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Title: Studies on anatoxin-a
Author: Chit, K. N.
Awarding Body: University College of Swansea
Current Institution: Swansea University
Date of Award: 1986
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Anatoxin-a was isolated from Anabaena flos-aquae NRC-44-1 and some of its characteristics have been studied. The highest concentration of the toxin (≈ 0.2 mg/g dry weight cells), was found in cultures 14 days after incubation, though this could be increased by modification of the growth medium. For example, decreasing the ratio of K^+ to Ca^2+ or replacing NaO_3 in the medium by NH_4C1 increased the concentration of anatoxin-a in the cyanobacterial cultures. An investigation into possible precursors for anatoxin-a revealed that, of those tested, L-[U-^14C] ornithine was incorporated into anatoxin-a most efficiently (0.021%). Furthermore, both labelling and isotope dilution experiments showed that putrescine is an intermediate in the biosynthetic pathway to anatoxin-a. The presence of ornithine decarboxylase activity in cell-free extracts of Anabaena flos-aquae NRC-44-1 was investigated and its properties have been investigated. Highest activity was found in cultures 14 days after incubation, coincident with the highest concentrations of anatoxin-a. The optimum pH of the enzyme was pH 8 and an examination of the Km for ornithine of the enzyme, and partial purification on DEAE-cellulose suggested that there may be two ornithine decarboxylases in Anabaena flos-aquae NRC-44-1. Ornithine decarboxylase was found to be 100 times less active in the non-toxic strain, Anabaena flos-aquae 1403/13f. Plasmid DNA was isolated from the toxic strain (Anabaena flos-aquae NRC-44-1). The molecular weight of that plasmid was determined on 1% agarose gel using Hind III digested fragments of λ DNA as a molecular weight marker, and it was found to be approximately 10Kb (6.5 Md). On the other hand, plasmid DNA could not be detected in the non-toxic strain (Anabaena flos-aquae 1403-13f). Furthermore, using isolated DNA from Anabaena flos-aquae NRC-44-1, the transformation of the non-toxic Anabaena flos-aquae into toxic strain, has been demonstrated. Finally, a possible biosynthetic route from the amino acid ornithine, leading to anatoxin-a, involving ornithine decarboxylase, and with putrescine as one of the intermediates, is suggested.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available