Partial Purification and Characterization of Antifungal Peptides Produced by Bacillus amyloliquefaciens PEP3 Against Phytophthora capsici

Main Article Content

Hairul A. Roslan
Ahmad Husaini
Samuel Lihan
Mohd F. Kota


The prevalence of antibiotic-resistant microorganisms has triggered the exploration for novel antimicrobial compounds. Peptides are biological molecules that are found in all living organisms. They play key roles in many biological processes including organism’s defense. We have previously isolated Bacillus species (Bacillus amyloliquefaciens PEP3) that showed antagonistic properties against fungi. Metabolites precipitates obtained from B. amyloliquefaciens PEP3 showed antifungal activity against plant pathogenic fungus, Phytophthora capsici. Further analysis showed that these metabolites were stable in the pH range of 6 to 8 and temperatures of between 25°C to 85°C after which it began to lose its activity. Polyacrylamide gel analysis indicated that the molecular weight of the metabolites to be approximately 5 kDa. The presence of biosynthetic lipopeptides producing genes namely ituD, srfA, fenD and Ipa-14 genes were detected via the use of specific primers. The metabolites were then isolated via methanol extraction, however none of the major lipopetides family were detected using liquid chromatogrpahy-mass spectrometry (LC-MS). Nevertheless, the results derived from this work suggested that B. amyloliquefaciens PEP3 to have biocontrol research value and warrants further analysis.

Article Details

How to Cite
Roslan, H. A., Husaini, A., Lihan, S., & Kota, M. F. (2020). Partial Purification and Characterization of Antifungal Peptides Produced by Bacillus amyloliquefaciens PEP3 Against Phytophthora capsici. Applied Science and Engineering Progress, 13(1), 56–66. Retrieved from
Research Articles


[1] J. E. E. U. Hellberg, M. A. Matilla, and G. P. C. Salmond, “The broad-spectrum antibiotic, zeamine, kills the nematode worm Caenorhabditis elegans,” Frontiers in Microbiology, vol. 6, p. 137, Feb. 2015.

[2] I. A. Abd El Daim, P. Häggblom, M. Karlsson, E. Stenström, and S. Timmusk, “Paenibacillus polymyxa A26 Sfp-type PPTase inactivation limits bacterial antagonism against Fusarium graminearum but not of F. culmorum in kernel assay,” Frontiers in Plant Science, vol. 6, p. 368, May 2015.

[3] R. Bunet, R. Riclea, L. Laureti, L. Hôtel, C. Paris, J. M. Girardet, D. Spiteller, J. S. Dickschat, P. Leblond, and B. Aigle, “A single Sfp-type phosphopantetheinyl transferase plays a major role in the biosynthesis of PKS and NRPS derived metabolites in Streptomyces ambofaciens ATCC23877,” PLoS One, vol. 9, no. 1, p. e87607, 2014.

[4] N. A. Zainudin, B. Condon, L. De Bruyne, C. Van Poucke, Q. Bi, W. Li, M. Höfte, and B. G. Turgeon, “Virulence, host-selective toxin production, and development of three Cochliobolus phytopathogens lacking the Sfp-type 4’-phosphopantetheinyl transferase Ppt1,” Molecular Plant-Microbe Interactions, vol. 28, no. 10, pp. 1130–1141,Oct. 2015.

[5] M. F. Kota, A. A. S. A. Husaini, S. Lihan, M. H. M. Hussain, and H. A. Roslan, “In vitro antagonism of Phytophthora capsici and Fusarium solani by bacterial isolates from Sarawak,” Malaysian Journal of Microbiology, vol. 11, no. Special Issue 2, pp. 137–143, 2015.

[6] A. F. de Faria, D. Stéfani, B. G. Vaz, Í. S. Silva, J. S. Garcia, M. N. Eberlin, M. J. Grossman, O. L. Alves, and L. R. Durrant, “Purification and structural characterization of fengycin homologues produced by Bacillus subtilis LSFM-05 grown on raw glycerol,” Journal of Industrial Microbiology & Biotechnology, vol. 38, no. 7, pp. 863–871, Jul. 2011.

