Malgré l’arsenal antifongique existant aujourd’hui, les mycoses superficielles sont en constante progression de par le monde et notamment dans le bassin caribéen. Nous nous sommes focalisés sur les pathologies qui posent, en Guadeloupe, de nombreux problèmes de résistance ou de rechute vis-à-vis des antifongiques actuels, à savoir : les dermatophyties, le Pityriasis versicolor (Malassezia sp.), les candidoses et les scytalidioses. Suite à ce constat, nous nous sommes tournés vers la flore guadeloupéenne où ont été sélectionnées dix plantes sur des critères ethnobotaniques, chimiotaxonomiques ou encore d’observations naturalistes avec un double objectif : trouver de nouveaux extraits antifongiques d’une part et, d’autre part, étudier leur composition et vérifier leur innocuité. Le screening antifongique in vitro des extraits c-hexane, EtOH et EtOH/eau (1:1, v/v) a été réalisé vis-à-vis de 4 dermatophytes, 1 Malassezia sp., 5 Candida spp. et 1 Scytalidium sp. Les extraits ont également été testés vis-à-vis d’un autre pathogène, Pneumocystis jirovecii responsable de la pneumocystose pulmonaire. Quatre plantes : Bursera simaruba, Cedrela odorata, Enterolobium cyclocarpum et Pluchea carolinensis ont été retenues afin de définir leurs cytotoxicités puis de procéder à l’isolement des composés responsables de leur activité antifongique par bioguidage. Cedrela odorata a montré une activité significative vis-à-vis de Pneumocystis jirovecii due en partie à la (+)-catéchine. Concernant les mycoses superficielles, Bursera simaruba et Cedrela odorata présentent une activité due à une synergie de composés non identifiés par bioguidage alors que Pluchea carolinensis et Enterolobium cyclocarpum doivent respectivement leurs activités à des flavonoïdes sulfatés et à des saponosides triterpéniques. Faisant suite à cette étude phytochimique et pharmacologique, la formulation des extraits sous forme de gels et vernis a été développée. Ainsi, cette étude permet d’apporter une réponse originale et efficace aux pathologies ciblées. / Despite the existing arsenal of antifungals today, superficial fungal infections have increased over the world and especially in the Caribbean basin. We focused our work on diseases that pose, in Guadeloupe, many problems of resistance or recurence towards current antifungals : dermatophytosis, Pityriasis versicolor (Malassezia sp.), Candidosis and Scytalidiosis. Following this observation, we were interested in the flora of Guadeloupe where ten plants were selected on natural observation, ethnobotanical or chemotaxonomical criteria with a dual purpose: to find new antifungal extracts on the one hand, and secondly, to study their composition and verify their safety. The in vitro screening of c-hexane, EtOH and EtOH/water (1:1, v/v) extracts, was made towards four dermatophytes, one Malassezia sp., five Candida spp. and one Scytalidium sp. The extracts were also tested on another pathogen, Pneumocystis jirovecii which is responsible of pneumonia. Four plants: Bursera simaruba, Cedrela odorata, Enterolobium cyclocarpum and Pluchea carolinensis were chosen to define their cytotoxicities and then we proceed to the isolation of active compounds by bioguiding. Cedrela odorata showed significant activity on Pneumocystis jirovecii and (+)-catechin was found to be partly responsible of this activity. Concerning the research on the superficial mycoses, Bursera simaruba and Cedrela odorata’s activities were due to a synergy of compounds unidentified by bioguiding while Pluchea carolinensis and Enterolobium cyclocarpum owe their activities to sulfated flavonoids and triterpene saponins, respectively. Following to these phytochemical and pharmacological studies, the drug formulation of the extracts has been developed. Thus, this study could be an original and effective response to the targeted disease.

