Bactérias quitinolíticas desenvolvem papel fundamental no ciclo de nutrientes através da degradação da quitina no ecossistema marinho. A hidrólise da quitina é realizada por quitinases, as quais podem ser utilizadas em processos biotecnológicos. O estudo teve como objetivo avaliar as condições ótimas de cultivo de bactérias quitinolíticas selecionadas do gênero Aeromonas e a degradação de quitina durante 96 horas de cultivo. Além disso, caracterizá-las pelo sequenciamento total do gene 16S rRNA e por MLSA. O estudo mostrou que os isolados representam duas espécies do gênero Aeromonas. Os isolados comportaram-se de diferentes maneiras em relação às condições de cultivo pré-determindas. Os halos de hidrólise de quitina não estão relacionados à atividade enzimática ou aos produtos de hidrólise de quitina quantificados. As maiores atividades de quitobiosidases e endoquitinases foram observadas em 24 horas e de N-acetil-glicosaminidases em 96 horas. Os perfis enzimáticos diferem entre os isolados e ao longo das 96 horas de cultivo mostrando diversidade enzimática. / Chitinolytic bacteria have key role in nutrient cycling through the chitin degradation in the marine ecosystem. The hydrolysis of chitin is performed by chitinases, which can be used in biotechnological processes. The study aimed to evaluate the optimal conditions for cultivation of selected chitinolytic bacteria of the Aeromonas genus and the degradation of chitin during 96 hours of culture. Moreover, characterize them by 16S rRNA gene full sequencing and MLSA. The study showed that the isolates represent two species of the Aeromonas genus. The isolates had different behavior in relation under conditions pre-determined. The halos of chitin hydrolysis were not related to enzymatic activity or to their products of chitin hydrolysis quantified. The hightest activities of chitobiosidases and endochitinases were observed in 24 hours and the N-acetyl-glicosaminidases in 96 hours. The enzyme profiles differ between isolates and during 96 hours of cultivation showing diversity enzyme.

A produção brasileira de alimentos cresce em ritmo vertiginoso, havendo previsão de expansão nos próximos anos. Nota-se, porém, que o modelo atual empregado ao controle de doenças e pragas agrícolas, tem causado diversos problemas de ordem ambiental, social e econômica. Uma alternativa à redução de tais impactos, tem sido a implementação do controle biológico no modelo de manejo. Neste trabalho, avaliou-se o controle biológico do fungo fitopatogênico Botrytis cinerea por compostos produzidos pela linhagem ASBV-1T. Os screenings \"in vitro\" demonstraram que a linhagem produz quitinases e metabólitos secundários ativos, conhecidamente descritos como fatores importantes ao controle de fitopatógenos. A caracterização parcial do complexo enzimático, indicou que as quitinases produzidas pela linhagem ASBV-1T apresentam maior atividade em meio alcalino (pH 6.8-10.0), temperatura de 30°C e possuem estimativa de peso molecular superior a 100 kDa. Os ensaios \"in vivo\", realizados em morangos comerciais (cv. Oso Grande) demonstraram a efetividade dos subprodutos bioativos de caráter ionóforos (monactina, dinactina, trinactina, tetranactina e valinomicina) em controlar a infestação de B. cinerea, durante a fase de pós-colheita, em condições de armazenamento. Por meio da suplementação do meio de cultivo por sais inorgânicos, a via biossintética responsável pela expressão dos compostos de interesse pode ser ativada. A adição de MgS04 resultou em um aumento de 400% na produção de macrotetralídeos e 20% de valinomicina. Contudo, a expressão dos macrotetralídeos foi completamente inibida pela adição de ZnSO4. dobrando a produção de valinomicina. De acordo com os estudos de taxonomia polifasica, o isolado ASBV-1T, apresenta caracteristicas quimiotaxonômicos e moleculares pertinentes ao gênero Streptomyces, formando uma nova linha filética, no subclado das espécies S. albolongus, S. cavourensis subsp. cavourensis e S. celluloflavus. Diante do supra citado, propõem-se que o isolado seja reconhecido como a linhagem tipo de Streptomyces araujoniae sp. nov. Os resultados obtidos neste trabalho, permitem afirmar que a linhagem S. araujoniae sp. nov. apresenta potencial de ação contra o fitopatógeno B. cinerea, podendo auxiliar no controle desta doença durante a fase de pós-colheita da cultura de morangos. Ainda resta, para estudos futuros, o desenvolvimento de formulação específica para o emprego destes compostos no manejo da cultura, bem como ensaios toxicológicos, assegurando a viabilidade de aplicação de tais compostos. / The Brazilian food production has been growing fast in recent years and is expected to expand even more. However, the models used for the control of pests and diseases in agriculture has been widely questioned because they cause problems of environmental, social and economic. To reducing the use of chemical agents, has been the implementation of biological control to the management model to reduce this problematic. In this study, we evaluated the biological control of fungal pathogen Botrytis cinera of active compounds produced by strain ASBV-1T. In vitro assays indicated the presence of chitinase and antifungal metabolites in compounds produced by strain ASBV-1T and may been contributed to the antagonistic activity againts the fungal pathogenic. Partial characterization of the enzymatic complex showed the highest production of chitinase under alkalines conditions at pH (6.8-10.0), 30°C and has estimated molecular weight above 100 kDa. The test in vivo, preformed in commercial strawberries (cv. Oso Grande) demonstrated the effectiveness of bioactive products of character ionophores (monoactin, dinactin, trinactin, tetranactin and valinomycin) controlling the infestation of B. cinerea during the post-harvest, in store conditions. The biosynthetic pathway responsible for the expression of the compounds of interest can be activated by supplementations of the culture medium by inorganic salts mainly with MgSO4 resulted in increse of 400% in the production of macrotetralides and 20% valinomycin. However, the expression of the macrotetralides was inhibited when added ZnSO4, bending the production of valinomycin. According to polyphasic taxonomic studies, the isolated ASBV-1T has chemotaxonomic and molecular characteristics of the genus Streptomyces, which formed a new phyletic line, in subcalde of the S. albolongus, S. cavourensis subsp. cavourensis e S. celluloflavus species. Then propose that the isolate is recognized as the type strain of Streptomyces araujoniae sp. nov. The results obtained in this study revealed that S. araujoniae sp. nov. has action potential against the pathogen B. cinerea, can help control this disease during the post-harvest strawberries. Remains for future studies, the development of specific formulation of the employment of such compounds to crop management and toxicological tests, ensuring the feasibility of using such compounds.

