Orientador: Telma Teixeira Franco / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-07-27T08:22:39Z (GMT). No. of bitstreams: 1 Ribeiro_AnaPauladosSantos_M.pdf: 2171899 bytes, checksum: 7cac906a30aa8e097dcc2c5be3d20c69 (MD5) Previous issue date: 2000 / Resumo: Enzimas quitinolíticas, como as quitinases, presentes nos reinos animal, vegetal e fúngico, constituem um importante grupo de enzimas associadas com o metabolismo e degradação de substratos insolúveis como a quitina. Neste trabalho foi realizado um estudo para a verificação da influência da fonte de carbono livre (glicose e lactose) e das condições de cultivo (agitação e pH) na produção de quitinase da linhagem de Trichoderma sp. T 6. As fermentações foram realizadas a 27°C, retirando-se amostras a cada 12 horas ao longo de 72 horas de fermentação para determinação da atividade quitinolítica, proteolítica, açúcares redutores e pH. Foi avaliado o efeito das três variáveis sobre a produção de quitinase por Trichoderma sp. T6. As melhores condições encontradas para o meio contendo glicose, foram agitação de 200 rprn, pH 6,0,0,3% de glicose atingindo uma média de produção de enzima de 1,92 U/rnL. Para os meios de cultura contendo lactose, a máxima produção de quitinase se deu nos ensaios 5,6 e 7 (0,5% lactose) com 0,76,0,75 e 0,85 U/rnL, respectivamente. Foi observada formação de proteases desde o início da fermentação nos ensaios contendo lactose. A hidrólise enzimática com o caldo bruto do ensaio de máxima produção quitinolítica foi acompanhada por 8 horas produzindo dois oligossacarídeos predominantes, o mono e o di-acetilquitobiose / Abstract: Chitynolytics enzymes, as chitinases, constitute an important group of enzymes related with metabolism and degradation of insoluble substract as chitin present in animal, plants and fungi. In this work studies of carbon source (glucose and lactose) and cultivation conditions (agitation and pH) to produce chitinase by Trichoderma sp. T6 were carried out. The effect of carbon source, agitation and pH upon chitinase production by Trichockrma sp. T6 was investigated. Fermentations were performed at 27°C, and samples were withdraw every 12 hours during 72 hours of fermentation in order to assay both chitinolytic and proteolytic activity, as well as reducing sugar and pH. The optimal conditions for chitinase production found in glucose medium were 200 rpm (agitation), 6,0 (pH) and 0,3% (glucose) achieving an average value of 1,92 V/roL. Liquid culture medium with lactose showed maximum production of chitinase at 5th, 6th and 7th experiments (0,5% lactose) with 0,76, 0,75 and 0,85 V/roL, respectively. lt was observed that proteases were formed at beginning of fermentation in the experiments using lactose. Chitin hydrolysis by the raw broth of maximum chitinolytic activity was followed for 8 hours and the two predominant oligosaccharides, were mono and di-acetylchitobiose, respectively / Mestrado / Desenvolvimento de Processos Químicos / Mestre em Engenharia Química