A celulose é a mais abundante fonte de carbono presente na madeira e nos resíduos agrícolas, e a sua hidrólise completa é realizada pela ação sinergística de diferentes enzimas, como: as endo-1,4-ß-D-glucanase, exo-1,4-ß-glucanase e ß-glucosidase ou celobiase. O presente trabalho descreve algumas propriedades fisiológicas e bioquímicas do sistema ß-glucosidásico do fungo termofílico Scytalidium thermophilum. Tal fungo foi isolado originalmente do solo da Índia e gentilmente cedido pelo Dr. G. Straastma (Holanda). O meio M8 favoreceu a produção das ß-glucosidases. Entre os açúcares testados como fonte de carbono, avicel e celobiose foram os melhores indutores das ß-glucosidases extracelular e micelial. Quando o fungo foi crescido em dois estágios, observou-se inicialmente a repressão da síntese por glicose e a indução por avicel ou celobiose. Utilizando-se ciclo-heximida, observou-se a síntese \"de novo\" das proteínas. A ß-glucosidase extracelular foi purificada utilizando-se um fracionamento protéico e uma coluna de troca-iônica DEAE-celulose, de onde foram obtidos duas atividades enzimáticas denominadas ß-glucosidases I e II. A ß-glucosidase I foi aplicada em coluna de troca iônica CM-celulose, enquanto que a ß-glucosidase II foi aplicada em Sephadex G-100. A ß-glucosidase I foi purificada 2 vezes com 4.0% de recuperação, ao passo que a ß-glucosidase II foi purificada 2,4 vezes com 2.0% de recuperação. A ß-glucosidase micelial foi purificada utilizando-se um choque térmico, fracionamento protéico, coluna de filtração Sephadex G-100 e uma coluna troca-iônica DEAE-celulose. Foi purificada 23 vezes com recuperação de 25%. A ß-glucosidases extracelular I e micelial apresentaram um temperatura ótima aparente de 70 e 60°C e um pH de 5.5 e 6.0, respectivamente. Ambas enzimas foram inibidas por Ag+2 e Hg+2. A ß-glucosidases extracelular I e micelial possuem um peso molecular de 40.7 kDa e 39kda (SDS-Page) e 57 kDa e 33,8 kda (Sephadex G-100), respectivamente. A ß-glucosidase extracelular I foi capaz de hidrolisar PNP-glu, PNP-xil, celobiose, xilana e CMC, enquanto que a ß-glucosidase micelial hidrolisou PNP-glu, PNP-fuc, PNP-xil, PNPgal, ONPG e lactose. Ambas enzimas foram ativadas por glicerol a 1M. A ß-glucosidase extracelular I foi ativada por xilose, frutose e lactose, e se mostrou resistente a glicose 50mM, enquanto que a ß-glucosidase micelial foi ativada por glicose e xilose. ß-glucosidases extracelular I e micelial apresentaram um PI de 4.0 e 6.5, respectivamente. Os parâmetros cinéticos estimados para a ß-glucosidase extracelular I foram de Km 4,33 e 0,342mM e Vmáx de 5,37 e 2,0µmoles/min/mg prot. para celobiose e PNP-glu, respectivamente. O valor de Ki (Constante de Inibição) foi de 71mM para glicose. Para a ß-glucosidase micelial, os valores de Km e Vmáx foram de 0,29mM e 13,27µmoles/min/mg prot; 0,5 mM e 7,25µmoles/min/mg prot e 1,61 mM e 4,12µmoles/min/mg prot para os substratos PNP-glu, PNP-fuc e celobiose, respectivamente. Na presença de glicose e xilose os valores de Km e Vmáx foram de 1,26mM e 40,04 µmoles/min/mg.prot, e 1,33mM, e 30,49 µmoles/min/mg prot, respectivamente para o PNP-glu. O valor de Ki (Constante de Inibição) foi de 1,32mM para celobiose. A análise dos produtos de hidrólise das ß-glucosidases extracelular I e micelial foram anlisadas em TLC, e revelaram que ambas enzimas realizam hidrólise quando celobiose foi utilizada a 10mM, e transglicosilação quando celobiose foi utilizada a 250mM. Os resultados aqui apresentados demonstram importante papel importante do Scytalidium como produtor de ß-glucosidase com potencial na sacarificação enzimática da celulose. / Cellulose is the most abundant carbon source found in woods and waste residues. In nature the complete hidrolysis of cellulose occurs by the sinergistic action of several enzymes such endo-1,4-ß-D-glucanase, exo-1,4-ß-glucanase e ß-glucosidase or cellobiase. The present work describe some physiological and biochemical properties of ß-glucosidase system from thermophilic fungus Scytalidium thermophilum. The fungus was gift to Dr. Straastma (Mushroom Experimental