Production, characterization and utilization of the bacteriocin produced by Enterococcus faecalis B9510 : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University, Palmerston North, New Zealand

Abstract

Bacteriocins are antimicrobial proteins and peptides produced by bacteria, antagonistic to other bacterial species but not affecting the producer species. The bacteriocins from Lactic Acid Bacteria (LAB) are particularly well-studied and exploited as safe and natural food preservatives. The current research aimed to identify and characterize a bacteriocin-like substance produced by Enterococcus faecalis B9510, a local isolate from silage. The production of this antimicrobial during the growth phase, destruction of antimicrobial activity by proteolytic enzymes and self-immunity of the producer strain indicated that the antimicrobial is a bacteriocin. The bacteriocin was heat-labile as the antimicrobial activity was destroyed by heating at 60 ºC for one hour. This bacteriocin was also found to lyse sensitive cells. To further characterize the bacteriocin to the protein level, the producer strain was grown in a completely defined medium, devoid of any proteins and peptides, to facilitate downstream processing. Purification was done by passing the culture supernatant through 10 kDa and 30 kDa ultrafiltration membranes. The 30 kDa ultrafiltration retentate showed antimicrobial activity and was then subjected to SDS-PAGE. The in-gel bacteriocin activity was then determined by incorporating dead cells of a sensitive strain Lactococcus lactis ssp. cremoris 2144 in a parallel SDS-PAGE gel, which was renatured after the electrophoresis. After renaturation a zone of clearance was observed around the active band, at approximately 35 kDa. The active band was excised and analyzed by mass spectrometry. The results revealed that the amino acid sequence matched a known bacteriocin enterolysin A. This was confirmed when the enterolysin A gene was amplified from the producer strain using PCR followed by DNA sequencing. The earlier studies on enterolysin A primarily focused on the structural gene, and primary structure of enterolysin A. No information is available on the function of neighbouring genes of the enterolysin A structural gene. An attempt was made in the current study to elucidate the function of genes found in close proximity to the structural gene, with the aim to find the immunity gene. Experiments were also conducted to find the mode of action of enterolysin A. Earlier studies have reported that enterolysin A is an endopeptidase which degrades the cell walls of sensitive Gram-positive bacteria. However, the cleavage site within the cell wall moiety has not been reported. The current study has revealed that enterolysin cleaves a peptide bond between D-glutamic acid and L-alanine in the stem peptide and N-bacteriocin produced by Enterococcus faecalis B9510 iv terminus of L-lysine and C-terminus of D-aspartic acid within the interpeptide bridge of peptidoglycan units of sensitive bacterial strains. Furthermore, transmission electron microscopy of enterolysin A treated cells gave new insight into the morphology of damaged cells. The antimicrobial spectrum of enterolysin A already reported was also extended to other species in the current study. The results revealed that in addition to its activity against the bacterial species already reported, enterolysin A is also active against Lactobacillus helveticus, Lactobacillus casei and Lactobacillus delbrueckii ssp. bulgaricus. To conclude the project, enterolysin A was coated on polyethylene film. This film was found to effectively control the growth of L. casei and thus can be incorporated into antimicrobial packaging against spoilage microorganisms.