Memoria para optar al título profesional de Ingeniero Agrónomo Mención Fitotecnia / Uno de los problemas más importantes de manejo dentro de un green de golf, es el manejo del fieltro, que se presenta como una capa entre la parte verde de la planta y la superficie del suelo. La presencia del fieltro le da al green características poco deseables a la hora de jugar, también le confiere una mayor susceptibilidad a plagas y enfermedades, baja conductividad hidráulica, mayor susceptibilidad a estrés por frío o calor, etc. Como la capa de fieltro está mayoritariamente compuesta por tejido en descomposición, se planteó la hipótesis que al aplicar Trichoderma harzianum esta capa disminuiría su grosor ya que este hongo es capaz de descomponer la materia orgánica presente en el fieltro. El estudio se realizó en un vivero de pasto de green en el Prince of Wales Country Club, en la Comuna de La Reina desde Noviembre del 2009 a Enero del 2010, utilizando un diseño experimental de bloques completos al azar con cinco repeticiones. El objetivo es evaluar diferentes dosis de aplicación de Trichoderma harzianum en la descomposición de la materia orgánica y la reducción de la capa de fieltro, por lo que se probaron dos dosis del producto comercial HARZTOP más un testigo y se realizaron tres mediciones: altura de capa de fieltro, contenido de materia orgánica en el fieltro y una medición de consolidación. Los resultados arrojaron una disminución de un 40% en la altura del fieltro con el uso del producto comercial, llegando a una altura mínima promedio de 11 mm. La materia orgánica, contrario a lo planteado en la hipótesis, presentó un significativo incremento, se cree que la causa de esto es un aumento en el desarrollo de las raíces estimulado por la presencia del hongo, lo que aumentó la cantidad de residuo orgánico en el fieltro. Finalmente la prueba de consolidación dio como resultado que a mayor fieltro, mayor presión mayor es la deformación de la superficie y mayor es el tiempo que le toma recuperarse y volver a su estado inicial. Esto influye negativamente al green, ya que se presentan hundimientos en la superficie de las pisadas de los jugadores, provocando desvíos en la trayectoria de la pelota sobre el green. / In this assay it was tested different amounts of a product which have Trichoderma harzianum as active ingredient with the purpose of a thatch reduction. These applications were done on Agrostis stolonifera nursery in the months of November 2009 until January 2010. The hypothesis raised was that the fungus has the ability to degrade organic matter that decomposes the thatch given a decreasing in the thickness of the layer. It was used a complete randomized block with five repetitions. The thatch height in millimeters, organic matter content in the thatch and a consolidation test were evaluated. The results show up a 40% thatch height reduction with the use of the commercial product, reaching up to 11 mm as a mean height. Organic matter, contrary to what was stated in the hypothesis presented a significant increase. Finally the consolidation test gave as a result that at a higher thatch, higher is the game surface deformation and at a higher pressure the surface deformation is also superior and besides, it takes some time to recover and come back to its initial state.

No description available.

A novel type of model substrates, i.e. immobilized p-aminophenyl-β-D-cellooligosaccharides, was developed and used in the study of exocellulases. The two major cellobiohydrolases from Trichoderma reesei, CBH I and CBH II were used as representative enzymes. p-Aminophenyl derivatives of cellobiose (PAPG₂), cellotriose (PAPG₃), and cellotetraose (PAPG₄) were synthesized from the reaction of p-nitrophenol and peracetylated glycosyl bromide of the corresponding cellooligosaccharides under the phase-transfer catalyzed conditions, followed by deacetylation and catalytic hydrogenation. p-Aminophenyl cellooligosaccharides were then tethered via their amino functional groups to N-hydroxy succinimide-activated agarose. The ability of CBH I and CBH II to associate with and catalyze the hydrolysis of reducing end tethered cellooligosaccharides was tested. CBH I catalyzed the hydrolysis of free PAPG₂ but CBH II did not. Both CBH I and CBH II reversibly bound, but did not hydrolyze, immobilized PAPG₂. Hence, the immobilized PAPG₂ was tested for the affinity chromatographic application. PAPG₂ is shown to be an effective ligand for the chromato graphic fractionation of cellobiohydrolases (CBHs). The PAPG₂-derivatized agarose specifically retained the CBH component of relatively complex cellulase mixtures. The purity of the resulting CBH preparation was comparable to that of corresponding enzyme preparations obtained using more traditional thioglycoside-based affinity ligands. The application of PAPG₂ as an affinity ligand suggests that the immobilized reducing end-blocked ligand associate with the T. reesei CBHs in a catalytically nonproductive mode. The catalytic activity for the hydrolysis of free and immobilized arylcellodextrins by the CBH I and CBH II were determined. CBH II attacked free and immobilized PAPG₃ and PAPG₄ in a typical exo manner in which cellobiose is a major hydrolytic product released from the nonreducing end. The rate of hydrolysis increases with increasing chain length suggesting the extended binding sites (at least 4 binding sites). Like CBH II, CBH I preferentially cleaved immobilized PAPG₃ and PAPG₄ at a second glycosidic linkage from the nonreducing end; the rate of hydrolysis increases as a function of chain length. However, it attacked free aryl-cellodextrins in a random manner. The rate of hydrolysis increases only from PAPG₂ to PAPG₃ and significantly drops in PAPG₄. This suggests that CBH I interacts with free and immobilized substrates in different modes. / Graduation date: 1999