A produção brasileira de alimentos cresce em ritmo vertiginoso, havendo previsão de expansão nos próximos anos. Nota-se, porém, que o modelo atual empregado ao controle de doenças e pragas agrícolas, tem causado diversos problemas de ordem ambiental, social e econômica. Uma alternativa à redução de tais impactos, tem sido a implementação do controle biológico no modelo de manejo. Neste trabalho, avaliou-se o controle biológico do fungo fitopatogênico Botrytis cinerea por compostos produzidos pela linhagem ASBV-1T. Os screenings \"in vitro\" demonstraram que a linhagem produz quitinases e metabólitos secundários ativos, conhecidamente descritos como fatores importantes ao controle de fitopatógenos. A caracterização parcial do complexo enzimático, indicou que as quitinases produzidas pela linhagem ASBV-1T apresentam maior atividade em meio alcalino (pH 6.8-10.0), temperatura de 30°C e possuem estimativa de peso molecular superior a 100 kDa. Os ensaios \"in vivo\", realizados em morangos comerciais (cv. Oso Grande) demonstraram a efetividade dos subprodutos bioativos de caráter ionóforos (monactina, dinactina, trinactina, tetranactina e valinomicina) em controlar a infestação de B. cinerea, durante a fase de pós-colheita, em condições de armazenamento. Por meio da suplementação do meio de cultivo por sais inorgânicos, a via biossintética responsável pela expressão dos compostos de interesse pode ser ativada. A adição de MgS04 resultou em um aumento de 400% na produção de macrotetralídeos e 20% de valinomicina. Contudo, a expressão dos macrotetralídeos foi completamente inibida pela adição de ZnSO4. dobrando a produção de valinomicina. De acordo com os estudos de taxonomia polifasica, o isolado ASBV-1T, apresenta caracteristicas quimiotaxonômicos e moleculares pertinentes ao gênero Streptomyces, formando uma nova linha filética, no subclado das espécies S. albolongus, S. cavourensis subsp. cavourensis e S. celluloflavus. Diante do supra citado, propõem-se que o isolado seja reconhecido como a linhagem tipo de Streptomyces araujoniae sp. nov. Os resultados obtidos neste trabalho, permitem afirmar que a linhagem S. araujoniae sp. nov. apresenta potencial de ação contra o fitopatógeno B. cinerea, podendo auxiliar no controle desta doença durante a fase de pós-colheita da cultura de morangos. Ainda resta, para estudos futuros, o desenvolvimento de formulação específica para o emprego destes compostos no manejo da cultura, bem como ensaios toxicológicos, assegurando a viabilidade de aplicação de tais compostos. / The Brazilian food production has been growing fast in recent years and is expected to expand even more. However, the models used for the control of pests and diseases in agriculture has been widely questioned because they cause problems of environmental, social and economic. To reducing the use of chemical agents, has been the implementation of biological control to the management model to reduce this problematic. In this study, we evaluated the biological control of fungal pathogen Botrytis cinera of active compounds produced by strain ASBV-1T. In vitro assays indicated the presence of chitinase and antifungal metabolites in compounds produced by strain ASBV-1T and may been contributed to the antagonistic activity againts the fungal pathogenic. Partial characterization of the enzymatic complex showed the highest production of chitinase under alkalines conditions at pH (6.8-10.0), 30°C and has estimated molecular weight above 100 kDa. The test in vivo, preformed in commercial strawberries (cv. Oso Grande) demonstrated the effectiveness of bioactive products of character ionophores (monoactin, dinactin, trinactin, tetranactin and valinomycin) controlling the infestation of B. cinerea during the post-harvest, in store conditions. The biosynthetic pathway responsible for the expression of the compounds of interest can be activated by supplementations of the culture medium by inorganic salts mainly with MgSO4 resulted in increse of 400% in the production of macrotetralides and 20% valinomycin. However, the expression of the macrotetralides was inhibited when added ZnSO4, bending the production of valinomycin. According to polyphasic taxonomic studies, the isolated ASBV-1T has chemotaxonomic and molecular characteristics of the genus Streptomyces, which formed a new phyletic line, in subcalde of the S. albolongus, S. cavourensis subsp. cavourensis e S. celluloflavus species. Then propose that the isolate is recognized as the type strain of Streptomyces araujoniae sp. nov. The results obtained in this study revealed that S. araujoniae sp. nov. has action potential against the pathogen B. cinerea, can help control this disease during the post-harvest strawberries. Remains for future studies, the development of specific formulation of the employment of such compounds to crop management and toxicological tests, ensuring the feasibility of using such compounds.