Station, The Netherlands). The culture medium M8 enhance the production of ß-glucosidase. Among carbohydrates tested as carbon source, avicel and cellobiose were the best inducers of ß-glucosidase extracellular and mycelial. When the fungus was grown in two stages, observed the repression by glucose, and induction by avicel or cellobiose. The presence of cycloheximide inhibited the syntesis of ß-glucosidase, suggesting that the enzyme produced in the presence of indutors required \"de novo\" synthesis. Extracellular ß-glucosidase was purified using the precipitation with 75% amonium sulfate, ion exchange cromatography column DEAE-cellulose, and were obtained two activities: ß-glucosidase extracellular I and II. The ß-glucosidase I was applied to a CM-cellulose colunm, while ß-glucosidase II was applied to a Sephadex G-100 colunm. The ß-glucosidase II was purified two times and 4% yield, and the ß-glucosidase II was purified 2,4 times and 2% yield. The mycelial ß-glucosidase was purified using the termic treatment, a precipitation with 75% amonium sulfate followed by Sephadex G-100 and DEAEcellulose. The enzyme was purified 23 time with 23% yield. The ß-glucosidase extracellular II and mycelial shown optima of temperature and pH of 60°C and 70°C, 4.4 and 6.0, respectively. Hg+2 and Ag+2 ions were strong inhibitors of ß-glucosidase extracellular I and mycelial. The molecular weight of ß-glucosidase extracellular I and mycelial was stimated as 40.7 KDa and 39KDa (SDS-PAGE) and 57kDa and 33.8 kDa (Sephadex G-100). The ß-glucosidase extracellular I hydrolyzed PNP-glu, PNP-xyl, cellobiose,xylan and CMC, while ß-glucosidase mycelial hydrolyzed PNP-fuc, PNP-xyl, PNP-gal, ONPG and lactose. Both enzymes were activeted by glycerol 1M. The ß-glucosidase extracellular I was activeted by xylose, fructose and lactose, and show strong at glucose 50mM. The ß-glucosidase mycelial was activeted by glucose and xylose. ß-glucosidase extracellular I and mycelial shows PI 4.0 and 6.5, respectively. The kinects studies reveled for ß-glucosidase extracellular I a Km of 4,33 and 0,342mM and Vmáx of 5,37 and 2,0µmoles/min/mg prot for cellobiose and PNP-glu, respectively. The Ki values obtained from Dixon plots was 71mM for glucose. To ß-glucosidase mycelial the Km and and Vmáx were 0,29mM e 13,27µmoles/min/mg prot; 0,5 mM e 7,25µmoles/min/mg prot and 1,61 mM and 4,12µmoles/min/mg prot for PNP-glu, PNPfuc and cellobiose, respectively. Using xylose or glucose the Km and Vmáx was 1,26mM e 40,04 µmoles/min/mg.prot, and 1,33mM, e 30,49 µmoles/min/mg prot, respectively for PNP-glu. The Ki values obtained from Dixon plots was 1,32mM using cellobiose. The products of hydrolisis of cellobiose by the action of purified enzymes glucosidase extracellular I and mycelial were analised in thin-layer-cromatography, and show hydrolisis of cellobiose at 10mM,and transglycosilation reaction when cellobiose was using at 250mM. The intrinsic biochemical and regulatory properties the ß-glucosidase system of Scytalidium support the idea that organism may be useful for biotechnological applications.

A celulose é a mais abundante fonte de carbono presente na madeira e nos resíduos agrícolas, e a sua hidrólise completa é realizada pela ação sinergística de diferentes enzimas, como: as endo-1,4-ß-D-glucanase, exo-1,4-ß-glucanase e ß-glucosidase ou celobiase. O presente trabalho descreve algumas propriedades fisiológicas e bioquímicas do sistema ß-glucosidásico do fungo termofílico Scytalidium thermophilum. Tal fungo foi isolado originalmente do solo da Índia e gentilmente cedido pelo Dr. G. Straastma (Holanda). O meio M8 favoreceu a produção das ß-glucosidases. Entre os açúcares testados como fonte de carbono, avicel e celobiose foram os melhores indutores das ß-glucosidases extracelular e micelial. Quando o fungo foi crescido em dois estágios, observou-se inicialmente a repressão da síntese por glicose e a indução por avicel ou celobiose. Utilizando-se ciclo-heximida, observou-se a síntese \"de novo\" das proteínas. A ß-glucosidase extracelular foi purificada utilizando-se um fracionamento protéico e