Characterization of bacteriocin 423 produced by Lactobacillus pentosus

[摘要] ENGLISH ABSTRACT:Worldwide, bacteriocins, particularly those produced by food-related lactic acid bacteria, arereceiving attention due to the possible use of these peptides as natural preservatives in food,replacing potentially harmful chemical preservatives.Bacteriocins are ribosomally synthesized proteins or peptides that inhibit closely relatedmicroorganisms. Most bacteriocins produced by lactic acid bacteria are small, heat resistantpeptides that inhibit other Gram-positive bacteria, including food-borne pathogens such asListeria monocytogenes, Bacillus cereus, Clostridium perfringens and Staphylococcus aureus,but do not inhibit Gram-negative bacteria, molds or fungi. Bacteriocins are produced asinactive prepeptides that become active after the N-terminal leader peptide is cleaved off.Small heat resistant bacteriocins are either lantibiotics (Class I), containing unusual posttranslationallymodified amino acids, or peptides that are non-Ianthionines (Class II). TheClass II bacteriocins are further divided into four different groups: Class lIa, the anti-listerialbacteriocins containing the YGNGV consensus sequence in the N-terminal of the protein,Class lib, bacteriocins consisting of two peptides, Class IIc, bacteriocins that are secreted viathe sec pathway, and Class lid, bacteriocins that do not belong in the previous threesubgroups.A bacteriocin producing lactic acid bacterium was isolated in our laboratory fromtraditionally home fermented South African sorghum beer. The producing bacterium wasfound to be a facultative heterofermentative Lactobacillus sp. and was identified asLactobacillus plantarum or Lactobacillus pentosus by using the API 50 CHL carbohydratefermentation system and numerical analysis of total soluble cell protein patterns. RAPD-PCRanalysis identified the strain as L. plantarum, but 16S rRNA sequencing confirmed itsidentification as L. pentosus.The bacteriocin, first designated plantaricin 423 and later bacteriocin 423, was identified asa Class lIa small heat resistant anti-listerial bacteriocin containing the YGNGV consensusmotif. Bacteriocin 423 inhibited a variety of Gram-positive bacteria, including Lactobacillusspp., Leuconostoc spp., Oenococcus oeni, Pediococcus spp., Enterococcus spp.,Propionibacterium spp., Staphylococcus spp., Bacillus spp., Clostridium spp. and Listeria spp.The bacteriocin was inactivated by proteolytic enzymes and active over a wide pH range (pH1-10). Bacteriocin 423 lost 50 % of its activity after autoclaving for 15 min at 121°C, but wasnot affected by lesser heat treatments.Bacteriocin production was increased by optimizing the growth medium, which consisted ofglucose, tryptone, yeast extract, potassium phosphate, sodium acetate, ammonium citrate, manganese sulphate, Tween 80 and casamino acids.The bacteriocin was found to be plasmid-encoded. Genetic analysis of the bacteriocinoperon indicated a high percentage of homology to the operon of another Class lIabacteriocin, pediocin PA-1, although the structural genes of the two bacteriocins weremarkedly different. The structural gene of bacteriocin 423 was amplified by PCR and clonedinto a yeastJE. coli vector between the ADH1 promoter and terminator sequences and fusedin-frame to the MFa1 secretion signal sequence. Saccharomyces cerevisiae transformed withthis plasmid expressed the bacteriocin.The sequence of prebacteriocin 423 (MMKKIEKL TEKEMANIIGGKYYGNGVTCGKHSCSVNWGOAFSCSVSHLANFGHGKC) is similar, but not identical to any other reported Class lIaanti-listeria I peptide.

[发布机构] Stellenbosch University