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 sulfatepolyacrylamide 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 ␣-chymotrypsin but insensitive to ␣-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 IIb bacteriocins according to the classification of Klaenhammer.

Purification and characterization of a bacteriocin produced by Lactobacillus acidophilus IBB 801

“…1996 ), amylovorin L471 ( De Vuyst et al . 1996a ), crispacin A ( Tahara & Kanatani 1997) and lactobin A ( Contreras et al . 1997 ) only inhibit a few Lactobacillus spp.…”

“…Lactobacillus amylovorus is known to produce amylovorin L471 ( De Vuyst et al . 1996a ) and lactobin A ( Contreras et al . 1997 ).…”

Antimicrobial Protein Produced by Vaginal Lactobacillusacidophilus that inhibits Gardnerella vaginalis

“…Even intensive attempts to extract protein from Tween 80 with several organic solvents failed. Other investigators attempted to disrupt lactocin micelles from Tween 80 using methanol-chloroform and ethanol-diethyl ether extraction of fatty acids to separate the two proteins, 4.5 and 6 kDa 6 .…”

“…To separate protein agglomerated with Tween 80, the sample was defatted three times with chloroform/methanol in a ratio of 2:1 6 . The aqueous and chloroform/methanol layers were air dried, dissolved in a small amount of PBS, and tested for antibacterial activity.…”

“…Several investigators have isolated and partially purified bacteriocin from different species of lactobacilli. Most of these investigations were conducted with nonhuman strains, predominantly isolated from food [1][2][3][4][5][6][7][8][9] .…”