uma coluna de troca-iônica DEAE-celulose, de onde foram obtidos duas atividades enzimáticas denominadas ß-glucosidases I e II. A ß-glucosidase I foi aplicada em coluna de troca iônica CM-celulose, enquanto que a ß-glucosidase II foi aplicada em Sephadex G-100. A ß-glucosidase I foi purificada 2 vezes com 4.0% de recuperação, ao passo que a ß-glucosidase II foi purificada 2,4 vezes com 2.0% de recuperação. A ß-glucosidase micelial foi purificada utilizando-se um choque térmico, fracionamento protéico, coluna de filtração Sephadex G-100 e uma coluna troca-iônica DEAE-celulose. Foi purificada 23 vezes com recuperação de 25%. A ß-glucosidases extracelular I e micelial apresentaram um temperatura ótima aparente de 70 e 60°C e um pH de 5.5 e 6.0, respectivamente. Ambas enzimas foram inibidas por Ag+2 e Hg+2. A ß-glucosidases extracelular I e micelial possuem um peso molecular de 40.7 kDa e 39kda (SDS-Page) e 57 kDa e 33,8 kda (Sephadex G-100), respectivamente. A ß-glucosidase extracelular I foi capaz de hidrolisar PNP-glu, PNP-xil, celobiose, xilana e CMC, enquanto que a ß-glucosidase micelial hidrolisou PNP-glu, PNP-fuc, PNP-xil, PNPgal, ONPG e lactose. Ambas enzimas foram ativadas por glicerol a 1M. A ß-glucosidase extracelular I foi ativada por xilose, frutose e lactose, e se mostrou resistente a glicose 50mM, enquanto que a ß-glucosidase micelial foi ativada por glicose e xilose. ß-glucosidases extracelular I e micelial apresentaram um PI de 4.0 e 6.5, respectivamente. Os parâmetros cinéticos estimados para a ß-glucosidase extracelular I foram de Km 4,33 e 0,342mM e Vmáx de 5,37 e 2,0µmoles/min/mg prot. para celobiose e PNP-glu, respectivamente. O valor de Ki (Constante de Inibição) foi de 71mM para glicose. Para a ß-glucosidase micelial, os valores de Km e Vmáx foram de 0,29mM e 13,27µmoles/min/mg prot; 0,5 mM e 7,25µmoles/min/mg prot e 1,61 mM e 4,12µmoles/min/mg prot para os substratos PNP-glu, PNP-fuc e celobiose, respectivamente. Na presença de glicose e xilose os valores de Km e Vmáx foram de 1,26mM e 40,04 µmoles/min/mg.prot, e 1,33mM, e 30,49 µmoles/min/mg prot, respectivamente para o PNP-glu. O valor de Ki (Constante de Inibição) foi de 1,32mM para celobiose. A análise dos produtos de hidrólise das ß-glucosidases extracelular I e micelial foram anlisadas em TLC, e revelaram que ambas enzimas realizam hidrólise quando celobiose foi utilizada a 10mM, e transglicosilação quando celobiose foi utilizada a 250mM. Os resultados aqui apresentados demonstram importante papel importante do Scytalidium como produtor de ß-glucosidase com potencial na sacarificação enzimática da celulose. / Cellulose is the most abundant carbon source found in woods and waste residues. In nature the complete hidrolysis of cellulose occurs by the sinergistic action of several enzymes such endo-1,4-ß-D-glucanase, exo-1,4-ß-glucanase e ß-glucosidase or cellobiase. The present work describe some physiological and biochemical properties of ß-glucosidase system from thermophilic fungus Scytalidium thermophilum. The fungus was gift to Dr. Straastma (Mushroom Experimental Station, The Netherlands). The culture medium M8 enhance the production of ß-glucosidase. Among carbohydrates tested as carbon source, avicel and cellobiose were the best inducers of ß-glucosidase extracellular and mycelial. When the fungus was grown in two stages, observed the repression by glucose, and induction by avicel or cellobiose. The presence of cycloheximide inhibited the syntesis of ß-glucosidase, suggesting that the enzyme produced in the presence of indutors required \"de novo\" synthesis. Extracellular ß-glucosidase was purified using the precipitation with 75% amonium sulfate, ion exchange cromatography column DEAE-cellulose, and were obtained two activities: ß-glucosidase extracellular I and II. The ß-glucosidase I was applied to a CM-cellulose colunm, while ß-glucosidase II was applied to a Sephadex G-100 colunm. The ß-glucosidase II was purified two times and 4% yield, and the ß-glucosidase II was purified 2,4 times and 2% yield. The mycelial ß-glucosidase was purified using the termic treatment, a