Isolation and identification of antifungal peptides from Bacillus BH072, a novel bacterium isolated from honey

• Published in: Microbiological research 2013-11, Vol.168 (9), p.598-606
• Main Authors: Zhao, Xin, Zhou, Zhi-jiang, Han, Ye, Wang, Zhan-zhong, Fan, Jie, Xiao, Hua-zhi
• Format: Article
• Language: English
• Subjects: ammonium sulfate; anion exchange chromatography; Antifungal Agents - chemistry; Antifungal Agents - isolation & purification; Antifungal Agents - pharmacology; Antifungal peptide; antifungal properties; antimicrobial peptides; Ascomycota - drug effects; Aspergillus niger; Aspergillus niger - drug effects; Bacillus; Bacillus (bacteria); Bacillus - classification; Bacillus - genetics; Bacillus - isolation & purification; Bacillus - metabolism; bacteria; Bacterial Typing Techniques; Botrytis cinerea; Chromatography, Gel; Chromatography, Ion Exchange; Cluster Analysis; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; DNA, Ribosomal - chemistry; DNA, Ribosomal - genetics; fermentation; flagellin; gel chromatography; genes; honey; Honey - microbiology; Identification; iturin; Mass Spectrometry; methanol; Molecular Weight; new subspecies; nucleotide sequences; Peptides - chemistry; Peptides - isolation & purification; Peptides - pharmacology; Phylogeny; polymerase chain reaction; Purification; Pythium; Pythium - drug effects; ribosomal DNA; RNA, Ribosomal, 16S - genetics; Sequence Analysis, DNA
• ISSN: 0944-5013; 1618-0623
• DOI: 10.1016/j.micres.2013.03.001

A bacterial strain BH072 isolated from a honey sample showed antifungal activity against mold. Based on morphological, biochemical, physiological tests, and analysis of 16S rDNA sequence, the strain was identified to be a new subspecies of Bacillus sp. It had a broad spectrum of antifungal activity against various mold, such as Aspergillus niger, Pythium, and Botrytis cinerea. Six pairs of antifungal genes primers were designed and synthesized, and ituA, hag, tasA genes were detected by PCR analysis. The remarkable antifungal activity could be associated with the co-production of these three peptides. One of them was purified by 30–40% ammonium sulfate precipitation, Sephadex G-75 gel filtration and anion exchange chromatography on D201 resin. The purified peptide was estimated to be 35.615kDa and identified to be flagellin by micrOTOF-Q II. By using methanol extraction, another substance was isolated from fermentation liquor, and determined to be iturin with liquid chromatography–mass spectrometry (LC–MS) method. The third possible peptide encoded by tasA was not isolated in this study. The culture liquor displayed antifungal activity in a wide pH range (5.0–9.0) and at 40–100°C. The result of the present work suggested that Bacillus BH072 might be a bio-control bacterium of research value.