Identification of an Antifungal Bacillus Strain and Studies on Its Antifungal Active Ingredients
Strain NK-2 which has broad spectrum of antifungal activity against eight kinds of phytopathogenic fungi was identified as Bacillus amyloliquefaciens by analysis of partial 16S rDNA, gyrA gene, and rpoB gene sequences. The crude protein, obtained from NK-
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Identification of an Antifungal Bacillus Strain and Studies on Its Antifungal Active Ingredients Yingde Shen, Ruibin Liu, Yuehua Chen and Jun Cai
Abstract Strain NK-2 which has broad spectrum of antifungal activity against eight kinds of phytopathogenic fungi was identified as Bacillus amyloliquefaciens by analysis of partial 16S rDNA, gyrA gene, and rpoB gene sequences. The crude protein, obtained from NK-2 culture, exhibited inhibitory activity on mycelial growth and conidial spore germination of Fusarium oxysporum. The crude proteins were further purified by Sephadex G-200 and the antifungal fraction was checked by SDS-PAGE. The putative active protein was then analyzed by Tandem mass spectrum. The result revealed that the target protein was high similar to protein YhfE from B. amyloliquefaciens FZB 42. Then we cloned the yhfE (GenBank accession:HM143898) from NK-2 genome and expressed it in Escherichia coli BL21. The purified recombinant YhfE protein could also inhibit the spore germination and mycelial growth of F. oxysporum. We have found for the first time, to our knowledge, that YhfE has antifungal activity.
Keywords Antifungal protein Bacillus amyloliquefaciens Mass spectrometry YhfE
The authors Y. Shen and R. Liu contributed equally to this paper. Y. Shen Y. Chen J. Cai (&) Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, College of Life Science, Nankai University, Tianjin 300071, People’s Republic of China e-mail: [email protected] R. Liu Tianjin Taihe Pharmaceutical Co. Ltd., Tianjin 300270, People’s Republic of China
T.-C. Zhang et al. (eds.), Proceedings of the 2012 International Conference on Applied Biotechnology (ICAB 2012), Lecture Notes in Electrical Engineering 251, DOI: 10.1007/978-3-642-37925-3_175, Ó Springer-Verlag Berlin Heidelberg 2014
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175.1 Introduction Bacillus amyloliquefaciens which is closely related to Bacillus subtillis has extensively been studied as biological control agents of plant pathogens, and it can produce a range of antimicrobial compounds, for example cyclic lipopeptides [1], polyketides [2], proteins, and so on. Many investigators have devoted their time and energy to search for the cyclic lipopeptides including iturins [3, 4], fengycins [5], and surfactines [5, 6]. Some bacterial antifungal proteins have also been isolated successfully from B. amyloliquefaciens, such as baciamin [7], b-Glucanase [8], and cellulose [9]. However, it is much less abundantly compared with that on bacterial antifungal peptides. In this paper, strain NK-2 with broad antifungal spectrum was identified as B. amyloliquefaciens using methods of molecular biology [10–12]. In order to probe what substances were responsible for its antifungal activity, we analyzed the putative active protein by Tandem Mass Spectrometry (MS-MS). Fortunately, the protein could be found in database and had high similar to protein YhfE of B. amyloliquefaciens FZB 42. To verify its antifungal activity, yhfE gene from NK-2 genome was expresse
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