precipitation with 75% amonium sulfate followed by Sephadex G-100 and DEAEcellulose. The enzyme was purified 23 time with 23% yield. The ß-glucosidase extracellular II and mycelial shown optima of temperature and pH of 60°C and 70°C, 4.4 and 6.0, respectively. Hg+2 and Ag+2 ions were strong inhibitors of ß-glucosidase extracellular I and mycelial. The molecular weight of ß-glucosidase extracellular I and mycelial was stimated as 40.7 KDa and 39KDa (SDS-PAGE) and 57kDa and 33.8 kDa (Sephadex G-100). The ß-glucosidase extracellular I hydrolyzed PNP-glu, PNP-xyl, cellobiose,xylan and CMC, while ß-glucosidase mycelial hydrolyzed PNP-fuc, PNP-xyl, PNP-gal, ONPG and lactose. Both enzymes were activeted by glycerol 1M. The ß-glucosidase extracellular I was activeted by xylose, fructose and lactose, and show strong at glucose 50mM. The ß-glucosidase mycelial was activeted by glucose and xylose. ß-glucosidase extracellular I and mycelial shows PI 4.0 and 6.5, respectively. The kinects studies reveled for ß-glucosidase extracellular I a Km of 4,33 and 0,342mM and Vmáx of 5,37 and 2,0µmoles/min/mg prot for cellobiose and PNP-glu, respectively. The Ki values obtained from Dixon plots was 71mM for glucose. To ß-glucosidase mycelial the Km and and Vmáx were 0,29mM e 13,27µmoles/min/mg prot; 0,5 mM e 7,25µmoles/min/mg prot and 1,61 mM and 4,12µmoles/min/mg prot for PNP-glu, PNPfuc and cellobiose, respectively. Using xylose or glucose the Km and Vmáx was 1,26mM e 40,04 µmoles/min/mg.prot, and 1,33mM, e 30,49 µmoles/min/mg prot, respectively for PNP-glu. The Ki values obtained from Dixon plots was 1,32mM using cellobiose. The products of hydrolisis of cellobiose by the action of purified enzymes glucosidase extracellular I and mycelial were analised in thin-layer-cromatography, and show hydrolisis of cellobiose at 10mM,and transglycosilation reaction when cellobiose was using at 250mM. The intrinsic biochemical and regulatory properties the ß-glucosidase system of Scytalidium support the idea that organism may be useful for biotechnological applications.

Malgré l'arsenal antifongique existant aujourd'hui, les mycoses superficielles sont en constante progression de par le monde et notamment dans le bassin caribéen. Nous nous sommes focalisés sur les pathologies qui posent, en Guadeloupe, de nombreux problèmes de résistance ou de rechute vis-à-vis des antifongiques actuels, à savoir : les dermatophyties, le Pityriasis versicolor (Malassezia sp.), les candidoses et les scytalidioses. Suite à ce constat, nous nous sommes tournés vers la flore guadeloupéenne où ont été sélectionnées dix plantes sur des critères ethnobotaniques, chimiotaxonomiques ou encore d'observations naturalistes avec un double objectif : trouver de nouveaux extraits antifongiques d'une part et, d'autre part, étudier leur composition et vérifier leur innocuité. Le screening antifongique in vitro des extraits c-hexane, EtOH et EtOH/eau (1:1, v/v) a été réalisé vis-à-vis de 4 dermatophytes, 1 Malassezia sp., 5 Candida spp. et 1 Scytalidium sp. Les extraits ont également été testés vis-à-vis d'un autre pathogène, Pneumocystis jirovecii responsable de la pneumocystose pulmonaire. Quatre plantes : Bursera simaruba, Cedrela odorata, Enterolobium cyclocarpum et Pluchea carolinensis ont été retenues afin de définir leurs cytotoxicités puis de procéder à l'isolement des composés responsables de leur activité antifongique par bioguidage. Cedrela odorata a montré une activité significative vis-à-vis de Pneumocystis jirovecii due en partie à la (+)-catéchine. Concernant les mycoses superficielles, Bursera simaruba et Cedrela odorata présentent une activité due à une synergie de composés non identifiés par bioguidage alors que Pluchea carolinensis et Enterolobium cyclocarpum doivent respectivement leurs activités à des flavonoïdes sulfatés et à des saponosides triterpéniques. Faisant suite à cette étude phytochimique et pharmacologique, la formulation des extraits sous forme de gels et vernis a été développée. Ainsi, cette étude permet d'apporter une réponse originale et efficace aux pathologies ciblées.