Bactérias quitinolíticas desenvolvem papel fundamental no ciclo de nutrientes através da degradação da quitina no ecossistema marinho. A hidrólise da quitina é realizada por quitinases, as quais podem ser utilizadas em processos biotecnológicos. O estudo teve como objetivo avaliar as condições ótimas de cultivo de bactérias quitinolíticas selecionadas do gênero Aeromonas e a degradação de quitina durante 96 horas de cultivo. Além disso, caracterizá-las pelo sequenciamento total do gene 16S rRNA e por MLSA. O estudo mostrou que os isolados representam duas espécies do gênero Aeromonas. Os isolados comportaram-se de diferentes maneiras em relação às condições de cultivo pré-determindas. Os halos de hidrólise de quitina não estão relacionados à atividade enzimática ou aos produtos de hidrólise de quitina quantificados. As maiores atividades de quitobiosidases e endoquitinases foram observadas em 24 horas e de N-acetil-glicosaminidases em 96 horas. Os perfis enzimáticos diferem entre os isolados e ao longo das 96 horas de cultivo mostrando diversidade enzimática. / Chitinolytic bacteria have key role in nutrient cycling through the chitin degradation in the marine ecosystem. The hydrolysis of chitin is performed by chitinases, which can be used in biotechnological processes. The study aimed to evaluate the optimal conditions for cultivation of selected chitinolytic bacteria of the Aeromonas genus and the degradation of chitin during 96 hours of culture. Moreover, characterize them by 16S rRNA gene full sequencing and MLSA. The study showed that the isolates represent two species of the Aeromonas genus. The isolates had different behavior in relation under conditions pre-determined. The halos of chitin hydrolysis were not related to enzymatic activity or to their products of chitin hydrolysis quantified. The hightest activities of chitobiosidases and endochitinases were observed in 24 hours and the N-acetyl-glicosaminidases in 96 hours. The enzyme profiles differ between isolates and during 96 hours of cultivation showing diversity enzyme.

Chitinases are a diverse family of proteins occurring in plants. Their function varies considerably, with certain chitinases having been associated with development. The majority however, are pathogenesis related (PR) proteins that have been shown to play a role during plant pathogen interactions. This has lead to many investigations on the use of chitinases in providing transgenic disease resistance. These studies are usually done using a constitutive expression system. This however stands in contrast with the natural defense system were PR gene expression is usually only upregulated when the plant is exposed to abiotic and/or biotic stress factors. The constitutive expression is therefore not ideal as it increases ‘cost’ penalties due to the energy being spent expressing the gene. In this study however, an inducible expression system was applied using a stress inducible promoter AtGSTF6 derived from Arabidopsis thaliana, to drive Lupinus albus IF3 chitinase expression when the plants are under pathogen attack. The construct AtGSTF6-IF3 was inserted into the binary vector pCAMBIA 2300 and transformed into Nicotiana Tabacum cv JR6 by Agrobacterium-mediated transformation. To demonstrate the functionality of such a construct, an expression study was done on transgenic N. Tabacum to determine transcription and in vitro chitinase enzyme activity. The data revealed that IF3 chitinase gene transcription from lupin plants was achieved in N. Tabacum. Nine of the twelve lines that tested positive for chitinase gene transcription after hydrogen peroxide treatment, showed increased chitinase activity. With the success of showing increased chitinase activity, these lines were subjected to a detached leaf assay with Rhizoctonia solani AG2, which causes leaf target spot disease. The assay showed that six of the nine lines identified as having increased chitinase activity showed reductions in lesion areas. More specifically, three of the four lines showing more than a five-fold increase in chitinase activity compared to the untransformed N. Tabacum, showed significant lesion reduction. The AtGSTF6-IF3 construct can therefore be recommended to increase disease resistance in N. Tabacum towards Rhizoctonia solani AG2 after showing both expression and increased disease resistance in certain transgenic lines. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Plant Science / unrestricted

No description available.