[7] P. Il Kim, J. Ryu, Y. H. Kim, and Y.-T. Chi, “Production of biosurfactant lipopeptides Iturin A, fengycin and surfactin A from Bacillus subtilis CMB32 for control of Colletotrichum gloeosporioides,” Journal of Microbiology and Biotechnology, vol. 20, no. 1, pp. 138–145, Jan. 2010.

[8] A. L. Moyne, R. Shelby, T. E. Cleveland, and S. Tuzun, “Bacillomycin D: An iturin with antifungal activity against Aspergillus flavus,” Journal of Applied Microbiology, vol. 90, no. 4, pp. 622–629, Apr. 2001.

[9] S. Zhang, Y.-X. Wang, L.-qiang Meng, J. Li, X.-yu Zhao, X. Cao, X.-Ling Chen, A.-xue Wang, and J.-Fu Li, “Isolation and characterization of antifungal lipopeptides produced by endophytic Bacillus amyloliquefaciens TF28,” African Journal of Microbiology Research, vol. 6, no. 8, pp. 1747–1755, 2012.

[10] H. Schagger, “Tricine-SDS-PAGE,” Nature Protocols, vol. 1, no. 1, pp. 16–22, 2006.

[11] I. Gromova and J. Celis, “Protein detection in gels by silver staining: A procedure compatible with massspectrometry,” in Cell Biology. 3rd ed., Massachusetts: Academic Press, 2006, pp. 219–223.

[12] S. Bringans, S. Eriksen, T. Kendrick, P. Gopalakrishnakone, A. Livk, R. Lock, and R. Lipscombe, “Proteomic analysis of the venom of Heterometrus longimanus (Asian black scorpion),” Proteomics, vol. 8, no. 5, pp. 1081–1096, Mar. 2008.

[13] F. Leães, R. Velho, D. Caldas, J. Pinto, S. Tsai, and A. Brandelli, “Influence of pH and temperature on the expression of sboA and ituD genes in Bacillus sp. P11,” Antonie Van Leeuwenhoek, vol. 104, July 2013.

[14] P. Fickers, “Antibiotic compounds from Bacillus : Why are they so amazing?,” American Journal of Biochemistry and Biotechnology, vol. 8, no. 1, pp. 38–43, 2012.

[15] H. Cawoy, D. Debois, L. Franzil, E. De Pauw, P. Thonart, and M. Ongena, “Lipopeptides as main ingredients for inhibition of fungal phytopathogens by Bacillus subtilis/amyloliquefaciens,” Microbial Biotechnology, vol. 8, no. 2, pp. 281–295, Mar. 2015.

[16] I. Mora, J. Cabrefiga, and E. Montesinos, “Cyclic lipopeptide biosynthetic genes and products, and inhibitory activity of plant-associated Bacillus against phytopathogenic bacteria,” PLoS One, vol. 10, no. 5, p. e0127738, 2015.

[17] A. Gotor-Vila, J. Usall, R. Torres, C. Solsona, and N. Teixidó, “Enhanced shelf-life of the formulated biocontrol agent Bacillus amyloliquefaciens CPA-8 combining diverse packaging strategies and storage conditions,” International Journal of Food Microbiology, vol. 290, pp. 205–213, 2019.

[18] Z. Gao, B. Zhang, H. Liu, J. Han, and Y. Zhang, “Identification of endophytic Bacillus velezensis ZSY-1 strain and antifungal activity of its volatile compounds against Alternaria solani and Botrytis cinerea,” Biological Control, vol. 105, pp. 27–39, 2017.

[19] J. Yuan, W. Raza, Q. Shen, and Q. Huang, “Antifungal Activity of Bacillus amyloliquefaciens NJN-6 Volatile Compounds against Fusarium oxysporum f. sp. cubense,” Applied and Environmental Microbiology, vol. 78, no. 16, pp. 5942– 5944, Aug. 2012.

[20] S. P. Chowdhury, J. Uhl, R. Grosch, S. Alquéres, S. Pittroff, K. Dietel, P. Schmitt-Kopplin, R. Borriss, and A. Hartmann, “Cyclic lipopeptides of Bacillus amyloliquefaciens subsp. plantarum colonizing the lettuce rhizosphere enhance plant defense responses toward the bottom rot pathogen Rhizoctonia solani,” Molecular Plant-Microbe Interactions, vol. 28, no. 9, pp. 984–995, Sep. 2015.

