Antibacterial Activity and Purification of Bacteriocin Produced by Brevibacillus laterosporus SA14

A bacteriocin producing strain, Brevibacillus laterosporus SA14 was isolated from an air sample at Walailak University, Thailand. The antibacterial activity of Brev. laterosporus SA14 was assayed by the cross streak and agar well diffusion method. Results from the cross streak method showed that it can inhibit both Gram positive and Gram negative bacteria. While results from the agar well diffusion method showed that Micrococcus luteus TISTR884 exhibited higher sensitivity to bacteriocin produced by Brev. laterosporus SA14 than other indicator strains. The highest amount of bacteriocin produced by Brev. laterosporus SA14 was detected during day 8 to day 10 of incubation. The crude protein exhibited bacteriocin activity of 800 AU/ml and did not affect cell membranes of Staphylococcus aureus TISTR516 and MRSA142. The bacteriocin activity increased from 25 AU/ml in supernatant to 800 AU/ml in crude protein after ammonium sulfate precipitation, corresponding to 88 % recovery and 32-fold purification. The fraction number was 39 to 41 from the Sp-sepharose fast flow column chromatography expressed highest bacteriocin activity with 44.8 % recovery and 128-fold purification. The partially purified bacteriocin was revealed as a single band on SDS-PAGE gel with a molecular weight of 6.9 kDa. The active fractions were finally pooled and purified by Reverse Phase High Performance Liquid Chromatography (RP-HPLC). The result showed a single peak (retention time of 3.466 min) with antibacterial activity. doi: 10.14456/WJST.2016.6