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Title: Microbiological studies of some peptide antibiotics
Author: Duncan, Margaret J.
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 1971
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Introduction: Among the penicillin-producing organisms which are members of the Class Fungi Imperfecti are Penicillium chrysogenum and the Cephalosporium sp. C-91. The former can produce a series of extracellular penicillins (I) with non-polar side-chains (R.CO) which vary with the side-chain precursor added to the fermentation medium. The Cephalosporium sp. produces only two extracellular antibiotics of this type and both have a δ-(D-α-aminoadipyl) side-chain. One is penicillin N (II) and the other cephalosporin C (III). Early studies established that the biogenetic precursors of the ring systems of both benzylpenicillin and cephalosporin C were L-cysteine and L-valine, and that the side-chains were derived from phenylacetic acid and α-aminoadipic acid respectively. The tripeptide δ-(α-aminoadipyl)cysteinylvaline (ACV,IV) was isolated from the mycelium of P. chrysogenum (Arnstein & Morris, 1960) and subsequently detected in the Cephalosporium sp. also (Smith et al., 1967). This suggested that δ-(α-aminoadipyl)cysteinylvaline might be an intermediate in the biosynthesis of the penicillins and of cephalosporin C. The difficulty of assessing the role of possible intermediates such as ACV in penicillin or cephalosporin biosynthesis has depended partly on the existence of permeability barriers. The development of broken-cell or cell-free systems in which these barriers have been removed and in which all or part of the biosynthetic pathways can be followed is thus clearly important. Cell-free or broken-cell systems have been used to study the biosynthesis of several peptide antibiotics. These systems have been prepared by mechanical rupture of the cells, or by enzymic digestion of the cell wall to form protoplasts, which were then easily disrupted by osmotic shock. Dissolution of the cell wall of B. licheniformis and of B. brevis by muramidase has led to cell-free systems capable of synthesising bacitracin and tyrocidine respectively (Ishihara, Sasaki & Shimura, 1968; Fujikawa, Suzuki & Kurahashi, 1966). However, protoplasts from fungi have proved more difficult to prepare (Villaneuva, 1965). Snail enzyme, an impure preparation containing approximately thirty enzymes, has commonly been used to prepare protoplasts from yeast cells. Cells from different strains and of different ages vary markedly in their susceptibility to snail enzyme, but young cells are in general more susceptible than older ones. Aims and Scope of the Present Investigation: The main aim was to prepare protoplasts from the Cephalosporium sp. and to study their properties with the intention of using them as the starting material for the preparation of an active cell-free extract. Towards this end it was decided to find out which phase of a culture of Cephalosporium sp. C-91 produced penicillin N and cephalosporin C at maximal rates, and the morphological appearance of the mycelium in this phase so that protoplasts could be prepared from this type of culture. Finally, it was hoped that a cell-free extract obtained from protoplasts would be able to carry out all or some of the steps associated with antibiotic biosynthesis.
Supervisor: Newton, G. G. F. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available