Purification and Characterization of Antifungal Lipopeptide from Bacillus amyloliquefaciens BI2
Bacillus amyloliquefaciens BI2 isolated from straw feed was shown to be antagonistic against Aspergillus flavus. Crude extract of antagonistic substance was obtained by ultrafiltration, nanofiltration, acid precipitation, and solvent extraction and it was
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Purification and Characterization of Antifungal Lipopeptide from Bacillus amyloliquefaciens BI2 Depei Wang, Kele Li, Yingying Wang, Ying Yang and Jian Zhang
Abstract Bacillus amyloliquefaciens BI2 isolated from straw feed was shown to be antagonistic against Aspergillus flavus. Crude extract of antagonistic substance was obtained by ultrafiltration, nanofiltration, acid precipitation, and solvent extraction and it was stable to high temperature, different pH value, and several proteinases. Then the pure substance was isolated and purified by Sephadex G-15 column chromatography and prepared thin-layer chromatography. Oil-film collapsing, emulsification of toluene and on-plate hydrolysis experiments showed the compound was cyclic peptide, and its molecular structure was determined by UV absorption spectrum and infrared absorption spectrum following the amino acid analysis, ESI–MS and MS–MS. It showed that isolated compound consists of three homologues with molecular mass 1,008, 1,022 and 1,036 Da, and bearing a cyclic structure. Meanwhile, it also showed that antifungal substance had absorption peak at 206 nm. All of those indicated that the isolated substance belongs to the homologues of surfactin.
Keywords Bacillus amyloliquefaciens BI2 Antifungal substance Purification Lipopeptide
48.1 Introduction Aspergillus flavus is one of the major of spoilage organisms of several moist grains and nut products such as peanuts, corn, and certain nuts. Aflatoxins which are highly toxic, mutagenic, and carcinogenic to human being and animals are its secondary metabolites. Meanwhile, A. flavus is also a human pathogen, which can D. Wang (&) K. Li Y. Wang Y. Yang J. Zhang College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, People’s Republic of China e-mail: [email protected]
T.-C. Zhang et al. (eds.), Proceedings of the 2012 International Conference on Applied Biotechnology (ICAB 2012), Lecture Notes in Electrical Engineering 249, DOI: 10.1007/978-3-642-37916-1_48, Ó Springer-Verlag Berlin Heidelberg 2014
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cause aspergillosis and onychomycosis [1, 2]. Therefore, there is interest in developing biological control methods which can inhibit the growth of pathogen and decrease toxin content. Bacillus species are the ideal candidates to use as biocontrol agents because they are safe and easily cultivated in a low-cost culture medium. In addition to, their spore can tolerate adverse conditions and many physicochemical factors such as high temperature and ultraviolet radiation. Previous studies showed that isolations of Bacillus sp. have been used successfully in the field of biological control of A. flavus. Bacillus sp., can synthesize a variety of metabolites with antibacterial and/or antifungal activity. Bacteria of the genus Bacillus produce a variety of antibiotics which are classified as ribosomal or nonribosomal and which mostly have a molecular weight of less than 2,000 Da. Nonribosomally synthesized circular oligopeptides contain the lipopeptide
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