Tesi sul tema "Acremonium"

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior<br>Este trabalho descreve o estudo quÃmico de trÃs espÃcies do gÃnero Ananas (A. comosus, A. bracteatus e A. lucidus) e dos fungos endofÃticos Acremonium curvulum e Fusarium oxysporum, isolados de A. lucidus. A investigaÃÃo fitoquÃmica das raÃzes, folhas e talos de A. bracteatus levou ao isolamento de trÃs compostos micromoleculares: cumataquenina (1), glicosÃdeo do sitosterol (2) e sitosterol (3). Das folhas da espÃcie A. lucidus foram isolados a mistura de trÃs monoacilglicerois (4) e o 5-(hidroximetil)-2-furaldeÃdo (5). O estudo dos Ãcidos graxos das folhas e raÃzes de A. bracteatus e A. lucidus, bem como de A. comosus, A. bracteatus e A. lucidus cultivadas in vitro foi realizado. Vinte e um Ãcidos graxos, na forma de Ãsteres metÃlicos, foram identificados nas oito amostras analisadas, sendo 16 (76%) Ãcidos graxos saturados e 5 (24%) insaturados. A prospecÃÃo quÃmica do fungo A. curvulum levou ao isolamento dos compostos 2-hidroxipropanoato de feniletila (6), ergosterol (7) e triptofol (8). A analise dos extratos de F. oxysporum levou ao isolamento do composto tetra(S)-butirolactona (9) e do (4R*, 5S*)-5-hidroxihexan-4-olida (71%) e (4R*, 5R*)-5-hidroxihexan-4-olida (29%) (10), estes na forma de uma mistura diastereoisomerica. A determinaÃÃo estrutural dos metabÃlitos secundÃrios isolados foi realizada empregando-se as tÃcnicas espectromÃtricas de infravermelho, ressonÃncia magnÃtica nuclear de hidrogÃnio e carbono-13, incluindo tÃcnicas bidimensionais (COSY, HMQC e HMBC) e espectrometria de massa, alÃm de comparaÃÃo com dados descritos na literatura.<br>The chemical study of three Ananas species (A. comosus, A. bracteatus and A. lucidus) and the endophytic fungi Acremonium curvulum and Fusarium oxysporum, both isolated from A. lucidus, are described. The phytochemical investigations of roots, leaves and steams from A. bracteatus yielded the isolation of three micro molecular compounds: kumatekenin (1), glycosilated sitosterol (2) and sitosterol (3). From leaves of A. lucidus it was isolated a mixture of three monoacylglicerols (4) and 5-(hydroxymethyl)-2-furaldehyde (5). The fatty acid composition of leaves and roots from A. bracteatus and A. lucidus, as well as in vitro A. comosus, A. bracteatus and A. lucidus was investigated. Twenty-one fatty acids, as methyl ester derivatives, were identified in eight studied samples, 16 (76%) of them saturated and 5 (24%) unsaturated acids. From the fungus A. curvulum three compounds were isolated: phenetyl 2-hydroxypropionate (6), ergosterol (7) and tryptophol (8). The chemical investigation of the organic extracts from F. oxysporum led to the isolation of tetra(S)-butirolactone (9) and the diastereoisomeric mixture of (4R*,5S*)-5-hydroxyhexan-4-olide (71%) and (4R*,5R*)-5-hydroxyhexan-4-olide (29%) (10). Structure elucidation of the isolated compounds was done by spectrometric analysis such as infrared, 1H and 13C NMR, including bidimensional techniques (COSY, HMQC e HMBC), mass spectrometry as well as literature comparison.

Orientador: Gil Eduardo Serra<br>Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos<br>Made available in DSpace on 2018-07-23T22:32:53Z (GMT). No. of bitstreams: 1 Andrietta_MariadaGracaStupiello_D.pdf: 4521244 bytes, checksum: 4888bd9055f2da9976f68ec3bac3eafd (MD5) Previous issue date: 1998<br>Resumo: Este trabalho teve como objetivo estudar um meio de cultivo industrial para produção de cefalosporina C, utilizando a linhagem C-1 O de Cephalosporium acremonium (A TCC 11550) em experimentos conduzidos em frascos agitados. Foram testadas diferentes fontes de proteína padronizadas, de uso indicado para fermentações industriais, fornecidas pela QUEST INTERNACIONAL - Divisão SHEFFIELD, constituídas à base de soja (Hy-Soy), lacto albumina (Edamin'S), caseína (N-Z Amine A e N-Z Amine As), milho (Hydrolyse Com Gluten ) e algodão (Hydrolyse Cottonseed). As fontes protéicas foram adicionadas a um meio basal, em quantidade equivalente a 4g/1 de nitrogênio total. O meio de cultivo que apresentou uma maior produção de cefalosporina C ( 0,63 gll) foi formulado à base de soja, sendo o meio à base de caseína (N-Z Amine A) o que apresentou uma menor produção do antibiótico. A partir disto foi feito um estudo de cinética da produção de cefalosporina C com esses dois meios de cultivo. Os resultados confirmaram a soja como melhor substrato para produção do antibiótico e mostraram também, ser esta a matéria prima mais produtiva, uma vez que com 144 horas de fermentação houve a produção de 0,93 gll de cefalosporina C. A caseína precisou de 192 horas de fermentação para produzir apenas 0,64 gll do antibiótico. Para os dois substratos testados foi verificada ainda a degradação do antibiótico após o pico de máxima produção. Em uma segunda etapa do trabalho foi avaliada a associação de fontes protéicas na formulação do meio de cultivo. Além de Hy-Soy e Edamin'S, que foram as que apresentaram os melhores resultados na primeira fase, foi introduzido um extrato protéico padronizado, à base de soja (Samprosoy), que é produzido no Brasil pela SANTISTA ALIMENTOS divisão SAMBRA. Neste experimento foram formulados sete diferentes meios de cultivo contendo 4 g/l de N total. Os resultados mostraram um sinergismo na associação de Samprosoy, Hy-Soy e Edamin'S, com uma maior produção do antibiótico (1,43 g/l em 144 horas). Esta composição foi utilizada como ponto central (36 g/l de saca rose, 27 gI1 de glicose e 4 g/l de N total) na primeira fase da otimização do meio de cultivo utilizando planejamento fatorial completo de dois níveis e três variáveis. As variáveis estudadas foram a concentração de sacarose, glicose e nitrogênio, e a variável resposta foi a concentração de cefalosporina C no caldo fermentado. Os resultados obtidos mostraram que maiores concentrações de glicose e nitrogênio levam a um aumento na produção do antibiótico, sendo a variação da concentração de sacarose não significativa na produção da cefalosporina C. Em uma segunda fase da otimização do meio de cultivo o ponto central foi deslocado de modo que esse meio apresentasse maiores concentrações de glicose e nitrogênio (37 g/l de glicose e 6 g/l de N total) e menor quantidade de sacarose (26 g/l). Confirmou-se que altas concentrações de glicose (acima de 45 g/l) e nitrogênio (acima de 6 g/l) direcionam a um aumento na produção, e que a concentração de sacarose não influencia no aumento da produção do antibiótico. Em uma última etapa foi conduzida uma fermentação simultânea em fermentador e em frascos agitados para o estudo do efeito da aeração na produção de cefalosporina C. Parte do meio preparado e inoculado no fermentador foi transferido para frascos Erlenmayer. Os resultados mostram que no fermentador a quantidade de cefalosporina C produzida foi 2,82 vezes maior que as obtidas em frascos agitados. A diferença certamente está associada ao nível constante da concentração de oxigênio no meio de fermentação, possível de se controlar no fermentador, mas não em frascos agitados.<br>Abstract: The purpose of this work was to study an industrial growing medium for cephalosporin C production in a shaker, using the C-1 O strain of Cephalosporuium acremonium (A TCC 11550). Different sources of standard protein have been tested, all of which recommended for industrial fermentation, and provided by QUEST INTERNACIONAL¬SHEFFIELD Division, soy-based (Hy Soy), Lactoalbumin (Edamin'S), casein (N-Z Amine and N_Z Anime A), com (Hidrolyse Com Gluten) and cotton (Hydrolyse Cottonseed). The protein sources were added to a basic medium amounting to 4 g/l of the total nitrogen. The growing medium that reached the highest cephalosporin C production (0.63 g/l) was soy-based, and the medium that had the lowest amount of the antibiotic was the casein-based (N-Z Amine A). These two growing mediums were then used for a kinetic study of cephalosporin C production. The results have confirmed soybean as the best substratum for the antibiotic production as well as the most productive raw material, since there was a 0.93 g/l cephalosporin C production within 144 hours of fermentation. The casein took 192 hours of fermentation to produce only 0.64 g/l of the antibiotic. It has been observed for both substrata that there was a degradation of the antibiotic after the maximum production peak was reached. In the second stage of the trial, the association of protein sources in the growing medium formulation was evaluated. In the addition to Hy-Soy and Edamin'S, proteins that presented the best results in the first stage, a standard soy-based proteinic extract (Samprosoy) produced in Brazil by SANTISTA ALIMENTOS - SAMBRA division was introduced. Seven different growing mediums containing 4 g/l of total N were formulated. The results have showed a synergism in the association among Samprosoy, Hy-Soyand Edamin'S, with a higher antibiotic production (1,43 g/l within 144 hours). This composition was used as a central point (36 g/l sucrose, 27 g/l glucose and 4 g/l of total N) at the first optimization stage of growing medium a complete two-Level and three-variable factorial design. The variable studied was the sucrose, glucose and nitrogen concentration, and the response variable was the cephalosporin C concentration in the fermented broth. The results have shown that higher glucose and nitrogen concentration have led to an increase in the antibiotic production, even though the variation in sucrose concentration was insignificant for cephalosporin C production. In the second stage of growing medium optimization the central point was dislocated in order to present higher glucose and nitrogen concentrations (37 g/l of glucose and 6 g/l of N total) and lower sucrose concentration (26 g/l). It has been confirmed that high glucose (over 45 g/l) and nitrogen (over 6 g/l) concentration result in an increase in the production, and that sucrose concentration has no effect in the increase of antibiotic production. In the last stage, a simultaneous fermentation in a fermenter and in a shaker was performed for the purpose of studying the aeration effect in cephalosporin C production. Part of the medium prepared and inoculated in the fermenter was transferred to Erlenmeyer flasks. The results have shown that in the fermenter, the amount of cephalosporin C produced was 2.82 times higher than in the shaker. The difference is surely associated with the constant oxygen concentration level in the fermentation medium, which was possible to the controlled in the fermenter but not in the shaker.<br>Doutorado<br>Doutor em Tecnologia de Alimentos

