Orientador: Jose Augusto Rosario Rodrigues / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-07T19:55:40Z (GMT). No. of bitstreams: 1 Zampieri_LuizArthur_M.pdf: 1219190 bytes, checksum: 50f1c90b246b806bd16bb30df4ecbbaf (MD5) Previous issue date: 2006 / Resumo: Neste trabalho estudou-se a bioxidação do sesquiterpeno valenceno (C15H24), produzindo o bioflavorizante nootkatona (C15H22O), buscando as condições ótimas para obtenção do máximo rendimento. Esta reação foi realizada utilizando dois sistemas enzimáticos distintos: o sistema lacase/mediador (LMS) de Trametes versicolor com mediador HBT (hidroxibenzotriazol) ou TEMPO (tetrametilpiperidin-N-oxil) e o sistema utilizando o complexo enzimático do citocromo P-450 de Chaetomium globosum. Outros microorganismos testados foram Botrytis cinerea, Mortierella isabellina e Mortierella ramaniana. As diversas variáveis (pH, tempo de reação, concentrações de enzima, mediadores e indutores, condições de aeração, entre outras) envolvidas nas respectivas reações foram estudadas através de planejamento fatorial e modelagem de superfície de resposta. A utilização do sistema LMS de Trametes versicolor mostrou ser uma ferramenta viável para obtenção de nootkatona a partir de valenceno, embora tenhamos obtido rendimento inferior (17%, agitador orbital em pequena escala e 15 % em escala preparativa, sob aeração externa) descrito na literatura (25%, sob aeração, escala preparativa), o que torna nosso procedimento pouco viável para utilização em maior escala, apesar disso, os resultados foram condizentes com os obtidos em reações semelhantes descritos na literatura científica, onde o rendimento de sistemas LMS dificilmente ultrapassa os 15%. O sistema enzimático CYP-450 apresentou rendimento inferior ao sistema lacase/mediador e, este último sistema, HBT mostrou ser um mediador mais eficiente que TEMPO. / Abstract: In this work, we study the sesquiterpene valencene bioxidation (C15H24), which produces the nootkatona biological flavor substance (C15H22O), in an attempt to achieve the best conditions for optimum yield. This reaction was carried out using two different enzymatic systems: the Trametes versicolor laccase-mediator system with HBT mediators (hydroxybenzotriazol), or TEMPO (tetramethyl-piperidine-N-oxide), and the system using the Chaetomium globosum cythocrome P-450 enzymatic complex. Other microorganisms were tested, such as Botrytis cinerea, Mortierella isabellina and Mortierella ramaniana. The different variables involved in the respective reactions were studied by means of factorial planning and modeling the response system. The use of the Trametes versicolor LMS system proved to be a viable tool to obtain nootkatone from valencene, although we have obtained an inferior yield (17% with orbital agitator in small scale and 15% in preparatory scale, under external aeration) in comparison with the highest value described in literature (25% under aeration). Thus, our procedure presents little viability for large scale use, although results were in agreement with those obtained in similar reactions described in scientific literature, in which the yield produced by LMS systems rarely exceeds 15%. The CYP-450 enzymatic system presented lower yield in comparison with the laccase-mediator system and in the latter, HBT turned out to be more efficient than TEMPO. / Mestrado / Quimica Organica / Mestre em Química

This study investigated the natural durability of 8 and 60 year old Eucalyptus bosistoana (coast grey box). The sample’s heartwood compounds were extracted with an optimised extraction process and then incorporated into agar. Trametes versicolor (white rot) and Gloeophyllum trabeum (brown rot) fungi were grown upon these agars and their growth rate was used to assess the fungicidal abilities of the extracts. The extraction method of cell wall compounds was optimised. An Accelerated Solvent Extraction system (ASE) was used with the following settings: • 2 cycles per sample • 70°C extraction temperature • 50% rinse • 5 minute static time Ethanol was found to extract the compounds of the highest fungicidal activity. Ethanol was found to extract similar amounts to water (~13% of dry weight for a 60 year old sample), however analysis of both water and ethanol extracts with a FTIR spectrometer, found that they were of different chemical composition. A difference in fungicidal activity of extracts was found between the 8 year old and 60 year old samples. There was a large difference in the percentage of extracts present between the samples as well as the type of compounds present, shown by FTIR.

