Isolation and Characterization of a Broad Spectrum Bacteriocin from Bacillus amyloliquefaciens RX7

• Published in: BioMed research international 2016-01, Vol.2016 (2016), p.1-7
• Main Authors: Lee, Ji Yoon, Oh, Ju Kyoung, Kim, Sang Hoon, Balolong, Marilen P., Lim, Kong Boon, Kang, Dae-Kyung
• Format: Article
• Language: English
• Subjects: Amino acids; Ammonium sulphate; Analysis; Anti-Infective Agents - isolation & purification; Anti-Infective Agents - pharmacology; Antibiotics; Antimicrobial agents; Bacillus; Bacillus amyloliquefaciens; Bacillus amyloliquefaciens - chemistry; Bacillus amyloliquefaciens - genetics; Bacillus halodurans; Bacteria; Bacteriocins - isolation & purification; Bacteriocins - pharmacology; Candida albicans; Candida albicans - drug effects; Cellulose acetate; Chymotrypsin; Food; Glycine; Gram-negative bacteria; Gram-positive bacteria; High performance liquid chromatography; Hydrogen-Ion Concentration; Listeria; Listeria monocytogenes; Listeria monocytogenes - drug effects; Microorganisms; Molecular Weight; Peptides; Precipitation (Meteorology); Proteolysis - drug effects; RNA; RNA, Ribosomal, 16S - genetics; Soil Microbiology; Temperature
• ISSN: 2314-6133; 2314-6141
• DOI: 10.1155/2016/8521476

We isolated a Bacillus strain, RX7, with inhibitory activity against Listeria monocytogenes from soil and identified it as Bacillus amyloliquefaciens based on 16S rRNA gene sequencing. The inhibitory activity was stable over a wide range of pH and was fully retained after 30 min at 80°C, after which it decreased gradually at higher temperatures. The activity was sensitive to the proteolytic action of α-chymotrypsin, proteinase-K, and trypsin, indicating its proteinaceous nature. This bacteriocin was active against a broad spectrum of bacteria and the fungus Candida albicans. Direct detection of antimicrobial activity on a sodium dodecyl sulfate-polyacrylamide gel suggested an apparent molecular mass of approximately 5 kDa. Ammonium sulfate precipitation and anion-exchange and gel permeation chromatography integrated with reverse phase-high-performance liquid chromatography were used for bacteriocin purification. Automated N-terminal Edman degradation of the purified RX7 bacteriocin recognized the first 15 amino acids as NH2-X-Ala-Trp-Tyr-Asp-Ile-Arg-Lys-Leu-Gly-Asn-Lys-Gly-Ala, where the letter X in the sequence indicates an unknown or nonstandard amino acid. Based on BLAST similarity search and multiple alignment analysis, the obtained partial sequence showed high homology with the two-peptide lantibiotic haloduracin (HalA1) from Bacillus halodurans, although at least two amino acids differed between the sequences. A time-kill study demonstrated a bactericidal mode of action of RX7 bacteriocin.