Orientadores: Francisco Maugeri Filho, Gonçalo Amarante Guimarães Pereira<br>Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos<br>Made available in DSpace on 2018-08-20T20:25:34Z (GMT). No. of bitstreams: 1 Goldbeck_Rosana_D.pdf: 2685408 bytes, checksum: 40606034d66dbd81493dcd1bbec17eaa (MD5) Previous issue date: 2012<br>Resumo: O etanol vem novamente despertando de modo crescente a atenção de pesquisadores, empresas e governo, devido ao aumento do preço do petróleo e perspectivas de esgotamento das fontes não-renováveis de combustíveis fósseis, bem como, preocupações de natureza ambiental, relacionadas à emissão de substâncias que comprometem o meio ambiente. O estabelecimento de metas extremamente ambiciosas para aumento do consumo do etanol nos próximos anos requer um aumento substancial da produção de etanol e, nesse sentido, estimula a pesquisa e o desenvolvimento de tecnologias que permitem o uso de novas matérias-primas na produção de etanol, como a biomassa lignocelulósica. No entanto a ampliação desta tecnologia é limitante devido ao alto custo das enzimas, indicando desta forma a importância da busca por novas fontes de enzimas capazes de contribuir para este processo. Em face disto, este trabalho teve como objetivo estudar as propriedades lignocelulolíticas de micro-organismos silvestres isolados de diversas regiões brasileiras, bem como realizar um estudo genético visando à produção de bioetanol. Inicialmente foi realizada uma seleção das cepas que apresentaram capacidade de produção de celulases, a partir de micro-organismos silvestres isolados de diversas regiões do país. Após a seleção, a cepa nomeada AAJ6 foi selecionada como potencial produtor de celulases e identificada molecularmente como Acremonium strictum. Posteriormente, foram realizados estudos de recuperação, purificação e caracterização enzimática das enzimas produzidas pelo microorganismo em estudo. A precipitação com acetona 60% foi o método que resultou em melhores porcentagens de recuperação, registrando 80,67% de recuperação para as endoglucanases (CMCase), 65% para a atividade de papel de filtro (FPase) e 25% para celobiase. Em relação à purificação, a resina Q-Sepharose foi selecionada como mais eficiente para a purificação das enzimas do complexo celulase produzidas por Acremonium strictum. Quanto à caracterização enzimática, as faixas de temperatura e pH estudadas não tiveram diferença significativa (p<0,05) em relação à atividade de endoglucanase (CMCase). Já para a atividade de FPase e celobiase, a faixa temperatura ótima foi de 54 a 57 °C e o pH ótimo foi de 4,7. Para a b-glicosidase, apenas a temperatura foi significativa, favorecendo temperaturas mais elevadas (54 a 57 °C) para a atividade enzimática. Paralelamente conduziram-se fermentações para produção de celulases empregando diferentes substratos, tanto substratos comerciais (carboximetilcelulose, SERVACEL® e AVICEL®) como bagaços de cana-de-açúcar pré¿tratados com diferentes intensidades. O bagaço de cana submetido a um pré-tratamento leve (12 kgf/cm²; 188,5°C) foi o que melhor induziu o micro-organismo em estudo a produzir as maiores atividades celulolíticas em comparação aos demais substratos estudados, registrando valores máximos de CMCase de 134,42 U/L em 240 horas de fermentação, 10,82 U/L de FPase em 192 horas, 27,72 U/L de celobiase em 96 horas e 3,48 U/L de b-glicosidase em 240 horas. Com o avanço da biotecnologia e da biologia molecular, a identificação de genes presentes num determinado micro-organismo já se tornou essencial. Em face disto, foi realizado o sequenciamento 454 do genoma do Acremonium strictum e dois genes de celulases foram identificados, sendo um gene de endoglucanase da família 74a e um gene de b-glicosidase. Estas enzimas foram isoladas, sequenciadas e clonadas em E.coli através do vetor pGEM-T Easy de forma que futuros trabalhos possam abordar os produtos de expressão destas enzimas em Saccharomyces cerevisiae visando à produção de bioetanol de segunda geração<br>Abstract: Ethanol has increasingly attracted the attention of researchers, companies and governments due to the increase in oil prices and prospects of depletion of nonrenewable fossil fuels, as well as environmental concerns related to emissions of substances that compromise the environment. Excessively ambitious goals for the increased consumption of ethanol in the for the years ahead requires a substantial increase in ethanol production and, accordingly, encourages research and development of technologies that allow the use of new raw materials for ethanol production, such as lignocellulosic biomass. However the expansion of this technology is limited due to the high cost of enzymes, thus indicating the importance of searching for new sources of enzymes able to contribute to this process. In the face of this, the present work intended to study the lignocellulolytic properties of wild microorganisms isolated from various regions of Brazil as well as conducting a genetic study aimed at producing bioethanol. Initially a selection of strains that were capable of producing cellulases was carried out. Than, the the selected strain, named AAJ6 and molecularly identified as Acremonium strictum, was shown to be a potential producer of cellulases. Subsequently, studies were performed for recovery, purification and characterization of the enzymes produced by this microorganism. Precipitation with 60% acetone was the method that led to improved recovery percentages, about 80%, for the endoglucanases (CMCase), 65% for filter paper activity (FPase) and 25% for cellobiase. With regard to purification, choromatographic column with Q-Sepharose resin was selected as the most efficient for the purification of the cellulase enzyme complex produced by Acremonium strictum. As enzymatic characterization, the temperature and pH ranges studied did not differ significantly (p<0.05) compared to the activity of endoglucanase (CMCase). As for the cellobiase and FPase activity, the optimum temperature range was 54 to 57 °C and optimum pH was 4.7. For the b-glucosidase, only temperature was significant, favoring higher temperatures (54 to 57 °C) for enzyme activity. Parallel fermentations were conducted for cellulase production using different cellulosic substrates (carboxymethylcellulose, SERVACEL® and AVICEL ®) and sugarcane bagasse pretreated with different intensities. Bagasse that underwent t mild pretreatment (12 kgf/cm², 188.5 °C) was the best inducer for microorganism under study, and led to the highest cellulolytic activities, being the maximum values 134.42 U/L U/L for CMCase after 240 hours of fermentation, 10.82 U/L for FPase after 192 hours, 27.72 U/L for cellobiase after 96 hours and 3.48 U/L for b-glucosidase after 240 hours. At the current stage of biotechnology and molecular biology, the identification of genes present in a given micro-organism has become essential. In view of this, the 454 sequencing of the genome of Acremonium strictum was carried out and two cellulase genes were identified, being an endoglucanase of the family 74a gene and b-glucosidase gene. These enzymes were isolated, sequenced and cloned into E. coli using the pGEM-T Easy vector so that future work can address the expression products of these enzymes in Saccharomyces cerevisiae in order to produce second generation bioethanol<br>Doutorado<br>Engenharia de Alimentos<br>Doutora em Engenharia de Alimentos

The biological control of plant pests with beneficial microbes has become increasingly important over the last decades. Soil microbes such as fungi and bacteria colonise the roots of plants and promote their growth. Some beneficial microbes can trigger a weak plant defence response that enhances the immune response of the plant at subsequent pathogen attacks and therefore increase the resistance of the plant to other invaders. This mechanism is called “priming”. While biocontrol agents are applied against a variety of plant pests fundamental knowledge of the molecular mechanisms of plant-microbe interactions is still lacking. Especially molecular studies on the role of resistance genes in the interaction of plants with beneficial endophytic fungi are rare. In this study it was investigated how the fungal biocontrol agent Acremonium alternatum affects the development of the clubroot pathogen Plasmodiophora brassicae within the plant host Arabidopsis thaliana. Clubroot is a devastating disease in crop plants such as cabbage and rapeseed and causes abnormal root growth that leads to so called “club roots”. P. brassicae develops within the plant roots and forms resting spores that are very durable and stay infective in soils for up to 2 decades. The control of clubroot by chemical means is difficult and the disease continues to spread on all continents and was also found in Saxony, Germany in recent years. In 2 preliminary studies the co-inoculation of clubroot plants with the fungus A. alternatum resulted in reduced clubroot symptoms in Chinese cabbage and Arabidopsis. It was therefore hypothesised that A. alternatum induces resistance mechanisms in the plant and thus enhances immunity. The focus of this study was to test this hypothesis by carrying out expression analyses on root tissue of infected Arabidopsis plants. For this the plants were inoculated with spores of P. brassicae and A. alternatum before RNA was extracted from the roots, followed by cDNA synthesis and quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-qPCR). A microarray of root tissue of infected Arabidopsis plants was carried out to depict the events at the stage of initial root hair infection with the clubroot pathogen. The findings from the gene expression analyses were verified for 2 genes with Arabidopsis mutants that are defective in the respective gene and with 2 overexpressor lines. Clubroot symptoms were assessed by rating the root galls according to their stage of development. The overall plant health was further evaluated by recording the developmental stage of the plants (generative vs. vegetative), stem lengths and plant biomass. In addition, 2 local varieties of the economically important crop plant rapeseed (Brassica napus var. Ability and var. Visby) were investigated with qRT-PCR and by recording the disease parameters just described. A second goal of this study was to assess the general biocontrol potential of the yet relatively unknown endophyte A. alternatum in terms of enzymatic activity and competitive behaviour against other phytopathogenic fungi. The potential of this fungus for the use in integrative pest management was investigated. The results presented here are novel findings for this fungus and have not been studied before. The microarray from Arabidopsis roots revealed that the clubroot pathogen P. brassicae suppresses its recognition by pathogen receptors of the plant and thus prevents the host to induce resistance mechanisms. The fungus A. alternatum boosted the level of the pathogen recognition-related genes BAK1 and FLS2 and thus helped to establish early plant defence responses. PCR analyses confirmed that these early responses led to salicylic acid-dependent resistance in the plants which was maintained for several days as shown by elevated levels of the PATHOGENESIS-RELATED gene PR1. Marker genes for an alternative resistance pathway that is mediated over the plant signals jasmonate and ethylene were not activated in Arabidopsis. The co-inoculation of Arabidopsis plants with the endophyte A. alternatum resulted in a significant reduction of clubroot symptoms by up to 24%. In rapeseed the reduction of disease symptoms was 19% and 28% when the plants were treated with a crude cell wall extract of A. alternatum before inoculation with the clubroot pathogen. PCR analyses from Arabidopsis showed a strong response of pathogen recognition genes to the cell wall extract and spores of the endophytic fungus. In rapeseed all of the investigated pathogen recognition genes were upregulated after the endophyte treatment but not with the clubroot pathogen. Together with the PCR results from the microarray these findings suggest that A. alternatum primes its host plant and enhances the resistance of the plant towards P. brassicae. In addition, the fungus increased biomass, stem lengths and survival rates of clubroot-infected plants. In vitro tests revealed that the endophyte can solubilise phosphate and is not very competitive against other phytopathogenic fungi such as Aspergillus or Fusarium which is likely an effect of the relatively slow growth of the endophyte on agar plates. From this study it can be concluded that i) the fungus Acremonium alternatum induces resistance mechanisms in Arabidopsis and 2 Brassica napus cultivars and facilitates the recognition of the clubroot pathogen Plasmodiophora brassicae; ii) that Arabidopsis and Brassica react differently to this beneficial microbe, a fact that has been observed for Plasmodiophora and other microorganisms as well; iii) living spores are not necessary for clubroot biocontrol in rapeseed as a crude cell wall extract reduces symptoms more efficiently. Overall the endophyte A. alternatum is a very promising candidate for the use in integrative pest management in plant strengtheners or as biocontrol agent<br>Die biologische Kontrolle von Pflanzenkrankheiten gewinnt zunehmend an Bedeutung. Bodenbewohnende Mikroben wie Pilze oder Bakterien kolonisieren die Wurzeln von Pflanzen und fördern deren Wachstum. Einige dieser förderlichen Mikroben aktivieren eine schwache Abwehrreaktion in der Pflanze die sich verstärkt bei einer weiteren Infektion mit einem Krankheitserreger. Dieser Mechanismus, den man “Priming” nennt, führt zu einer verbesserten Resistenz der Pflanze gegenüber Pflanzenpathogenen. Obwohl natürliche Schädlingsbekämpfer bereits gegen eine Vielzahl an Krankheiten eingesetzt werden, weiss man über grundsätzliche molekulare Mechanismen dieser Pflanzen-Mikroben-Interaktionen nur wenig. Besonders die Rolle von Resistenzgenen ist bisher wenig erforscht, welche bei der Beziehung zwischen Pilzen und Pflanzen eine Rolle spielen. In der hier vorliegenden Arbeit wurde untersucht, wie der endophytische Pilz Acremonium alternatum die Entwicklung des Krankheitserregers Plasmodiophora brassicae in der Pflanze Arabidopsis thaliana beeinflusst. Die Kohlhernie, ausgelöst von P. brassicae, ist eine verheerende Krankheit die u. a. bei Kohl und Raps auftritt und Wurzelgallen, so genannte “Hernien”, hervorruft. Der Krankheitserreger entwickelt sich im Wurzelsystem der Pflanze und bildet Dauersporen, die bis zu 20 Jahre lang im Boden infektiös überdauern können. Ein Eindämmen der Krankheit mit Pflanzenschutzmitteln ist durch den komplexen Lebenslauf des Erregers sehr schwierig, das führte zu einer weltweiten Verbreitung der Kohlhernie. Auch in Sachsen wurden in den letzten Jahren Fälle von Kohlhernie gemeldet. Wie 2 Studien zeigen, führt die Ko-Inokulation von Kohlhernie-erkrankten Pflanzen mit A. alternatum zu einer Verringerung der Symptome in Chinakohl und Arabidopsis. Es wurde daher die Hypothese aufgestellt, dass der Pilz Resistenzmechanismen in der Pflanze anschaltet und damit ihre Immunität erhöht. Um diese Hypothese zu testen, wurden in der hier vorliegenden Studie Genexpressionsanalysen an infizierten Arabidopsiswurzeln durchgeführt. Dafür wurden die Pflanzen zunächst mit Sporen des Kohlhernieerregers und des Pilzes inokuliert, es wurde RNA aus den Wurzeln extrahiert, in cDNA umgeschrieben und diese mittels quantitativer Reverse-Transkriptase-Polymerasenkettenreaktion (RT-qPCR) untersucht. Ein Microarray von Wurzeln infizierter Pflanzen wurde durchgeführt um die Ereignisse abzubilden, die sich zeitnah nach der Infektion in den Wurzeln abspielen. Die Ergebnisse der Genexpressionsanalysen wurden dann an Arabidopsismutanten, die einen Gendefekt im jeweiligen Gen haben, und an Überexprimierer-Pflanzen verifiziert. Kohlherniesymptome an Pflanzen wurden durch eine Kategorisierung der Schadsymptome erfasst. Die allgemeine Pflanzengesundheit sowie der Entwicklungsstand der Pflanze, Stengellängen und das Frischgewicht wurden bestimmt. Zusätzlich wurden 2 Rapssorten, die in Sachsen angebaut werden, untersucht im Hinblick auf die Krankheitsenwicklung und die Reguation von Abwehrgenen. Ein weiteres Ziel dieser Arbeit war es das Biokontrollpotential des bisher schlecht untersuchten Pilzes A. alternatum zu bestimmen. Dazu wurde in vitro die Enzymaktivität des Pilzes getestet sowie seine Konkurrenzfähigkeit gegenüber anderen pflanzenpathogenen Pilzen. Das Potential des Pilzes für die Anwendung im integrierten Pflanzenschutz wurde getestet. Die hier präsentieren Ergebnisse stellen neue Erkenntnisse dar, die für diesen Pilz noch nie untersucht wurden. Der Microarray von Arabidopsiswurzeln zeigte, dass der Kohlhernieerregers die Erkennung durch die Pflanze verhindert und damit Abwehrmechanismen verhindert. Der Pilz A. alternatum förderte die Aktivität der pflanzlichen Erkennungsrezeptoren FLS2 und BAK1 und setzte damit die Erkennung von P. brassicae in Gang. PCR-Analysen ergaben, dass diese früh induzierten Abwehrmechanismen zu einer systemischen Resistenz in der Pflanze führte durch die Aktivierung des Pathogenese-relevanten Gens PR1. Genmarker, die die Aktivität eines alternativen, von Jasmonat und Ethylen vermittelten Abwehrweges anzeigen, waren nicht ativiert. Die Ko-Inokulation von Arabidopsis mit dem Endophyten führte zu einer signifikanten Reduktion der Krankheitssymptome um 24%. In Raps betrug die Reduktion 19% und 24% wenn die Pflanzen vor der Kohlhernie-Infektion mit einem Zellwandextrakt des Pilzes behandelt wurden. Mittels PCR konnte gezeigt werden, dass Gene für das Erkennen von Pathogenen in der Wurzel von Arabidopsis auf den Zellwandextrakt und Sporen des Pilzes reagieren. In Raps wurden alle der untersuchten Erkennungsgene aufreguliert nach der Infektion mit A. alternatum, nicht jedoch bei der Infektion mit P. brassicae. Zusammenfassend lässt sich sagen, dass der endophytische Pilz A. alternatum die Wirtspflanze auf eine folgende Infektion vorbereitet (Priming) und systemische Abwehr-mechanismen in der Pflanze induziert, wenn diese mit Kohlhernie infiziert ist. Außerdem treibt der Pilz das Sprosswachstum voran, erhöht die Biomasse und fördert das Überleben von Kohlhernie-infizierten Pflanzen. In vitro-Tests ergaben, dass der Endophyt Kalziumphosphat löslich machen kann und wenig kompetitiv gegenüber Pflanzenpathogenen wie Aspergillus oder Fusarium ist. Dies ist vermutlich mit dem langsameren Wachstum des Endophyten im Gegensatz zu den anderen Pilzen zu erklären. Aus den Ergebnissen dieser Arbeit lassen sich folgende Schlüsse ziehen: i) der endophytische Pilz Acremonium alternatum induziert Resistenzmechanismen in Arabidopsis und Raps und und fördert die Erkennung des Kohlhernieerregers Plasmodiophora brassicae; ii) Arabidopsis und Raps reagieren unterschiedlich auf diesen förderlichen Pilz, ein solcher Unterschied wurde bereits für Plasmodiophora und andere Mikroben beschrieben; iii) lebende Sporen des Pilzes sind nicht notwendig um Krankheitssymptome der Kohlhernie in Raps zu verringern, ein Zellwandextrakt von A. alternatum ist dafür besser geeignet. Ganz allgemein lässt sich sagen, dass der endophytische Pilz Acremonium alternatum ein sehr vielversprechender Kandidat ist für den Einsatz im integrierten Pflanzenschutz in Pflanzenstärkungsmitteln oder als Biokontrollorganismus