Submitted by Mario BC (mario@bc.ufrpe.br) on 2017-03-14T13:04:27Z No. of bitstreams: 1 Jose Aldo Teixeira da Silva.pdf: 972040 bytes, checksum: d6bba973ec0295e20860c1178753f8e3 (MD5) / Made available in DSpace on 2017-03-14T13:04:27Z (GMT). No. of bitstreams: 1 Jose Aldo Teixeira da Silva.pdf: 972040 bytes, checksum: d6bba973ec0295e20860c1178753f8e3 (MD5) Previous issue date: 2015-10-26 / Cassava has economic importance in Brazil and abroad, for their food importance. The Northeast is the region that most increased this crop in Brazil, but farmers use few technologies, resulting in diseases and decline in production. The crop is attacked for numerous pathogens as Fusarium solani and Scytalidium lignicola, that causes diseases such as cassava root rot and cassava black root, affecting the commercial part of plant, the tubera. Several research, support the use of practices that enable the production and support the environment, so the use of biocontrol has been used, particularly with the use of Trichoderma spp. However, little research reporting the physiological mechanisms that are activated by plants when exposed to this interaction, pathogen-plant-antagonist such as plant response to infection diseases. Thus, the objective this work was to verify the potential antagonism of Trichoderma spp. against F. solani and S. lignicola, and evaluating the physiological response of plants exposed to the pathosystem. We analyzed in vitro inhibition of mycelial growth of F. solani and S. lignicola to select the best Trichoderma antagonist with potential direct these pathogens. By the method of use of basal medium with colloidal chitin as the only carbon source, it was used to evaluate the best Trichoderma production of chitinase. In greenhouse, the selected Trichoderma was compared against the use of plant resistance inducer in vivo inhibition assessing the severity of disease infested plants after 92 days of growth. Then we evaluated the enzyme of antioxidative complex (peroxidase ascorbate, catalase, peroxidase and polyphenyl oxidase). All strains were inhibited the growth of pathogens. However, the best strain against F. solani was T. hamatum (6656), and has been for S. lignicola was T. harzianum (3086), with values of 88.91 and 80.78% growth mycelial respectively, being designated as the best candidates for treatments in the greenhouse. For chitinase production evaluation, all Trichoderma tested were positive, highlighting the T. aureoviride (5158) to produce 6.70 U mL-1, being selected candidate for inhibiting the severity of pathogens in greenhouse. All Trichoderma selected for in vivo testing against the severities of diseases, presented efficiency compared to the control with the presence of the pathogen. However, the T. aureoviride (5158), showed the blunt values for both pathogens. The results of the enzymes of the plants, treatments that stopped inoculation of Trichoderma showed satisfactory results, collaborating with the answer in a greenhouse, however, again treating with the strain (5158), showed the best values, especially the production of the enzymes peroxidase and ascorbate peroxidase. Therefore, concludes the efficiency of Trichoderma aureoviride (5158), for use as biocontrol of root rot and black rot of cassava as induced resistance to plant pathogens, contributing to the production of enzymes antioxidatives. / A mandioca tem grande expressão econômica no Brasil e no mundo, pela sua importância alimentar. O Nordeste é a região que mais aumentou a produção dessa cultura no Brasil, porém, os produtores usam poucas tecnologias, acarretando em possíveis desenvolvimentos de doenças e queda na produção. A cultura sofre muitas perdas na produção pela ação de inúmeros fitopatógenos, sendo os do solo os mais severos, o Fusarium solani e Scytalidium lignicola, ganham ênfase por causarem doenças como a podridão radicular e negra da mandioca, respectivamente, afetando diretamente a parte comercial da planta, a tubera. Muitas pesquisas, apoiam o uso de práticas que viabilizem a produção e favoreçam o ambiente, assim, a utilização de biocontroladores vem ganhando destaque, principalmente fungos do gênero Trichoderma spp., por suas características como antagonistas de uma gama de fitopatógenos. Todavia, há poucas pesquisas que relatam os mecanismos fisiológicos que são ativados pelas plantas, quando expostos a essa interação, patógeno-planta-antagonista, como resposta vegetal a infecção de doenças. Desse modo, objetivou-se verificar o potencial antagônico de dez Trichoderma spp. aos patógenos F. solani e S. lignicola, e avaliar a resposta fisiológica das plantas exposta a esse patossistema. Foi analisado, in vitro, a inibição do crescimento micelial do F. solani e S. lignicola em meio batata-dextrose-ágar, para selecionar o melhor Trichoderma com potencial antagonista direto a esses patógenos. Através do método da utilização de meio basal com quitina coloidal, sendo a única fonte de carbono, foi utilizada para avaliar o melhor Trichoderma para produção de quitinase. Em casa de vegetação, foi comparado os Trichoderma selecionados, contra o indutor de resistência, in vivo, avaliando a inibição das doenças infestadas nas plantas, após 92 dias de crescimento. Em seguida, avaliou-se a produção de enzimas das plantas dos tratamentos in vivo, do complexo oxidativo (ascorbato peroxidase, catalase, peroxidase e polifeniloxidase). Todas as estirpes apresentaram inibição do crescimento dos fitopatógenos. Porém, a melhor estirpe contra o F. solani foi o T. hamatum (6656), e para o S. lignicola foi o T. harzianum (3086), com valores de 88,91 e 80,78% de inbiçao de crescimento micelial, respectivamente, sendo designados como os melhores candidatos para tratamentos em casa de vegetação. Para a avaliação de produção de quitinase, todos os Trichoderma testados foram positivos, destaque dado ao T. aureoviride (5158) por produzir 6,70 U mL-1, sendo selecionado para candidato da inibição da severidade dos patógenos em casa de vegetação. Todas os Trichoderma selecionados para teste in vivo contra as severidades das doenças, apresentaram eficiência em comparação ao controle com a presença do patógeno. No entanto, o T. aureoviride (5158), foi a que apresentou valores contundentes para os dois patógenos. Dos resultados das enzimas das plantas, os tratamentos que detiveram a inoculação dos Trichoderma apresentaram resultados satisfatórios, colaborando com a resposta em casa de vegetação, contudo, novamente o tratamento com a estirpe (5158), foi a que apresentou melhores valores, com destaque a produção das enzimas ascorbato peroxidase e peroxidase. Portanto, conclui-se a efiência do Trichoderma aureoviride (5158), para uso como biocontrolador da podridão radicular e da podridão negra da mandioca, pois induziu resistência a planta aos fitopatógenos, colaborando com a produção das enzimas antioxidativas.