The traditional filter paper assay for saccharifying cellulase originally described by M. Mandels et al (1976) has been modified to make possible low activity determinations of Trichoderma cellulases. The enzymatic activity appears to decline during a prolonged incubation period if no precautions have been taken. By means of adding bovine serum albumin and potassium chloride as protein stabilizers and sodium azide as an antimicrobial agent filter paper activities in the range from 0.02 to 0.37 (IUPAC assay, 1987) can be estimated by extending the incubation time up to 20 hours. Filter paper activity values obtained by this method may be compared to those obtained by the IUPAC assay by using a conversion factor from 1.4 to 1.7. Acid whey and sweet whey permeate have been investigated as media for growth and metabolite production by Trichoderma reesei QM 9414 using shake flask cultures and spore inocula. In the case of acid whey the mycelial growth after 2 weeks is 13 mg dry weight /ml substrate. The specific growth rate is 0.29/day. The fungus appears to metabolize the whey protein the first 2 weeks. The alkalinity of acid whey rises continuously over a three week period up to a pH of 8.5. In the case of whey permeate the maximal mycelial weight gain is 4.4 mg/ml which appears after 8 days. A rise in net soluble protein level comes after 3-5 days and reaches a maximum value of 0.23 mg/ml after 2 weeks. The pH of whey permeate rises continuously to 7.5 after 3 days and then slowly declines. The net production of cellulases is low on both media. Dilution 1:6 of the acid whey, supplementation with ammonium sulfate and pHadjustments did not enhance the production of cellulases. Acid whey supports a significant growth and sweet whey permeate shows potential for extracellular protein production. A literature review surveys the composition and uses of acid whey, environmental aspects of whey wastes, the fungus Trichoderma reesei, the mode of action of the Trichoderma reesei cellulase system and the structure of cellulose in cotton and wood. / Graduation date: 1994

<i>Trichoderma harzianum </i>and <i>Aneurinobacillus migulanus </i>were tested separately and in combination as potential biological control agents against <i>F. oxysporum </i>f. sp. <i>gladioli </i>on gladiolus in soil-less culture.  The major secondary metabolites produced by <i>Trichoderma harzianum </i>against <i>F. oxysporum </i>f. sp. <i>gladioli </i>in the treated gladiolus corms as well as in the potato dextrose broth were studied.  The impact of re-application times on the effectiveness of <i>Trichoderma harzianum </i>and <i>Aneurinobacillus migulanus </i>against gladiolus corm rot and Gladiolus production in soil-less culture was examined. Results showed that <i>T. harzianum </i>proved to use different mechanisms against <i>F. oxysporum </i>f. sp. <i>gladioli </i>depending on  the inoculation method. <i>T. harzianum </i>and <i>A. migulanus </i>reduced host defence enzymes.  Also, they increased GA ₃ and IAA content in the corms.  SEM showed that the suppressive mechanisms of <i>T. harzianum </i>and <i>A. migulanus </i>differed <i>T. harzianum </i>appeared to operate through a combination of antibiosis and substrate competition, 6-n-pentyl-6H-pyran-2-one (6PP) and harzianic acid (HA) were found as the main metabolites secreted by <i>T. harzianum.  </i>A significant positive correlation was revealed between qPCR and CFU for both <i>T. harzianum </i>and <i>F. oxysporum </i>f. sp. <i>gladioli.  </i>Multiple applications of <i>T. harzianum </i>and <i>A. migulanus </i>re- application was a successful strategy for suppression of <i>F. oxysporum </i>f. sp. <i>gladioli.  </i>Briefly, <i>T. harzianum </i>was more effective than <i>A. migulanus </i>in <i>F. oxysporum </i>f. sp. <i>gladioli </i>suppression and Gladiolus growth enhancement in soil-less culture. The mixing strategy between <i>T. harzianum </i>and <i>A. migulanus </i>failed.