Os produtos naturais marinhos são apontados com uma das fontes de substâncias bioativas mais importantes para a descoberta de novos fármacos. Neste ambiente, os organismos estão em constante interação ecológica por meio da produção de metabólitos secundários. Fungos endofíticos e cianobactérias representam grupos de micro-organismos que realizam a biossíntese de substâncias com características químicas únicas e atividades biológicas potentes. Entretanto, quando retirados de seu habitat natural, esses seres microbianos geralmente perdem sua capacidade metabólica através de um fenômeno denominado silenciamento gênico, no qual genes biossintéticos deixam de ser transcritos devido a motivos ainda indeterminados. Esse mecanismo genético é intermediado, dentre outros fatores, pelas enzimas DNAmetiltransferase (DNA-MT) e Histona-desacetilase (HDAC). Deste modo, seus inibidores têm sido utilizados com sucesso para promover a eliciação de substâncias que não seriam produzidas em condições laboratoriais. Outra importante abordagem na pesquisa de produtos naturais têm sido a desreplicação baseada na fragmentação (MS/MS) para identificação de substâncias ou análogos. As redes moleculares (molecular networking) constituem uma nova abordagem na qual dados de espectrometria de massas são agrupados de acordo com as semelhanças entre os padrões de fragmentação, formando famílias de moléculas, permitindo a rápida visualização do perfil químico de várias amostras ao mesmo tempo. Deste modo, este trabalho apresenta o isolamento e identificação de metabólitos inéditos a partir de fungos endofíticos e cianobactérias oriundos do ambiente marinho. Para isto, técnicas de eliciação epigenética foram utilizadas em ambos os grupos de organismos, e a desreplicação via redes moleculares foi utilizada em cianobactérias. Fungos endofíticos associados à macroalga vermelha Bostrychia tenella foram alvo de estudos químicos e epigenéticos. As linhagens Xylaria sp. e Nigrospora oryzae foram submetidas ao cultivo em meio sólido arroz, o que resultou no isomento da substância citocalasina D e de um derivado potencialmente inédito da griseofulvina. A linhagem Penicillium decaturense foi cultivada em meio líquido PDB resultando no isolamento da 10,11-deidrocurvularina e possíveis análogos. Experimentos com inibidores epigenéticos (butirato de sódio e procaína) promoveram a modulação do perfil químico desta linhagem, ao estimular a produção de metabólitos não expressos em condições normais de cultivo. Ainda, a linhagem Acremonium sp. produziu várias substâncias quando cultivada em meio de líquido Czapek sob a influência de procaína, sendo uma delas potencialmente inédita e derivada da classe de metabólitos das brevianamidas. Frações orgânicas da cianobactéria Schizothrix sp., coletada no Panamá, foram analisadas em LC-MS/MS e os dados gerados foram utilizados para a criação de redes moleculares. Este estudo resultou na identificação dos metabólitos barbamida, hectoclorina, curacinas A e D, curazole, acetato de malingamida D, dolastatina 10 e carmaficina B. Ainda, análogos das substâncias curazole, dolastatina D e dois análogos inéditos das carmaficinas foram propostos. A cianobactéria Moorea producens JHB, coletada na Jamaica, foi submetida ao cultivo sob influência do ii composto butirato de sódio, e produziu dois metabólitos inéditos, propostos de acordo com os dados de fragmentação, como sendo derivados da jamaicamida e da hectoclorina, num tipo de biossíntese cruzada. Portanto, este trabalho confirma os fungos endofíticos e cianobactérias marinhos como promissores quanto a exploração do metabolismo secundário.<br>Marine natural products are pointed out as one of the most important sources of bioactive compounds for drug discovery. In this environment, organisms are in constantly interaction ecological through the production of secondary metabolites. Endophytic fungi and cyanobacteria represent groups of microorganisms that perform biosynthesis of substances with unique chemical features and potent biological activities. However, when removed from their natural habitat, these microbial beings generally lose their metabolic capacity through a phenomenon called gene silencing, in which biosynthetic genes are no longer transcribed due to reasons still undetermined. This genetic mechanism is brokered, among other factors, by the enzyme DNA methyltransferase (DNA-MT) and histone deacetylase (HDAC). Thus, their inhibitors have been used successfully to promote the elicitation of substances that would not be produced under laboratory conditions. Another important approach in the natural products research field have been dereplication based on the fragmentation (MS/MS) for the identification of substances or analogues. The molecular networking is a new approach in which data from mass spectrometry are grouped according to the similarities between the patterns of fragmentation, forming families of molecules, allowing rapid visualization of the chemical profile of several samples simultaneously. Thus, this work presents the isolation and identification of novel metabolites from endophytic fungi and cyanobacteria originating from the marine environment. For this propose, epigenetic elicitation techniques were used in both groups of organisms and the molecular networks via dereplication was used in cyanobacteria. Endophytic fungi associated with red seaweed Bostrychia tenella were subjected to chemical and epigenetic studies. Xylaria sp. and Nigrospora oryzae strains were cultured in solid medium rice, resulting in isolation of substance of cytochalasin D and a potentially novel derivative of griseofulvin. Penicillium decaturense strain was grown in PDB liquid medium resulting in the isolation of 10,11- deidrocurvularina and possible analogues. Experiments with epigenetic inhibitors (sodium butyrate and procaine) promoted the modulation of the chemical profile of this strain, to stimulate the production of metabolites not expressed under normal culture conditions. Moreover, Acremonium sp. produced various substances when grown in liquid medium under the influence of Czapek procaine, one of novel and potentially derived from the class of metabolites brevianamides. Organic fractions of the cyanobacteria Schizothrix sp., collected in Panama, were analized by LC-MS/MS and the data generated were used to create molecular networks. This study resulted in the identification of metabolites barbamide, hectochlorin, curacins A and D, curazole malyngamide D acetate, dolastatin 10 and carmaphycin B. Also, analogs of curazole, dolastatin 10 and carmaphycins A and B have been proposed. Cyanobacteria Moorea producens JHB, collected in Jamaica, was grown under the influence of sodium butyrate, and produced two new proposed metabolites in accordance with the fragmentation data as being derived from jamaicamide and hectochlorin, in a sort of crossed biosynthesis. Therefore, this work corroborates marine endophytic fungi and cyanobacteria as promising for exploration of secondary metabolism.

