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Made available in DSpace on 2014-06-11T19:32:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-08Bitstream added on 2014-06-13T20:04:39Z : No. of bitstreams: 1 terrasan_crf_dr_rcla.pdf: 2923287 bytes, checksum: 6f131c106722e1d7349212e5619e3b4f (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Xilanases e β-xilosidases são as principais enzimas responsáveis pela degradação da xilana, o segundo principal constituinte da parede celular vegetal. Tanto a produção destas enzimas por micro-organismos, quanto a caracterização bioquímica das mesmas têm sido amplamente estudadas devido às suas inúmeras aplicações biotecnológicas. Neste trabalho, as principais xilanase e β-xilosidase produzidas por uma linhagem de Penicillium janczewskii em meio de cultura líquido foram purificadas por métodos clássicos de purificação de proteínas, sendo, posteriormente, caracterizadas bioquimicamente. A xilanase apresentou atividade ótima em pH 6,0 e a 65 °C e a β-xilosidase em pH 5,0 e a 75 °C. Ambas as enzimas apresentaram características interessantes principalmente com relação à atividade ótima de ambas as enzimas em elevadas temperaturas, a prolongada estabilidade da β-xilosidase a 60 °C, e a estabilidade da xilanase em pH mais alcalinos, considerando-se algumas de suas possíveis aplicações. Visando uma futura aplicação, a produção destas enzimas foi avaliada em cultivos sólidos utilizando-se como substrato o bagaço de malte, resíduo da indústria cervejeira. As melhores condições para produção enzimática foram: utilização da umidade inicial do substrato de 50% fornecida com solução de sais de Vogel, tempo de cultivo de sete dias a 25 °C para produção de xilanases e a 20 °C para produção de b-xilosidases. O material fermentado apresentou aumento no teor protéico, na quantidade de alguns aminoácidos essenciais e ausência de micotoxinas. Ainda com um enfoque ambiental o filtrado de cultura de P. janczewskii, produzido em condições anteriormente selecionadas, foi aplicado no biobranqueamento de uma polpa kraft de eucalipto pré-branqueada com oxigênio, sendo realizados ensaios para seleção da concentração de xilanases e do tempo de reação / Xylanases and β-xylosidases are the main enzymes responsible for the degradation of xylan, the second main constituent of plant cell walls. The production of these enzymes by microorganisms, and their biochemical characterization has been extensively studied due to their wide range of biotechnological applications. In this work, the main xylanase and β-xylosidase produced in liquid cultures by a Penicillium janczewskii strain were purified by classical methods of protein purification, and further biochemically characterized. The xylanase showed optimal activity at pH 6.0 and 65 °C and β-xylosidases at pH 5.0 and 75 °C. Considering some possibilities of applications, the enzymes presented interesting characteristics especially in relation to the optimal activity at high temperatures, the prolonged stability of the β-xylosidase at 60 °C, and the stability of the xylanase in alkaline pH. Aiming at a future application, the production of these enzymes was investigated in solid state fermentation using brewer’s spent grain, a residue of the brewing industry, as substrate. The optimized conditions were: 50% initial substrate moisture supplied by Vogel’s salt solution, culturing for 7 days at 25 °C for xylanase production and at 20 °C for b-xylosidase production. The fermented substrate showed increase in protein content, in the amount of some essential amino acids, and absence of mycotoxins. Maintaining the environmental focus, the P. janczewskii crude filtrate, produced under previously selected conditions, was applied in the biobleaching of eucalyptus kraft pulp pre-bleached with oxygen. Trials were conducted for the selection of xylanase concentration and reaction time

