinese ginseng possesses anti-fungal, anti-viral, translation-inhibiting and ribonuclease activities. Life Sci., 68(7):739–749. [doi:10.1016/S0024-3205(00)00970-X]

  25. Ngai, P.H.K., Zhao, Z., Ng, T.B., 2005. Agrocybin, an antifungal peptide from the edible mushroom Agrocybe cylindracea. Peptides, 26(2):191–196. [doi:10.1016/j.peptides.2004.09.011]

  26. Okigbo, R.N., 2005. Biological control of postharvest fungal rot of yam (Dioscorea spp.) with Bacillus subtilis. Mycopathologia, 159(2):307–314. [doi:10.1007/s11046-004-2454-8]

  27. Peypoux, F., Besson, F., Michel, G., 1980. Characterization of a new antibiotic of iturin group bacilloycin D. J. Antibiot., 33(10):1146–1149.

  28. Peypoux, F., Pommier, M.T., Marion, D., Ptak, M., Das, B.C., Michel, G., 1986. Revised structure of mycosubtilin, a lipidolipid antibiotic from B. subtilis. J. Antibiot., 39(5): 636–641.

  29. Peypoux, F., Bonmatin, J.M., Wallach, J., 1999. Recent trends in the biochemistry of surfactin. Appl. Microbiol. Biotechnol., 51(5):553–563. [doi:10.1007/s002530051432]

  30. Pressey, R., 1997. Two isoforms of NP24: a thaumatin-like protein in tomato fruit. Phytochemistry, 44(7):1241–1245. [doi:10.1016/S0031-9422(96)00667-X]

  31. Sengupta, S., Banerjee, A.B., Bose, S.K., 1971. γ-Glutamyl and D- or L-peptide linkages in mycobacillin, a cyclic peptide antibiotic. Biochem. J., 121:839–846.

  32. Stein, T., 2005. Bacillus subtilis antibiotics: structures, syntheses and specific functions. Mol. Microbiol., 56(4): 845–857. [doi:10.1111/j.1365-2958.2005.04587.x]

  33. Theis, T., Marax, F., Salvenmoser, W., Stahl, U., Meyer, V., 2005. New insight into the target site and mode of action of the antifungal protein of Aspergillus giganteus. Res., 156(1):47–56. [doi:10.1016/j.resmic.2004.08.006]

  34. Tsuge, K., Akiyama, T., Shoda, M., 2001. Cloning, sequencing, and characterization of the iturin A operon. J. Bacteriol., 183(21):6265–6273. [doi:10.1128/JB.183.21.6265-6273.2001]

  35. Vogelsang, R., Barz, W., 1993. Purification, characterization and differential hormonal regulation of a β-1,3-glucanase and two chitinases from chickpea (Cicer arietinum L.). Planta, 189(1):60–69. [doi:10.1007/BF00201344]

  36. Wang, H., Ng, T.B., 2002. Isolation of cicadin, a novel and potent antifungal peptide from juvenile cicadas. Peptides, 23(1):7–11. [doi:10.1016/S0196-9781(01)00573-3]

  37. Wang, H., Ng, T.B., 2004. Eryngin, a novel antifungal peptide from fruiting bodies of the edible mushroom Pleurotus eryngii. Peptides, 25(1):1–5. [doi:10.1016/j.peptides.2003.11.014]

  38. Wang, S.Y., Wu, J.H., Ng, T.B., Ye, X.Y., Rao, P.F., 2004. A non-specific lipid transfer protein with antifungal and antibacterial activities from the mung bean. Peptides, 25(8):1235–1242. [doi:10.1016/j.peptides.2004.06.004]

  39. Wulff, E.G., Mguni, C.M., Mortensen, C.N., Keswani, C.L., Hockenhull, J., 2002. Biological control of black rot (Xanthomonas campestris pv. campestris) of brassicas with an antagonistic strain of Bacillus subtilis in Zimbabwe. Eur. J. Plant Pathol., 108(4):317–325. [doi:10.1023/A:1015671031906]

  40. Wurms, K., Greenwood, D., Sharrock, K., Long, P., 1999. Thaumatinlike protein in kiwi fruit. J. Sci. Food. Agric., 79(11):1448–1452. [doi:10.1002/(SICI)1097-0010(199908) 79:11〈1448::AID-JSFA381〉3.0.CO;2-3]

  41. Xie, D., Peng, J., Wang, J., Hu, J., Wang, Y., 1998. Purification and properties of antifungal protein X98III from Bacillus subtilis. Acta Microbiologica Sinica, 38(1): 13–19 (in Chinese).

  42. Ye, X.Y., Ng, T.B., 2000. Mungin, a novel cyclophilin-like antifungal protein from the mung bean. Biochem. Biophys. Res. Commun., 273(3):1111–1115. [doi:10.1006/bbrc.2000.3067]

  43. Ye, X.Y., Ng, T.B., 2002a. Isolation of a new cyclophilin-like protein from chickpeas with mitogenic, antifungal and anti-HIV-1 reverse transcriptase activities. Life Sci., 70(10):1129–1138. [doi:10.1016/S0024-3205(01)01473-4]

  44. Ye, X.Y., Ng, T.B., 2002b. Isolation of a novel peroxidase from French bean legumes and first demonstration of antifungal activity of a non-milk peroxidase. Life Sci., 71(14):1667–1680. [doi:10.1016/S0024-3205(02)01925-2]

  45. Ye, X.Y., Wang, H.X., Ng, T.B., 1999. First chromatographic isolation of an antifungal thaumatin-like protein from French bean legumes and demonstration of its antifungal activity. Biochem. Biophys. Res. Comun., 263(1):130–134. [doi:10.1006/bbrc.1999.1166]

  46. Ye, X.Y., Wang, H.X., Ng, T.B., 2000. Sativin, a novel antifungal miraculin-like protein isolated from legumes of the sugar snap Pisum sativum var. macrocarpon. Life Sci., 67(7):775–781. [doi:10.1016/S0024-3205(00)00672-X]

  47. Ye, X.Y., Ng, T.B., Tsang, P.W.K., Wang, J., 2001. Isolation of a homodimeric lectin with antifungal and antiviral activiral activities from red kidney bean (Phaseolus vulgaris) activities. J. Protein Chem., 20(5):367–375. [doi:10. 1023/A:1012276619686]

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Correspondence to Qian Yang.

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Project supported by the Hi-Tech Research and Development Program (863) of China (No. 2003AA241140) and the Natural Science Foundation of Heilongjiang Province, China (No. C200522)

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Li, J., Yang, Q., Zhao, L. et al. Purification and characterization of a novel antifungal protein from Bacillus subtilis strain B29. J. Zhejiang Univ. Sci. B 10, 264–272 (2009) doi:10.1631/jzus.B0820341

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Key words

  • Bacillus subtilis
  • Antifungal protein
  • Purification

CLC number

  • Q81