Isolation and characterisation of an antimicrobial peptide from Enterococcus B9510 : a thesis in partial fulfilment of the requirements for the degree of Master of Technology in Biotechnology at Massey University, Palmerston North, New Zealand

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This work deals with the antimicrobial activity of bacteriocins produced by Enterococcus B9510. The greatest importance demonstrated by this work was the range of activity exhibited by this bacterium, across both Gram positive and negative pathogens. This is not typical for bacteriocins, which tend to inhibit only closely related bacteria in similar environments. While attempts to identify the peptide sequence were unsuccessful the overall results of this investigation are enough to suggest that the bacteriocin from Enterococcus B9510 was novel and warrants further investigation. Bacteriocins are small peptides that demonstrate antimicrobial properties and are produced by a number of lactic acid bacteria, including Enterococcus sp.. Bacteriocins are important because they are a possible alternative for food preservation. As a natural product they could be used to improve customer perceptions, due to the reduced need for chemical preservatives in products. From a range of lactic acid bacteria which were tested tor antimicrobial activity across a selection of pathogens the strain Enterococcus B9510, which was a local isolate from bovine rumen, was selected due to its high level of activity. Using 16S ribosomal DNA (rDNA) analysis and sequence alignment with the BLAST network service, B9510 showed the highest homology (97%) to Enterococcus faecalis in the genome database. Fermentation trials were carried out in order to maximise the concentrations of bacteriocins produced. In a controlled pH environment, cellular growth and activity was highest at pH 5.5; however, the effect of low pH showed a higher relative activity at pH 4, possibly due to pH interactions with the bacteriocin. Multi-factor trials were carried out to find the effect of glucose concentration, salt concentration and air saturation on the bacteriocin production. High salt concentrations showed a reduced production of bacteriocins and low cellular growth whereas aeration had little effect on growth but some affect on bacteriocin activity, possibly due to a variation in stress on the bacteria. Glucose concentration effected cellular growth rates, which may be a significant factor in the production of the antimicrobial activity. Purification of the bacteriocins was carried out using two methods. The pH binding method of attaching the bacteriocins to the cell wall by neutralising the pH of the fermentation broth was unsuccessful over a range of pH values, pH 6-9. However, the resin binding method was successful in binding the bacteriocins from a cell free broth. An ion-exchange resin (Macro-Prep CM) was used to purify the active fraction and remove a large pigment component associated with the fermentation media. High Performance Liquid Chromatography provided an effective means to isolate samples for molecular weight determination and N-terminal sequencing. Antimicrobial activity of the isolated fraction was tested under a range of environmental conditions including temperature, salt concentration and pH. Bacteriocin activity was still present after being held at 100°C for an hour. Optimal activity was observed at pH 3, but activity was shown by the peptide over the range of pH 1.7 - 11. Low levels of salt (<200 mM) increased the activity of the peptides; however, high concentrations (≥500 mM) reduced the effectiveness of the bacteriocin. Enzymes which acted upon peptide bonds were effective at inactivating the bacteriocin, while non-peptidase enzymes were ineffective. Testing against thirteen different bacteria, including E. coli, Listeria and Bacillus, showed that the peptide was active against both Gram positive and Gram negative bacteria. Mass determination showed that the active peptide was between 1.27 and 1.35 kDa. Attempts to fully sequence the purified peptide were unsuccessful, however, an N-terminal sequence of LMPPYGVIMFF was predicted, which had a molecular weight of 1.314 kDa. This sequence showed no significant homology to any known sequences in the protein databases using the BLAST network service.
Bacteriocins, Enterococcus, Food -- Microbiology