Philosophiae Doctor - PhD / Microbial nitrile hydratases (NHases) are important industrial enzymes that catalyse the conversion of nitriles to the corresponding amides. A thermostable, cobalt-type Bacillus sp. RAPc8 NHase was previously cloned and expressed in E. coli. In this study, the primary aim was to determine the molecular structure of Bacillus sp. RAPc8 NHase. The heterotetrameric enzyme was purified to near homogeneity using heatpurification, hydrophobic interaction chromatography and ion exchange chromatography. Purified NHase was crystallised using the hanging-drop vapourdiffusion method. Crystals produced in the presence of 30% PEG 400, 0.1M MES pH 6.5 and 0.1M magnesium chloride were selected for X-ray diffraction studies. These crystals diffracted well, with diffraction spots visible beyond 2.4Å, with little mosaicity. At 2.5Å, the data were 93% complete. The crystal structure of Bacillus sp. RAPc8 NHase was solved via molecular replacement using the crystal structure of Pseudonocardia thermophila NHase as a search model. The final refined structure had good refinement statistics and geometry. The overall fold was very similar to that of previously determined NHase structures. Bacillus sp. RAPc8 NHase was most similar to Bacillus smithii NHase (0.355År.m.s.d.) and least similar to Rhodococcus sp. R312 NHase (1.191Å r.m.s.d.). One cobalt atom per heterodimer was bound to a typical NHase metal-binding motif, with post-translationally modified cysteine residues among the ligands to the metal. The substrate-binding and catalytic cavity of Bacillus sp. RAPc8 NHase was identified and described in detail. Surface representation of the structure revealed an extended, curved solvent accessible channel with access to bulk solvent from two locations in the heterodimer. The amino-acid residues forming the channel were identified and the geometric dimensions measured. Enzyme inhibition kinetics indicated that benzonitrile was a potent uncompetitive inhibitor of NHase. This information was used to aid the genetic engineering of aromatic substrate specificity into Bacillus sp. RAPc8 NHase. Site-directed mutants of NHase were prepared using the Quickchange mutagenesis procedure. Mutant W76G showed a two to three fold decrease in benzonitrile inhibition compared with the wild-type. Analysis of the substrate channel of this mutant NHase showed an 11% increase in volume and a 20% increase in inner surface area compared to that of the wild-type NHase. Due to the lack of other significant differences between the two structures (an r.m.s.d. of only 0.101Å was observed), this difference was thought to be responsible for the decrease in benzonitrile inhibition. A structure-modelling based approach for assessing the likely structural differences that may result as a result of a specific mutation was suggested and tested. This approach may be of value in future mutagenesis work. / South Africa

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Thèse (M.E.S.R.)--Université du Québec à Chicoutimi, 1991. / Document électronique également accessible en format PDF. CaQCU

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O estudo tem como objetivo geral: entender como as atividades desenvolvidas pela FIEMG construÃram e mobilizaram as capacidades para o desenvolvimento da Simbiose Industrial. Simbiose Industrial - SI ocorre atravÃs das trocas fÃsicas de materiais, de energia, de Ãgua, e/ou subprodutos entre indÃstrias (geograficamente prÃximas) em uma abordagem coletiva para a vantagem competitiva, melhorando assim o desempenho ambiental das empresas, bem como diminuindo seus custos totais (CHERTOW, 2000; CHERTOW, 2007). Dentre diversos casos de SI no mundo, destacam-se o caso do Parque industrial de Kalundborg na Dinamarca e o National Industrial Symbiosis Programme â NISP na Inglaterra. No Brasil, A FederaÃÃo das IndÃstrias do Estado de Minas Gerais â FIEMG coordena o Programa Mineiro de Simbiose Industrial â PMSI, versÃo adaptada no programa de simbiose britÃnico. Foram entrevistadas representantes de sete empresas de diferentes setores e regiÃes do estado alÃm de quatro atores: a FederaÃÃo das IndÃstrias do Estado de Minas Gerais, o ÃrgÃo ambiental, um sindicato setorial e uma associaÃÃo de catadores. A anÃlise dos dados procura identificar os elementos presente no framework de Spekkink (2014): Contextos, Capacidades Institucionais, Atores, InteraÃÃes e Redes de Simbioses na realidade mineira. Os resultados apresentam como a FIEMG utiliza suas capacidades institucionais na promoÃÃo do PMSI; como os atores sÃo envolvidos na simbiose atravÃs de suas crenÃas, desejos e oportunidades; como ocorrem as interaÃÃes entre esses atores e os resultados em forma de rede de simbioses. Como conclusÃes, esta pesquisa constata a importÃncia do promotor da SI e sua articulaÃÃo com os atores, principalmente os sindicatos setoriais; a fraca influÃncia dos contextos de governanÃa, econÃmico e fÃsico na promoÃÃo do PMSI; a fraca participaÃÃo de atores como o ÃrgÃo ambiental no processo de simbiose e as barreiras que surgem nas trocas entre as empresas. / The main objective of this study is to understand how the activities developed by FIEMG â the Federation of Industries of the State of Minas Gerais, have built and mobilized the capacities for the development of Industrial Symbiosis. Industrial symbiosis - IS occurs through physical exchange of materials, energy, water, and/or by-products among (geographically close)industries in a collective approach to increase competitive advantage, thus improving the environmental performance of companies as well as reducing their total costs (Chertow, 2000; Chertow, 2007). Among several cases of IS in the world, we highlight the case of Kalundborg Industrial Park in Denmark and the National Industrial Symbiosis Programme - NISP in England. In Brazil, FIEMG coordinates the Mining Industrial Symbiosis Programme - PMSI, which is based on the British symbiosis program. Representatives of seven companies from different sectors and regions of the state were interviewed, as well as four actors: the Federation of Industries of the State of Minas Gerais, the environmental agency, an employerâs association and a cooperative of waste pickers. Data analysis aims at identifying the elements present in Spekkink framework (2014) Contexts, Institutional Capacities, Actors, Interactions and Network of Symbiotic Exchanges in mining reality. The results show how FIEMG uses its institutional capacity to promote the PMSI; how the actors are involved in symbiosis through their beliefs, desires and opportunities; the way the interactions occur between these actors and the results as a symbiosis network. This study evidences the importance of the IS promoter and its connection with the actors, especially the employerâs associations; the little influence of governance, economic and physical contexts in promoting PMSI; the low participation of some actors, as the environmental agency, in the symbiosis process and the barriers that come from the exchanges between companies.