RODRIGUES, A. C. P. Estudo Químico de Ananas (A. comosus, A. bracteatus e A. lucidus) e dos Fungos Endofíticos (Acremonium curvulum e Fusarium oxysporum) isolados de Ananas lucidus. 2009. 180 f. Tese (Doutorado em Química Orgânica) - Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2009.<br>Submitted by José Jairo Viana de Sousa (jairo@ufc.br) on 2014-10-08T20:05:35Z No. of bitstreams: 1 2009_tese_acprodrigues.pdf: 12048896 bytes, checksum: f798174e6a53a436dc24e7f852985f85 (MD5)<br>Approved for entry into archive by José Jairo Viana de Sousa(jairo@ufc.br) on 2014-10-09T19:00:26Z (GMT) No. of bitstreams: 1 2009_tese_acprodrigues.pdf: 12048896 bytes, checksum: f798174e6a53a436dc24e7f852985f85 (MD5)<br>Made available in DSpace on 2014-10-09T19:00:26Z (GMT). No. of bitstreams: 1 2009_tese_acprodrigues.pdf: 12048896 bytes, checksum: f798174e6a53a436dc24e7f852985f85 (MD5) Previous issue date: 2009<br>The chemical study of three Ananas species (A. comosus, A. bracteatus and A. lucidus) and the endophytic fungi Acremonium curvulum and Fusarium oxysporum, both isolated from A. lucidus, are described. The phytochemical investigations of roots, leaves and steams from A. bracteatus yielded the isolation of three micro molecular compounds: kumatekenin (1), glycosilated sitosterol (2) and sitosterol (3). From leaves of A. lucidus it was isolated a mixture of three monoacylglicerols (4) and 5-(hydroxymethyl)-2-furaldehyde (5). The fatty acid composition of leaves and roots from A. bracteatus and A. lucidus, as well as in vitro A. comosus, A. bracteatus and A. lucidus was investigated. Twenty-one fatty acids, as methyl ester derivatives, were identified in eight studied samples, 16 (76%) of them saturated and 5 (24%) unsaturated acids. From the fungus A. curvulum three compounds were isolated: phenetyl 2-hydroxypropionate (6), ergosterol (7) and tryptophol (8). The chemical investigation of the organic extracts from F. oxysporum led to the isolation of tetra(S)-butirolactone (9) and the diastereoisomeric mixture of (4R*,5S*)-5-hydroxyhexan-4-olide (71%) and (4R*,5R*)-5-hydroxyhexan-4-olide (29%) (10). Structure elucidation of the isolated compounds was done by spectrometric analysis such as infrared, 1H and 13C NMR, including bidimensional techniques (COSY, HMQC e HMBC), mass spectrometry as well as literature comparison.<br>Este trabalho descreve o estudo químico de três espécies do gênero Ananas (A. comosus, A. bracteatus e A. lucidus) e dos fungos endofíticos Acremonium curvulum e Fusarium oxysporum, isolados de A. lucidus. A investigação fitoquímica das raízes, folhas e talos de A. bracteatus levou ao isolamento de três compostos micromoleculares: cumataquenina (1), glicosídeo do sitosterol (2) e sitosterol (3). Das folhas da espécie A. lucidus foram isolados a mistura de três monoacilglicerois (4) e o 5-(hidroximetil)-2-furaldeído (5). O estudo dos ácidos graxos das folhas e raízes de A. bracteatus e A. lucidus, bem como de A. comosus, A. bracteatus e A. lucidus cultivadas in vitro foi realizado. Vinte e um ácidos graxos, na forma de ésteres metílicos, foram identificados nas oito amostras analisadas, sendo 16 (76%) ácidos graxos saturados e 5 (24%) insaturados. A prospecção química do fungo A. curvulum levou ao isolamento dos compostos 2-hidroxipropanoato de feniletila (6), ergosterol (7) e triptofol (8). A analise dos extratos de F. oxysporum levou ao isolamento do composto tetra(S)-butirolactona (9) e do (4R*, 5S*)-5-hidroxihexan-4-olida (71%) e (4R*, 5R*)-5-hidroxihexan-4-olida (29%) (10), estes na forma de uma mistura diastereoisomerica. A determinação estrutural dos metabólitos secundários isolados foi realizada empregando-se as técnicas espectrométricas de infravermelho, ressonância magnética nuclear de hidrogênio e carbono-13, incluindo técnicas bidimensionais (COSY, HMQC e HMBC) e espectrometria de massa, além de comparação com dados descritos na literatura.

Les composés de type pyrrocidine sont des polykétides PKS-NRPS possédant des activités biologiques intéressantes comme antifongique ou antibiotique. La synthèse totale de ces composés est un réél challenge comme il est constitué de 10 centres chiraux et d'un macrocycle complexe. L'étude de leur biosynthèse pourrait être d'une aide importante afin de comprendre le mécanisme de formation de cette structure spéciale, et en particulier l'étape de la cyclisation complexe.Le précurseur linéaire de ces polykétides étant composé par une chaine nonakétide (partie PKS) et d'une L-tyrosine (partie NRPS), des hypothèses sur leur biosynthèse ont été émises dans cette thèse. Des expériences d'incorporation de précurseurs marqués ou non vont être réalisées dans différents milieux de culture en vue d'obtenir des informations sur cette biosynthèse, et plus précisément le passage du précurseur linéaire vers la structure polycyclique complexe. En parallèle, des supplémentations des cultures d'A. zeae avec des dérivés de la tyrosine seront faites dans le but d'obtenir des analogues pouvant avoir des activités biologiques nouvelles ou meilleures que les pyrrocidines<br>Pyrrocidine and its related compounds are PKS-NRPS polyketides having biological interests such as antifungal or antibiotic activities. The total synthesis of these entities has been challenging since the family of hirsutellone is composed by 10 chiral centers and a complex macrocycle. Studying the biosynthesis of these compounds can be an asset for the comprehension of this special molecular structure, especially for the complex cyclization step. Knowing that the linear precursor of these molecules is constituted by a nonaketide chain (PKS part) and by an L-tyrosine (NRPS part), hypotheses about the biosynthesis of hirsutellone-related compounds have been developed in this thesis. Some incorporation experiments of labeled or unlabeled compounds has been done in different culture media in order to have more information about this biosynthesis, in particular the conversion of the linear precursor into this complex polycyclic structure. In parallel, the supplementation of L-tyrosine derivatives will help us to get some analogs of pyrrocidine which can have new or better activities than natural products

Orientador: João Lucio de Azevedo<br>Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia<br>Made available in DSpace on 2018-07-19T15:48:28Z (GMT). No. of bitstreams: 1 Vialta_Airton_D.pdf: 7068425 bytes, checksum: 35be95d571d938d79e669c7ef9161e46 (MD5) Previous issue date: 1994<br>Doutorado<br>Doutor em Genetica

Se estudiaron los géneros Scedosporium, Acremonium, Phialemonium, Lecythophora y Paecilomyces. En Scedosporium se seleccionó la mejor herramienta para la tipificación de S. aurantiacum y S. prolificans. En los otros géneros se estudiaron la identificación morfológica y molecular para establecer el espectro de especies y sus relaciones filogenéticas. Los resultados indican que el sistema basado en la secuenciación multilocus es la mejor metodología para la tipificación de Scedosporium. Se ha establecido el espectro de las especies asociadas a muestras clínicas de los Estados Unidos, siendo Acremonium kiliense, el complejo Acremonium sclerotigenum-Acremonium egyptiacum, Acremonium implicatum, Phialemonium obovatum, Phialemonium curvatum, Lecythophora hoffmannii, Cephalotheca foveolata y Lecythophora mutabilis las especies más encontradas. Se propone el nuevo género Phialemoniopsis. Se transfiere Sarcopodium oculorum y Phialemonium curvatum a Phialemoniopsis, y Acremonium atrogriseum y Taifanglania inflata a Phialemonium. Se proponen las nuevas especies: Phialemoniopsis cornearis, Phialemoniopsis pluriloculosa, Phialemonium globosum, Lecythophora luteo-rubra, Lecythophora cateniformis y Paecilomyces lavendulus.<br>Scedosporium, Acremonium, Phialemonium, Lecythophora and Paecilomyces were studied. In Scedosporium, the study has focussed on choosing the best tool for the characterization of S. aurantiacum and S. prolificans. As for the other genera, studies have focussed on morphological and molecular identification. The results showed that the system based on multilocus sequencing is the best methodology for the characterization of Scedosporium. By correlating the results obtained by morphological and molecular techniques, we have been able to establish the species associated with clinical samples from the United States, Acremonium kiliense, complex Acremonium sclerotigenum-Acremonium egyptiacum, Acremonium implicatum, Phialemonium obovatum, Phialemonium curvatum, Lecythophora hoffmannii, Cephalotheca foveolata and Lecythophora mutabilis being the most found species. We proposed Phialemoniopsis as a new genus. We transferred Sarcopodium oculorum and Phialemonium curvatum to Phialemoniopsis, and Acremonium atrogriseum and Taifanglania inflata to Phialemonium. We proposed the new species: Phialemoniopsis cornearis, Phialemoniopsis pluriloculosa, Phialemonium globosum, Lecythophora luteo-rubra, Lecythophora cateniformis and Paecilomyces lavendulus.

Thesis (MSc)--Stellenbosch University, 2013.<br>ENGLISH ABSTRACT: Humanity is currently dependant on fossil fuels as an energy source. Increasing economic development and industrialization is, however, raising the demand for this unsustainable energy source. This increased pressure on dwindling reserves and growing concern over detrimental environmental effects associated with the use of these fuels have sparked great interest in the development of alternative sources. Bioethanol has surfaced as a good alternative to fossil fuels, as it can be produced from cheap, abundant, renewable, non-food sources. Bioethanol is also carbonneutral, i.e. utilisation thereof leaves the net level of carbon dioxide in the atmosphere unperturbed. Lignocellulose, more specifically its cellulose fraction, has been identified as a possible feedstock for the production of bioethanol. The use of lignocellulose as feedstock will allow for a more sustainable supply and much needed energy security. Lignocellulosic feedstocks can be divided into two main categories, i.e. wastes from processes other than fuel production and crops grown specifically for fuel production. Cereal crops such as triticale have been identified as good industrial crops for the production of energy. Triticale’s higher biomass yield, moderate water and nutrient requirements, steadily increasing area of cultivation and main use as an animal feed and not a human food source, makes it attractive as feedstock for the production of bioethanol. The combined activity of endoglucanases, exoglucanases and β-glucosidases is needed to hydrolyse crystalline cellulose to fermentable sugars. The high cost of these enzymes is, however, the most significant barrier to the economical production of bioethanol from cellulosic biomass. A promising strategy for a reduction in costs is the production of these cellulolytic enzymes, hydrolysis of biomass and fermentation of the resulting sugars to bioethanol in a single process step via a cellulolytic microorganism. The development of such a consolidated bioprocessing (CBP) organism can be achieved by the introduction of cellulolytic activity into a noncellulolytic microorganism that is able to ferment glucose to ethanol. Saccharomyces cerevisiae is a good host candidate for CBP as this yeast’s high tolerance towards ethanol and its use in industrial applications has been established. The enzymatic activities of endoglucanases and exoglucanases are, however, inhibited by the build-up of cellobiose during the hydrolysis of cellulose. This effect may be alleviated with the introduction of a better functioning β-glucosidase into the system. β-Glucosidases hydrolyse cellobiose to glucose, alleviating the inhibition on the enzymatic activities of endoglucanases and exoglucanases. Despite advances in enzyme production systems and engineering enzymes currently in use for higher stability and activity, there is still a demand to expand the current collection of enzymes. Bioprospecting for novel cellulolytic enzymes focuses on specific environment, with high turnover rates of cellulosic material or extreme conditions, such as the composting process. These enzymes are becoming more attractive compared to their mesophillic counterparts due to their potential industrial applications and the fact that they represent the lower natural limits of protein stability.<br>AFRIKAANSE OPSOMMING: Die mensdom is hoofsaaklik van fossielbrandstowwe as 'n energiebron afhanklik. Toenemende ekonomiese ontwikkeling en industrialisasie verhoog egter die aanvraag na hierdie onvolhoubare energiebron. Druk op kwynende reserwes en groeiende kommer oor die nadelige gevolge vir die omgewing wat met die gebruik van hierdie brandstowwe gepaard gaan, het tot groot belangstelling in die ontwikkeling van alternatiewe bronne gelei. Bio-etanol is 'n goeie alternatief vir fossielbrandstowwe, want dit kan van goedkoop, vollop, hernubare nievoedselbronne geproduseer word. Bio-etanol is ook koolstof-neutraal; die gebruik daarvan laat die netto vlak van koolstofdioksied in die atmosfeer onverstoord. Lignosellulose, en meer spesifiek die sellulose fraksie, is as moontlike grondstof vir die vervaardiging van bio-etanol geïdentifiseer. Die gebruik van lignosellulose as grondstof sal meer volhoubare voorsiening en broodnodige energie-sekuriteit verseker. Sellulose grondstowwe kan in twee hoof kategorieë verdeel word, nl. Newe produkteafval van prosesse anders as brandstofproduksie en gewasse wat spesifiek vir brandstofproduksie gekweek word. Graangewasse soos korog is geïdentifiseer as 'n goeie industriële gewas vir die produksie van energie. Korog se hoër biomassa opbrengs, matige water en voedingstofvereistes, groeiende bewerkingsgebied en die gebruik as 'n veevoergewas eerder as 'n menslike voedselbron, maak dit aantreklik as 'n grondstof vir die vervaardiging van bio-etanol. Die gesamentlike aktiwiteit van endoglukanases, eksoglukanases en β-glukosidases is nodig om kristallyne sellulose tot fermenteerbare suikers te hidroliseer. Die hoë koste van hierdie ensieme is egter die grootste hindernis vir die ekonomiese produksie van bio-etanol vanaf sellulosiese biomassa. 'n Belowende koste verminderingstrategie is die produksie van hierdie sellulolitiese ensieme, die hidrolise van biomassa, en die fermentasie van die suikers na bio-etanol in 'n enkelstap-proses via 'n sellulolitiese mikro-organisme. Die ontwikkeling van so 'n gekonsolideerde bioprosesserings (CBP) organisme kan deur die uitdrukking van sellulolitiese aktiwiteite in 'n nie-sellulolitiese mikro-organisme wat wel in staat is om glukose na etanol om te fermenteer, gerealiseer word. Saccharomyces cerevisiae is 'n goeie kandidaat gasheer vir CBP, omdat hierdie gis ‘n hoë verdraagsaamheid teenoor etanol toon en sy gebruik in industriële toepassings gevestig is. Die ensiematiese aktiwiteite van endoglukanases en eksoglukanases word egter deur die ophoop van sellobiose gedurende die hidrolise van sellulose geïnhibeer. Hierdie effek kan met die byvoeging van meer effektiewe β-glukosidases verlig word. β-Glukosidases hidroliseer sellobiose na glukose en verlig dus die inhibisie op die endoglukanase en eksoglukanase ensiematiese aktiwiteite. Ten spyte van vooruitgang in ensiemproduksie stelsels en ensiemmodifiserings strategieë wat tans vir hoër stabiliteit en aktiwiteit in gebruik is, bestaan daar steeds 'n behoefte om die bestaande versameling van ensieme uit te brei. Bioprospektering vir nuwe sellulolitiese ensieme fokus op spesifieke omgewings, met hoë omsetkoerse van sellulose materiaal of omgewings met uiterste toestande, soos die komposterings-proses. Hierdie ensieme is besig om meer aantreklik in vergelyking met hul mesofieliese eweknieë te raak as gevolg van hul potensiele industriële toepassings en die feit dat hulle die laer natuurlike grense van proteïen-stabiliteit verteenwoordig.<br>Stellenbosch University and the Technology Innovation Agency for financial support

