An investigation into the biocatalytic application of the thermostable nitrile hydratase from the thermophilic strain Geobacillus pallidus RAPc8
Doctoral Thesis
2008
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University of Cape Town
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Nitrile hydratases (NHases) are bacterial metalloenzymes that catalyze the hydration of nitriles to their corresponding amides. The enzymes have been found in several microorganisms and participate in the metabolism of nitrile compounds as source of carbon and nitrogen. The commercial use of NHases is well recognized and has prompted research interest as well as the application of the enzymes in the manufacture of commodity amide chemicals. This has largely been due to the versatile nature of the enzymes, associated with their physiochemical properties and broad substrate specificity. However, the widespread application of nitrile-converting enzymes in the industrial processes has been restricted in part by the thermal instability of the mesophilic-derived enzymes, and thus there is an increased focus on NHases from thermophilic microorganisms. A novel moderately thermophilic microorganism, Geobacillus pallidus RAPc8, was isolated by our collaborators (Pereira and co-workers, 1998). The strain has an optimal growth temperature of 65oC and constutitively expresses a thermostable nitrile hydratase. The gene cluster containing the nitrile hydratase were cloned, sequenced, and inducibly expressed in E. coli BL21 (DE3) to levels of approximately 49 U/mg. The NHase was purified by four steps including heat treatment, ammonium sulfate precipitation, hydrophobic interaction chromatography and ion exchange chromatography.
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Includes abstract.
Includes bibliographical references (leaves 190-215).
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Chiyanzu, I. 2008. An investigation into the biocatalytic application of the thermostable nitrile hydratase from the thermophilic strain Geobacillus pallidus RAPc8. University of Cape Town.