Under cellulolytic conditions, the white-rot fungus Trametes versicolor produces cellobiose dehydrogenase (CDH), an enzyme with a number of biochemical properties that are potentially relevant to the degradation of lignin and cellulose. To clarify its biochemical properties, CDH was purified from cultures of T. versicolor. Two isoforms of CDH were found: a 97 kDa isoform with both heme and flavin cofactors, and an 81 kDa isoform with a flavin cofactor. Both isoforms of CDH were found to be quite non-specific in their reductive half reactions. The flavin enzyme catalyzed many of the same reactions as the heme/flavin enzyme, but less efficiently. The flavin isoform reduced Fe(III) and Cu(II) only at concentrations well above those found physiologically. Thus the heme/flavin enzyme, but not the flavin enzyme, could be involved in promoting and sustaining the generation of hydroxyl radicals (·OH) by Fenton's chemistry. / To characterize further the structural features of CDH, a genomic clone was isolated and sequenced. CDH was found to consist of 748 amino acids, without its predicted 19 amino acid signal peptide. Consistent with the domain structure of other CDHs, T. versicolor CDH appeared to be divided into an amino terminal heme domain and a carboxy terminal flavin domain, connected by a hydroxyamino acid-rich linker. Within the flavin domain, a putative cellulose-binding domain (CBD) was found by alignment to the hypothesized CBD of P. chrysosporium CDH. The CBD of CDH appeared to be structurally unrelated to other CBDs which have been reported. / A cDNA clone encoding T. versicolor CDH was isolated by RT-PCR. Using this clone, three vectors for the heterologous expression in Aspergillus oryzae of CDH were prepared. These vectors were built by performing in-frame fusions of the cDNA to control sequences from the highly expressed A. oryzae amylase gene. These vectors were transformed into A. oryzae and one strain was isolated which contained the expression construct DNA. / A rapid method for cloning cdh-like genes was developed. Using short stretches of amino acids completely conserved within T. versicolor and P. chrysosporium CDH, PCR primers were designed to amplify a homologous gene from other fungi. The primers were tested using genomic DNA of Pycnoporus cinnabarinus. A 1.8-kb fragment of P. cinnabarinus cdh was thereby amplified and cloned, and its sequence was determined. The three CDHs displayed very high homology at the amino acid level. / Finally, to probe the role of CDH in lignocellulose degradation by T. versicolor, a "knockout" vector was constructed consisting of a phleomycin-resistance cassette inserted into the protein coding sequence of cloned T. versicolor cdh. T. versicolor was transformed with the knockout vector and the transformants were analyzed for their CDH-producing phenotype. Three isolates were found that produced no detectable CDH. Biobleaching and delignification by the CDH(-) strains appeared to be unaffected, suggesting that CDH does not play an important role in these processes.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1965. / Typescript. Vita. Includes bibliographical references (leaves 121-127).

Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2008 Nov 30

Made available in DSpace on 2014-06-11T19:35:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-08-19Bitstream added on 2014-06-13T20:07:23Z : No. of bitstreams: 1 pietrobon_tao_dr_rcla.pdf: 1539030 bytes, checksum: e0afbbf1e01319cce93f6e55e70f4326 (MD5) / A espécie Polistes versicolor tem se mostrado um excelente modelo para o estudo com vespas eussociais primitivas neotropicais, pela distribuição ampla, número de trabalhos já realizados com ela, tipo de organização social e por pertencer a um gênero considerado chave na compreensão da evolução do comportamento social. O presente trabalho apresenta os resultados do estudo comportamental, associado à análise histoquímica da glândula ectomandibular e à descrição do processo de morte celular programada desta glândula. Apresenta também nota a respeito de infecção viral nas células epiteliais próximas à glândula ectomandibular desta vespa. Polistes versicolor apresenta colônia monogínica, em qualquer fase do desenvolvimento colonial. As subordinadas apresentam especialização momentânea, sendo que todos os indivíduos são capazes de desenvolver qualquer atividade, independente de idade ou casta. Os machos apresentam comportamentos de cuidado com a prole e manutenção do ninho, porém em freqüências bastante baixas. Os indivíduos de uma colônia podem ser divididos em 3 grupos de acordo com a função que desempenham. Estes grupos são variáveis de acordo com a fase do desenvolvimento da colônia. A glândula ectomandibular de P. versicolor apresenta proteínas, lipídios e glicoconjugados neutros, tanto em sua constituição, quanto no produto sintetizado, porém em concentrações que variavam individualmente, de acordo com a fase do desenvolvimento da colônia. A variação dos compostos da secreção pode estar relacionada a 2 densidade populacional no ninho, de adultas e/ou imaturos. Esta glândula apresenta sinais de início do processo de morte celular em indivíduos entre 20 e 25 dias de idade, apesar de observar-se muitas variações, indicando não haver um ciclo etário rígido. A morte celular programada desta glândula apresenta características apoptóticas... . / The species Polistes versicolor is known as an excellent model for the study of neotropical wasps because of its wide distribution, number of papers on it, kind of social organization and also because the genus Polistes is considered essential to the understanding of the evolution of the social behaviour. This work shows the results of behavioural study, associated to histochemical analysis of the ectal mandibular gland and the description of its programmed cell death process. It also presents a note on a viral infection found in the epithelial cells of this wasp. In