Os fungos entomopatogênicos, como Metarhizium anisopliae, têm despertado grande interesse como agentes no controle de insetos-pragas. Estas espécies de fungos são especializadas na secreção de um complexo enzimático constituído de proteases, lipases e quitinases, entre outras, estando relacionadas com patogenicidade e virulência. Neste contexto a foi analisada a secreção de quitinases pelos isolados IBCB 167, IBCB 360, IBCB 384 e IBCB 425 de M. anisopliae var. anisopliae, como identificado molecularmente. Contudo, alguns aspectos morfológicos analisados mostram pequenas diferenças entre estes isolados. Para produção de quitinases, os isolados foram cultivados em fermentação submersa na presença e ausência de indutores e em fermentação em estado sólido, tendo crisálida como substrato. A maior síntese de quitinase intracelular foi obtida para IBCB 425 no meio contendo extrato de levedura mais glicose (EG). Visando a produção da enzima extracelular e disponibilidade de fonte de carbono, o meio extrato de levedura mais crisálida (EC), sob agitação foi padronizado para fermentação submersa (FSbm). Os maiores níveis enzimáticos intracelulares para os isolados IBCB 167, IBCB 360, IBCB 384 e IBCB 425 foram obtidos entre 72 h e 216 h e para a forma extracelular entre 96h e 144h. A produção quitinásica em fermentação sólida (FSS) utilizando crisálida como fonte de carbono foi otimizada por delineamento composto central rotacional (DCCR, tendo como variáveis o tempo de crescimento e umidade. O melhor produtor de quitinase em FSS foi o isolado IBCB 360. A análise do secretoma mostrou um maior número de proteínas quando os isolados foram cultivados na presença do indutor, com destaque para o isolado IBCB 425. A maioria das proteínas secretadas pelos isolados IBCB 167 e IBCB 384, foram identificadas, estando entre elas a endo-N-acetyl--D-glucosaminidase, enzima do complexo quitinolítico. As quitinases produzidas pelos quatro isolados foram parcialmente purificadas em DEAE Celulose, obtendo-se dois picos de atividade quitinásica. O processo de purificação foi continuado para o IBCB 384 em coluna de exclusão molecular, obtendo se um único pico de atividade quitinásica, analisado por electrospray. A temperatura ótima de atividade para as quitinases produzida em FSS pelos isolados IBCB 167, IBCB 360, IBCB 384 e IBCB 425 variou de 50ºC a 60ºC e pH ótimo de atividade ficou na faixa de 5,0 - 5,5. As quitinases dos isolados mantiveram-se estáveis nas temperaturas de 30ºC e 40ºC e em uma ampla faixa de pH. Os sais BaCl2 e MnCl2 ativaram as quitinases dos isolados IBCB 167 e IBCB 425. Além disso, todas as quitinases foram tolerantes ao - mercaptoetanol. O comportamento cinético de todas as quitinases foi Michaeliano e a maior afinidade pelo substrato foi para a quitinase do isolado IBCB 360. Já os experimentos in vivo e in vitro mostraram que o isolado IBCB 425 foi melhor na fase pré-infecção e isolado IBCB 384 foi melhor na fase pós infecção, sendo o mais virulento. Portanto, os isolados M. anisopliae têm se mostrado como bons produtores de quitinases, com boa estabilidade a temperatura e pH além de outras características distintas que provavelmente estão relacionadas com o potencial de patogenicidade e virulência de cada isolado. / Entomopathogenic fungi such as Metarhizium anisopliae, have been attracting great interest as agents to control insect pests. These species of fungi are specialized in the secretion of an enzymatic complex consisting of proteases, lipases and chitinases, among others which are related to pathogenicity and virulence. In this context the secretion of chitinase by IBCB 167, IBCB 360, IBCB 384 and IBCB 425 isolated from M. anisopliae var. anisopliae molecularly identified, were analyzed. However, some structural features analyzed showed small differences among these isolates. For chitinase production, the isolates were grown in submerged culture in the presence and absence of inducers and under solid state fermentation with chrysalis as substrate. The enhanced synthesis of intracellular chitinase was obtained with IBCB 425 using yeast extract plus glucose (EG). Aiming to produce the extracellular enzyme and the carbon source available, the medium yeast extract and chrysalis (EC), was standardized to SbmF. The high intracellular enzymes levels produced by IBCB 167, IBCB 360, IBCB 384 and IBCB 425 isolates were obtained between 72 h and 216 h and for the extracellular form between 96h and 144h. The chitinase production in SSF using chrysalis as carbon source was optimized by CCRD, having the time of growth and moisture as variables. The best producer of chitinase in SSF was the isolated IBCB 360. The analysis of the secretome showed a great number of proteins when the isolates were grown in the presence of the inducer, especially for the isolated IBCB 425. Most of the proteins secreted by IBCB 167 and IBCB 384 isolates were identified. Among these proteins the endo-N-acetyl--D-glucosaminidase was identified, enzyme that participates in the chitinulitic complex. Chitinases produced by the isolates were partially purified on DEAE - cellulose, yielding two peaks of chitinase activity. The purification process was continued for IBCB 384 isolated using molecular exclusion chromatographic column, yielding only one peak of chitinase activity, analyzed by electrospray. The optimal temperature for the chitinase activity from IBCB 167, IBCB 360, IBCB 384 and IBCB 425 isolates obtained in SSF, ranged from 50 ºC to 60 ºC and the optimum pH of activity was 5.0 to 5.5. All chitinases were stable at 30 ºC and 40 ºC, and wide pH range. The BaCl2 and MnCl2 salts activated the chitinases of IBCB 167 and IBCB 425 isolates. In addition, all chitinases were mercaptoethanol tolerant. The kinetic behavior of all chitinases was Michaelian with the highest affinity to the substrate observed for the chitinase of isolated IBCB 360. The in vivo and in vitro experiments showed that isolated IBCB 425 was better in pre -infection phase and the isolated IBCB 384 was better in the post infection phase. Therefore, the M. anisopliae isolates were good producers of chitinases with interesting temperature and pH stability, and other different characteristics that are probably related to the potential pathogenicity and virulence of each isolate.