Spontaneous chlorate resistant mutants of Penicillium chrysogenum and Cephalosporium acremonium have been isolated. Putative genotypes of the P. chrysogenum mutants were identified using the phenotypic characterization of Cove (1979). This analysis was also attempted with C. acremonium, however it was found that the wild type organism could not grow on minimal media containing glucose and hypoxanthine and thus cnx and niaD mutants could not be differentiated using this method. Following the distinction of cnx and niaD mutants using cytochrome C reductase and purine hydroxylase I assays, a simple plate test using minimal media containing quinic acid as carbon source and inosine as nitrogen source was developed to analyse cnx and niaD mutants. Nonreverting (at less than 1 in 108) niaD mutants were isolated for later experiments. P. chrysogenum niaD mutant niaD19 was transformed to nitrate utilization at a frequency of up to 20 transformants/mug DNA using the Aspergillus nidulans niaD gene, up to nine/mug DNA with the A. niger niaD gene and up to three/mug DNA using the A. oryzae niaD gene. Vector constructs carrying the A. nidulans ans-1 sequence with the A niger niaD gene did not show increased transformation efficiency. Linearization of the A. niger niaD containing plasmid resulted in a two to three times increase in transformation frequency. Southern blot hybridization analysis demonstrated that vector sequences had integrated into the recipient genome. The control of heterologous niaD gene expression generally agreed with that found in the wild type strain, that is, induction by nitrate and repression in the presence of ammonium. The A. nidulans, A. niger and A. oryzae genes failed to transform C. acremonium niaD mutants. A DNA fragment containing the C. acremonium niaD gene was isolated by cross hybridization to the A. nidulans niaD gene. This fragment was sub-cloned into pUC18 (designated pSTA700), and showed back hybridization to C. acremonium wild type DNA exhibiting the expected hybridization pattern. When partially sequenced, it showed nucleotide and determined amino acid homology to the A. nidulans niaD gene and protein. The clone pSTA700 transformed a C. acremonium niaD mutant, CSG116 and P. chrysogenum STP19 to nitrate utilization at a frequency of up to 47 and six transformants/mug DNA, respectively. When heat shock was applied to C. acremonium protoplasts 10mins prior to the addition of DNA, transformation frequencies of up to 137/mug DNA were obtained. Southern analysis of transformants revealed multiple integration of vector sequences, with no detectable preference for the homologous niaD site. Some C. acremonium transformants showed a greatly increased nitrate reductase activity (up to 10 times wild type activity) when induced with nitrate. The niaD transformation system was successfully used to introduce unselected markers into C. acremonium such as hygromycin B and benomyl resistance. The co-transformation frequency was up to 25% when equal molar ratios of plasmids were used. Antibiotic biosynthetic genes isolated from C. acremonium (pcbC and cefEF) and A. nidulans (pcbC and penE) were introduced into C. acremonium CSG116 by co-transformation with the C. acremonium niaD gene or the construction of vectors containing both the C. acremonium niaD gene and antibiotic biosynthetic genes. Isolates transformed with the C. acremonium pcbC and cefEF genes exhibited an increase in antibacterial activity (greater than 20% compared to wild type) at frequencies of up to 10% and six percent of transformants, respectively. When A. nidulans constructs containing the pcbC and penE genes, but not the pcbC gene alone, were co-transformed into C. acremonium, up to 18% of transformants exhibited an increase in antibacterial activity. The presence of penicillin antibiotics could not be found in these transformants and thus the exact cause of this increase in antibacterial activity could not be determined.

En aquets tesis es vol enfocar en l'estudi dels géneres fúngics Acremonium, Arthrographis, Arthropsis, Ochroconis, Sarocladium, Scytalidium i Verruconis. Alguns d'ells són ocasionalment aïllats de mostres clíbiques, encara què, l'espectre real de les seves espècies en l'area clínica es poc coneguda, sumant-li la seva difícil identificació i complexa taxonomia. Es van estudiar un total de 248 aïllats, 131 obtingust de mostres clíniques o de sòls i 117 corresponents a soques tipus o de referència de col.leccions internacionals de cultius. La caracterizació fenotípica dels aïllats es va realitzar a traves de l'observació de les seves característiques macroscópiques i microscópiques. La identificació molecular, mitjacant la seqüenciació de les regions ribosomals ITS i LSU. Per obtenir una millor resolució filogenética entre algunes espècies, es van seqüenciar regions parcials d'altres gens (actina, tubulina, factor de elongació, RNA polimerasa i quitina sintasa). L'activitat in vitro d'alguns antifúngics va a ser evaluata front a espècies de Arthrographis, Arthropsis, Ochroconis i Scytalidium. Apart de les espècies comunment conegudes com a patògens oportunistes A. kalrae i V. gallopava, altres espècies no conegudas previament a partir de mostres clíniques vàren ser identificades (Arthropsis hispanica, Sarocladium terricola, Scytalidium cuboideum i Ochroconis cordanae). Incluint les noves espècies descrites aquí: Acremonium dimorphosporum, A. moniliforme, Arthrographis chlamydospora, A.<br>pseudostrictum i S. subulatum. Adicionalment, quatre géneres nous, Acremoniopsis, Brunneomyces, Cervusimilis i Collarina i 16 nous taxons, Acremoniopsis suttonii, Acremonium asperulatum, A. citrinum, A: parvum, A: pilosum, A. variecolor, Brunneomyces brunnescens, B. hominis, B. europaeus, Cervusimilis alba, Collarina aurantiaca, Ochroconis icarus, Sarocladium gamsii, S. implicatum, S. summerbellii i S. terricola, vàren ser descrits de mostres de diferents origens. En general, les proves de sensibilitat antifúngica mostrar que la terbinafina va ser la droga més activa davant les espècies avaluades, excepte per S. cuboideum, on el posaconazol va a mostrar la millor activitat En esta tesis nos enfocamos en el estudio de los géneros fúngicos Acremonium, Arthrographis, Arthropsis, Ochroconis, Sarocladium, Scytalidium y Verruconis. Algunos de ellos son ocasionalmente aislados de muestras clínicas, sin embargo, el espectro real de sus especies en el área clínica es poco conocido, sumado a su difícil identificación y compleja taxonomía. Se estudiaron un total de 248 aislados, 131 obtenidos de muestras clínicas o de suelos y 117 correspondientes a cepas tipo o de referencia de colecciones internacionales de cultivos. La caracterización fenotípica de los aislados se realizó a través de la observación de sus características macroscópicas y microscópicas, y la identificación molecular, mediante la secuencación de las regiones ribosomales ITS y LSU. Para obtener una mejor resolución filogenética entre algunas especies, se secuenciaron regiones parciales de otros genes (actina, tubulina, factor de elongación, RNA polimerasa II y quitina sintasa). La actividad in vitro de varios antifúngicos fue evaluada frente a especies de Arthrographis, Arthropsis, Ochroconis y Scytalidium. Aparte de las especies comunmente reportadas como patógenos oportunistas, Arthrographis kalrae y Verruconis gallopava, otras especies no reportadas previamente a partir de muestras clínicas fueron identificadas (Arthropsis hispanica, Sarocladium terricola, Scytalidium cuboideum y Ochroconis cordanae), incluyendo las nuevas especies descritas aquí:<br>Ochroconis olivacea, O. ramosa, Sarocladium bifurcatum, S. hominis, S. pseudostrictum y S. subulatum. Adicionalmente, cuatro géneros nuevos, Acremoniopsis, Brunneomyces, Cervusimilis y Collarina, y 16 nuevos taxones, Acremoniopsis suttonii, Acremonium asperulatum, A. citrinum, A. parvum, A. pilosum, A: variecolor, Brunneomyces brunnescens, B. hominis, B. europaeus, Cervusimilis alba, Collarina aurantiaca, Ochroconis icarus, Sarocladium gamsii, S. implicatum, S. sumerbellii y S. terricola, fueron descritos de muestras de diversos orígenes. En general, las pruebas de sensibilidad antifúngica revelaron que la terbinafina fué la droga más activa frente a las especies evaluadas, excepto para S. cuboideum, donde el posaconazol mostró la mejor actividad. In this thesis, we have studied the fungal genera Acremonium, Arthrographis, Arthropsis, Ochroconis, Sarocladium, Scytalidium and Verruconis. Some of them are occasionally recovered from clinical specimens, but the real spectrum of their species in the clinical setting is poorly known. Furthermore, the scarce morphological differentiation of their species make difficult their identification and taxonomy. A total of 248 isolates were studied. 131 obtained from clinical or soil samples and 117 corresponding to type or reference strains from international culture collections. Phenotypical characterization was performed by the observation of their macroscopic and microscopic features in artificial media. Molecular identification was assessed by sequencing of two ribosomal regions (ITS and partial LSU). To obtain deeper phylogenetic resolution of some species, fragments of different loci were also used (actin, tubulin, translation elongation factor, RNA polymerase II and chitin syntase). The in vitro activity of several antifungal drugs was evaluated against Arthrographis, Arthropsis, Ochroconis and Scytalidium species. Apart from the commonly reported opportunistic species Arthrographis kalrae and Verruconis gallopava, other species never associated before to clinical specimens were identified (Arthropsis hispanica, Sarocladium terricola, Scytalidium cuboideum, Ochroconis

Infection of tall fescue (<i>Festuca arundinacea</> Schreb.) with the endophyte fungus (<i>Acremonium coenophialum</i>, Morgan-Jones and Gam) has been associated with toxicity symptoms observed in cattle. The overall objective was to investigate the influence of endophyte infection on growth and chemical composition of tall fescue and the toxicity of endophyte-infected (EI) tall fescue to cattle. In a greenhouse study with pairs of genetically identical EI and non-infected (NI) ‘Kenhy’ tall fescue clones, concentration of N, Ca, Mg, Al, B, Mn and Zn was higher and K and S was lower in NI, compared to EI tall fescue. Insect resistance was higher in EI, compared to NI. Yield and chemical composition of high and low EI tall fescue were measured at four growth stages (stockpiled, prebloom, bloom and regrowth after harvest at bloom), two sites (Glade Spring and Blackstone) and three rates of N fertilization (0, 40 and 80 kg/ha) in a field study. Tall fescue grown at Glade Spring was higher in N, Mg, Al, Cu, Fe and Mn, compared to Blackstone. Nitrogen fertilization increased N, Mg, Ca, B, Cu, Na, Zn and decreased NDF, ADF, cellulose, P and S concentration in tall fescue. Neutral detergent fiber, ADF, cellulose, lignin, Fe and Na were higher in low, compared to high EI tall fescue. Concentrations of Cu, Na and Zn in stockpiled and Ca, Cu, Na and Zn in bloom-cut tall fescue hay were below dietary requirements for 227-kg steers. A disc meter was also evaluated for use in predicting yield of tall fescue. The meter is useful for non-destructive estimation of yield. Three feeding studies were conducted with steers (6/treatment/year). Diets were orchardgrass/alfalfa hay, spring-cut EI tall fescue hay, spring-cut EI tall fescue silage and fall-cut EI tall fescue silage. Serum prolactin and cholesterol were depressed in steers fed fescue hay and silages, compared to steers fed orchardgrass/alfalfa hay. Differences in mineral composition of hay and silage were reflected in serum minerals in steers. Ergopeptine alkaloids in EI tall fescue may have contributed to the depression of serum prolactin. The spring-cut silage contained the highest concentration of ergopeptine alkaloids, compared to other diets. Steers fed the spring-cut tall fescue silage had the lowest basal and thyrotropin-releasing hormone stimulated prolactin compared to steers fed the other diets.<br>Ph. D.