any phase of its colonial development, P. versicolor, shows monoginic colonies. The subordinates present momentary specialization, being all individuals able to develop any activity, independently of their age or caste. The males extend a care behaviour towards the progeny and the nest, however this behaviour is not so frequent. Copulation between possible siblings were observed. The histochemical pattern of ectal mandibular gland evidenced the presence of proteins, lipids and neutral glicoconjugates in concentrations that vary according to the colony development. The variation in the secretion compounds may be related to the population density of adults and/or immature individuals in the nest. Signs of the programmed cell death process were observed in individuals among 20 and 25 days old, although many variations were observed, indicating that there was not a strict age cycle. The programmed cell death characteristic found in this gland renders apoptotical and non-apoptotical features, like observed in different adult insects. Finally, a case of viral infection by Nucleopolyhedrovirus in epithelial 4 cells, near the ectal mandibular gland, and haemocytes, probably derived from its preys, was described with a discussion about probable infection ways.

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Parasitic weeds of the family Orobanchaceae attach to the roots of host plants via haustoria capable of drawing nutrients from host vascular tissue. Species in this family span the spectrum of host nutrient dependency, allowing comparisons that provide insight into parasite adaptation. A key aspect of this is the relationship between parasite metabolism and the metabolite profile of its host. To what extent does the metabolite profile of the parasite depend on that of the host? Do parasites that differ in host-dependency also differ in their metabolism or do they use common metabolic strategies? These questions were addressed using comparative profiling of primary metabolites to gain insight into carbon and nitrogen assimilation by the obligate holoparasite Phelipanche aegyptiaca and the facultative hemiparasite Triphysaria versicolor. First, metabolite profiles of these parasites and their hosts were compared during the key life stages before and after haustorial attachment. Second, the impact of specific variations in host metabolism was analyzed for P. aegyptiaca growing on Arabidopsis thaliana hosts that had mutations in amino acid metabolism but otherwise identical genetic backgrounds. Comparison of P. aegyptiaca and T. versicolor metabolite profiles identified substantial differences in the stages spanning the transition from pre-haustorial development through post-haustorial feeding. Each parasite species is distinct from the other and from their hosts. For parasites growing on host lines that differ in amino acid content, the size of P. aegyptiaca tubercles decreased when grown on the aap6 mutant line, which has decreased levels of asparagine in the phloem sap compared to the wild type. However, altered amino acid levels in other lines did not impact P. aegyptiaca growth, indicating that this parasite has ability to compensate for variation in host metabolic composition. This research highlights the importance of aspartate and asparagine to early post-attachment metabolism in both P. aegyptiaca and T. versicolor and through host deficiencies possibly associated with decreased growth in P. aegyptiaca. Overall, this work provides insights both into the metabolism of parasitic plants and lays the foundation for the development of new metabolism-based control strategies. / Ph. D. / Parasitic weeds of the plant family Orobanchaceae attach to the roots of host plants via haustoria. Parasite haustoria embed into the host plant and are capable of drawing nutrients from host vascular tissue. Species in this family span the spectrum of the extent to which a parasitic plant may depend on its host for nutrients. This allows comparisons that provide insight into the ways in which parasites adapt. A key aspect of this is the relationship between the metabolite profile of the parasite and the metabolite profile of the host. To what extent does the metabolite profile of the parasite depend on that of the host? Do parasites that differ in host-dependency also differ in their metabolism or do they use common metabolic strategies? These questions were addressed using comparative profiling of primary metabolites to gain insight into carbon and nitrogen assimilation by the obligate parasite Phelipanche aegyptiaca (which cannot perform photosynthesis) and the facultative parasite Triphysaria versicolor (which can perform photosynthesis). First, metabolite profiles of these parasites and their hosts were compared during the key life stages before and after haustorial attachment. Second, the impact of specific variations in host metabolism was analyzed for P. aegyptiaca growing on Arabidopsis thaliana hosts. These hosts had mutations in enzymes related to amino acid metabolism but otherwise identical genetic backgrounds. Comparison of P. aegyptiaca and T. versicolor metabolite profiles identified substantial differences in the stages spanning the transition from pre-haustorial development through post-haustorial feeding. Each parasite species is distinct from the other and from their hosts. For parasites growing on host lines that differ in amino acid content, the size of P. aegyptiaca tubercles decreased when grown on the aap6 mutant line, which has decreased levels of asparagine in the phloem sap compared to the wild type. However, altered amino acid levels in other lines did not impact P. aegyptiaca growth, indicating that this parasite has ability to compensate for variation in host metabolic composition. Overall, this work provides insights both into the metabolism of parasitic plants and lays the foundation for the development of new metabolism-based control strategies.