Phytohormones act as signals to regulate plant growth and development by modulation of gene expression in response to internal developmental cues or external environmental stimuli, such as light and pathogen infection. There are five major classes of phytohormones, viz. auxins, cytokinins, gibberellins, ethylene and abscisic acid. Of these, cytokinins, 6N substituted adenine derivatives, are of special importance owing to their possible diverse roles in plant growth and development. They induce cell division, cell expansion in cotyledon, chloroplast and etioplast development, suppression of apical dominance and senescence, and differentiation of in vitro cultured cells. However, very little is known about the mechanism of cytokinin action at the molecular level. Cytokinins have been demonstrated to modulate the expression of genes coding for several enzymes including nitrate reductase, ribulose-l,5-bisphosphate carboxylase, RNA polymerase I, and pathogenesis-related (PR) enzymes, i.e. chitinases and β-1,3- glucanases. One of the important questions regarding cytokinin regulation of enzyme activities and/or the accumulation of their corresponding proteins and mRNAs is how the cytokinin signal is transduced. There is considerable evidence from earlier reports demonstrating that pathogens alter hormone physiology of the host plant and it has been proposed that the infection-associated enzyme changes might be mediated by phytohormones. In the present study, two PR enzymes, viz. cucumber chitinase and β-l,3-glucanase, have been chosen to examine the mode of regulation of their gene expression by cytokinins, including the identification of cytokinin signal transduction components. Plant chitinases and glucanases are important enzymes in plant defense mechanisms against fungal pathogens as they degrade the major fungal cell wall components, chitin and β-1,3-glucan, respectively. Besides their role in plant defense, they are known to be involved in diverse physiological and developmental processes, such as embryogenesis, seed germination and flower development, and are also developmentally and hormonally regulated. Initially, in order to study the effects of various cytokinins on chitinase and β-1,3-gIucanase enzyme activities and their gene expression, cotyledons excised from seven-day-old dark-grown cucumber seedlings were treated with water, and cytokinins, viz. benzyladenine, kinetin, zeatin and zeatin riboside. It was observed that chitinase and β-l,3-ghucanase enzyme activities and their transcripts were induced to varying extents following treatments of cotyledons with the cytokinins tested. However, a maximum increase in enzyme activities and their transcript levels was noticed in zeatin-treated cotyledons. Therefore, zeatin was used for further studies. The main objective of the present study was to investigate the cytokinin signal transduction mechanism involving the induction of expression of chitinase and β-1-3-glucanase. In order to obtain insights into the downstream components of the cytokinin-signaling pathway, effects of several agonists and antagonists of the signal transduction components on zeatin-induced chitinase and β-l,3-glucanase activities, and their protein and transcript levels were monitored by enzyme assay, by immunoblot analysis, and by northern analysis, respectively. Treatment of excised dark-grown cucumber cotyledons with staurosporine, a broad spectrum protein kinase inhibitor, reduced the zeatin-induced chitinase and β-l,3-glucanase enzyme activities and the accumulation of their proteins and transcripts. On the other hand, treatment with sodium fluoride, a general inhibitor of protein phosphatases, stimulated the basal chitinase and β-1,3-glucanase enzyme activities and their protein and transcript accumulation, whereas it had no effect on the zeatin-induced enzyme activities and their protein and transcript accumulation. These findings suggested that protein phosphorylation is critical in the cytokinin induction of expression of chitinase and β-l,3-glucanase. Since Ca2+ is known to be an important second messenger in several plant signal transduction pathways, the possible involvement of Ca2+ in the cytokinin-induced expression of chitinase andβ-l,3-glucanase was examined. The results of the present investigation showed that the chitinase and β-1,3-ghicanase activities and their proteins and transcripts were appreciably increased by exogenous CaCl2 treatment in control cotyledons. Treatment of cotyledons with zeatin plus CaCl2 did not result in a further increase in either these enzyme activities or their protein and transcript accumulation as compared to zeatin or CaCl2 treatment alone. The lack of additivity of zeatin plus CaCl2 treatment indicated a common mechanism of action of zeatin and Ca2+ in the induction of these enzyme activities and their gene expression. To test the occurrence of influx of extracellular Ca2+ by cytokinin, cotyledons were treated with the plasma membrane Ca2+ channel blocker, verapamil, and Ca2+ ionophore A23187. Verapamil treatment inhibited the zeatin-induced chitinase and β-1,3-ghicanase enzyme activities and their protein and transcript accumulation. An increase in the intracellular Ca2+ levels by means of Ca2+ ionophore treatment resulted in a significant increase in basal chitinase and β-l,3-glucanase activities and their protein and transcript accumulation. These results suggested that an influx of extracellular Ca2+ leading to increased levels of cytosolic Ca2+ is required for the cytokinin induction of expression of these enzymes. The correlation of chitinase and β-1,3-glucanase enzyme activities and their protein and transcript accumulation in the zeatin-treated cotyledons suggested that the cytokinin zeatin stimulates chitinase and β-l,3-glucanase accumulation at the mRNA level and that the increase in enzyme activities is due to an increase in the amount of the enzyme protein and not by the activation of the existing enzyme. Further, the effect of zeatin on both the enzyme activities and their transcript levels under conditions that inhibit protein synthesis was studied. Treatment of excised dark-grown cucumber cotyledons with cycloheximide, an inhibitor of protein synthesis, in the presence of zeatin, completely nullified the stimulatory effect of zeatin. These results indicated the requirement of cytokinin-induced enhanced concurrent protein synthesis in the observed stimulation of chitinase and β-l,3-glucanase enzyme activities as well as their transcript accumulation Ca2+ In an attempt to isolate the full length cucumber β-l,3-glucanase cDNA from a cucumber cDNA library, we isolated and sequenced one cDNA clone, which was 978 bp long and had a potential polyadenylation signal A ATA A starting 172 bases before the polyadenylation tail A deduced amino acid sequence of the cDNA, which was 242 amino acids in length, apparently encoded a partial β-amyrin synthase. Sequence comparison of the deduced partial amino acid sequence of cucumber β-amyrin synthase with other known plant β-amyrin synthase sequences available in databases revealed significant homologies to β-amyrin synthases from Panax, Pisum and Glycyrrhiza. Southern blot analysis indicated that there was only one β-amyrin synthase gene in the cucumber genome. RT-PCR analysis performed on total RNA isolated from zeatin- and salicylic acid-treated cotyledons using forward and reverse primers designed from the internal regions of the cDNA showed that the transcript levels of β-amyrin synthase were enhanced by both zeatin and salicylic acid. In conclusion, we have demonstrated that chitinase and β-l,3-glucanase accumulation is stimulated by exogenous cytokinin treatment of excised cucumber cotyledons, and this effect is correlated with the content of chitinase and β-1,3-glucanase transcripts as judged by northern analyses. Further, the findings reported in the thesis suggested that Ca2+ influx from extracellular space, protein phosphorylation by staurosporine-sensitive protein kinase(s) and concurrent protein synthesis are required for the signaling of cytokinin-induced expression of both these pathogenesis-related enzymes.

