Isolation, purification, and amino acid sequence of lactobin A, one of the two bacteriocins produced by Lactobacillus amylovorus LMG P-13139

Abstract

Lactobacillus amylovorus LMG P-13139, isolated from corn steep liquor, produces two bactericidal peptides with respective estimated molecular masses of 4.5 and 6.0 kDa upon denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antimicrobial activity detected in the fermentation supernatant fraction of L. amylovorus LMG P-13139 was heat stable (20 min, 121°C), displayed a narrow inhibitory spectrum, and was sensitive to proteinase K, trypsin, and α-chymotrypsin but insensitive to α-amylase, lysozyme, catalase, and lipase. The 4.5-kDa bacteriocin was purified and characterized and designated lactobin A. Lactobin A was isolated as a floating pellicle from culture supernatant brought to 35% saturation with ammonium sulfate. Upon this ammonium sulfate treatment, crude lactobin A was incorporated, together with Tween 80 as a major contaminant, in high- molecular-mass complexes sized at approximately 670 kDa by gel filtration chromatography. Contaminating fatty acids were removed from these micelles by a simple one-step methanol-chloroform extraction without loss of activity. Both inhibitory peptides were separated in an isocratic isopropanol gradient on a PepRPC 5/5 reversed-phase column, and both peptides retained activity towards Lactobacillus helveticus ATCC 15009 upon separation. Lactobin A has a molecular mass determined by electrospray mass spectrometry of 4,879 ± 0.69 Da. Its peptide chain contains 50 unmodified amino acids, of which 26% are glycine residues and 40% are hydrophobic residues (A, V, L, I, and P). It displays the highest structural homology (42% identity and 28% similarity) with the lafX gene product, encoded by the second open reading frame of the lactacin F operon. These data strongly indicate that lactobin A belongs to the class lib bacteriocins according to the classification of Klaenhammer.