Purification and characterization of a novel bacteriocin produced by Enterococcus faecalis strain RJ-11

Abstract

Lactic acid bacteria exhibiting activity against the gram-positive bacterium Bacillus subtilis were isolated from rice bran. One of the isolates, identified as Enterococcus faecalis RJ-11, exhibited a wide spectrum of growth inhibition with various gram-positive bacteria. A bacteriocin purified from culture fluid, designated enterocin RJ-11, was heat stable and was not sensitive to acid and alkaline conditions, but it was sensitive to several proteolytic enzymes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that enterocin RJ-11 had a molecular weight of 5,000 in its monomeric form. The amino acid sequence determined for purified enterocin RJ-11 exhibited high levels of similarity to the sequences of enterocins produced by Enterococcus faecium.

FIG. 1.

Bioassay for proteins with antibacterial activity after Tricine-SDS-PAGE. Crude proteins in culture fluids of strains RJ-11 (lane 1), RB-3 (lane 2), RJ-10 (lane 3), SJ-16 (lane 4), and YJ-35 (lane 5) were fractionated by Tricine-SDS-PAGE (16% polyacrylamide gel). Proteins with antibacterial activity were visualized by overlaying soft nutrient agar containing cells of L. monocytogenes SUB635 on the gel and incubating it at 30°C overnight. The numbers on the left indicate the positions of molecular weight markers.

FIG. 2.

Production of bacteriocin by E. faecalis RJ-11. Cells were cultured in a flask (•) or in a jar fermentor (○). At appropriate intervals, cell densities (A) and antibacterial activities in the culture fluid (B) were determined. O.D 660, optical density at 660 nm.

FIG. 3.

Elution profiles of proteins with antibacterial activity. Crude proteins in culture fluid were eluted from a Sephadex G-50 column (20 by 700 mm) by using citrate buffer in the absence (A) or in the presence (B) of 0.1% Tween 80. Each fraction contained 4.5 ml of the eluate.

FIG. 4.

Tricine-SDS-PAGE analysis of purified enterocin RJ-11. Proteins were fractionated by Tricine-SDS-PAGE (16% polyacrylamide gel). The resulting gel was stained with Coomassie brilliant blue (A) or assayed for activity against L. monocytogenes (B). The samples used were crude proteins salted out from culture fluid and then dialyzed (lane 1) and proteins in the fraction of the first active peak (lane 2) and the second active peak (lane 3) eluted by Sephadex G-50 gel permeation chromatography.

FIG. 5.

Amino acid sequence of enterocin RJ-11 and comparison with the sequences of enterocins L50A and L50B. The sequence containing 44 amino acid residues determined for purified enterocin RJ-11 was deduced from four sets of data analyzed independently. The sequences of enterocins L50A and L50B were obtained from the DDBJ database (accession numbers of AJ223633 and Y16413, respectively). Residues identical to residues of enterocin L50A and/or enterocin L50B are indicated by a black background.