Orientador: Helia Harumi Sato / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-06T15:33:09Z (GMT). No. of bitstreams: 1 Fleuri_LucianaFrancisco_D.pdf: 2217112 bytes, checksum: 6e5410649f00f4a57c5ec78f758b6ebb (MD5) Previous issue date: 2006 / Resumo: O presente trabalho visou o estudo da produção, purificação e aplicação de b-1,3 glucanases, proteases e quitinases. A linhagem Cellulosimicrobium cellulans 191 foi utilizada para o estudo da produção de b-1,3 glucanases e quitinases e as linhagens B26 e C. cellulans 191 foram utilizadas para a produção de proteases, em meios de cultivo contendo diferentes indutores. Foram realizados planejamentos fatorias 23, e os fatores estudados foram: pH inicial, temperatura (oC) e agitação dos frascos. No planejamento experimental para a produção de b-1,3 glucanase foi verificado maior produção da enzima (0,64 U/mL) em meio de cultivo A composto por 2,0 g/L de (NH4)2SO4; 0,2 g/L de MgSO4.7H2O e 10 g/L de parede celular de levedura em tampão fosfato 0,2 M, pH 8,5, após 24 h de fermentação, a 33oC e 200 rpm. Nos planejamentos experimentais para a produção de protease pelas linhagens B26 e 191 foi verificado maior produção da enzima em meio de cultivo B composto por 2,0 g/L de (NH4)2SO4; 0,2 g/L de MgSO4.7H2O e 80 g/L de levedura seca utilizada como indutor em tampão fosfato 0,15 M, pH 6,5, após 30 h de fermentação a 20oC e 200 rpm, sendo obtido 5,01 U/mL e 4,25 U/mL, respectivamente. No planejamento experimental para a produção de quitinase foi verificado maior produção da enzima (7,06 U/mL) em meio de cultivo C composto por 4,0 g/L de extrato de levedura; 2,0 g/L de triptona; 4,0 g/L de MgSO4.7H2O; 1,2 g/L de KH2PO4; 2,8 g/L de K2HPO4 e 15 g/L de quitina neutralizada utilizada como indutor, com pH inicial de 5,5 após 72 h de fermentação a 25oC e 200 rpm. Todos os modelos obtidos foram preditivos e significativos a um nível de confiança de 95%. No estudo de produção das enzimas da linhagem C. cellulans 191 em fermentador de 5 L, a maior produção de b-1,3 glucanase, utilizando meio de cultivo A e 1,5 vvm e 3 vvm, foi respectivamente 0,32 U/mL e 0,72 U/mL, após 24 h de fermentação a 30oC. Na produção de protease em fermentador de 5 L, utilizando meio de cultivo B e 1,5 vvm foi obtido 1,87 U/mL e 2,34 U/mL, respectivamente, após 6 h e 30 h de fermentação a 30oC; enquanto que com 3 vvm, foi obtido 4,89 U/mL e 6,14 U/mL de protease após 6 h e 33 h de fermentação, a 30oC. Em fermentador de 5 L, a maior produção de quitinase em meio de cultivo C foi de 4,19 U/mL com 1,5 vvm após 168 h de fermentação e 4,38 U/mL de quitinase após 144 h de fermentação com 3 vvm, a 25oC. No estudo de produção de b-1,3 glucanases, proteases e quitinases da linhagem C. cellulans 191 em frascos agitados, em meios de cultivo A, B e C, foram produzidos respectivamente, 1,12 U/mL de b-1,3 glucanase; 4,2 U/mL de protease e 6,9 U/mL de quitinase. A b-1,3 glucanase (45 KDa) foi purificada 11,83 vezes com rendimento de 25% em resina de troca iônica DEAE-Sephadex A50. Na purificação das proteases em resina de troca iônica DEAE-Sephadex A50 foram obtidas três frações de proteases denominadas P1, P2 e P3. A fração P3 apresentou duas bandas de massas moleculares de 14 e 16 KDa em eletroforese SDS-PAGE. A quitinase (61 KDa) foi purificada cerca de 6,65 vezes com rendimento de 46,61% em resina de filtração em gel Sepharose CL4B200. A b-1,3 glucanase purificada apresentou atividade de lise de diversas leveduras e foi capaz de formar protoplastos da levedura Saccharomyces cerevisiae KL-88. O pré-tratamento das leveduras com protease P3 purificada não aumentou a lise das leveduras com a ß-1,3 glucanase. A quitinase purificada foi capaz de lisar células de algumas espécies de fungos em suspensão aquosa, mas não foi capaz de inibir, o crescimento dos fungos em placas de ágar batata dextrose. A preparação bruta de quitinase apresentou halo de inibição do crescimento de alguns fungos estudados. Os produtos formados pela reação da b-1,3 glucanase purificada sobre a laminarina e da protease purificada sobre a levedura seca apresentaram capacidade antioxidante / Abstract: The aim of this work was to study the production, purification and application of b-1,3 glucanases, proteases and chitinases. The strain Cellulosimicrobium cellulans 191 was used to study the production of b-1,3 glucanases and chitinases and strains B26 and C. cellulans 191 for the production of proteases, using culture media containing different inductors. 