Thesis (Ph. D.)--Oregon State University, 1991. / Includes mounted photographs. Typescript (photocopy). Includes bibliographical references (leaves 111-122). Also available on the World Wide Web.

Univ., Diss., 2010--Marburg.

Berlin, Freie Univ., Diss., 2004. / Dateiformat: zip, Dateien im PDF-Format. Computerdatei im Fernzugriff.

Berlin, Freie Universiẗat, Diss., 2004. / Dateiformat: zip, Dateien im PDF-Format.

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Orientador: Adilma Regina Pippa Scamparini / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-01T16:52:25Z (GMT). No. of bitstreams: 1 Portilho_Marcia_D.pdf: 19156956 bytes, checksum: 0c4b5deb0e33d7fbd57626c1eaa7d976 (MD5) Previous issue date: 2002 / Resumo: Determinadas bactérias do gênero Agrobacterium, isoladas de amostras de solo e não patogênicas, são produtoras de dois polissacarídeos extracelulares: a succinoglicana, ácida e solúvel, e a goma curdulana, um polímero neutro e insolúvel. Este último, aprovado pelo Food and Drug Administration (FDA) - USA - em 1996, vem sendo empregado na indústria de alimentos, devido à sua capacidade de formar um excelente gel, firme e resistente. O presente trabalho foi realizado com o objetivo de elaborar um meio para a obtenção do inóculo e avaliar aspectos relacionados à produção do polissacarídeo insolúvel de duas linhagens de Agrobacterium sp (ATCC 31749 e IFO 13140). No meio de cultivo para formação do inóculo microbiano, um extrato de levedura de procedência comercial foi definido como fonte de nitrogênio e de sais. Foram feitos testes com glucose, sacarose e melaço de cana-de-açúcar em diferentes concentrações como fonte de carbono. Verificou-se que o carboidrato teve um efeito estimulante no crescimento microbiano. O meio elaborado com o extrato de levedura e sacarose foi o que apresentou melhores resultados, quanto ao crescimento, para as duas linhagens. Os inóculos microbianos produzidos a partir deste meio mostraram-se adequados para a produção do polissacarídeo insolúvel em um meio de cultivo proposto na literatura. As amostras de polissacarídeo insolúvel, obtidas das linhagens avaliadas, foram comparadas a uma amostra comercial do polissacarídeo insolúvel (curdulana) através de testes de solubilidade, cromatografia líquida de alta eficiência, espectrometria no infravermelho e capacidade de formar gel. As duas linhagens produziram um polissacarídeo com características muito semelhantes a curdulana comercial. Glucose de milho, glucose de mandioca e maltose de milho foram empregadas no meio de fermentação para a produção do polissacarídeo insolúvel, como substituintes da fonte de carbono no meio proposto na literatura. As duas linhagens apresentaram produção do polissacarídeo com rendimentos iguais ou superiores aos citados na literatura (que são de aproximadamente 50,00%). A linhagem ATCC 31749 apresentou melhor produção com o emprego da maltose de milho, com rendimento de 84,68%, enquanto a linhagem IFO 13140 produziu melhor a partir da glucose de milho, com 50,00% de rendimento (sobre os açúcares redutores). / Abstract: Some bacteria of Agrobacterium genus isolated from soil samples and not pathogenic produce two extracellular polysaccharides: an acid and soluble succinoglycan and curdlan gum, an insoluble and neutral polymer. The last one, which received the approval of the FDA - Food and Drug Administration - USA - in 1996, is usually used in food industry because of its property of a fmn, resilient and excellent gel. The present work was carried on in order to elaborate a medium to obtain a inoculum and evaluate aspects related to the production of an insoluble polysaccharide in two strains of Agrobacterium sp (ATCC 31749 and IFO 13140). It was used an yeast extract, commercially available as a medium, in order to create a microbial inoculum, defined as a nitrogen and salts sources. Tests were made using glucose, sucrose and sugar cane molasses in different concentrations as carbon source. The medium elaborated with yeast extract and sucrose presented the best results related to growth in both strains. The inoculums produced by that culture were suitable to the production of insoluble polysaccharides in the medium proposed in the literature. The samples of insoluble polysaccharide obtained from the evaluated strains were compared to a commercial sample of insoluble polysaccharide (curdlan) through tests of solubility, high-pressure liquid chromatography, infrared spectrometry and ability to produce gel. Both strains produced a polysaccharide whose features were very similar to the commercial curdlan. Maize glucose, cassava glucose and maize maltose were used as a fermentation medium to the production of the insoluble polysaccharide as a substitute of carbon source in the medium proposed in the literature. Both strains produced the polysaccharide and the results were the sameor higher than the literature (50,00%). The strain ATCC 31749 presented the best production using maize maltose (productivity of 84,68%) and strain IFO 13140produced better through maize glucose (productivity of 50,00%). / Doutorado / Doutor em Ciência de Alimentos

