Novel activity evaluation and subsequent partial purification of antimicrobial peptides produced by Bacillus subtilis LFB112

The antibacterial activity of antimicrobial peptides produced by strain LFB 112 was determined using the cup-plate method and a standard curve of penicillin. An effective method to evaluate the antimicrobial activity of antibacterial peptides was established. Three active fractions, Fraction A (5.0–5.5 kDa), Fraction B (3.0–3.5 kDa) and Fraction C (400–800 Da), were separated by anion-exchange, reversed phase liquid chromatography (RPLC) and gel filtration chromatography. Fraction A contained an antimicrobial peptide; its partial sequence (LVQSPNGNFAASFVLDGTK), determined by liquid chromatography-mass spectrometry (LC-MS), was homologous with antimicrobial peptide LCI. In addition, there were three major ingredients in fraction B (3,371.811, 3,442.379 and 3,486.596 Da, respectively), and several small peptides (400–800 Da) in fraction C. After purification, the potencies of fractions A, B and C were 188.6, 11,217.26 and 10,290.63 U/mg, respectively. Furthermore, antimicrobial peptides in fraction B, apparently different from known antimicrobial peptides produced by Bacillus subtilis (based on molecular weight), and small (molecular weight 400–800 Da) antimicrobial peptides produced by B. subtilis LFB112 were also identified. Antimicrobial peptides produced by strain LFB112 with high potency against several undesirable bacteria were identified. These compounds have potential as alternatives to known antibiotics.