23 factorial experimental designs were performed and the factors studied were: initial pH, temperature (oC) and flask rotatory speed. In the experimental design for the production of b-1,3 glucanase, greater enzyme production (0.64 U/mL) was obtained in culture medium A containing 2.0 g/L (NH4)2SO4; 0.2 g/L MgSO4.7H2O and 10 g/L cell wall yeast in 0.2 M phosphate buffer, pH 8.5, after 24 h of fermentation at 33oC and 200 rpm. In the experimental design for the production of protease by strains B26 and 191, greater enzyme production was obtained in culture medium B containing 2.0 g/L (NH4)2SO4; 0.2 g/L de MgSO4.7H2O and 80 g/L dry yeast in 0.15 M phosphate buffer, pH 6.5, after 30 h of fermentation at 20oC and 200 rpm, with yields of 5.01 U/mL and 4.25 U/mL, respectively. In the experimental design for the production of chitinase, greater enzyme production (7.06 U/mL) was obtained in culture medium C containing 4.0 g/L yeast extract; 2.0 g/L tryptone; 4.0 g/L MgSO4.7H2O; 1.2 g/L KH2PO4; 2.8 g/L K2HPO4 and 15 g/L of neutralized chitin with an initial pH of 5.5, after 72 h of fermentation at 25oC and 200 rpm. All models obtained were predictive and significant at a confidence level of 95%. In the study on the production of enzymes from C. cellulans strain 191 in a 5 L fermenter, the highest productions of b-1,3 glucanase in medium A with 1.5 vvm and 3.0 vvm were 0.32 U/mL and 0.72 U/mL respectively, after 24 h of fermentation at 30oC. In the production of protease in a 5 L fermenter using culture medium B and 1.5 vvm, 1.87 U/mL and 2.34 U/mL were obtained after 6 h and 30 h respectively of fermentation at 30oC, whilst with 3 vvm, 4.89 U/mL and 6.14 U/mL of protease were obtained after 6 h and 33 h respectively of fermentation at 30oC. In a 5 L fermenter, the highest production of chitinase from C. cellulans strain 191 using 1.5 vvm was 4.19 U/mL after 168 h of fermentation, whilst with 3 vvm, the production was 4.38 U/mL of chitinase after 144 h of fermentation at 25oC. In the study on the production of b-1,3 glucanases, proteases and chitinases from C. cellulans strain 191 in shaken flasks and culture media A, B and C, 1.12 U/mL of b-1,3 glucanase; 4.2 U/mL of protease and 6.9 U/mL of chitinase were produced respectively. In the purification study, the b-1,3 glucanase (45 KDa) was purified 11.83 times with a yield of 25% using a DEAE-Sephadex A50 ion-exchange resin. In the purification of the proteases using the DEAE-Sephadex A50 ion-exchange resin, three protease fractions were obtained named P1, P2 and P3. Fraction P3 presented two proteins with molecular weights of 14 and 16 KDa in SDS-PAGE electrophoresis. The chitinase (61 KDa) was purified about 6.65 times with a yield of 46.61% in a Sepharose CL4B200 gel filtration resin. The purified b-1,3 glucanase presented lysis activity against several yeasts and was able to form protoplasts from the Saccharomyces cerevisiae KL-88 yeast. Pre-treatment of the yeasts with the purified protease P3 did not increase cell lysis by the b-1,3 glucanase. The purified chitinase was able to lyse the cell walls of some fungal species in aqueous suspension, but was not able to inhibit the growth of these fungi on potato dextrose agar plates. The crude chitinase preparation presented growth inhibition halos for some of the fungi studied. The products formed from the reaction between the purified b-1,3 glucanase and laminarin and between the purified protease and the dry yeast presented antioxidant power / Doutorado / Doutor em Ciência de Alimentos

Os fungos entomopatogênicos, como Metarhizium anisopliae, têm despertado grande interesse como agentes no controle de insetos-pragas. Estas espécies de fungos são especializadas na secreção de um complexo enzimático constituído de proteases, lipases e quitinases, entre outras, estando relacionadas com patogenicidade e virulência. Neste contexto a foi analisada a secreção de quitinases pelos isolados IBCB 167, IBCB 360, IBCB 384 e IBCB 425 de M. anisopliae var. anisopliae, como identificado molecularmente. Contudo, alguns aspectos morfológicos analisados mostram pequenas diferenças entre estes isolados. Para produção de quitinases, os isolados foram cultivados em fermentação submersa na presença e ausência de indutores e em fermentação em estado sólido, tendo crisálida como substrato. A maior síntese de quitinase intracelular foi obtida para IBCB 425 no meio contendo extrato de levedura mais glicose (EG). Visando a produção da enzima extracelular e disponibilidade de fonte de carbono, o meio extrato de levedura mais crisálida (EC), sob agitação foi padronizado para fermentação submersa (FSbm). Os maiores níveis enzimáticos intracelulares para os isolados IBCB 167, IBCB 360, IBCB 384 e IBCB 425 foram obtidos entre 72 h e 216 h e para a forma extracelular entre 96h e 144h. A produção quitinásica em fermentação sólida (FSS) utilizando crisálida como fonte de carbono foi otimizada por delineamento composto central rotacional (DCCR, tendo como variáveis o tempo de crescimento e umidade. O melhor produtor de quitinase