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Title: Nitroaryl reductase : purification from Saccharomyces cerevisiae and use in biotransformations
Author: Blackie, Josie A.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1998
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It is well known that baker's yeast (Saccharomyces cerevisiae) can be used as a reagent for the reduction of a variety of functional groups, especially the enantioselective reduction of carbonyl groups to the corresponding optically active alcohols. In the context of this thesis it has been shown that aromatic nitro compounds undergo reduction with S. cerevisiae to the corresponding anilines under conditions of neutral pH, room temperature and in an aqueous medium. Mechanistic studies employing putative intermediates, suggest that nitroso and hydroxylamino species are involved on the reduction pathway. Further insights into the mechanism have been gained by investigating the reduction of a series of dicyanonitroaromatic substrates. In all cases, in addition to reduction of the nitro group, the nitrile group ortho to the nitro group undergoes conversion to the amide, via a proposed heterocyclic intermediate. The broad range of nitroaromatic substrates reduced by the baker's yeast prompted us to commence purification of the enzyme(s) responsible for the biotransformations. Ten litre fermentations were used in order to provide a sufficient quantity of cells for the subsequent purification steps. The use of size exclusion, anion exchange, hydrophobic interaction and affinity chromatography led to partial purification of the nitroaryl reductase enzymes. The reduction of 1,4-dinitrobenzene to 4-nitroaniline provided an ideal enzyme assay for the production of kinetic data, the enzyme demonstrating Michaelis-Menten kinetics. Subsequent to the successful optimisation of the purification protocol. Marina Alexeeva (University of Edinburgh) has completed the purification of three nitroaryl reductases with molecular weights of approximately 50 KDa, 45 KDa as judged by SDS-PAGE.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available