Este trabalho procurou avaliar algumas isozimas envolvidas com o processo de degradação de polissacarídeos da parede celular vegetal, encontradas em Aspergillus versicolor, fungo pertencente a um gênero onde a multiplicidade de componentes enzimáticos com a mesma atividade oscila de uma a três. Duas ß-xilosidases foram purificadas por meio de DEAE-celulose e precipitação com sulfato de amônia. Ambas mostraram-se enzimas que, quando deglicosiladas por PNGaseF, apresentam mesmos mapas trípticos. A glicosilação mostrou-se importante para a manifestação de diferenças bioquímicas relacionadas às interações com ambiente eletrolítico adjacente, visto as mudanças das curvas de pH e alterações de comportamento frente a sais de íons metálicos, tão bem como para a manutenção do funcionamento das enzimas. Também foi verificado que as diferentes formas glicosiladas periplásmicas são produzidas em meios distintos (xilana e xilose), cujos pH finais corresponderam aos pH das enzimas encontradas. Por meio de zimogramas, verificou-se que estas não eram produzidas de imediato, mas selecionadas ao longo do tempo de cultivo. Esta seleção foi inicialmente independente do pH, visto este, mesmo tamponado, ser corrigido pelo fungo, de modo a se apresentar, ao final de 48h, correlato com o da enzima selecionada. Exame cromatográfico em Concanavalina-A das enzimas deglicosiladas por EndoH mostraram que a oriunda de xilana tem aporte maior de ramificações biantenárias, que são ricas em galactose. Ambas apresentaram mapas trípticos iguais. A ß-xilosidase induzida por xilana apresentou proporções de manose, galactose e glucose iguais a 69,68 : 30,25 : 0,056 %, respectivamente, enquanto que ß-xilosidase induzida por xilose apresentou proporções iguais a 85,35 : 14,54 : 0,094%, respectivamente, resultado que corrobora com a resultado anterior. Duas ß-glucosidases de superfície micelial foram purificadas por meio de DEAE-Sephacel e DEAE-celulose. Ambas mostraram-se mais semelhantes entre si que as ß-xilosidases, e independentes de fonte de carbono ou pH. No entanto, apresentaram diferenças frente à inibição por celobiose (acentuada a partir de 10mM para ß-glucosidase I e 20mM para B-glucosidase II) e, embora sutis, a pH. Após serem submetidas a 45 ºC por 30min (temperatura que induziu segunda conformação estável) mostraram curvas de pH muito distintas, que foram eliminadas nas enzimas submetidas ao mesmo experimento após deglicosilação por PNGaseF. Ambas apresentaram mapas trípticos iguais. A ß-glucosidase I mostrou-se constituída de Man:Gal:Glu em proporções iguais a 78,36%:21,61%:0,033% do carboidrato total, respectivamente, e a ß-glucosidase II, Man:Gal:Glu iguais a 83,59%:16,38%:0,028%, respectivamente. Ambas também apresentaram sinergismo quando juntas. Sobre celobiose, foi verificado apenas em pH acima de 5.5. Sobre celooligossacarídeos, manifestou-se em pH 5,25. A ß-glucosidase I foi menos ativa que a ß-glucosidase II quando em ausência de glucose e celobiose na solução de reação inicial, situação na qual o contrário foi verdadeiro. O sinergismo foi drasticamente eliminado após deglicosilação por PNGaseF. Não se sabe se este resultado foi oriundo de mudanças cinéticas provocadas pela deglicosilação ou de uma possível eliminação de agregação anteriormente existente entre glicosiladas. O componente ß-glicosidásico extracelular foi purificado por meio de DEAE-Sephacel e Octyl-Sepharose, e se revelou um heteroagregado de fosfatase ácida com ß-glicosidase, que se mostrou estável e ativo em ampla gama de temperaturas e pH, com ótimos de 55ºC e 5,5, respectivamente. Entretanto, os perfis das curvas foram distintos entre as enzimas componentes. Somente concentrações superiores a 0,8mM de cloreto de cobalto apresentaram efeito desagregador, podendo ser empregado em conjunto com DEAE-celulose para purificação das enzimas isoladas. Quando separadas, fosfatase mostrou-se 260% mais ativa e ß-glicosidase, 50% menos ativa. Nesta condição, as faixas de pH se restringiram, acidificando-se, localizando-se entre pH 4,0 e 5,5. Aparentemente, a agregação reforça a atividade