In this study, Scytalidium thermophilum laccase gene was first cloned into E. coli and then heterologously expressed in A. sojae. S. thermophilum is a thermophilic fungus with an important role in determining selectivity of compost produced for growing Agaricus bisporus. S. thermophilum laccase gene was first cloned by Novo Nordisk Bio Tech, Inc. in 1998. This laccase gene (lccS) has an open reading frame of 2092bp. It is composed of five exons punctuated by four small introns. The coding region, excluding intervening sequences is very GC-rich (60.8% G+C) and encodes a preproenzyme of 616 amino acids: a 21 amino acid signal peptide and a 24 amino acid predicted propeptide. lccS gene was amplified using specific primers to exclude the signal and pro-peptide coding regions and ligated to expression vector pAN52-4. The recombinant plasmid was used to transform Aspergillus sojae ATCC11906 (pyrG-). Heterologuos expression was observed in glucose-containing media, under the control of the glyceraldehydes 3-phosphate dehydnogenese promoter and the secretion signal of glucoamylase gene. Laccase gene is an important step towards the high level expression of this enzyme in a GRAS eucaryotic host and for further biotransformation and enzyme engineering studies. In this study also bioinformatic analysis of N-terminal and C-terminal propeptide cleavage sites of fungal proteins including laccases were studied.

Scytalidium thermophilum is a termophilic fungus that effectively produces the extracellular enzyme catalase phenol oxidase (CATPO). The enzyme is distinct among catalases with its bifunctionality of oxidising phenolic compounds in the absence of H2O2. CATPO is capable of oxidizing catechol, chlorogenic acid, caffeic acid and catechin which are ortho &ndash / diphenolic compounds. Diphenolic compounds are known as strong antioxidants. Catalase is one of the important antioxidant enzymes. Therefore, in this thesis the effect of CATPO on the final antioxidant capacity of the oxidized products was analysed. Antioxidant capacity measurements of oxidized and unreacted phenolic compounds were done using the two widely used methods TEAC and FRAP. CATPO oxidized catechol showed 2.4 fold increase when compared to its nonoxidized form, which was highest among others. Catechol was followed by caffeic acid, chlorogenic acid, and catechin. This finding is new to the literature and may be of importance to the antioxidant mechanism of organisms. Results have also shown that the most well known phenol oxidases, laccase and tyrosinase, do not result in such high increases in antioxidant capacity upon oxidation of the substrates tested. Due to this finding, as a possible means of applying CATPO to increase the antioxidant capacity of products daily consumed, tea was selected. Tea is the second most consumed beverage after water and it is known to possess high amounts of flavanols. Green tea is rich in catechins whereas black tea is a rich source of theaflavins and thearubigins. Fermentation is a critical process for production of good quality tea and is the key step differing between green and black tea production. During this process phenol oxidases catalyze the oxidation of polyphenolic compounds present in tea leaves to their corresponding o-quinones. Utilization of CATPO in tea samples resulted in an increase in antioxidant capacity and its effect was enhanced by an increase in brewing time. Interestingly, the addition of sugar decreased antioxidant capacity. Laccase and tyrosinase were ineffective in increasing the antioxidant capacity of tea samples.

