Date of Award


Document Type

Master Thesis

Degree Name

Masters of Science (Research)


Biological Sciences

First Advisor

Dr. Olivia McAuliffe

Second Advisor

Dr. Aidan Coffey

Third Advisor

Prof. R. Paul Ross


Antibiotics are of the utmost importance in the fight against pathogenic bacteria, however, in recent years there has been a continual increase in the development of antibiotic resistance among bacteria. As a result it has become essential to develop new technologies to combat these pathogens. One such technology is the application of phage lysins as antimicrobial agents against pathogenic infections. A promising phage lysin is LysK, a 495 amino-acid protein derived from Staphylococcal Phage K. LysK can lyse a wide range of staphylococcal species including MRSA. It is also capable of killing both live and dead cells increasing its potential as an effective therapeutic antimicrobial.

Bacteriocins and other antimicrobial peptides have also attracted attention as possible antimicrobial agents. These compounds may be used in isolation or in combination with currently used antimicrobial compounds to exert their toxic effects on the pathogenic cell One such compound is Enterolysin A (EntL),a 343aa lytic enzyme produced by Enterococcus faecalis DPC5280. EntL is a metallopetidase which can kill a range of Gram positive bacteria including Lactococci sp. In the current study, the phage lysin LysK and its truncated 165aa derivative Cl, which consists of the catalytic cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP) domain, were purified by ion exchange chromatography. These were further processed via size exclusion chromatography which ensured a high degree of purification. The native EntL protein, which encodes the bacteriocin Enterolysin A, was purified by immobilized metal affinity

chromatography (IMAC). Secondary structure analysis was performed on all three proteins (LysK, Cl and EntL) using both experimental and predictive techniques. Whilst the results were not in complete agreement they did both suggest each protein involved was composed predominantly of P-sheet and coil consisting in the least amount of a-helix. Thermal denaturation experiments were performed using circular dichroism. LysK was the only protein which showed refolding capabilities suggesting the amidase and cell wall binding domains play a role in protein refolding capabilities. The ability of a protein to refold is a property which can increase its shelf life which is beneficial for manufacturing and storage of an antimicrobial agent. Crystallization parameters were determined for the CHAP domain. 2M Am sulphate, 2% PEG 400, Cacodylate pH 7.5 gave long needle like crystals which took approximately a month to grow at room temperature.


Master of Science by Jennifer Garry B.Sc.

Moorepark Food Research Centre, Teagasc, Fermoy, Co. Cork, and

Department of Biological Sciences, Cork Institute of Technology, Bishopstown, Cork.

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