Date of Award

2012

Document Type

Master Thesis

Degree Name

Masters of Science (Research)

Department

Biological Sciences

First Advisor

Dr. Aidan Coffey

Abstract

The bacteriophage K-derived peptidase CHAPk (Cysteine/Histidine-dependant amidohydrolase peptidase) can be produced from E. coli XL 1-Blue. Standard operating procedures for the production, purification and quality assessment of the peptidase were developed and set out in detail in this thesis. CHAPk exhibits strong antistaphylococcal properties and the purified enzyme was evaluated for its potential to eliminate S. aureus from the bovine udder and from human skin. As a teat dip CHAPk reduced the levels of S. aureus by three to five log cycles on both animal and human surfaces. CHAPk activity in milk was evaluated using challenge assays against the bovine mastitis isolate S. aureus DPC5246. The enzyme was found to eliminate cells in 10% resconstituted skim milk, however, its antistaphylococcal activity in raw milk was not as good. Given that in nature, many bacterial cells are not necessarily in the exponential phase of growth, activity of CHAPk against stationary-phase S. aureus was also evaluated. Activity on stationary-phase cells was evaluated over a period of 5 weeks using three S. aureus strains, DPC5246, Newbould 305 and Xen 29 and it was revealed that when aged at 30°C, cultures older than 3 days could be up to 2-fold less sensitive to the endolysin; there was less of a difference when cells were aged at 4°C. Finally, it was found that the process of lyophilization consistently reduced the concentration and activity of the CHAPk enzyme regardless of storage conditions of the powder.

Access Level

info:eu-repo/semantics/openAccess

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