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Title: The role of nitroreductases, nitrilases and nitrile hydratase in breakdown of aromatic compounds in Rhodopseudomonas palustris
Author: Farsi, Reem
ISNI:       0000 0004 6347 8669
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2017
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Aromatic compounds are among the most persistent and prevalent toxic pollutants in the environment. Biotransformation and bioremediation processes can convert these compounds into non-toxic compounds and valuable products. The purple phototrophic bacterium Rhodopseudomonas palustris (R. palustris) has the ability to degrade a wide range of aromatic compounds and the identification of several nitroreductases, nitrilases and a nitrile hydratase (NHase) in the genome sequence indicates an ability to degrade nitroaromatic and nitrile compounds respectively. The candidate nitroreductases RPA1711 and RPA4285 were recombinantly produced in E.coli and the purified proteins were assayed for their ability to reduce the nitro-groups of ligands to amino groups by UV-Spectrophotometry. Michaelis-Menten constants (Km) for RPA1711 of 0.21 mM and 0.13 mM were obtained for 2,4 Dinitrotoluene (2,4-DNT) and 2,6-Dinitrotoluene (2,6 DNT) respectively, while RPA4285 had higher Km values with 2,4-DNT of 0.76 mM and with 2,6-DNT of 0.41 mM. On the other hand, nitriles were utilised by nitrilases and a nitrile hydratase and both aromatic and aliphatic nitriles were degraded. The RPA0599, RPA1563 and RPA4166 nitrilases were purified and their activities were determined by the release of ammonia using the continuous coupled assay, phenol-hypochlorite, ophthalaldehyde (OPA) methods. 4-cyanopyridine was a preferred substrate. Furthermore, a NHase of two subunits RPA2805-2806 and NHase with activator (P14K) RPA2805-RPA2806-RPA2807 was also purified and characterised. Benzonitrile was a preferred substrate for the NHase. Exploring the ability of R. palustris to degrade nitroaromatic and nitrile pollutants might help improve bioremediation biotechnology.
Supervisor: Kelly, David J. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available