Bacteriocin leader pepides are currently receiving much attention due to their possible
functions. It is predicted that these leaders prevent cytoplasmic toxicity within the
producer organism by rendering the bacteriocin inactive. Leucocin A, a class IIa
bacteriocin produced by Leuconostoc gelidum UAL187-22 is synthesized with a 24
amino acid leader pepide which is cleaved during extracellular translocation. The
antimicrobial activity of the leucocin A precursor, pre-leucocin A, was determined to
gain insight into whether, the presence of a leader peptide has an impact on anti-listerial activity. The leucocin A and pre-leucocin A genes were generated by PCR of L. gelidum
UAL187-22 plasmid DNA. Recombinant plasmids, pLcaA and pPreLcaA were isolated
by cloning the amplified genes into the Escherichia coli pMAL.c2 vector, and by
screening transformant colonies using blue white selection methods. The malE-LcaA
and malE-preLcaA fusion genes were expressed, and resulting maltose binding fusion
proteins, were purified using amylose affinity chromatography. Fractions collected,
contained partially pure forms of MBP-LcaA (46.433 kDa) and MBP-preLcaA (49.088
kDa) fusion proteins. Following Factor Xa digestion, the MBP affinity tag was
removed; and recombinant peptides, leucocin A and pre-leucocin A were further
purified by reverese phase high performance liquid chromatography. It was determined
that leucocin A was eluted with a retention time of 24.893, while pre-leucocin A was
eluted with a retention time of 31.447. Fractions of pure leucocin A and pre-leucocin A
were thereafter assayed for activity using a deferred antagonism assay, with Listeria
monocytogenes being the indicator strain. Pre-leucocin A tested positive for
antimicrobial activity. However, when compared to leucocin A it was found that the
leucocin A precursor inhibits Listeria to a lesser degree than leucocin A. The relative
bactericidal activities of leucocin A and pre-leucocin A was calculated at 6.0 x 10⁵ AU
and 4.0 x 10⁵ AU. Taking this into consideration, it was estimated that the leucocin A
precursor is ~66.667 % active as mature leucocin A. Hence the presence of a leader
peptide does not have an influence on leucocin A antimicrobial activity. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/10739 |
| Date | January 2008 |
| Creators | Reddy, Jiren. |
| Contributors | Beukes, Mervyn. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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