Endo-b-1,4 xylanases (EC. 3.2.1.8) are typically produced as a mixture of different hydrolytic enzymes such as b-1,4-xylosidase (EC. 3.2.1.37) , a-Larabinofuranosidases (EC. 3.2.1.55), and feruloyl esterase (EC 3.1.1.73) that hydrolyze xylan molecule, which constitutes 20-30% of the weight of wood and agricultural wastes. Thus, xylan, a renewable biomass, can be utilized as a substrate for the preparation of many products such as fuels, solvents and pharmaceuticals. Besides, xylanolytic enzymes themselves are also used in food,feed, textile industries and pre-bleaching of kraft. In the first part of the study, xylanolytic systems of a soil isolate Bacillus pumilus SB-M13 and a thermophilic fungus Scytalidium thermophilum were investigated. Production rate and type of xylanolytic changed depending on the carbon source and the microorganism. However, xylanolytic enzyme production was found to be sequential, in synergy and under the control of carbon catabolite repression for both microorganisms. In the second part, B. pumilus SB-M13 b-1,4 xylanase was purified and biochemically characterized. The enzyme was stable at alkaline pHs and highest activity was observed at 60&deg / C and pH 7.5. Enzyme Km and kcat values were determined as 1.87 mg/ml and 43,000 U/mg, respectively. B. pumilus SB-M13 and S .thermophilum a-L-arabinofuranosidases were also purified and biochemically characterized. Although produced from a mesophilic microorganism, B. pumilus SB-M13 arabinofuranosidase was quite thermostable. Moreover, unlike other fungi, S. thermophilum produced alkaline stable arabinofuranosidases. Both enzymes were multimeric, alkaline stable and most active at 70&deg / C and pH 7.0. However, when compared to S. thermophilum, catalytic power of B. pumilus SB-M13 arabinofuranosidase was higher.

In this study, the aim was identification and classification of the enzyme having phenol oxidase activity produced by a thermophilic fungus, Scytalidium thermophilum. For this purpose, enzyme production, purification, biochemical characterization and structural analysis by X-ray crystallography studies have been performed. At the beginning of the research, this enzyme was considered as a phenol oxidase and analyzed accordingly. However, during purification, amino acid sequencing and structural studies, the enzyme was shown to be a catalase, with an additional catechol oxidase activity. This novel bifunctional catalase-catechol oxidase (CCO) was purified 10 fold with 45 % yield by anion exchange and gel filtration chromatographies. CCO was determined as a tetrameric protein having total and subunit molecular weights of 320 and 80 kDa, respectively. Isoelectric point of CCO was verified as 5.0. CCO catalase and catechol oxidase activities were characterized in terms of their kinetic behavior at different pH and temperatures. Depending on the substrate specificity and inhibitor studies of CCO, the phenol oxidase activity was determined as catechol oxidase but not tyrosinase or laccase. The best crystallization condition for CCO was determined and X-ray diffraction data was collected at the Daresbury Synchrotron Radiation Source (United Kingdom) at 2.7 &Aring / resolution. The preliminary structure was solved by molecular replacement method using Penicilium vitale catalase structure. CCO was verified to have a tetrameric structure with two homodimers and a metal center in each polypeptide chain.

Scytalidium thermophilum is a thermophilic fungus with an important role in the composting process of mushroom cultivation. An extracellular phenol oxidase of Scytalidium thermophilum (STEP) with novel features was previously studied in our laboratory. This enzyme later turned out to be a catalase having phenol oxidase activity. The aim of this study was to clone Scytalidium thermophilum bifunctional catalase/phenol oxidase encoding gene and express the gene in Aspergillus sojae for future site directed mutagenesis studies. Scytalidium thermophilum catalase gene was first cloned into E. coli XL1 Blue MRF&rsquo / and then heterologously expressed in Aspergillus sojae ATCC11906. For that aim, the catalase gene was amplified using specific primers, excluding the signal and pro-peptide coding regions and amplified fragment was then cloned into E.coli XL1 Blue MRF&rsquo / and sequenced. It was observed that the cloned gene, named as catpo, was 10 amino acids different from the amino acid sequence of the S.thermophilum catalase gene formerly cloned by Novo Nordisk. The catpo gene encoding a mature protein of 681 amino acids was then ligated onto expression vector pAN52-4 and the recombinant plasmid was transformed into Aspergillus sojae ATCC11906. Heterologous expression was observed under the control of the glyceraldehydes 3-phosphate dehydrogenese promoter of Aspergillus nidulans and the secretion signal of the glucoamylase gene of Aspergillus niger. Catalase activity of the transformants reached at a level of 13206 U/g at the end of the fourth day of cultivation. However, this is still lower than the catalase activity of the gene donor strain of Scytalidium thermophilum.