celobiásica. Fosfatase foi ativa sobre farelo de trigo e fitato de sódio e adsorveu fortemente em farelo de trigo. ß-glicosidase apresentou atividade sobre farelo de trigo, xilana e Avicel (liberando apenas glicose desta última), revelando-se enzima com atividade celobioidrolásica inespecífica, embora ávida por celobiose. As atividades foram determinadas como oriundas do mesmo sítio catalítico. ß-glicosidase não foi seqüestrada por farelo de trigo quando desagregada, resultado invertido quando reagregada. O seqüestro por farelo de trigo, aparentemente, deveu-se a interações entre sítio catalítico da fosfatase e substrato. / This study explored enzyme multiplicity on hemicelllulose and cellulose degrading systems. It first demonstrates the differences of PNGaseF deglycosylation and EndoH deglycosylation on forms of two ß-glicosidase activities present on surface of mycelia from Aspergillus versicolor grown on several carbon sources. Aspergillus versicolor produces ß-xylosidases with different biochemical properties and different degree of glycosylation, when grown on xylan or xylose. Were investigated the biochemical properties of these ß-xylosidases after deglycosylation. The purified enzymes were deglycosylated with endo-H or PNGase F. After this treatment both enzymes migrated faster in PAGE exhibiting the same Rf. On SDS-PAGE both enzymes showed similar migration. The optima temperature of xylan-induced and xylose-induced ß-xylosidases was 45 ºC and 40 ºC, respectively, and of 35 ºC after deglycosylation. The xylan-induced enzyme was more active at acidic pH than the xylose-induced enzyme. After deglycosylation the optimum pH of both enzymes was 6.0. The thermal resistance of the enzymes at 55 ºC showed a half-life of 15 min and 9 min for xylose-and xylan-induced enzymes, respectively. After deglycosylation both exhibited half-lives of 7.5. Native enzymes exhibited different response to ions, while deglycosylated enzymes exhibited identical sensitivity to ions. Limited proteolysis yielded coincident profiles in SDS-PAGE for both deglycosylated enzymes. All data suggest that the two A.versicolor ß-xylosidases share a common polypeptide core with differential glycosylation, apparently responsible for their biochemical and biophysical differences. Aspergillus versicolor also produced ß-glucosidases with different biochemical properties and different degree of glycosylation independently of carbon source. ß-Glucosidase I differed from ß-glucosidase II principally considering the amount and composition of carbohydrate, sensitivity to ions and pH. The purified enzymes shared the same tripitic maps and molecular masses after deglycosylations. All results showed that the biochemical differences observed for two enzymes were directly linked to PNGaseF- deglycosylation. Considering that Rfs, elution profiles on Con-A and residual glycosylation of both enzymes treated with EndoH or PNGaseF were the same, but differed on the mannose/galactose ratio, we inferred differences on proportion of hybrid-type/high-mannose-type glycans. The significance of this glycoform diversity was stressed in analysis of the action of mixture of both ß-glucosidases on celooligosoccharides and on cellobiose. This synergism was abolished after PNGaseF deglycosylation. These results are the first to show synergism between glycoforms of glycosil-hydrolases, representing a new class of synergistic type. The work also described a new form of aggregation between enzymes. Generally, ß-glycosidases are described as soluble components, attached to cell wall or free in the culture medium. This work verified that it could be extracelular adsorbed to wheat straw when aggregated with an acid phosphatase. The results strongly suggested that phosphatase is the component responsible for the process of adsorption on the substrate. The disaggregation was cobalt mediated, being not observed for another ions. The aggregation state has positive effects on glycosidase activity, extending pH ratio and increasing hydrolysis velocity. The opposite was found to phosphatase activity.