Glicose e oxigênio são moléculas essenciais para a maioria dos organismos vivos. Além de sua importância nos processos de produção de energia - glicose como fonte de carbono e energia e oxigênio como aceptor dos elétrons doados por NADH e FADH2 - estes dois compostos funcionam como efetuadores, modulando vários processos metabólicos e fisiológicos nas células. Visto que a mitocôndria é um dos alvos afetados pelas disponibilidades destas duas moléculas, nós isolamos e seqüenciamos o genoma mitocondrial de Trichoderma reesei, um fungo multicelular empregado neste trabalho como sistema modelo. Foi estudado o efeito da variação de concentração de glicose e oxigênio sobre a expressão de transcritos do genoma mitocondrial, bem como sua implicação no metabolismo de glicose. São apresentadas análises da expressão gênica de aproximadamente 2000 transcritos de T. reesei submetido a concentrações limitantes de oxigênio dissolvido, realizadas com o emprego da técnica de microarrays de cDNA. Pelo menos 330 transcritos foram diferencialmente expressos em função da disponibilidade de oxigênio. Aqueles envolvidos nos processos de síntese protéica e divisão celular foram regulados negativamente, enquanto transcritos relacionados com funções de defesa celular e síntese de RNA foram positivamente regulados. Uma fração substancial de outros genes afetados pela baixa disponibilidade de oxigênio não possui, atualmente, funções celulares conhecidas. Esta observação deve contribuir para a posterior anotação funcional do genoma de T. reesei. Também foram identificados reguladores transcricionais diferencialmente expressos em baixas tensões de oxigênio. O perfil de expressão destes reguladores aponta-os como potenciais candidatos ao envolvimento com a expressão de genes afetados pela disponibilidade de oxigênio. / Glucose and oxygen are essential molecules in most of living organisms. In addition to their importance in production of energy - glucose as a carbon and energy source and oxygen as an acceptor of electrons donated by NADH and FADH2 - both molecules function as effectors modulating various metabolic and physiological processes in the cell. Because one of the targets affected by both molecules is the mitochondrion, we isolated and sequenced the mitochondrial genome of Trichoderma reesei, a multicellular fungus that is used in this study as a model system. The effect of varying the concentration of glucose and oxygen on the expression of the transcripts of the mitochondrial genome, and its implication on the metabolism of glucose, was studied. Gene-wide expression analyses of nearly 2000 transcripts of T. reesei under limited concentration of dissolved oxygen, using cDNA microarry technique, are presented. At least 330 transcripts were differentially expressed with respect to oxygen availability. Those involved in protein synthesis and cell division processes were downregulated, while transcripts involved in cell defense and RNA synthesis were upregulated. A substantive fraction of other anaerobically affected genes have currently unknown cellular roles, and these results should therefore contribute to further functional annotation of the genome. ln addition, we have identified transcriptional regulators that are differentially expressed at a low oxygen tensions. The expression profile of these regulators points them out as potential candidates involved in the expression of genes affected by oxygen availability.

In this research, the interfacial behavior of Trichoderma reesei CBHI and Thermomonospora fusca E��� cellulases were studied at synthetic surfaces. For this purpose, colloidal silica and polystyrene particles were used to prepare cellulase-particle suspensions that were analyzed by several solution-phase techniques. These included circular dichroism spectroscopy, size exclusion chromatography and filtration, and a spectrophotometric assay for cellulase activity. All techniques were performed in the presence and absence of particles. Circular dichroism spectroscopy (CD) and size exclusion chromatography showed, however, that binding did not occur between either cellulase and silica, presumably because silica is hydrophilic and negatively charged. Binding did occur between each cellulase and polystyrene, most likely mediated through hydrophobic associations. Cellulase-polystyrene complexes were not analyzed using CD because of high light absorption by the polystyrene nanoparticles. Upon adsorption to polystyrene, the activity of the E��� dropped about 95% relative to that of the free enzyme. While this substantial loss in activity may have been the result of binding being mediated through the catalytic domain, strong evidence supporting the thought that adsorption occurs through hydrophobic associations, mediated through the binding domain, suggests that structural or steric factors were partly responsible for the loss. / Graduation date: 1999