Made available in DSpace on 2016-06-02T19:55:29Z (GMT). No. of bitstreams: 1 DoutAAF.pdf: 3275307 bytes, checksum: f45ed0ce056382979fefde457b9cfe5f (MD5) Previous issue date: 2003-10-10<br>The scope of the current work was to investigate the effect of glucose, amonium acetate, DL-methionine, phosphorus, oleic acid and salt solution on the maximum specific growth rate (µmax), as well as the effect of sucrose and DL-methionine on the specific cefalosporin C production rate (µp), by Cephalosporium acremonium ATCC 48272, using factorial design and response surface techniques. The initial medium of fermentation was used according to factorial design and the experiments were carried out in shaker flasks at controlled temperature at 28 °C, initial pH of 7,0+0,1 and agitation speed 250 rpm. For both specific rate the variables involved were correlated by a fitted second order model. The canonic and surface response analysis showed a maximum specific growth rate of 0.22 h-1 obtained when glucose and amonium acetate concentration, were 4.2 , 2.5 g/L, respectively, and specific cefalosporin C production rate of 1.12 mgCPC/gcel.h when sucrose and DL-methionine concentration, were 10.5, 2.5 g/L, respectively, for a celular concentration of 14.0 g/L. The experimental design showed to be very efficcient in evaluating the bioprocess, as long as high values of µmax e µp were obtained in comparison with literature data. The feedforward neural network (FNN) was employed for modelling and simulation the effect of glucose, amonium acetate, DL-methionine, phosphorus, oleic acid and salt solution on the maximum specific growth rate. The network topologies were used with 6 neurons in the input layer, 22 hidden layer and 1 output layer. The data base for training the FNN was obtained from results of the factorial designs processed with a backpropagation learning algorithm. The surfaces response obtained by simulations for all range of glucose and amonium acetate concentrations showed inhibitory effect for the two substrates on the maximum specific growth rate. The results of the simulation are in good agreement with the experimental and statistical model results.<br>Neste trabalho verificou-se o efeito das variáveis concentração de glicose, acetato de amônio, DL-metionina, fósforo, ácido oléico e solução de sais na velocidade específica máxima de crescimento (µmax), assim como sacarose e DL-metionina na velocidade específica de produção do antibiótico cefalosporina C (µp), pelo fungo Cephalosporium acremonium ATCC 48272, usando como ferramenta estatística a técnica de planejamento experimental e a análise por superfície de resposta. Na otimização destas variáveis, os ensaios foram conduzidos de acordo com os planejamentos fatoriais, fracionário, completo e composto central, em fermentações em incubador rotatório, variando as concentrações iniciais dos nutrientes do meio, a 28 oC, pH 7,0+0,1, com agitação de 250 rpm. Para ambos os casos, um modelo ajustado de 2a ordem foi o que melhor correlacionou as variáveis. Os resultados da otimização mostraram que uma velocidade máxima de crescimento de 0,22 h-1 pode ser atingida para uma concentração de glicose e acetato de amônio em torno de 4,20 e 2,5 g/L, respectivamente, e uma velocidade específica de produção de cefalosporina C de 1,12 mgCPC/gcel.h para uma concentração de sacarose de 10,5 g/L e DL-metionina 2,5 g/L com uma massa celular média, para produção, de 14,0 g/L. O planejamento estatístico mostrou-se uma ferramenta bastante eficiente para avaliação do bioprocesso, pois com a realização dos ensaios propostos, foi possível obter valores de µmax e µp superiores aos encontrados na literatura. Desenvolveu-se um modelo baseado na técnica de redes neurais para a simulação de todo o domínio das concentrações de glicose, acetato de amônio, DL-metionina, fósforo, ácido oléico e solução de sais na velocidade específica máxima de crescimento. A rede que melhor representou os valores experimentais foi uma do tipo "feedforward", com três camadas e 22 neurônios na camada oculta, treinada com o algoritmo de retropropagação, por um conjunto de resultados obtidos através de ensaios do planejamento estatístico. As superfícies obtidas pela simulação em amplo domínio de concentrações de glicose e acetato de amônio, mostraram que o crescimento do microrganismo segue um modelo cinético de inibição por este dois nutrientes, prevendo altos valores de µmax para concentrações de glicose e acetato de amônio, comparáveis com os obtidos pelo modelo estatístico.

Orientador: Eleni Gomes<br>Banca: Maricy Raquel Lindenbah Bonfá<br>Banca: Heizir Ferreira de Castro<br>Banca: Adriana Aguiar Mendes<br>Banca: Tatiane Lembo<br>Resumo: As lipases apresentam atividades catalíticas específicas as quais permitem seu uso em diferentes setores da indústria, como de alimentos, tratamento de águas residuais, cosméticos, farmacêuticos e biocombutíveis. A imobilização dessas enzimas torna mais eficiente sua aplicação uma vez que preserva suas propriedades bioquímicas nas condições reacionais e facilita recuperação e reutilização. O objetivo deste trabalho foi caracterizar a lipase bruta produzida pelo fungo Acremonium-like ROG 2.1.9, purificá-la parcialmente e imobilizá-la em diferentes matrizes, incluindo resíduos agro-industriais, e sua utilização na produção de biodiesel por transesterificação enzimática. A enzima apresentou maior atividade a pH 7,0 e a 45°C e estabilidade na presença de n-hexano e metanol, com 92% e 40% de atividade residual, respectivamente, após 24 horas em presença daqueles compostos. A lipase mostrou ainda, especificidade por ácidos graxos de cadeia média como p-nitrofenil decanoato (C10:0), seguido de p-nitrofenil palmitato (C16:0). A enzima foi purificada parcialmente por precipitação fracionada com sulfato de amônio, com maior rendimento obtido na faixa entre 30-50% de saturação do sal. Após ser parcialmente purificada, a lipase foi imobilizada por adsorção em bagaço de cana e sabugo de milho não ativados, por meio de interação hidrofóbica em Sepabeads C18, Lewatit 105 e Lewatit 1600, por interação iônica em PEI-agarose, MANAE-sabugo de milho e MANAE-bagaço de cana e por ligação covalente...<br>Abstract: Lipases have specific catalytic activities which allow its use in different industry sectors such as food, waste water treatment, cosmetics, pharmaceuticals and biofuel. The efficiency, preservation of the biochemical properties, recovery and reuse of the activity of theses enzymes are increased by immobilization on an inert support.The objective of this study was to purify partially and characterize the lipase transesterification produced by Acremonium-like ROG 2.1.9, and to immobilize it on different matrices, including agro industrial waste. The enzyme showed the highest activity at pH 7.0 and at 45 °C and stability in the presence of n-hexane and methanol (92% and 40%, respectively), for 24 hours. The lipase also showed specificity for meddium-chain fatty acids, as p-nitrophenyl decanoate (C10: 0), followed by p-nitrophenyl palmitate (C16: 0). The enzyme was partially purified by fractional precipitation with ammonium sulfate, with higher yield in the range of 30-50% salt saturation. The partially purified lipase was immobilized by adsorption on sugarcane bagasse and on corn cob, through hydrophobic interaction on Sepabeads C18, Lewatit 105 and Lewatit 1600; by ionic interaction on PEI-agarose, MANAE-corn cobs and MANAE- cane bagasse and by multipoint covalent bond on Glyoxyl-cob corn and Glyoxyl-cane bagasse. The immobilization by ionic interaction on MANAE-cob afforded an increase of 100% in the activity of enzyme. The Lip-MANAE-cob derivative showed higher ionic strength, LipMANAE-bagasse was more stable thermally (45 °C) and Lip-PEI-agarose derivative was the most stable in ethanol (30% v / v). The activity of the MANAE-cob and MANAE-bagasse derivatives increased in the presence of ethanol, n-hexane and DMSO and in the presence of surfactants CTAB and Triton X-100. Based on the results, it was conclude that the use of agro-industrial waste (bagasse and corn cob) ...<br>Mestre

Some aspects of the presence of systemic endophytic fungi in agriculturally important New Zealand grasses were studied in relation to plant breeding. Seedling resistance to adult Argentine stem weevil feeding in perennial ryegrass, Italian ryegrass and tall fescue was found to be related to the presence of their respective Acremonium endophytes in the seed rather than to plant genetic resistance. In addition a study of perennial ryegrass revealed that this resistance was independent of endophyte viability. The seedling resistance conferred by the endophyte of Italian ryegrass was found to be beneficial for field establishment. This endophyte differs from that in perennial ryegrass and tall fescue in that it does not confer resistance to Argentine stem weevil on mature plants, but only on seedlings. The extent of plant genetic seedling tolerance to adult Argentine stem weevil feeding was limited to broad inter-specific differences, with tall fescue more tolerant than perennial ryegrass and both of these more tolerant than Italian ryegrass. This ranking corresponds with previous observations on feeding preference on mature plants. A study of factors affecting the concentration of endophyte mycelia in infected seed of perennial ryegrass revealed that plant genetic factors had little effect. The major factors studied were: 1) the endophyte concentration in the maternal parent plant directly influenced the endophyte concentration in the seed. 2) nitrogen fertilizer applications to a seed crop reduced the concentration of mycelia in the seed, with earlier applications having a greater effect. 3) application of the fungicide propiconazole (Tilt) to a seed crop reduced the endophyte concentration in the seed. 4) the endophyte concentration in the seed was found to directly influence the endophyte concentration in seedlings, six month old plants and that of seed harvested from a first year seed crop. As there have been no previous reports of tetraploid perennial ryegrass cultivars with endophyte an experiment was conducted to determine if these could be developed by the standard procedure of colchicine treatment. The results revealed that endophyte was retained following colchicine treatment.

El ascomiceto Monosporascus cannonballus Pollack et Uecker es uno de los principales hongos asociados al síndrome del "colapso", que afecta al cultivo de cucurbitáceas en España y otros países. Las ascosporas son el inóculo principal del hongo, quedando en el suelo tras la descomposición de las raíces afectadas, pudiendo ser extraídas del suelo mediante un proceso físico, que permite su cuantificación a lo largo del tiempo. Basándose en esta técnica, se han realizado varios estudios epidemiológicos que han permitido obtener resultados innovadores. Se ha estudiado la dinámica poblacional de las ascosporas de M. cannonballus en suelos con diferentes condiciones hídricas y de cultivo. En campos con cultivo de melón, se ha observado que el nivel de ascosporas alcanza un máximo siete meses después de la plantación (3-4 meses después del final del cultivo), para ir disminuyendo después progresivamente, hasta llegar a niveles similares a los iniciales a los doce meses de la plantación. En campos con encharcamiento invernal, se ha observado un descenso progresivo del nivel de ascosporas, constatando que éstas pueden sobrevivir en suelo al menos por un periodo de tres años, sin haber perdido su infectividad. M. cannonballus ha sido considerado como un hongo termófilo, típico de zonas desérticas y semiáridas; en este estudio se ha demostrado que es capaz de sobrevivir en zonas templadas y en condiciones de encharcamiento. Se ha realizado un estudio de cuantificación de ascosporas en suelo de campos de melón de varias zonas productoras de la Comunidad Valenciana, detectándose ascosporas de M. cannonballus en todos ellos. En el momento de aparición de síntomas de "colapso", se han observado diferencias significativas entre los campos y entre las zonas síntomáticas y asintomáticas, a favor de unas u otras, según los campos. Al comparar los niveles iniciales de ascosporas en suelo con los obtenidos dos o tres meses tras el final del cultivo, se han visto situaciones contradictori<br>Beltrán Martínez, R. (2006). Estudios epidemiológicos y de patogenicidad de Monosporascus cannonballus Pollack et Uecker [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/1865<br>Palancia