em FSS foi o isolado IBCB 360. A análise do secretoma mostrou um maior número de proteínas quando os isolados foram cultivados na presença do indutor, com destaque para o isolado IBCB 425. A maioria das proteínas secretadas pelos isolados IBCB 167 e IBCB 384, foram identificadas, estando entre elas a endo-N-acetyl--D-glucosaminidase, enzima do complexo quitinolítico. As quitinases produzidas pelos quatro isolados foram parcialmente purificadas em DEAE Celulose, obtendo-se dois picos de atividade quitinásica. O processo de purificação foi continuado para o IBCB 384 em coluna de exclusão molecular, obtendo se um único pico de atividade quitinásica, analisado por electrospray. A temperatura ótima de atividade para as quitinases produzida em FSS pelos isolados IBCB 167, IBCB 360, IBCB 384 e IBCB 425 variou de 50ºC a 60ºC e pH ótimo de atividade ficou na faixa de 5,0 - 5,5. As quitinases dos isolados mantiveram-se estáveis nas temperaturas de 30ºC e 40ºC e em uma ampla faixa de pH. Os sais BaCl2 e MnCl2 ativaram as quitinases dos isolados IBCB 167 e IBCB 425. Além disso, todas as quitinases foram tolerantes ao - mercaptoetanol. O comportamento cinético de todas as quitinases foi Michaeliano e a maior afinidade pelo substrato foi para a quitinase do isolado IBCB 360. Já os experimentos in vivo e in vitro mostraram que o isolado IBCB 425 foi melhor na fase pré-infecção e isolado IBCB 384 foi melhor na fase pós infecção, sendo o mais virulento. Portanto, os isolados M. anisopliae têm se mostrado como bons produtores de quitinases, com boa estabilidade a temperatura e pH além de outras características distintas que provavelmente estão relacionadas com o potencial de patogenicidade e virulência de cada isolado. / Entomopathogenic fungi such as Metarhizium anisopliae, have been attracting great interest as agents to control insect pests. These species of fungi are specialized in the secretion of an enzymatic complex consisting of proteases, lipases and chitinases, among others which are related to pathogenicity and virulence. In this context the secretion of chitinase by IBCB 167, IBCB 360, IBCB 384 and IBCB 425 isolated from M. anisopliae var. anisopliae molecularly identified, were analyzed. However, some structural features analyzed showed small differences among these isolates. For chitinase production, the isolates were grown in submerged culture in the presence and absence of inducers and under solid state fermentation with chrysalis as substrate. The enhanced synthesis of intracellular chitinase was obtained with IBCB 425 using yeast extract plus glucose (EG). Aiming to produce the extracellular enzyme and the carbon source available, the medium yeast extract and chrysalis (EC), was standardized to SbmF. The high intracellular enzymes levels produced by IBCB 167, IBCB 360, IBCB 384 and IBCB 425 isolates were obtained between 72 h and 216 h and for the extracellular form between 96h and 144h. The chitinase production in SSF using chrysalis as carbon source was optimized by CCRD, having the time of growth and moisture as variables. The best producer of chitinase in SSF was the isolated IBCB 360. The analysis of the secretome showed a great number of proteins when the isolates were grown in the presence of the inducer, especially for the isolated IBCB 425. Most of the proteins secreted by IBCB 167 and IBCB 384 isolates were identified. Among these proteins the endo-N-acetyl--D-glucosaminidase was identified, enzyme that participates in the chitinulitic complex. Chitinases produced by the isolates were partially purified on DEAE - cellulose, yielding two peaks of chitinase activity. The purification process was continued for IBCB 384 isolated using molecular exclusion chromatographic column, yielding only one peak of chitinase activity, analyzed by electrospray. The optimal temperature for the chitinase activity from IBCB 167, IBCB 360, IBCB 384 and IBCB 425 isolates obtained in SSF, ranged from 50 ºC to 60 ºC and the optimum pH of activity was 5.0 to 5.5. All chitinases were stable at 30 ºC and 40 ºC, and wide pH range. The BaCl2 and MnCl2 salts activated the chitinases of IBCB 167 and IBCB 425 isolates. In addition, all chitinases were mercaptoethanol tolerant. The kinetic behavior of all chitinases was Michaelian with the highest affinity to the substrate observed for the chitinase of isolated IBCB 360. The in vivo and in vitro experiments showed that isolated IBCB 425 was better in pre -infection phase and the isolated IBCB 384 was better in the post infection phase. Therefore, the M. anisopliae isolates were good producers of chitinases with interesting temperature and pH stability, and other different characteristics that are probably related to the potential pathogenicity and virulence of each isolate.