There is widespread interest in the biocatalytic production of enantiomerically pure D-amino acids for use in the synthesis of antibiotics, insecticides, herbicides, drug carriers and many other pharmaceuticals. Hydantoin-hydrolysing enzyme systems can be successfully utilised to stereoselectively convert racemic hydantoins into enantiomerically pure amino acid products. In fact, the use of microbial D-hydantoinase and D-stereoselective N-carbamoyl amino acid amidohydrolase activity to produce D-p-hydroxyphenylglycine from D,L-5-phydroxyphenylhydantoin has been described as one of the most successful biotechnological applications of enzyme technology developed to date. A need to utilise the novel biodiversity of South African microorganisms for the development of an indigenous process to produce enantiomerically pure amino acids was identified in 1995. Subsequently, the Rhodes Hydantoinase Group was established and several local hydantoin-hydrolysing microorganisms were isolated. The research in this study describes the isolation and selection of Agrobacterium tumefaciens RU-OR, which produced D-stereoselective hydantoinhydrolysing activity. Characterisation of the hydantoin-hydrolysing enzyme system of RU-OR revealed novel biocatalytic properties, and potential for the application of this strain for the biocatalytic production of D-amino acids. A fundamental understanding of the regulation of hydantoin-hydrolysing enzyme activity in A. tumefaciens RU-OR was established, and utilised to produce mutant strains with altered regulation of hydantoin-hydrolysing activity. These strains were used to further elucidate the mechanisms regulating the production of hydantoins-hydrolysing activity in A. tumefaciens RU-OR cells. Overproduction of hydantoinase and N-carbamoyl-D-amino acid amidohydrolase activity in selected mutant strains resulted in efficient conversion of D,L-5-p-hydroxyphenylhydantoin to D-p-hydroxyphenylglycine. Thus the establishment of a primary understanding of the hydantoin-hydrolysing enzyme system in A. tumefaciens RU-OR could be used to manipulate the hydantoin-hydrolysing activity in RU-OR cells to produce an improved biocatalyst. The isolation of A. tumfecaiens RU-OR genes encoding for hydantoin-hydrolysing activity revealed two separate N-carbamoyl-D-amino acid amidohydrolaseencoding genes (ncaR1 and ncaR2) in this bacterium with distinct chromosomal locations, nucleotide coding sequence and predicted primary amino acid sequence. The novel biocatalytic properties of the hydantoin-hydrolysing enzyme system in A. tumefaciens RU-OR and mutant derivatives present fascinating opportunities for further elucidation of the natural function, regulation and biocatalytic potential of hydantoin-hydrolysing enzymes.

The mechanism of glucoside hydrolysis by Agrobacterium β-glucosidase has been investigated through the study of linear free energy relationships and a-secondary deuterium kinetic isotope effects. A two-step mechanism has previously been proposed for this process, consisting of; (1) cleavage of the glucosidic bond and formation of a covalent glucosyl-enzyme intermediate ("glucosylation"), and (2) hydrolysis of the intermediate to yield free enzyme and glucose ("deglucosylation"). Values of kcat and Km were determined for enzymic hydrolysis of fifteen substituted phenyl β-D-glucopyranosides with leaving group pKa's ranging from 3.96 to 10.34. A linear free energy correlation of log(kcat) vs. leaving group pKa resulted in a concave-downward plot with a break near pKa 8, indicating a change in rate-determining step of a multistep reaction. The rates of hydrolysis of substrates with leaving group pKa's < 8 are independent of phenol structure, indicating that deglucosylation is rate-limiting. Glucosides with leaving group pKa's > 8 exhibit a linear dependence of hydrolysis rate upon pKa, and thus it is proposed that glucosylation is the rate-determining step for these substrates. The value of the Hammett reaction constant, ρ, is 1.6, indicating that cleavage of the glucosidic bond is significantly advanced at the transition state. The α-secondary deuterium kinetic isotope effects on hydrolysis of five substituted phenyl β-D-glucopyranosides were determined (deuterium substitution at the anomeric center), and the values were found to be segregated into two groups. The faster substrates (leaving group pKa < 8) exhibited kH/kD values of approximately 1.11, while values for the slower substrates averaged 1.06. These results support the hypothesis of a change in rate-determining step as leaving group pKa decreases. The magnitude of the isotope effect on glucosylation indicates a small amount of sp³ to sp² rehybridization at the transitionstate, which combined with the ρ value for this process suggests a substantial degree of nucleophilic participation of the enzymic carboxylate. 2-Deoxy-2-fluoro-D-glucosides with highly activated leaving groups are potent covalent inactivators of Agrobacterium β-glucosidase which operate by trapping the enzyme as its glucosyl-enzyme intermediate. Values of ki and Ki were determined for six substituted phenyl 2-deoxy-2-fluoro-β-D-glucopyranosides whose leaving group pKa's ranged from 3.96 to 7.18. A linear correlation was observed for both log(ki) and log(ki/Ki) vs. leaving group pKa, with ρ values of 2.0 and 2.7, respectively, which indicates that cleavage of the glycosidic bond is virtually complete at the transition state. Such an observation of a linear free energy relationship between the rate of enzyme inactivation and the electronic structure of the inactivator is rarely accomplished in enzymology. Preliminary investigation of the α-secondary deuterium kinetic isotope effect on enzyme inactivation by 2',4'-dinitrophenyl 2-deoxy-2-fluoro-β-D-glucopyranoside indicates that the effect is quite small, probably 0-5%. These results suggest that the inactivation proceeds via an essentially concerted mechanism and that the transition state has little oxocarbonium ion character. / Science, Faculty of / Chemistry, Department of / Graduate

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