Lau, Shong.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2007.<br>Includes bibliographical references (leaves 124-129).<br>Abstracts in English and Chinese.<br>Abstract of thesis --- p.i<br>碩士論文摘要 --- p.iii<br>Acknowledgement --- p.iv<br>Declaration --- p.v<br>Abbreviations --- p.vi<br>Genetic symbols --- p.viii<br>Table of content --- p.ix<br>List of figures --- p.xiii<br>List of tables --- p.xv<br>Chapter Chapter 1 --- Introduction --- p.1<br>Chapter 1.1 --- Thesis outline --- p.1<br>Chapter 1.2 --- A. chrysogenum --- p.3<br>Chapter 1.3 --- Antibiotic industry --- p.4<br>Chapter 1.4 --- Cephalosporins --- p.4<br>Chapter 1.5 --- CPC biosynthetic pathway --- p.5<br>Chapter 1.6 --- DAC --- p.8<br>Chapter 1.7 --- Aims of study --- p.10<br>Chapter Chapter 2 --- Construction of transformation cassettes --- p.11<br>Chapter 2.1 --- Introduction --- p.11<br>Chapter 2.2 --- Materials and Methods --- p.13<br>Chapter 2.2.1 --- Construction of cassette RTCAH --- p.13<br>Chapter 2.2.1.1 --- Cultivation of R. toruloides --- p.13<br>Chapter 2.2.1.2 --- Preparation of R. toruloides genomic DNA --- p.13<br>Chapter 2.2.1.3 --- PCR of R. toruloides CAH gene --- p.14<br>Chapter 2.2.1.4 --- Construction of pRTCAHhyr --- p.16<br>Chapter 2.2.2 --- Construction of cassette GHG --- p.17<br>Chapter 2.3 --- Results and Discussion --- p.18<br>Chapter 2.3.1 --- pGHG construction --- p.18<br>Chapter 2.3.2 --- pRTCAHhyr construction --- p.18<br>Chapter Chapter 3 --- Optimization of integrative transformation of A. chrysogenum --- p.22<br>Chapter 3.1 --- Introduction --- p.22<br>Chapter 3.2 --- Materials and Methods --- p.24<br>Chapter 3.2.1 --- Strain and culture medium --- p.24<br>Chapter 3.2.2 --- Reagents --- p.24<br>Chapter 3.2.3 --- Standard transformation procedures --- p.25<br>Chapter 3.2.3.1 --- Cell cultivation --- p.25<br>Chapter 3.2.3.2 --- Protoplast preparation --- p.25<br>Chapter 3.2.3.3 --- PEG mediated protoplast fusion --- p.27<br>Chapter 3.2.4 --- Examination of transformation parameters --- p.28<br>Chapter 3.3 --- Results and Discussion --- p.29<br>Chapter 3.3.1 --- Cell growth period --- p.30<br>Chapter 3.3.2 --- DNA concentration --- p.32<br>Chapter 3.3.3 --- PEG molecular weight --- p.35<br>Chapter 3.3.4 --- Transformation additives --- p.37<br>Chapter 3.3.5 --- Modified transformation protocol --- p.39<br>Chapter Chapter 4 --- Metabolic engineering of A. chrysogenum --- p.42<br>Chapter 4.1 --- Introduction --- p.42<br>Chapter 4.2 --- Materials and Methods --- p.43<br>Chapter 4.2.1 --- Transformation of A. chrysogenum --- p.43<br>Chapter 4.2.2 --- Screening of transformants --- p.43<br>Chapter 4.2.3 --- HPLC analysis --- p.43<br>Chapter 4.2.4 --- A. chrysogenum genomic DNA preparation --- p.44<br>Chapter 4.2.5 --- Genotyping by PCR --- p.45<br>Chapter 4.2.5.1 --- GHG transformants --- p.45<br>Chapter 4.2.5.2 --- RTCAH transformants --- p.47<br>Chapter 4.2.6 --- Genotyping of GHG transformants by Southern hybridization --- p.47<br>Chapter 4.2.6.1 --- Preparation of DIG-labeled GHG probe by PCR --- p.48<br>Chapter 4.2.6.2 --- PCR preparation of DIG-labeled Cef-EF probe --- p.48<br>Chapter 4.2.6.3 --- Restriction digestion of genomic DNA of GHG transformants --- p.49<br>Chapter 4.2.6.4 --- Agarose gel electrophoresis and DNA transfer to positively charged nylon membrane --- p.50<br>Chapter 4.2.6.5 --- Pre-hybridization and hybridization --- p.52<br>Chapter 4.2.6.6 --- Membrane washing and blocking --- p.52<br>Chapter 4.2.6.7 --- Membrane detection --- p.53<br>Chapter 4.2.7 --- Genotyping of RTCAH transformants by Southern hybridization --- p.53<br>Chapter 4.2.7.1 --- PCR preparation of DIG-labeled RTCAH probe --- p.54<br>Chapter 4.2.7.2 --- Restriction digestion of genomic DNA of RTCAH transformants --- p.54<br>Chapter 4.2.7.3 --- Agarose gel electrophoresis and Southern hybridization of RTCAH transformants --- p.55<br>Chapter 4.3 --- Results and Discussion --- p.56<br>Chapter 4.3.1 --- Hygromycin B screening and HPLC analysis of GHG transformants --- p.56<br>Chapter 4.3.2 --- Genotyping of GHG232 by PCR --- p.56<br>Chapter 4.3.3 --- Genotyping of GHG232 by Southern hybridization --- p.58<br>Chapter 4.3.4 --- Hygromycin B screening and HPLC analysis of RTCAH transformant --- p.61<br>Chapter 4.3.5 --- Genotyping of RTCAH transformants by PCR --- p.61<br>Chapter 4.3.6 --- Genotyping of RTCAH transformants by Southern hybridization --- p.63<br>Chapter Chapter 5 --- Characterization of modified strains and fermentation study --- p.65<br>Chapter 5.1 --- Introduction --- p.65<br>Chapter 5.2 --- Materials and Methods --- p.67<br>Chapter 5.2.1 --- Comparison of DAC production in GHG232 and RTCAH transformants by small-scale fermentation --- p.67<br>Chapter 5.2.2 --- CPC conversion assay --- p.67<br>Chapter 5.2.3 --- Evaluation of fermentation media --- p.68<br>Chapter 5.2.4 --- Factorial design for medium formulation --- p.68<br>Chapter 5.3 --- Results and Discussion --- p.71<br>Chapter 5.3.1 --- Evaluation of DAC production profiles of GHG232 and RTCAH transformants --- p.71<br>Chapter 5.3.2 --- CPC conversion assay --- p.79<br>Chapter 5.3.3 --- Medium formulation --- p.81<br>Chapter 5.3.4 --- Factorial design for medium formulation --- p.85<br>Chapter 5.3.4.1 --- CSL and NH4OAc --- p.85<br>Chapter 5.3.4.2 --- CaC03 --- p.91<br>Chapter 5.3.4.3 --- Sucrose --- p.94<br>Chapter 5.3.4.4 --- Starch and soy oil --- p.97<br>Chapter 5.3.4.5 --- Methionine --- p.99<br>Chapter Chapter 6 --- Conclusive remarks --- p.101<br>Appendix: Study of reusability of commercial plasmid extraction kit --- p.103<br>Chapter A1 --- Introduction --- p.103<br>Chapter A2 --- Materials and Methods --- p.105<br>Chapter A2.1 --- Preparation of competent E. coli DH5a cells --- p.105<br>Chapter A2.2 --- Transformation and cultivation ofE. coli DH5a cells --- p.106<br>Chapter A2.3 --- Column and solutions for plasmid DNA preparation --- p.107<br>Chapter A2.4 --- Plasmid preparation --- p.108<br>Chapter A2.5 --- Regeneration and storage of DNA extraction column --- p.109<br>Chapter A2.6 --- Yield and quality assessment of the prepared plasmid DNA --- p.109<br>Chapter A3 --- Results and Discussion --- p.111<br>Chapter A3.1 --- Plasmid DNA yield and purity comparison --- p.111<br>Chapter A3.2 --- Column regeneration and EtOH storage --- p.111<br>Chapter A3.2.1 --- DNA yield after column regeneration --- p.111<br>Chapter A3.2.2 --- Purity of plasmid DNA --- p.112<br>Chapter A3.2.3 --- Restriction digestion --- p.117<br>Chapter A3.2.4 --- E. coli DH5α cells transformation --- p.121<br>Chapter A4 --- Conclusive remarks --- p.123<br>Bibliography --- p.124

博士<br>國立陽明大學<br>生化暨分子生物研究所<br>94<br>Sugar oxidases and dehydrogenases are commercial importance as potential diagnostic reagents and industrial biocatalysts. Most of these enzymes are specific for a variety of mono- and disaccharides, and only a few enzymes are highly selective for oligosaccharides including galactose oxidase, cellobiose dehydrogenase , and glucooligosaccharide oxidase (GOOX). GOOX from Acremonium strictum was screened for potential applications in oligosaccharide acid production and carbohydrate detection. It is a monomeric glycoprotein with a covalently linked FAD. In addition to D-glucose, maltose, lactose, and cellobiose, this enzyme can react with malto- and cello-oligosaccharides, and hence the name of this novel oxidase. The broad substrate specificity of GOOX, particularly toward oligosaccharides, suggests that it may have great potential applicability. To facilitate further characterization and potential industrial use of A. strictum GOOX, we have cloned the encoding gene, which is composed of a 25-residue signal peptide and a 474-residue mature protein. The production of GOOX in P.pastoris can be increased dramatically by fermentation with fed-batch process and reach about 300 mg/L after 6 days induction. The protein crystals have been grown in 25﹪PEG monomethyl ether 550, 0.01 M ZnSO4, 0.1 M MES (pH 6.5). The protein structure is composed of FAD- and substrate-binding domains. This sugar oxidase possesses an open carbohydrate-binding groove, allowing the accommodation of higher oligosaccharides. Unexpectedly, the protein structure demonstrates the first known double attachment flavinylation, 6-S-cysteinyl, 8a-N1-histidyl FAD via the C6 atom and the 8a-methyl group of the isoalloxazine ring with Cys130 and His70, respectively. Mutation of the flavinylation residues, including H70A, H70S, H70C, H70Y and C130A mutants, decreased the kcat values by 50- to 600-fold but had little effect on the Km. The double mutant H70A/C130A was abolished the covalent FAD linkage and the FAD binding. Structural and mutational studies suggest that the covalent FAD attachment is able to enhance the oxidative power of the flavin. The complex structure reveals that GOOX may prefer a b-D-glucosyl residue at the reducing end with the conserved Tyr429 acting as a general base to abstract the OH1 proton in concert with the H1 hydride transfer to the flavin. We have used the primary deuterium and solvent kinetic isotope effects to determine the relative cleavage timing of the glucose OH1 and CH1 bonds in GOOX. The decreased activity by 50 fold and the kinetic isotope effects show that CH1 bond cleavage is more rate-limited in the Y429F mutant, consistent with the proposed proton shuttling by Tyr429. Measurement of the multiple isotope effects suggests that the primary and solvent isotope effects arise from the same chemical step, consistent with concerted cleavage of the glucose OH and CH bonds. This supports a hydride transfer mechanism for GOOX.

This project explored the feasibility of using fungi in a constructed wetland for the treatment of pulp mill effluent. The effluent is high in dissolved lignins (some of which are chlorinated), which have proven very difficult to degrade biologically. Mindful of work done with the (terrestrial) white rot fungi, especially Phanerochaete chtysosporium, the question is asked, Is there a fungus which can tolerate submerged conditions while degrading a significant amount of dissolved lignins? Two fungal species with lignin-degrading capability were isolated from submerged films in a log pond. These fungi have been evaluated for decolorization potential under different environmental conditions. Results of laboratory experiments show that one of these fungi, identified as Acremonium sp., was capable of 44% decolorization of pulp mill effluent under sterile, submerged, room temperature conditions. The fungal decolorization was evaluated both in floating cultures and as a film inoculated on wood chips. In addition, bench-scale examination of the potential of this fungus to decolorize pulp mill effluent in non-sterile conditions was completed.<br>Graduation date: 1994

碩士<br>國立臺灣大學<br>漁業科學研究所<br>105<br>The marine environment is extremely complex which contains a broad spectrum of fungal diversity. The algae of this study was collected from the seaboard near the National Museum of Marine Science and Technology, and the antimicrobial activity of the alga-derived fungi were screened. Among them, the crude extract of Acremonium tubakii NTU60, which was isolated from Ulva intestinalis, exibited significant antibacterial activity. In order to determine the best condition for enhancing chemical diversity, NTU60 were cultured in liquid- and solid-state fermentation. A series of separation and purification of the bioactive compound was thus carried out. Ten compounds, including cephaibols A－C (1－3), cephaibol E (4), glycocholic acid (5), cholic acid (6), glycochenodeoxycholate (7), glycodeoxycholsaeure (8), helvolic acid (9) and 1-Linoleoyl glycerol (10) were isolated from liquid- or solid-state fermented products of Acremonium tubakii NTU60. Cephaibol A (1) exhibited the activity of inhibiting Cryptococcus neoformans with a minimum inhibitory concentration (MIC) of 8 μg/mL, while Cephaibol B (2) and Helvolic acid (9) showed the activity of inhibiting Staphylococcus aureus with the MIC 16 μg/mL and 4 μg/mL, respectively. On the other hand, Cephaibol B (2) also showed cytotoxic activity against paclitaxel-resistant ovarian cancer cells (TOV-21G-RT) and hepatocellular carcinoma cells (SK-Hep-1), the half inhibitory concentrations (IC50) were 6.2 ± 0.1 μM and 3.1 ± 0.5 μM, respectively. Cephaibol E (4) showed the inhibitory effect on LPS-induced NO production murine brain microglial cell line (BV-2) with 5.3 ± 1.5 μM, without cytotoxic activity.