In the stamen (male reproductive tissue) of petunia 'Hurrah' flowers, the occurrence of SOD (superoxide dismutase) provided an effective anti-oxidative mechanism against superoxide production. Superoxide production and SOD activities at five developmental stages showed a positive correlation. The highest superoxide production and SOD activity in different parts of the stamen (anther, filament and pollen) were at stages with high metabolic activity: (i) during growing buds (in anthers and filaments) (ii) when flowers with predehiscent anthers were fully open (in pollen). In all parts of the stamen, SOD activity was the lowest at stage five (fully open flowers with dehiscent anthers), superoxide production was also lower at this stage with the exception of the pollen. The highest SOD activity was localized in anthers with the pollen, suggesting that the filaments only have a structural support function. SOD was examined on a native PAGE with regard to the isozymes present within the stamen of five developmental stages. Three isozymes, which were identified as Mn SOD, Fe SOD and Cu/Zn SOD by reactions with inhibitors, were commonly found at five developmental stages in crude extracts of anthers, filaments and pollen. The developmental stages with stronger isozyme bands on the native PAGE were consistent with the stages with higher SOD activities, and the Mn SOD and Fe SOD isozyme bands were more intense than Cu/Zn SOD bands, suggesting the activities of Mn SOD and Fe SOD in the crude extracts were much higher than Cu/Zn SOD. SOD from 1,000 stamens of dehiscent mature flowers was partially purified using ammonium sulphate fractionation and DEAE cellulose column chromatography. The purified bound fraction contained only one SOD isozyme on a native PAGE, which was shown to be a Mn SOD, as it is sensitive to neither hydrogen peroxide nor cyanide. The specific activity of the purified SOD was 66.5 U/mg and the yield of total activity was 3.0%. The progress of enzyme purification was monitored using SDS-PAGE and the bound fraction contained two major polypeptide bands. The purified enzyme activity was optimal in the range of neutral pH, but it was the highest at pH 7.8. Through incubation at various pH levels for 24 hours, favourable stability of the purified fraction was confirmed around a pH range of 7 to 8.5. The purified enzyme retained 87% of its initial activity at -20 ? after one month of storage, but at 4 ? only 38% of the initial activity remained after the same period of storage.

In the stamen (male reproductive tissue) of petunia 'Hurrah' flowers, the occurrence of SOD (superoxide dismutase) provided an effective anti-oxidative mechanism against superoxide production. Superoxide production and SOD activities at five developmental stages showed a positive correlation. The highest superoxide production and SOD activity in different parts of the stamen (anther, filament and pollen) were at stages with high metabolic activity: (i) during growing buds (in anthers and filaments) (ii) when flowers with predehiscent anthers were fully open (in pollen). In all parts of the stamen, SOD activity was the lowest at stage five (fully open flowers with dehiscent anthers), superoxide production was also lower at this stage with the exception of the pollen. The highest SOD activity was localized in anthers with the pollen, suggesting that the filaments only have a structural support function. SOD was examined on a native PAGE with regard to the isozymes present within the stamen of five developmental stages. Three isozymes, which were identified as Mn SOD, Fe SOD and Cu/Zn SOD by reactions with inhibitors, were commonly found at five developmental stages in crude extracts of anthers, filaments and pollen. The developmental stages with stronger isozyme bands on the native PAGE were consistent with the stages with higher SOD activities, and the Mn SOD and Fe SOD isozyme bands were more intense than Cu/Zn SOD bands, suggesting the activities of Mn SOD and Fe SOD in the crude extracts were much higher than Cu/Zn SOD. SOD from 1,000 stamens of dehiscent mature flowers was partially purified using ammonium sulphate fractionation and DEAE cellulose column chromatography. The purified bound fraction contained only one SOD isozyme on a native PAGE, which was shown to be a Mn SOD, as it is sensitive to neither hydrogen peroxide nor cyanide. The specific activity of the purified SOD was 66.5 U/mg and the yield of total activity was 3.0%. The progress of enzyme purification was monitored using SDS-PAGE and the bound fraction contained two major polypeptide bands. The purified enzyme activity was optimal in the range of neutral pH, but it was the highest at pH 7.8. Through incubation at various pH levels for 24 hours, favourable stability of the purified fraction was confirmed around a pH range of 7 to 8.5. The purified enzyme retained 87% of its initial activity at -20 ? after one month of storage, but at 4 ? only 38% of the initial activity remained after the same period of storage.

In this thesis, on the basis of the asymmetrical charge distribution of E. coli topoisomerase I, I developed a new rapid procedure to purify E. coli DNA topoismoerase I in the milligram range. The new procedure includes using both cation- and anion-exchange columns, i.e., SP-sepharose FF and Q-sepharose FF columns. E. coli topoisomerase I purified here is free of nuclease contamination. The kinetic constants of the DNA relaxation reaction of E. coli DNA topoisomerase I were determined as well. I also used isothermal titration calorimetry to investigate the energetics of DNA supercoiling by using the unwinding properties of DNA intercalators, ethidium and daunomycin. After comparing the enthalpy changes of these DNA intercalators binding to supercoiled and nicked or relaxed plasmid DNA pXXZ06, I determined the DNA supercoiling enthalpy is about 12 kcal/mol per turn of DNA supercoil, which is in good agreement with the previously published results.