Enhanced production, purification, and characterization of propionicin PLG-1, a bacteriocin produced by Propionibacterium thoenii P127

Propionibacterium thoenii strain P127 produces the bacteriocin propionicin PLG-1. Goals of this study were to increase the sensitivity of the standard well diffusion assay system for bacteriocin activity, to improve production of propionicin PLG-1 under controlled conditions in a fermenter, and to obtain the amino acid sequence and composition of the purified bacteriocin.; For the well diffusion assay, a 5-mm deep base layer that contained 2.5% agar, 0.85% NaCl and 0.1% Tween 80 was used. Plates were incubated at 37{dollar}spcirc{lcub}rm C{rcub}{dollar} for 2 h before adding bacteriocin samples to the wells. Lactobacillus delbrueckii ATCC 4797 was used as indicator strain, rather than Propionibacterium acidipropionici P5. Large, clear zones of inhibition could be measured after 12 h of incubation. Recovery of bacteriocin from the culture supernatant was improved by adding 0.1% Tween 80 to buffer used for dialysis and resuspension of precipitated protein.; Bacteriocin production was compared in six different media under controlled conditions in a fermenter: 12.5% beet molasses; 9% corn steep liquor; combinations of these media at 1:3, 1:1, and 3:1 vol:vol ratios; and the standard growth medium, sodium lactate broth. Cell populations reached 10{dollar}sp9{dollar} cells/ml in all media. Maximum production of propionicin PLG-1 was obtained in 3:1 beet molasses:corn steep liquor, and was 5 times greater than in sodium lactate broth. Measurable activity was detected after 4 days of culture incubation.; Fed-batch fermentations were conducted for 21 days in sodium lactate broth with regular feedings of sodium lactate. Average concentrations of viable cells were higher than in batch fermentations: 2.2 {dollar}times10sp9{dollar} cells/ml vs. 3.7 {dollar}times10sp8{dollar} cells/ml. Propionic acid concentrations were in excess of 30 g/l and acetic acid concentrations were over 10 g/l by the end of fed-batch fermentation. Bacteriocin activity ranged between 100 and 184 AU/ml in three fed-batch fermentations; in contrast 8 AU/ml was the highest titer obtained in batch fermentation. After reaching its maximum value at 16-17 days, bacteriocin activity dropped sharply with continued incubation. This suggests production of an inhibitor or of proteolytic activity.; Propionicin PLG-1 was purified to homogeneity by precipitation with 75% saturated ammonium sulfate followed by ion exchange column chromatography and reversed-phase high-performance liquid chromatography. According to amino acid composition analysis, propionicin PLG-1 is composed of 99 amino acid residues, of which 42% are hydrophobic (Ala, Ile, Leu, Val, and Pro); calculated molecular weight is 9,328 d. The N-terminal amino acid sequence is: NH{dollar}sb{lcub}2-{rcub}sp1{dollar}Asn-{dollar}sp2{dollar}Val-{dollar}sp3{dollar}Asp-{dollar}sp4{dollar}Ala(Thr)-{dollar}sp5 {lcub}rm Arg-{rcub}sp6{dollar}Thr(Cys)-{dollar}sp7{dollar}Ala(Thr)-{dollar}sp8{dollar}Arg{dollar}sp9{dollar}Thr(Ala)-{dollar}sp{lcub}1 0{rcub}{lcub}rm Pro{rcub}.{dollar} No homology of this sequence to sequences of other bacteriocins from lactic acid bacteria was seen in a search of the SWISS-PROT data bank.