The efficiency of cellulose hydrolysis under straight saccharification and simultaneous saccharification and fermentation (SSF) conditions was evaluated using three lignocellulosic materials (switchgrass, cornstover, and poplar), which had been pretreated with dilute sulfuric acid under conditions which optimized xylose concentrations in the prehydrolysate liquid. Yields of glucose, cellobiose and ethanol obtained from the pretreated feedstocks were measured over 168 hrs. The final theoretical conversions of cellulose from pretreated switchgrass, cornstover, and poplar in straight saccharification were 85-100% (average 94%), 84-100% (average 96%), and 75-100% (average 87%), respectively, while in SSF the conversions were 84-90% (average 87%), 91-96% (average 90%), 72%-82% (average 76%), respectively. The conversion rates of poplar in straight saccharification and SSF were significantly lower than those of switchgrass and cornstover. The effects of reaction parameters such as enzyme activity, cellulose availability, and yeast cell viability on the extent of hydrolysis in straight saccharification and SSF were also studied. Results indicate that the lower glucose or ethanol yields associated with some of the poplar were due to the recalcitrant nature of its cellulose. To compare accurately the efficiencies between straight saccharification and SSF, a direct method for determining the cellulose content of the feedstocks residues resulting from SSF experiments has been developed and evaluated. The method improves on classical cellulose assays by incorporating a yeast lysing enzyme to remove yeast glucans from the feedstocks residue prior to acid hydrolysis and subsequent quantification of cellulose derived glucose. A freeze-drying step was identified as necessary to render the SSF yeast cells susceptible to enzyme lysis. The method was applied to the analysis of the cellulose and yeast-glucan content of SSF residues from the three pretreated feedstocks. Cellulose assays employing the lysing enzyme preparation demonstrated relative errors up to 7.2% when yeast-associated glucan were not removed prior to analysis of SSF residues. Enzymatic lysis of SSF yeast cells may be viewed as a general preparatory procedure to be used prior to the subsequent chemical and physical analysis of SSF residues. / Graduation date: 1996

Fungal endophytes live inside plants without causing apparent symptoms of infection. All plant species surveyed thus far, including liverworts, mosses, seedless vascular plants, conifers, and angiosperms, harbor one or more endophytic fungi. Fungal endophytes can be divided into four groups including class 1, class 2, class 3 and class 4 endophytic fungi according to host range, colonization pattern, transmission, and ecological function. Class 2 fungal endophytes benefit their host by increasing environmental stress tolerance (i.e. water, temperature, salt) in a habitat-specific manner. In my study, class 2 fungal endophytes were studied from weedy plants growing in an environmentally stressed area: mine tailings from the Athabasca oil sands. This area is a vast hydrocarbon reserve in western Canada that supplies 10% of Canadian oil needs. Hydrocarbons are extracted from tar sands with hot water, alkali, and solvents. The tailing sands can later be remediated (by adding organic material and fertilizer) to establish new plant communities. Prior to remediation, tailing sands have extremely low content of organic carbon and available minerals, and are hydrophobic compared to unimpacted and remediated soils. Nevertheless, <i>Taraxacum</i> (dandelion) and <i>Sonchus</i> (sow-thistle) can colonize extracted tailing sands even prior to remediation. Preliminary results show that pioneer plants have similar fungal abundance as plants of unextracted treatments. Fungal endophytes were isolated from surface sterilized <i>Taraxacum</i> and <i>Sonchus</i> that had been growing upon unimpacted, remediated and extracted soil. Fungi isolated in this way included <i>Alternaria, Tricoderma, Fusarium</i> and an unidentified <i>Perithecial Ascomycote</i>. These endophytic fungi were used to inoculate tomato plants in a greenhouse trial to determine whether they confer stress tolerance to host plants, especially for drought and low mineral nutrition. Before exposing the tomato plants to environmental stresses, the specific endophytic fungal strains applied were successfully recovered from tomato plants originally inoculated with the same endophytic fungi. Although the other endophytic fungi turned out to be harmful to the tomato plants in the test, a <i>Trichoderma spp.</i> strain isolated from samples of extracted treatment appears to confer tolerance of tailing sands to the tomato plants. This <i>Trichoderma spp.</i> strain which we can call <i>TSTh20-1</i> was molecularly identified as <i>Trichoderma harzianum</i>. Despite an identification to species, all strains of <i>T. harzianum</i> are not necessarily identical regarding strain-specific attributes. Using similar techniques described here, it is possible to isolate and potentially use beneficial class 2 endophytic fungal strains for the remediation process in the Athabasca oil sands or to assist plant growth in other high stress environments.