Purification and Characterization of the Bacteriocin Thuricin Bn1 Produced by Bacillus thuringiensis subsp. kurstaki Bn1 Isolated from a Hazelnut Pest

A novel bioactive molecule produced by Bacillus thuringiensis subsp. kurstaki Bn1 (Bt-Bn1), isolated from a common pest of hazelnut, Balaninus nucum L. (Coleoptera: Curculionidae), was determined, purified, and characterized in this study. The Bt-Bn1 strain was investigated for antibacterial activity with an agar spot assay and well diffusion assay against B. cereus, B. weinhenstephenensis, L. monocytogenes, P. savastanoi, P. syringae, P. lemoignei, and many other B. thuringiensis strains. The production of bioactive molecule was determined at the early logarithmic phase in the growth cycle of strain Bt-Bn1 and its production continued until the beginning of the stationary phase. The mode of action of this molecule displayed bacteriocidal or bacteriolytic effect depending on the concentration. The bioactive molecule was purified 78-fold from the bacteria supernatant with ammonium sulfate precipitation, dialysis, ultrafiltration, gel filtration chromatography, and HPLC, respectively. The molecular mass of this molecule was estimated via SDS-PAGE and confirmed by the ESI-TOFMS as 3,139 Da. The bioactive molecule was also determined to be a heat-stable, pH-stable (range 6-8), and proteinase K sensitive antibacterial peptide, similar to bacteriocins. Based on all characteristics determined in this study, the purified bacteriocin was named as thuricin Bn1 because of the similarities to the previously identified thuricin-like bacteriocin produced by the various B. thuringiensis strains. Plasmid elution studies showed that gene responsible for the production of thuricin Bn1 is located on the chromosome of Bt-Bn1. Therefore, it is a novel bacteriocin and the first recorded one produced by an insect originated bacterium. It has potential usage for the control of many different pathogenic and spoilage bacteria in the food industry, agriculture, and various other areas.