碩士<br>國立臺灣大學<br>漁業科學研究所<br>106<br>In this study, a number of alga-derived fungal strains were isolated from marine algae collected from northeastern coast of Taiwan. In the preliminary antimicrobial screening against bacteria and fungi, including Escherichia coli, Staphylococcus aureus, Candida albicans and Cryptococcus neoformans, the ethyl acetate extracts of liquid (potato dextrose broth) and solid (brown rice) fermented products of Acremonium sp. NTU492, a fungus derived from the red alga Mastophora rosea, were found to exhibit significant growth inhibitory activity against C. albicans and C. neoformans. A series of fractionation and separation was thus undertaken, which has resulted in the isolation and purification of 15 compounds, and their structures were elucidated by spectral analysis. Among these, nine novel compounds included acrepeptins A−E (2−6), acremonisins A−B (12−13), (4R, 7R, 9S, E)-4,7-dihydroxy-9-propyl-2,3,4,7,8,9-hexahydro-2H-oxecin-1-one（14）and 4,4-dihydroxy-6,8-dimethoxy-5-methylisochroman-1-one (15). The other were known compounds, determined to be 8-deoxy-trichothecin (1), cyclo[L-alanyl-L-threonyl-(2S)-2-hydroxy-3-methylbutanoyl-L-isoleucyl-(2S)-2-hydroxy-3-methylbutan -oyl] (7), β-alanine, N-[N-[N-[N-[1-(2-hydroxy-4-methyl-1-oxopentyl)-L-prolyl]-L-isoleucyl]-N-methyl-L-valyl]-N-methyl-L-alanyl] (8), guangomide A (9), guangomide B (10) and brefeldin A (11). In terms of antibacterial activity, 8-deoxy-trichothecin (1) showed activity against Candida albicans and Cryptococcus neoformans with MIC = 2.0 μg/mL and 0.5 μg/mL, respectively; brefeldin A（11）showed activity against Candida albicans with MIC = 32 μg/mL; (4R, 7R, 9S, E)-4,7-dihydroxy-9-propyl-2,3,4,7,8,9-hexahydro-2H-oxecin-1-one（14）showed activity against Cryptococcus neoformans with MFC = 16 μg/mL. In respect of anticancer activity, 8-deoxy-trichothecin (1) showed activity against hepatocellular carcinoma cells (SK-Hep-1) and paclitaxel-resistant ovarian cancer cells (TOV-21G-RT), with IC50 = 2.1 and 1.8 μM, respectively; cyclo[L-alanyl-L-threonyl-(2S)-2-hydroxy-3-methylbutanoyl-L-isoleucyl-(2S)-2-hydroxy-3-methyl butanoyl] (7) showed activity against hepatocellular carcinoma cells and prostate cancer cells (PC-3), with IC50 = 3.7 and 6.6 μM respectively; brefeldin A（11）showed activity against hepatocellular carcinoma cells and prostate cancer cells with both IC50 lower than 1 μΜ; (4R, 7R, 9S, E)-4,7-dihydroxy-9-propyl-2,3,4,7,8,9-hexahydro-2H-oxecin-1-one（14）showed activity against hepatocellular carcinoma cells and prostate cancer cells with IC50 = 1.6 and 1.7 μM respectively. Finally, in regard to anti-inflammatory activity, acrepeptin A−C (2−4) inhibited 73.3, 58.6 and 77.2％ of NO production under the concentration of 20 μM. In summary, a total of 15 compounds were isolated, and their structures, chemical properties and biological activities were investigated in this study.

碩士<br>國立中興大學<br>植物病理學系<br>87<br>Abstract In 1996, commercial plantings of lettuce ( Lactuca sativa L. ) at Hsilo in Yunlin County were damaged by a disease previously unreported in Taiwan. Symptoms consisted of small, circular to irregular, yellowish- brown to brown lesions, occurring on minor and lateral veins of lower leaves. Lesions are often so numerous that they coalesce to form areas extending several centimeters along leaf veins. Infection may also result in a severe necrosis of lower leaf. A species of Acremonium was consistently isolated from disease portions on 2% ( w/v ) water agar. Pathogenicity was confirmed by culturing two representative isolates on potato dextrose agar ( PDA ) for 14 days, under a 12-hr light / 12-hr dark cycle, filtering the suspensions through cheesecloth, obtaining spore concentrations of 1.5×105 conidia and spraying suspensions onto thirty four crops including Compositae. Plants were incubated in a humid chamber. After 6 days, leaf spots similar to the original symptoms developed only on inoculated plants of six lettuce cultivars and tricolor chrysanthemum. Other inoculated plants remained symptomless. The inoculation test was repeated and results were the same. On malt extract agar, growth of the fungus is slow. Colonies reach 16.5-20 mm diameter for 10 days at 20-25℃, with pale orange to orange, pionnotal to slightly cottony mycelia. Phialides erect, very variable in length, simple or occasionally branched, septate, hyaline, 20-70μm.. Conidia borne singly but remaining in mucilaginous heads, hyaline, one-septate, smooth-walled, subcylindrical, not distinctly truncated, 11.25-17.5×2.5-5.0μm, L/W 3.5-4.5. Chlamydospores produced abundantly, globose to oval, hyaline, terminal or intercalary, single cell, 5-12.5μm.. The optimum temperature for mycelial growth of Acremonium sp. isolates AL-0818 and AL-0724 was at 24-28℃. The best temperature for both conidial germination and infection on lettuce was at 28℃. The results indicated that the fungus may be a new species of Acremonium on Lactuca sativa in the world. Fourteen carbohydrates and eighteen nitrogenous compounds were evaluated for their effects on mycelial growth of two isolates, AL-0818 and AL-0724 of Acremonium sp., the causal agent of brown spot of lettuce. Among those compounds, sucrose and galactose were more effective than other carbohydrates to enhance the growth of Acremonium sp. As to nitrogenous compounds, casein was the most effective in stimulating growth of the pathogen. The optimum ratio of sucrose and casein by weight was at ten. Sixteen pesticides were respectively added into the basal medium ( a modified Czapek*s medium containing sucrose and casein, SC-medium ) for indexing their suppressive effectiveness. Carbendazim at 250ppm and flutonlanil at 100ppm were not obviously effective in inhibiting the pathogen. In advance, carbendazim, flutonlanil, streptomycin sulfate, and neomycin sulfate were selected to make up a formulation of the selective medium. The relationship between concentrations of the four pesticides and the germ-tube growth length of the pathogen was analyzed for determining the optimal formulation that favored the growth of Acremonium sp. and suppressed the contamination of undesired microorganisms. Finally, Sucrose-casein semiselective medium (SC-semiselective medium) consisting of 30g sucrose, 3g casein, 1g K2HPO4, 0.5g KCl, 0.5g MgSO4*7H2O, 0.01g FeSO4*7H2O, 15g agar, 150ppm carbendazim, 50ppm flutonlanil, 200ppm streptomycin sulfate, 50ppm neomycin sulfate and 1L distilled water was hence formulated for the enumeration and isolation of Acremonium sp. from soil. The results showed that Acremonium sp. was able to be accurately detected from artificially and naturally infested soils by the use of SC semiselective medium. Population density of the pathogen in naturally infested field soil was 0-4.3×103 cfu/g soil by detection with SC semiselective medium. The pathogen population density of artificial infested soil decreased from 105 to 103 after two months in the field, and followed by eight months, the fungus could still be detected from the tested soil. High temperature did not favor the survival of the Acremonium sp. Therefore, population density of the fungus rapidly decreased with increment of the temperature. The soils collected from three kinds of localities affected markedly the survival of the pathogen. Especially, Chihu soil was not the most beneficial for its survival. Eleven organic materials (amendments) were added (0.5-2%, w/w) individually to soil artificially infested with the Acremonium sp. Most of them were significantly effective to enhance or maintain the survival of the pathogen in the soil. Disease incidence of lettuce brown spot was highly correlated with population density of the pathogen in soil. The optimum soil temperatures for the infection on lettuce were 20-28℃. Key words : Lettuce, brown spot of lettuce, host range, survival, new disease, and Acremonium sp.

碩士<br>國立嘉義大學<br>動物科學系研究所<br>97<br>The aim of this study is to investigate the effect of feed supplemented with Acremonium implicatum mycelium (one species of the winter worm summer grass) on the growth performance, carcass traits and immunity of the broilers and Taiwan fightcocks. The first part of the experiment: In the trial 1, sixty 1 wk Arbor acres chicks were randomly assigned into the treatments with the feed supplemented with A. implicatum mycelium 0, 0.1, 0.3 and 0.5% with three replicates for a 4-week feeding trial. The growth performance in all treatments had no difference as compared with control. In the trial 2, twenty nine 5wk Arbor acres male chicks were picked (0%, n=10; 0.1%, n=7; 0.3%, n=7; 0.5%, n=5) and fed as the original treatments in the individual cages for a 7-week feeding trial. The 5 wk BW of the 0.5% treatment were lower than control (P < 0.05). Although the 12 wk BW and 1-12 wk WG of the 0.3% treatment had no difference as compared with control, they were the highest among all the groups (P < 0.05), and were improved 4.8, 4.9%, respectively. The comb length of the 0.5% treatment were significantly lower than the control (P < 0.05). The carcass traits of all treatments had no difference as compared with control (P > 0.05). The second part of the experiment: In the trial 1, the experimental animals were divided into two batches. In the first batch and second batch, thirty six and eighty healthy 10 wk Taiwan fightcocks were randomly assigned into the treatments with feed supplemented with A. implicatum mycelium 0, 0.1, 0.5 and 1% with three and two replicates for a 10-week feeding trial. And the chickens in the first and second batch were fed in the individual cages and floor pens respectively. The 0.5 and 1% treatments could significantly increase 10-13 wk WG and FCR, and improve the comb appearance. In the trial 2, forty five 20 wk healthy male Taiwan fightcocks were randomly assigned into the treatments with feed supplemented with A. implicatum mycelium 0, 0.1, 0.5, 1 and 3% with three replicates in the individual cages for a 9-week feeding trial. The growth performance, carcass traits, immunity and leukocyte subpopulation had no difference as compared with control (P > 0.05). In conclusion, feed supplemented with 0.5% and 1% A. implicatum mycelium could improve the comb development and 10-13 wk WG and FCR of the Taiwan fightcocks.

博士<br>國立陽明大學<br>生化暨分子生物研究所<br>96<br>Glucooligosaccharide oxidase (GOOX) from Acremonium strictum catalyzes the oxidation of a variety of carbohydrates to the corresponding lactones with high selectivity for cello- and maltooligosaccharides. The crystal structures of GOOX alone or with substrates including 5-amino-5-deoxy-cellobiono-1,5-lactam, cellobiose, cellotriose, cellotetraose, and lactose, show the substrate preference with an open carbohydrate-binding groove allowing the accommodation of higher oligosaccharides. The complex structures also display the preference of a beta-D-glucosyl residue at the reducing end with the conserved Tyr429 acting as a general base to abstract the OH1 proton in concert with the H1 hydride transfer to the flavin N5. The Y429H/F mutants still have residual activities, but reduced to 2% and 4% of kcat observed for wild-type, respectively, indicating the important role in catalysis. Moreover, the first known bi-covalent flavinylation, a 6-S-cysteinyl, 8alpha-N1-histidyl FAD, is demonstrated which is cross-linked to Cys130 and His70. Single mutants of H70A/C/S/Y and C130A still retain the covalent FAD, indicating that flavinylation at these two sites is independent. The wild-type protein exhibits a midpoint potential of +126 mV. The H70A, H70C and C130A mutants with a redox potential of ~+70 mV, ~+106 mV and +61 mV, respectively, still possess lower activity, but the kcat values reduced to about 2%, 32%, and 5%, while the flavin reduction rate to 0.6%, 9% and 14%, compared to the wild-type GOOX. The C130A crystal structure provides direct evidence for a novel function of 6-S-cysteinylation in assistance of substrate binding. Finally, the double H70A/C130A replacement abolishes the covalent linkage, FAD binding and enzyme activity. The wild-type enzyme is more heat and guanidine-HCl resistant than the mutants. Therefore, the bi-covalent flavin linkage enhances not only the cofactor binding, the redox potential, the substrate binding but also the stability of the protein structure.