Purification of bacteriocin produced by lactic acid bacteria and mass production by recombinant E.coli

Abstract

To obtain antimicrobial peptide from a traditional fermented food, Lactococcus lactis subsp. lactis MY23 was isolated among hundreds of strains from Kimchi based on antimicrobial activity screening for Propionibacterium acnes. The culture broth of Lactococcus lactis subsp. lactis MY23 showed selective antimicrobial activity against a few pathogenic bacteria including Propionibacterium acnes, Listeria monocytogenes, and Rothia dentocariosa. The antimicrobial peptide, named lactococcin K, was purified to a 70.8 fold increase by Mono S column chromatography, Phenyl-Superose hydrophobic interaction chromatography, and NH2 normal-phase chromatography. The purified lactococcin K was inactivated by proteolytic enzymes such as trypsin and protease K, but not by lipase, lysozyme, or glucanase. The molecular size of the native lactococcin K was 5.5 kDa in tricine/SDS-PAGE active staining. The structural gene of lactococcin K, lcnK, was cloned from the whole genome of Lactococcus lactis subsp. lactis MY23 and was expressed in an Escherichia coli JM105. The recombinant bacteriocin produced by E. coli was more stable to heat and pH than that of the parent strain. The molecular mass calculated from the amino acid sequence of lcnK is a half of purified native lactococcin K and recombinant peptide from E.coli. The lcnK was amplified by PCR and was subcloned into pET-21c(+) expression vector (pTMY). The evaluation of antimicrobial activity of transformant containing the plasmid pTMY revealed that the recombinant peptide, lcnKⅡ, has a similar inhibitory activity against the indicator strain. These data suggest that the native form of lactococcin K is a homodimer. Because lcnKⅡ was easily digested with protease in E.coli host, we made a fusion protein containing lcnKⅡ and Maltose binding protein (MBP). The resulting plasmid, pBMBPB, containing MBP gene, lcnK and enterokinase cleavage-site was used to transform E. coli BL21(DE3). A fusion protein from E.coi BL21(DE3) containing th...