[21] K. Hyun-Gi, K. Jin-Cheol, C. Gyoung-Ja, L. Kwang-Youll, K. Hyun-Ju, H. Eul-Chul, M. Byung-Ju, and L. Seon-Woo, “Production of surfactin and iturin by bacillus licheniformis N1 responsible for plant disease control activity,” The Plant Pathology Journal, vol. 26, no. 2, pp. 170–177, Jun. 2010.

[22] S. R. Tendulkar, Y. K. Saikumari, V. Patel, S. Raghotama, T. K. Munshi, P. Balaram, and B. B. Chattoo, “Isolation, purification and characterization of an antifungal molecule produced by Bacillus licheniformis BC98, and its effect on phytopathogen Magnaporthe grisea,” Journal of Applied Microbiology, vol. 103, no. 6, pp. 2331–2339, Dec. 2007.

[23] K. Yokota, M. Yatsuda, E. Miwa, and K. Higuchi, “Comparative study on sample preparation methods for the HPLC quantification of iturin from culture supernatant of an antagonistic Bacillus strain,” Journal of the International Society for Southeast Asian Agricultural Sciences, vol. 18, pp. 70–75, Jan. 2012.

[24] S. H. Ji, N. C. Paul, J. X. Deng, Y. S. Kim, B.-S. Yun, and S. H. Yu, “Biocontrol activity of Bacillus amyloliquefaciens CNU114001 against fungal plant diseases,” Mycobiology, vol. 41, no. 4, pp. 234–242, Dec. 2013.

[25] K. M. Cho, S. Y. Hong, S. M. Lee, Y. H. Kim, G. G. Kahng, Y. P. Lim, H. Kim, and H. D. Yun, “Endophytic bacterial communities in ginseng and their antifungal activity against pathogens,” Microbial Ecology, vol. 54, no. 2, pp. 341–351, Aug. 2007.

[26] M. Grover, L. Nain, S. B. Singh, and A. K. Saxena, “Molecular and biochemical approaches for characterization of antifungal trait of a potent biocontrol agent Bacillus subtilis RP24,” Current Microbiology, vol. 60, no. 2, pp. 99–106, Feb. 2010.

[27] T.-B. Cui, H.-Y. Chai, and L.-X. Jiang, “Isolation and partial characterization of an antifungal protein produced by Bacillus licheniformis BS-3,” Molecules, vol. 17, no. 6, pp. 7336–7347, Jun. 2012.

[28] M. Gong, J. D. Wang, J. Zhang, H. Yang, X. F. Lu, Y. Pei, and J. Q. Cheng, “Study of the antifungal ability of Bacillus subtilis strain PY-1 in vitro and identification of its antifungal substance (iturin A),” Acta Biochimica et Biophysica Sinica (Shanghai), vol. 38, no. 4, pp. 233–240, Apr. 2006.

[29] D. Lin and A. Grossfield, “Thermodynamics of micelle formation and membrane fusion modulate antimicrobial lipopeptide activity,” Biophysical Journal, vol. 109, no. 4, pp. 750–759, Aug. 2015.

[30] H. B. Ayed, N. Hmidet, M. Béchet, M. Chollet, G. Chataigné, V. Leclère, P. Jacques, and M. Nasri, “Identification and biochemical characteristics of lipopeptides from Bacillus mojavensis A21,” Process Biochemistry, vol. 49, no. 10, pp. 1699– 1707, 2014.

[31] S. Kaewklom, S. Lumlert, W. Kraikul, and R. Aunpad, “Control of Listeria monocytogenes on sliced bologna sausage using a novel bacteriocin, amysin, produced by Bacillus amyloliquefaciens isolated from Thai shrimp paste (Kapi),” Food Control, vol. 32, no. 2, pp. 552–557, 2013.

[32] S. Sur and A. Grossfield, “Understanding the function of the cyclic antifungal lipopeptide fengycin using all-atom Md simulation,” Biophysical Journal, vol. 108, no. 2, p. 84a, 2015.

[33] M. K. Solanki, R. K. Singh, S. Srivastava, S. Kumar, P. L. Kashyap, and A. K. Srivastava, “Characterization of antagonistic-potential of two Bacillus strains and their biocontrol activity against Rhizoctonia solani in tomato,” Journal of Basic Microbiology, vol. 55, no. 1, pp. 82–90, Jan. 2015.